Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies

Chemokine Receptors and NeuroAIDS



Olimpia Meucci Editor

Chemokine Receptors and NeuroAIDS Beyond Co-Receptor Function and Links to Other Neuropathologies



Editor Olimpia Meucci Departments of Pharmacology and Physiology & Institute of Molecular Medicine and Infectious Disease Drexel University College of Medicine Philadelphia, PA USA [email protected]

ISBN 978-1-4419-0792-9 e-ISBN 978-1-4419-0793-6 DOI 10.1007/978-1-4419-0793-6 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2009930629 © Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

This book is dedicated in memory of my father, Dante Meucci, a surgeon of rare professional and human qualities who infused my three brothers and me with the passion for science, medicine, and caring for others; and to my mother, Rosaria Rago Meucci for showing me the joy of discoveries and always supporting my choices.

Preface

This is a remarkably timely volume for several distinct reasons. First, it captures an inflection point in the neurological consequences of the HIV pandemic. With the widespread use of highly active anti-retroviral therapy (HAART), devastating syndromes of HIV-associated dementia and vacuolar myelopathy have given way, respectively, to milder complications including cognitive impairment and painful peripheral neuropathy. The present book addresses mechanisms of these contemporary disorders. Second, there is an extensive discussion of pathways by which HIV infection, or its treatment, can lead to pain. Chemokines and their receptors constitute an integral part of this process, and this book contains the largest compilation to date, of concentrated information about the implication of chemokine biology in pain. Lastly, the book addresses chemokine receptor CXCR4 from multiple perspectives. This receptor and CXCL12, its ligand, participate in virtually every phase of biology from development, through organization and deployment of immune and nervous-system elements, infection, immunity, neurophysiology, adult neurogenesis, and functions of numerous tissue stem cells. The combination of these varied unique emphases renders this book highly topical. Beginning Sect. I, Kolson and colleagues provide a measured consideration of a wide variety of research methods from magnetic resonance spectroscopy (MRS) to analysis of HIV proteins in in  vitro systems. Fischer-Smith and Rappaport treat several overlapping topics, in effect yielding a “second opinion” about how to apply current research methods in the study of HIV-related neurological disease. Tan, Hoke, and Nath effectively and forcefully integrate clinical and pathogenetic data related to the crucial topic of HIV disease and peripheral neuropathy. Wigdahl’s group contributes an authoritative and accessible treatment of HIV-viral latency embellished with well-designed graphics to illustrate complex concepts. Klein and coworkers combine a lucid review of chemokine receptors responsible for leukocyte trafficking with the presentation of a novel hypothesis, drawn from their research, about the role of CXCL12/CXCR4 as organizers of CNS perivascular infiltrates. Section II opens with a fascinating discussion by Vergote, Overall, and Power on how proteolytic cleavage of CXCL12 by MMP2 mediates neurotoxicity via an aberrant ligand–receptor interaction with CXCR3. Rostene et al. provide a succinct summary of the role(s) of CXCR4 in activating nociceptors in the context of HIV vii

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infection plus anti-retroviral agent treatment and also discuss the function(s) of CCL2/CCR2 in generating neuropathic pain. This latter topic is amplified in Miller’s masterful discussion of CCR2 in neuropathic pain, in a chapter which also explains the roles of chemokine receptors in neural development and adult neurogenesis. Khan addresses the complex field of chemokine receptor interactions with cell-cycle regulatory components such as Rb and p53, as an entrée to comparing signaling to CXCR4 by HIV gp120 and the cardinal ligand CXCL12. Rubin contributes a subtle and comprehensive chapter on chemokine receptors in glioma, extending from cell biology to signaling to therapeutic application. Bezzi et  al. discuss the critical issue of glutamate as a gliotransmitter, and places CXCR4 in the context of astrocyte regulation of synaptic activity. They incorporate unexpected roles of TNFa and prostaglandins as signaling intermediates, and also cover fully the topic of glutamate exocytosis by astrocytes, and its dysregulation during neuroinflammatory pathologies including HIV-associated cognitive impairment. Limatola and colleagues elegantly dissect how stimulation of CX3CR1 on microglia leads to adenosine production, which acts through A1R adenosine receptor on neurons, mediating neuroprotection. Section III provides a very extensive and multifaceted treatment of the interactions between the chemokine and opioid system. These interactions affect the roles of chemokine receptors in pain, in HIV infections and immune-system functions, as well as in the emerging role of selected chemokines as neuromodulators/neurotransmitters – aspects that become particularly important when considering the prominent population of HIV-positive individuals who also abuse opiates. A contribution by Roger’s group opens the section; these authors focus on their pioneering work regarding bidirectional communication between chemokine and opioid receptors in immune cells and address the relevance of these interactions to HIV infection. Martin and Roy discuss the major mechanisms of dysregulation of innate immunity by morphine and its implications for HIV progression, while providing interesting insights into the role of these mechanisms in wound healing. Next is a comprehensive review by Hauser et al. about the unique regulation of glial cells by opiates and a detailed dissection of the respective contribution of astroglia and microglia to HIV neuropathology in patients with history of drug abuse. This section is concluded by the work of Sengupta and Meucci introducing novel mechanisms implicated in negative modulation of neuronal CXCR4 by opiates, which significantly alter CXCR4-mediated signaling in the brain. This pathway may have significant consequences for the physiological actions of the CXCR4 receptor in the nervous system and may also promote disease progression. These contributions represent major groups in this important field and fulfill a notable void in the current literature. Ohio, USA

Richard M. Ransohoff

Acknowledgments

First, I would like to thank all those who contributed to this book as they enthusiastically agreed to participate in spite of their demanding academic and/or clinical responsibilities. Without their help and friendship, this book would not exist. I feel fortunate to have them as colleagues. In particular, I would like to thank Richard Ransohoff, who provided invaluable insights when the project started, helped me put things in perspective, and kindly wrote the book overview. I also received tremendous and continuous support from Richard J. Miller; while I was in his lab at the University of Chicago, my interest for chemokines sparkled and he let me develop it without any limitation and help me gain independence. I have been lucky to find such great mentors, colleagues, and friends in Philadelphia as well. Some of them, Dennis Kolson, Jay Rappaport, and Brian Wigdahl, also contributed to the book with their own work, and I am grateful for their time and efforts. My gratitude also goes out to Ann Avouris, Neuroscience Editor at Springer, for guiding me with patience and knowledge through my first book experience. Ann and her assistants, Elisabeth Thompson and Melissa Higgs, made my job easier and more pleasant. It was a new challenge for me, but they made it fun. I would not be here without the support of the NIH, NIDA in particular, which has generously sponsored my work throughout these years. I am particularly grateful to Charles Sharp, who successfully walked me through my very first grant application as a “new investigator” in 2001. Though he recently retired, he is still very involved with science and always provides precious inputs. Likewise, all the other members of NIDA I have personally interacted with – Diane Lawrence, Albert Avila, David Shurtleff – are always available, supportive, and enthusiastic. Their positive attitude is contagious and helps science move forward. Special thanks also go out to all the members of my research group that supported this project as needed and nicely coped with my limited availability to them when dealing with any book-related issues. By now they should have returned to be my main priority, so I hope we have fully resumed our regular lab meetings and one-on-one discussions. Last but not least, I wish to thank my husband and son: they cherish me and make me laugh, and expect nothing but the same in return. Thank You All Philadelphia, USA

Olimpia Meucci

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Contents

1 Introduction................................................................................................

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Section I 2 HIV Neuroinvasion: Early Events, Late Manifestations........................ Maria F. Chen, Samantha Soldan, and Dennis L. Kolson

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3 HIV Co-receptors: The Brain Perspective............................................... Tracy Fischer-Smith and Jay Rappaport

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4 HIV Infection and the PNS....................................................................... Kevin Tan, Avindra Nath, and Ahmet Hoke

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5 HIV Latency and Reactivation: Role in Neuropathogenesis................. Anupam Banerjee, Michael R. Nonnemacher, and Brian Wigdahl

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6 HIV Coreceptors and Their Roles in Leukocyte Trafficking During Neuroinflammatory Diseases................................... 119 Robyn S. Klein and Erin E. McCandless Section II 7 Chemokine Proteolytic Processing in HIV Infection: Neurotoxic and Neuroimmune Consequences......................................... 149 David Vergote, Christopher M. Overall, and Christopher Power 8 Chemokines and Chemokine Receptors in the Brain............................. 173 Stéphane Mélik Parsadaniantz, Ghazal Banisadr, Philippe Sarret, and William Rostène

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  9 Chemokine Signaling in the Nervous System and Its Role in Development and Neuropathology............................... 191 Richard J. Miller 10  Modulation of Neuronal Cell Cycle Proteins by Chemokine Receptors and Its Role in the Survival of Postmitotic Neurons........... 221 Muhammad Z. Khan 11 Chemokines and Primary Brain Tumors............................................... 253 Shyam S. Rao, Mahil Rao, Nicole Warrington, and Joshua B. Rubin 12 Chemokines as Neuromodulators: Regulation of Glutamatergic Transmission by CXCR4-Mediated Glutamate Release From Astrocytes....................................................................................... 271 Corrado Calì, Julie Marchaland, Osvaldo Mirante, and Paola Bezzi 13 Role of CX3CL1 in Synaptic Activity and Neuroprotection................ 301 Davide Ragozzino, Clotilde Lauro, and Cristina Limatola Section III 14 Interaction Between Opioid and Chemokine Receptors in Immune Cells: Implications for HIV Infection................................. 319 Christine Happel, Changcheng Song, Mathew J. Finley, and Thomas J. Rogers 15 Chronic Morphine’s Role on Innate Immunity, Bacterial Susceptibility and Implications in Wound Healing............................... 337 Josephine Martin and Sabita Roy 16 Opioids, Astroglial Chemokines, Microglial Reactivity, and Neuronal Injury in HIV-1 Encephalitis.......................................... 353 Kurt F. Hauser, Nazira El-Hage, Annadora J. Bruce-Keller, and Pamela E. Knapp 17 Regulation of Neuronal Chemokine Receptor CXCR4 by m-Opioid Agonists and Its Involvement in NeuroAIDS................... 379 Rajarshi Sengupta and Olimpia Meucci About the Editor.............................................................................................. 399 About the Book................................................................................................. 401 Index.................................................................................................................. 403

Contributors

Anupam Banerjee Department of Microbiology and Immunology, Drexel University, 2900 Queen Lane, Philadelphia, PA, USA Paola Bezzi Department of Cellular Biology and Morphology (DBCM), Faculty of Medicine, University of Lausanne, UNIL – Bugnon, Rue du Bugnon 21, CH-1015 Lausanne, Switzerland Annadora J. Bruce-Keller Division of Basic Research, Pennington Biomedical Research Center, Louisiana State University Baton Rouge, LA, USA Maria F. Chen Department of Neurology, Hospital of the University of Pennsylvania, 3 West Gates, 3400 Spruce Street, Philadelphia, PA, USA Cali Corrado Department of Cellular Biology and Morphology (DBCM), Faculty of Medicine, University of Lausanne, UNIL – Bugnon, Rue du Bugnon 21, CH-1015 Lausanne, Switzerland Nazira El-Hage Department of Pharmacology & Toxicology, Virginia Commonwealth University, School of Medicine, 1217 East Marshall Street, Richmond, VA, USA Mathew J. Finley Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, PA, USA Tracy Fischer-Smith Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, Philadelphia, PA, USA

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Christine Happel Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, PA, USA Kurt F. Hauser Department of Pharmacology and Toxicology, Virginia Commonwealth University School of Medicine, 1217 East Marshall Street, Richmond, VA, USA Ahmet Hoke Neuromuscular Division, Department of Neurology, Johns Hopkins University, Path 509, 600 N. Wolfe St., Baltimore, MD, USA Muhammad Z. Khan Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA, USA Robyn S. Klein Department of Internal Medicine, Division of Infectious Diseases, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO, USA Pamela E. Knapp Department of Anatomy and Neurobiology, Virginia Commonwealth University School of Medicine, 1217 East Marshall Street, Richmond, VA, USA Dennis L. Kolson Department of Neurology, Hospital of the University of Pennsylvania, 3 West Gates, 3400 Spruce Street, Philadelphia, PA, USA Clotilde Lauro Department of Physiology and Pharmacology, University Sapienza, Piazzale A. Moro, 5, 00185 Rome, Italy Cristina Limatola Department of Physiology and Pharmacology, University Sapienza, Piazzale A. Moro, 5, 00185 Rome, Italy Julie Marchaland Department of Cellular Biology and Morphology (DBCM), Faculty of Medicine, University of Lausanne, UNIL – Bugnon, Rue du Bugnon 21, CH-1015 Lausanne, Switzerland Josephine Martin Department of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 312 Church Street SE, Minneapolis, MN, USA Erin E. McCandless Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO, USA Olimpia Meucci Department of Pharmacology and Physiology & Microbiology and Immunology, Drexel University College of Medicine, New College Building, 8804, 245 N 15th St., Philadelphia, PA, USA

Contributors

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Richard J. Miller Department of Molecular Pharmacology and Biological Chemistry, Northwestern University School of Medicine, 303 E Chicago Avenue, Chicago, IL, USA Osvaldo Mirante Department of Cellular Biology and Morphology (DBCM), Faculty of Medicine, University of Lausanne, UNIL – Bugnon, Rue du Bugnon 21, CH-1015 Lausanne, Switzerland Avindra Nath The Johns Hopkins University, 600 N. Wolfe St., Room: 509 Pathology, 600 N. Wolfe St., Baltimore, MD, USA Michael R. Nonnemacher Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, USA Christopher M. Overall Department of Biochemistry & Molecular Biology, Center for Blood Research, University of British Columbia, 4th Floor, Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, BC, Canada Stéphane Mélik Parsadaniantz INSERM U 732-UPMC, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France Christopher Power Department of Medicine (Neurology), 6-11 Heritage Medical Research Centre, University of Alberta, Edmonton, AB, Canada T6G 2S2 Davide Ragozzino Istituto Fisiologia Umana, Piazzale A. Moro 5, I00185 Roma, Italy Richard M. Ransohoff Neuroinflammatory Research Center, Lerner Research Institute/NC30, 9500 Euclid Avenue, Cleveland, OH, USA Mahil Rao Department of Pediatrics, St. Louis Children’s Hospital, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO, USA Shyam S. Rao Department of Radiation Oncology, Department of Pediatrics, St. Louis Children’s Hospital, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO, USA Jay Rappaport Department of Neuroscience, Temple University School of Medicine, 1900 N. 12th St., Biology Life Science Bldg., Rm. 246, Philadelphia, PA, USA

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Thomas J. Rogers Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, PA, USA William Rostène INSERM U732-UPMC, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France Sabita Roy Department of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 312 Church Street SE, Minneapolis, MN, USA Joshua B. Rubin Department of Pediatrics, St. Louis Children’s Hospital, Washington University School of Medicine, Campus Box 8208, 660 South Euclid Avenue, St. Louis, MO, USA Philippe Sarret Département de Physiologie et Biophysique, Faculté de Médecine et des Sciences de la Sante, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, QC, Canada Rajarshi Sengupta Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 N 15th St., Philadelphia, PA, USA Samantha Soldan Department of Neurology, Hospital of the University of Pennsylvania, 3 West Gates, 3400 Spruce Street, Philadelphia, PA, USA Changcheng Song Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, PA, USA Kevin Tan Department of Neurology, National Neuroscience Institute, 11 Jalan Tan Tock Seng, Singapore, Republic of Singapore David Vergote Department of Medicine, 6-11 Heritage Medical Research Centre, University of Alberta, Edmonton, AB, Canada T6G 2S2 Nicole Warrington Department of Pediatrics, St. Louis Children’s Hospital, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO, USA Brian Wigdahl Department of Microbiology & Immunology (G44), Drexel University, 2900 Queen Lane, Philadelphia, PA, USA

Chapter 1

Introduction

During the last two decades chemokine receptors have consistently been in the spotlight albeit for different reasons. They have “evolved” from coordinators of inflammatory/immune responses to HIV co-receptors, mediators of organogenesis and development, cancer metastases inducers, and neuromodulators. However, it seems that our knowledge of the physiological and pathological roles of this large subfamily of G-protein-coupled receptors has not yet reached a plateau. Novel and essential biological functions of chemokine receptors are continuously been discovered and each of these new discoveries has led to alternative therapeutic strategies that show promise in the treatment of several pathologies, including AIDS and AIDS-related conditions. HIV neuropathology is a complex disease, whose development and progression depends on the interplay of an array of host and viral factors. Although much has been learned about how the HIV virus may enter and damage the nervous system, the events ultimately leading to neurological dysfunction are still not clear and many questions remain about the factors that affect disease progression. Modern antiretroviral therapies have significantly changed the manifestations of the HIVinduced neurological syndrome, however they have also introduced novel problems, such as ambiguity about the nature of the minor motor cognitive disorders, alteration of the natural disease process (a confounding factor with respect to neuropathogenesis), and, importantly, drug toxicity. Furthermore, owing to their relatively low efficacy to crossing the blood–brain barrier and the presence of viral reservoirs, these drugs still do not provide full protection from HIV entry and replication in the brain. Unfortunately, even though combinatorial antiretroviral therapies have reduced the incidence and severity of the HIV neurological complications, their prevalence has increased due to the longer life span of treated patients. Excellent publications have previously covered these and other important aspects of HIV neuropathogenesis (see for example “The neurology of AIDS,” 2005 – Oxford University Press), which are not reviewed in the present book. Our focus has been on unresolved or emerging issues concerning the role of chemokine receptors in neuronal injury and HIV neuropathology – including their ability to regulate fundamental neuronal and glial functions, and their role in neurovirulence and neurotoxicity. Although the importance of these molecules in the CNS is now apparent, O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_1, © Springer Science+Business Media, LLC 2010

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further research is required to design effective pharmacological agents that specifically target the brain chemokine system without major side effects. To this end, specific topics have been reviewed by international experts with the ultimate goal of encouraging future investigation in the most controversial areas and fostering interaction between clinicians and basic scientists. A secondary goal of this volume is to increase awareness about differences in disease progression in certain patient populations, specifically drug abusers, which may also help identifying novel therapeutic strategies. Due to space and time limitations some important (still developing) topics could not be covered in this volume. A prime example is the recent “re-discovery” of CXCR7 as a new binding site for the chemokine SDF/CXCL12. However, at this time, very little is known about the protein expression and function of this chemokine receptor in cells of the CNS in mammals. The lack of a brain phenotype in CXCR7-deficient animals, the overlapping deficits found in mice lacking CXCR4 or CXCL12, and the apparent absence of typical signaling via CXCR7 independent of CXCR4, would suggest that this receptor does not play a primary role in the signaling triggered by SDF/CXCL12 in the CNS. However, it may act as a regulator of CXCR4 activity. Different groups are currently investigating this issue, which clearly deserves to be closely monitored. Conversely, other subjects that were previously not fully recognized and have only recently reached a research stage that would benefit from discussion have been given more attention. For instance, the last section of this book is dedicated to the interaction between chemokine and opioid receptors in both immune and nervous systems. In recent years this has been a very active area of research as HIV infection and drug abuse are tightly correlated; drug abuse is a co-morbidity factor for AIDS and seems to promote progression to neuroAIDS. In developed countries, drug abusers account for about a third of newly infected HIV-patients. Furthermore, the interaction between chemokine and opioid receptors is also relevant to the involvement of chemokines in neuropathic pain. The uniqueness of this publication lies in pursuing the idea that HIV infection may interfere with the mundane effects of chemokines at different levels and lead to neuropathology by altering the normal function of chemokine receptors in the CNS. This would imply that chemokine receptors are to be investigated in different ways (i.e. as membrane receptors with multiple functions in neural cells, regulators of innate immune responses, or HIV co-receptors) depending on whether the focus is on neuronal deficits or viral entry processes. It also means that discoveries in this field would support progress of therapeutic interventions for other neuropathologies. Olimpia Meucci

Section I

Chapter 2

HIV Neuroinvasion: Early Events, Late Manifestations Maria F. Chen, Samantha Soldan, and Dennis L. Kolson

2.1 Clinical Manifestations and Epidemiology of HIV Infection of the Nervous System Although the widespread use of HAART has significantly improved neurological outcomes in individuals infected with HIV-1, a relatively high risk (~30%) for developing neurocognitive dysfunction caused by HIV replication within cellular reservoirs (macrophages/microglia) in the CNS remains (McArthur 2004; Roc et al. 2007; Sacktor 2002). Furthermore, damage to the peripheral nervous system (PNS) in HIV-infected individuals is probably equally prevalent, reflecting the effects of antiretroviral drug toxicity and persistent HIV replication in similar peripheral cellular reservoirs (Keswani et al. 2002; McArthur 2004). HIV-associated neurocognitive disorders (now collectively referred to as HAND) can present with a spectrum of severity: HIV-associated dementia (HAD) and less severe forms that have been categorized by selective criteria based upon both behavioral and neuropsychological test performance, minor cognitive motor disorder (MCMD), HIVassociated mild neurocognitive disorder (MND), and asymptomatic neurocognitive impairment (ANI) (Antinori et al. 2007). Generally, HAD manifests as a subcortical dementia characterized by psychomotor slowing, behavioral changes, and deficits in memory, abstraction, information processing, verbal fluency, decision-making, and attention; also, its progression is relatively slow (years). These cognitive impairments suggest pathological involvement of the fronto-striato-thalamo-cortical circuits (Woods et al. 2004), and recent studies have demonstrated that synaptic and dendritic damage within the hippocampus and putamen is highly correlated with the degree of cognitive impairment (Moore et al. 2006). Moreover, pathological studies have demonstrated that the mere presence of antemortem neurocognitive impairment is predictive of the M.F. Chen, S. Soldan, and D.L. Kolson (*) Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA email: [email protected]

O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_2, © Springer Science+Business Media, LLC 2010

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pathological diagnosis of HIV encephalitis at death (positive predictive value = 95%) (Cherner et al. 2002). Despite the strong correlations between structural brain damage and the severity of neurocognitive impairment in HIV infection, these clinical deficits are not absolutely irreversible. The milder HAND disorders do not uniformly progress to HAD nor do those with HAD always present as milder disorders (Ellis et  al. 2007). Furthermore, a significant number of individuals with HAD (up to 22%), irrespective of the use of HAART, can revert to normal or greatly improved cognitive function, for as yet undefined reasons (McArthur 2004; Sacktor 2002). To date, however, the most obvious factor altering the natural history of cognitive dysfunction in HIV infected individuals is HAART. Prior to the widespread use of HAART in the United States (1996), approximately 20% of HIV-positive individuals suffered from HAD and up to 40% suffered from milder HAND disorders (McArthur 2004; Sacktor 2002; Sacktor et al. 2001). Since then, however, the incidence of HAD has decreased (~8%), while its prevalence has increased slowly, possibly due to increased survival and associated vulnerability of the aging brain to effects of even low-level HIV replication (Becker et al. 2004; Bhaskaran et al. 2008; McArthur 2004; Sacktor 2002; Sacktor et al. 2001; Valcour and Paul 2006; Valcour et al. 2004). This longer life expectancy may also partly explain the trend of increased presentation of HAD in individuals with CD4 T-cell counts greater than 200 cells/mm3, which was rare during the pre-HAART era, when cognitive dysfunction was much more frequently associated with severe immunosuppresion (Bhaskaran et al. 2008; Ellis et al. 1997) or generally poor health associated with anemia and low weight (McArthur et al. 1993) (Sacktor et al. 2001). Within HIV infected populations of sub-Saharan Africa (Uganda), the prevalence of HAD has been estimated at 31% (72% in this cohort were HAART-naïve) (Wong et al. 2007), which is near the pre-HAART prevalence of HAD in the United States. Improvement in HAND with HAART administration has been documented in cohort studies in North America, Europe, Australia, and elsewhere (d’Arminio Monforte et al. 2004; Dore et al. 2003; Gray et al. 2001; May et al. 2007; Robertson et al. 2004; Sacktor et al. 2003; Sacktor et al. 2000; von Giesen et al. 2002), including sub-Saharan Africa (Sacktor et al. 2006). If our experience in developed countries accurately predicts the natural history of HAND in post-HAART individuals in other such regions of the world, we can anticipate persistence of at least milder HAND syndromes throughout these regions of the world as in developed countries. It appears that the major changes in the natural history of HAND syndromes in the post-HAART era are slower and more variable progression, less predictable progression to death, and significant improvement in some subsets of patients. In addition, concern about the increasing incidence of peripheral neuropathy, because of the prolonged use of nucleoside reverse transcriptase inhibitors (NRTs) common to HAART regimens, is increasing (Cherry and Wesselingh 2003), and a concern about neurotoxicity of HAART in the brain has been raised (Schweinsburg et al. 2005). Thus, the natural history of neurological complications of HIV infection is changing in the post-HAART era (Brew 2004) and effective treatment will most likely require additional preventative adjunctive therapies to HAART and continued efforts at reducing neurotoxicity of antiretroviral compounds.

2  HIV Neuroinvasion: Early Events, Late Manifestations

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2.2 Biology of HIV Infection and Invasion of the Brain HIV and the related simian immunodeficiency virus (SIV) are retroviruses that when introduced into non-natural hosts cause profound CD4 T-lymphocyte depletion, chronic immune activation, fatigue of T-cell responses and, eventually, immune failure. Efficient infection of cells by HIV requires surface co-expression of chemokine receptors (primarily CXCR4 and CCR5 on T-lymphocytes and CCR5 (rarely CXCR4) on macrophages) and the CD4 receptor. Binding of native HIV envelope glycoprotein (gp120) trimers to CD4 occurs first, and results in a gp120 conformational rearrangement that exposes the chemokine receptor binding site on gp120, allowing it to engage the chemokine receptor (Doms 2004). After this binding of gp120 to the chemokine receptor, the noncovalently associated fusion peptide, gp41, is exposed and inserted into the target cell membrane in the process of fusion, which delivers the infectious particle to the target cell cytoplasm for completion of the replication cycle (reverse transcription, integration, and production of new infectious virions). Replication of HIV within the CNS appears to drive neuropathogenesis of HAND, although defining the relationships between virus replication (cellular targets, genotype/phenotype of neurotropic strains, regional distribution, level of replication) and stages of either structural damage or neurocognitive dysfunction has been difficult. Based on neuropathological studies that demonstrate predominant HIV (or SIV in macaques) expression in perivascular macrophages, entry of HIV into the CNS appears to occur via infected circulating monocytes (Budka 1991; Wiley et  al. 1986; Williams et  al. 2001), and this can occur early (within 1–2  weeks) after virus enters into the host (Davis et al. 1992; Gray et  al. 1993). Monocytes (infected and noninfected) can pass through capillary endothelial cells via classical transendothelial migration, a process involving movement through endothelial intercellular junctions (diapedesis, reviewed in (Maslin et  al. 2005)), and possibly also through transcellular migration (pinocytosis (Liu et  al. 2002) (Lossinsky et  al. 1991)) through the endothelial cell (although the later is controversial). The process of monocyte recruitment and migration into the CNS during HIV infection is regulated by a complex cascade of selective induction of multiple adhesion molecules on both monocytes and endothelial cells (EC) (reviewed in (Maslin et  al. 2005)). Several adhesion molecules (E-selectin on monocytes; sialomucin CD34, VCAM-1, ICAM-1, P-selectin glycoprotein ligand-1 (PSGL-1 (Marshall et al. 2003)) on EC and others) can function to promote initial monocyte adhesion (rolling, loose adhesion) to ECs and several of these are induced by proinflammatory cytokines such as TNFa and IL-8 and (Baumheter et  al. 1993). Infection of monocytoid cells is associated with increased expression of VLA-4 (a4B1 integrin), which then more strongly tethers these cells to EC via binding to VCAM (Birdsall et  al. 1994). Expression of each of these is induced by beta chemokine CCL-2/ MCP-1 (monocyte chemoattractant protein-1). Further strengthening of monocyte adhesion is enhanced by binding of monocyte LFA-1 to EC ICAM-1 (van Buul and

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Hordijk 2004), and diapedesis through EC/EC junctions is promoted by homophilic PCAM/PCAM interactions and CD99/CD99 interactions between moncocytes and EC (Mamdouh et  al. 2003; Schenkel et  al. 2002). Expression of each of these adhesion molecules is regulated by chemokines and/or cytokines involved in inflammatory responses in the CNS (Meager 1999). Chemokine expression by EC (CCL2 CXCL1, IL-8, CXC3CL1) can also selectively bind monocytes through chemokine receptor binding with cytokines expressed on the EC surface (Ebnet et al. 1996; van Buul and Hordijk 2004; Weber et al. 1999). Some of these chemokines are tethered to the EC surface by heparin sulfate proteoglycans, and CX3CL1 itself is normally directly tethered to the EC surface (Goda et al. 2000). Monocytes expressing high levels of the fractalkine receptor CXC3CR1 (CD14lo/CD16hi) can preferentially bind to EC expressing fractalkine (CX3CL1), while CD14hi/CD16lo monocytes preferentially bind to EC expressing the CCR2/ MCP-1 receptor (Ancuta et al. 2003; Geissmann et al. 2003; Maslin et al. 2005). Notably, Pulliam et al. (1997) have shown that increased expression of CD16 on peripheral monocytes is associated with the presence of HAD, consistent with the hypothesis that immune activation of peripheral monocytes during HIV infection is associated with increased monocyte trafficking into the CNS and an effector action of these cells in the pathogenesis of HAD (Gartner 2000). A more recent study involving a distinct patient cohort confirmed high monocyte CD16 expression in AIDS patients with and without HAD, and further demonstrated that elevated plasma levels of lipopolysaccharide (LPS) and activated monocytes are indeed associated with HAD (Ancuta et al. 2008). Infected monocytes that migrate into the brain can accumulate within the endothelial cell basement membrane to differentiate into macrophages (Nottet et  al. 1996). These perivascular macrophages are generally thought to become the major CNS reservoirs for HIV replication, from which sheds virus subsequently infects other macrophages (Rempel et al. 2008). The HIV strains that have been isolated from CSF and brain tissue, as well as functional envelope sequences amplified from these tissue compartments nearly uniformly express the characteristics of preferred use of the CCR5 chemokine coreceptor and tropism for macrophages (Gorry et  al. 2001; Ohagen et  al. 2003; Peters et  al. 2004; Peters et  al. 2007). The tropism of HIV within the CNS and throughout peripheral tissues is determined primarily by the cellular coexpression of the CD4 and CCR5 (and/or CXCR4) receptors. The published literature indicates that, for all naturally occurring primary HIV-1 envelopes, this binding of gp120 to chemokine receptors requires the initial binding of gp120 to CD4 to “uncover” the chemokine receptor binding site followed by binding of the “triggered” envelope to CCR5 or CXCR4 (discussed in (Edwards et al. 2001)). In contrast, some naturally-occurring SIV strains express gp120 that can bind directly to chemokine receptors in the absence of CD4 (Borsetti et  al. 2000; Edinger et  al. 1999). Furthermore, gp120 expressed by some laboratory-adapted (cell line passaged) HIV strains such as IIIB/LAI and others can acquire the ability to bind chemokine receptors in the absence of CD4 through mutation (LaBranche et  al. 1999). For this reason, the source of gp120 proteins (naturally occurring vs. laboratoryadapted) and virions used in in vitro studies of gp120/target cell interactions is critical

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for validating the biological relevance of such model systems, particularly those involving HIV neuropathogenesis. Despite several reports of detection of HIV genomic sequences in neurons in vivo (Bagasra et al. 1996; Torres-Munoz et al. 2008; Torres-Munoz et al. 2001) and a plausible infection mechanism mediated by chemokine receptors (CXCR4 or CRR5) and independent of CD4 in neurons (Rottman et  al. 1997; Sanders et  al. 1998), the body of work showing lack of viral protein and RNA in neurons generally supports the absence of productive HIV infection of neurons in vivo (Achim et  al. 1994; Glass et  al. 1995; Takahashi et  al. 1996; Williams et  al. 2001). The absence of infection of neurons supports an indirect mechanism of neuronal injury through release of soluble neurotoxins from infected and/or activated macrophages/ microglia and astrocytes, although released viral proteins (gp120, Tat) might also directly contribute (reviewed in (Mattson et al. 2005)).

2.3 HIV Neuropathogenesis: Human and Primate Studies The pathological hallmark of HIV infection in the brain, termed HIV encephalitis, is characterized by the presence of myelin pallor, reactive astrocytosis, infiltration of predominantly monocytic cells, and multinucleated giant cells (MNGC), which are the unique effect of HIV-driven fusion of macrophages/microglia (Budka 1989; Navia et al. 1986; Wiley and Achim 1994). Postmortem studies have demonstrated that morphological changes in neurons (dendritic simplification and vacoulization, loss of synaptic density) and loss of neurons are commonly found in the brains of HAND patients ((Asare et  al. 1996; Everall et  al. 1994; Masliah et  al. 1992a; Masliah et al. 1992b; Masliah et al. 1997; Sa et al. 2004; Wiley et al. 1991), reviewed in (Ellis et al. 2007)). Damage appears to occur early in the basal ganglia, thalamus, and central white matter (Navia et al. 1986; Petito 1988), where HIV antigen is commonly detected (Kure et al. 1990a; Kure et al. 1990b; Park et al. 1990) but degeneration ultimately involves the entire brain. Several of these studies have focused on specific brain regions and neuronal subtypes. The type of neuronal damage observed includes the following: loss of dendritic arborizations of the dentate granule and hilar basket cells, CA3 and CA1 hippocampal pyramidal cells (Sa et al. 2004), and frontal cortical and hippocampal interneurons (Fox et al. 1997; Masliah et  al. 1992b), as well as dropout of neurons in frontal, temporal, and parietal cortex (Everall et al. 1994; Wiley et al. 1991). One study reported loss of oxytocin-producing neurons in the paraventricular hypothalamic nucleus in a study of 20 AIDS patients (4 with suspected HAD), although opportunistic brain infections were present in most of these patients, making a direct relationship between HIV replication and neurodegeneration unclear (Purba et  al. 1993). Thus, neuronal damage induced by HIV infection of the brain affects multiple brain regions and neuronal subtypes, but the factors that determine neuronal vulnerability are only partially understood. Because pathological features of HAND determined postmortem do not provide a picture of how damage is acquired over time, investigators have focused on the use

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of neuroimaging analyses of infected individuals and pathological and neuroimaging analyses of SIV-infected macaques to study early effects of brain infection. Two macaque models (Macaca mulatta, rhesus; Macaca nemestrina, pigtail) of SIV infection have been effectively used for studying the virus-triggered pathways of neurodegeneration that lead to cognitive dysfunction, and for characterizing early events in pathogenesis. Several groups studying the pigtail SIVE model use an immunosuppressing viral swarm (SIV/Delta B670) either with or without co-infection with a CNS-adapted molecularly cloned SIV strain, SIV/17E-Fr to induce SIVE (Bonneh-Barkay et al. 2008; Mankowski et al. 2002). Use of the SIV/ Delta B670 swarm alone results in a somewhat variable and delayed neurodegeneration, similar to the natural history of HAND in humans, while coinfection with SIV/17E-Fr typically produces SIVE in up to 90% of inoculated animals within 3  months (Mankowski et  al. 2002). The rhesus model typically involves use of either SIVmac251 swarm (a dual macrophage- and T-cell line-tropic swarm) or SIVmac239 (a molecular T-cell line-tropic clone of SIVmac251), which induce SIVE in ~30% of inoculated animals in 2  years (Fuller et  al. 2004; Lentz et  al. 2008). These models have shown that SIV infection is associated with infection of perivascular macrophages, robust astrocytosis, multinucleated giant cell formation, infiltration of CD4+ and CD8+ T cells (CD4+ T cells predominate), and natural killer (NK) cells (Mankowski et al. 2002). An initial burst of SIV replication occurs within the CNS, followed by a period of relative quiescence, and subsequent reactivation of virus replication in the end stages of AIDS and SIVE. Furthermore, CSF/ plasma ratios of CXCL2/MCP-1 are consistently higher throughout the course of infection in those animals eventually developing SIVE (Mankowski et al. 2002). In the SIV-rhesus macaque model, similar to HIV infection in humans, entry into the brain is observed early after systemic virus inoculation (7 days for SIV entry) (Chakrabarti et al. 1991). Using calbindin as a neuronal marker specific for GABAergic neurons, and synaptophysin as a marker for presynaptic membranes, investigators showed that macaques sacrificed 14  days after infection sustained significant damage to GABAergic neuronal cell bodies and synapses in the frontal cortex (Gonzalez et al. 2000). Fragmentation and shrinkage of calbindin-immunoreactive neurons and loss of synaptophysin were even more prominent in macaques sacrificed 2 years after infection, indicating that damage to these neurons occurs early and probably throughout the chronic course of infection. In addition, reactive astrogliosis marked by enhanced GFAP expression was also noted early in infection and throughout the disease course, although at least one HIV study has shown that the degree of astrogliosis does not correlate with the presence or severity of neuronal damage (Masliah et al. 1992a). Early neuronal damage detected by immunohistochemistry has been confirmed by brain magnetic resonance spectroscopy (MRS) analysis of the neuronal marker N-acetylaspartate (NAA) (commonly expressed as an NAA/creatine ratio; NAA/ Cr) in the acute and chronic phases of infection in SIV-infected macaques (Fuller et al. 2004; Greco et al. 2004; Lentz et al. 2005; Lentz et al. 2008; Williams et al. 2005). In a macaque model involving CD8+ T lymphocyte depletion along with SIV inoculation, Williams et al. (2005) demonstrated a reduction in NAA/Cr in the

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frontal cortex within 10 weeks of infection in animals developing SIV encephalitis (SIVE). Neuronal damage was confirmed by quantitative immunohistochemical studies that showed a significant loss of synaptophysin in the frontal cortex. There was a biphasic increase in the percentage of circulating CD14+ monocytes that coexpressed CD16 as well as the CD14lo/CD16hi monocyte subset, which occurred immediately (7–14  days) after infection and again prior to or with the onset of AIDS. The early monocyte increase occurred concomitantly with the initial decrease in NAA, and the CD14lo/CD16hi monocyte subset consistently harbored SIV proviral DNA. A follow up study by Lentz et al. (2008) also showed a decrease in both GABA/Cr and Glutamate/Cr ratios in SIV-infected macaques with and without SIVE, indicating injury to inhibitory and excitatory neurons, respectively. Other studies have shown that an increase in the myoinositol (MI)/Cr ratio (marker of astrocytic activation) often occurs prior to an NAA/Cr decrease (Greco et  al. 2004), indicating an early CNS inflammatory response prior to neuronal injury. Interestingly, antiretroviral drug administration had a significant effect on the NAA/ Cr decrement, which was at least partially reversible by administration of non-CNS penetrating antiretroviral drugs 28  days after infection, although whether this is associated with a recovery of synaptophysin expression is unclear. In all animals studied, no structural changes were detected by conventional Magnetic resonance imaging (MRI) at any time point. These studies suggest that MRS can detect early neuronal damage in SIV infection of the CNS, similar to studies in HIV-infected individuals, and that antiretroviral therapy that reduces systemic virus replication and monocyte activation in the circulation can attenuate neuronal damage. However, the effects of long-lived SIV replication within the CNS compartment are more difficult to address in these short-term studies. Similarly, in HIV-infected individuals, several studies using brain MRS have demonstrated changes in brain metabolites occurring early in infection that correlate with worsening neurological function. Brain NAA/Cr ratios have been found to be significantly reduced in HAD patients, indicating neuronal loss (Chang et  al. 1999a; Chang et al. 1999b; Chang et al. 2003; Meyerhoff et al. 1993; Tracey et al. 1996). Increases in glial-associated metabolites such as choline and myoinositol (which are elevated during gliosis or membrane turnover that occurs with glial activation) were more sensitive in detecting clinically milder disease early in infection (Chang et al. 1999a; Yiannoutsos et al. 2004)). Increases in choline and myoinositol reverted with response to HAART (Chang et al. 1999b), indicating that virus replication, both within and outside of the CNS, contribute to glial activation. HAART is able to partially reverse neurologic impairment in HAD, and HAART regimens that express higher CNS penetration are more effective in reducing cerebrospinal fluid (CSF) viral loads and improving neurological performance (Ances and Ellis 2007; Letendre et al. 2004; Marra et al. 2003). Together with the aforementioned macaque MRS studies, these studies suggest that suppression of virus replication within the peripheral circulation and CNS compartments are necessary for maximum protection against neuronal damage, probably by decreasing virus-induced glial cell activation and trafficking. They also suggest that macaque SIV models can be very useful for testing neuroprotection treatment approaches.

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In both HIV infection and SIV infection elevations of CCL2 in the cerebrospinal fluid (CSF) tend to precede the development of signs of neurological dysfunction, consistent with a proposed role for CCL2 in promoting neurodegeneration through enhancement of monocyte trafficking and establishing a resident population of infected macrophages within the CNS (Zink et al. 2001) (Williams et al. 2001).

2.4 Mechanisms of HIV-Induced Neurodegeneration: Neurotoxicity of HIV Proteins One of the predominant hypotheses of how infected microglia and macrophages can directly mediate neurotoxicity is by the release of viral proteins such as gp120 and Tat, which then bind to receptors on neurons (Brenneman et al. 1988; Mattson et al. 2005). Neurotoxicity resulting from exposure to recombinant gp120 has been confirmed in multiple in vitro model systems (Alirezaei et al. 2007; Bennett et al. 1995; Brenneman et al. 1988; Dawson et al. 1993; Dreyer et al. 1990; Dreyer et al. 1999; Lannuzel et al. 1995; Meucci and Miller 1996), although the mechanisms by which such toxicity is induced remain controversial (Bachis and Mocchetti 2004; Gonzalez-Scarano and Martin-Garcia 2005; Kaul et  al. 2001). Using Scatchard analyses, Hesselgesser et  al. (1998) demonstrated binding of gp120 (HIV IIIB strain) to human neuronal CXCR4 (kD = 54 nM), which was associated with induction of apoptosis. Several other studies have indirectly addressed gp120/chemokine receptor interactions in neurotoxicity model systems. Zhang et al. (2003) demonstrated the ability of anti-gp120 antibodies and antibodies against CCR5 and CXCR4 to reduce (20– 80%) gp120 toxicity in exposed, non-differentiated human neuronal cells. Meucci et al. (1998) showed that anti-gp120 (IIIB) antibodies reduced toxicity of recombinant gp120 by 45% in purified primary rat hippocampal neurons co-cultured with an astrocyte feeder layer. Zheng et al (Zheng et al. 1999a) examined the ability of virions from laboratory-passaged X4 HIV strains (MN, IIIB, Lai) and several R5 strains (JR-FL, Bal, ADA, DJV, MS-CSF) to induce apoptosis in human fetal neurons in mixed neuronal/glial cultures. Surprisingly, virions from each strain induced neuronal apoptosis (X4 virions more so than R5 virions) in a manner that was blocked by an anti-CXCR4 antibody. These and other similar studies suggest that complex interactions between gp120 and cellular surface binding moieties in cultured cells can lead to effects that may or may not be linked to gp120/chemokine receptor binding in neurons. In support of indirect effects of gp120 on neuronal survival, Kaul and Lipton (Kaul and Lipton 1999) provided evidence that gp120 neurotoxicity in primary rat neuronal cultures depends upon the presence of macrophages/microglia, through which gp120 can induce neurotoxin release after engaging chemokine receptors (and CD4) (reviewed in (Kaul et al. 2001)). Signaling initiated by gp120/ macrophage chemokine receptor interactions is thought to result in activation of the p38 MAPK proapoptotic pathway, because pharmacologic inhibition of p38 MAPK can abrogate gp120-induced apoptosis (Kaul and Lipton 1999).

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There are also reports of gp120 interacting directly with the N-methyl-daspartate receptor (NMDAR) in neurons and activating death pathways (Fontana et al. 1997; Gemignani et al. 2000; Pattarini et al. 1998; Pittaluga et al. 1996; Xin et al. 1999). These studies indicate that gp120 and peptide-fragments of gp120 are able to bind to NMDA receptors at the glycine-binding site (on the NR1 subunit) to activate the receptor and induce release of neuropeptides or neurotransmitters. However, it is not clear whether such gp120 effects are associated with neurotoxicity (Gemignani et  al. 2000). It thus seems likely that selected recombinant gp120 proteins can induce neurotoxicity by several mechanisms: direct toxic effects mediated by interactions with neuronal receptors and indirect effects mediated through interactions with glial cells. In addition to gp120, the HIV-1 transactivating protein, Tat, is thought to be released by virus producing cells either during lysis or by active secretion (Chang et al. 1997; Ensoli et al. 1993). Similar to addition of gp120, addition of recombinant Tat protein to neuronal cultures can induce neuronal apoptosis (Kruman et al. 1998; Magnuson et  al. 1995; Nath et  al. 1996; New et  al. 1998). Tat, (86–104 amino-acids in length in its naturally occuring two-exon form; 72 amino acids in length in the laboratory-adapted IIIB strain (one exon), has been shown to be released from HIV-infected T lymphocytic cell lines, and it can be detected in the serum of a minority of HIV-infected individuals (Ensoli et  al. 1990; Westendorp et  al. 1995). In our review of the literature, we found no clear evidence of the release of Tat by HIV-infected primary macrophages in vitro. A study by Tardieu et al. (1992) demonstrated Tat immunoreactivity in the human U937 monocytic cell line after infection with HIV-1 in co-cultures with primary human neuronal/glial cell populations. Although release of Tat from the infected U937 cells was not demonstrated, immunhistochemical labeling demonstrated Tat and gp120 expression associated with the extension of necrosis in neurons and astrocytes, which suggested the possibility of release of both Tat and gp120 by the infected U937 cells. In other studies, Tat transcripts and Tat protein have been identified in the brains of patients with HAD or those with HIV encephalitis (Hudson et al. 2000; Nath et al. 2000; Wesselingh et al. 1993; Wiley et al. 1996). Soluble Tat protein has been shown to bind via its basic region (located at amino acid position 48–57) to heparan sulfate proteoglycans on cell surfaces or in extracellular matrix, where it is protected from degradation (Chang et al. 1997). Binding to heparin or heparinase results in the release of Tat from the extracellular matrix and allows it to bind to integrins (Barillari et al. 1993). Tat can also bind to the low density lipoprotein receptor-related protein (LRP) on neurons (Chang et al. 1997; Eugenin et al. 2007; Evans et al. 2007; Liu et al. 2000), and such binding prevents LPR-mediated clearance of its natural ligands, which include amyloid precursor protein, amyloid beta protein, apolipoprotein E4, and alpha-2-macroglobulin. The accumulation of these natural ligands in the extracellular space of the brain has been shown in other neurodegenerative diseases, which suggests a possible mechanism by which Tat could induce extracellular protein deposition in the brain. Although Tat may directly interact with receptors on neurons, the major pathway for Tat-mediated neurotoxicity in vitro is thought to occur through a direct interaction

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with neuronal membranes, resulting in depolarization (Nath 2002). By causing an initial release of calcium from intracellular IP3 sensitive pools, Tat can activate nonNMDA-glutamate and NMDA receptors and induce calcium influx into neurons (Haughey et al. 1999; Kruman et al. 1998; Li et al. 2004; Magnuson et al. 1995). Tat-induced disruption of calcium homeostasis can result in the production of reactive oxygen species (ROS), leading to oxidative stress, mitochondrial dysfunction and apoptosis (Mattson et al. 2005). Thus, as for gp120, there are multiple mechanisms by which Tat could potentially induce neurotoxicity, although nearly all of the published studies have focused on direct effects on neurons. Nonetheless, indirect neuromodulating effects of Tat could be mediated through Tat modulation of glial cell cytokine and chemokine production (induction of CCL2, CXCL8, CXCL10, CCL3, CCL4 and CCL5), inhibition of astrocyte glutamate scavenging, and disruption of the blood–brain barrier (reviewed in King et  al. (2006)). The ability of recombinant Tat to induce expression of multiple chemokines from glia suggests a mechanism by which HIV replication in the CNS (with release of Tat) could modulate multiple steps in neurodegeneration through effector functions of induced chemokines (monocyte transendothelial migration, glial cell activation, and direct neurotoxicity).

2.5 Mechanisms of HIV-Induced Neurodegeneration: Roles for Chemokines and Chemokine Receptors Chemokines and chemokine receptors expressed within the CNS have central roles in HIV neuropathogenesis, from the function of chemokine receptors in mediating infection in the macrophage/microglial reservoir (Collman and Yi 1999; Doms 2000; Martin-Garcia et al. 2002) to other possible pathogenic effects of chemokine receptor-mediated signaling activation in neurons and glia, which are supported by a rapidly growing body of published studies. Studies of cerebrospinal fluid (CSF) in cohorts of HAND patients have revealed significant elevations of CCL2/MCP-1 and CXCL10/IP-10 (Cinque et al. 2005; Kelder et al. 1998; Mankowski et al. 2004) and elevated levels of CCL2 in SIV infected macaques that develop SIVE (Zink et  al. 2001). Because neurons express multiple chemokine receptors (Coughlan et  al. 2000; Horuk et  al. 1997; Lavi et  al. 1997; Meucci et  al. 2000; Miller and Meucci 1999; Rottman et  al. 1997), they are potentially functionally altered by exposure to induced chemokines during HIV/SIV infection. Alpha chemokines, which bind CXCR chemokine receptors, are normally expressed in all major cell types in the brain (macrophages/microglia, astrocytes, neurons, endothelial cells) and, upon binding to their cognate receptor, they induce signaling through a Gi protein-dependent decrease in cyclic AMP and an increase in intracellular calcium. Among those found at elevated levels in the brain or CSF of individuals with HAD are CXCL12/SDF-1 alpha and CXCL10 (Rostasy et al. 2003) (Cinque et al. 2005). On the other hand, beta chemokines (which bind CCR receptors) are expressed at relatively low levels under physiological conditions in the normal brain. CXCL12/

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SDF-1 alpha, an alpha chemokine that binds CXCR4, is produced by macrophages, astrocytes, and neurons in the brain, and an increase in CXCL12 transcripts has been found in the brain tissue of individuals with HIV encephalitis (Zhang et al. 1998). Signaling in neurons via CXCL12 exposure has been shown to produce either neuroprotective or neurotoxic responses, depending upon the experimental conditions (Kaul and Lipton 1999; Khan et  al. 2008; Zheng et  al. 1999b). It has been shown to enhance synaptic transmission, induce AKT/protein kinase B, or activate caspase-3 under different conditions (Kaul and Lipton 1999; Zheng et al. 1999b). CXCL12 can undergo proteolytic cleavage by matrix metallic proteinases (specifically MMP-2) (McQuibban et al. 2001), which changes its coreceptor specificity from CXCR4 to CXCR3 and also enhances its neurotoxicity (Zhang et  al. 2003) (Vergote et al. 2006). Activation of CXCR3 in neurons by its natural ligand CXCL10 results in elevations in intracellular calcium and activation of caspase-3 leading to neuronal apoptosis (Sui et al. 2004; Sui et al. 2006). CNS beta chemokine expression is also altered in HIV infection and these chemokines may also result in a protective or a destructive milieu (Schmidtmayerova et al. 1996). Among the beta chemokines that are expressed at increased levels during HIV infection of the CNS are CCL2, MIP-1 alpha, MIP-1 beta, and RANTES/CCL5 (Kelder et al. 1998), although the association of MIP-1 alpha, MIP-1 beta and CCL5 with HAND is unclear (Letendre et al. 1999). In vitro studies show that MIP-1 alpha/ beta can protect hippocampal neurons from gp120-induced apoptosis (Kaul and Lipton 1999; Meucci et al. 1998). CCL5 also protects neurons against gp120-induced damage, although CCL2 does not (Meucci et  al. 1998). In contrast, the beta chemokine CCL2 appears to have a detrimental effect in CNS infection. Elevated CSF CCL2 expression is associated with an increased risk of HAND (Kelder et al. 1998; Ragin et al. 2006; Sevigny et al. 2004; Sevigny et al. 2007). This increased risk might reflect CCL2’s role as a potent monocyte chemoattractant in the CNS (Gonzalez et al. 2002; Monteiro de Almeida et al. 2006). Its expression induced in microglia activated by interferons and in astrocytes activated by IL-1beta and TNFalpha (Andjelkovic et al. 2000; McManus et al. 2000). Of interest, it has been suggested that the neuroprotective effects of RANTES are mediated by the induction of CCL2 (Eugenin et al. 2003). Collectively, these results suggest that the fluctuations in the ambient chemokine concentrations within the brain during the course of HIV infection have varied effects in neurons, both temporally and regionally, depending upon the local neuronal subpopulations that are exposed to activated/infected glia. Finally, the unique chemokine, fractalkine/CX3CL1, which belongs to the Cx3C chemokine family, is also elevated in the CSF of individuals with HAND (Pereira et al. 2001). The tethering of CX3CL1 to EC cells in the brain can mediate monocyte attachment, which could promote transendothelial migration of monocytes to the CNS, suggesting a role in enhancing HIV neuropathogenesis (Ancuta et  al. 2003; Geissmann et al. 2003; Maslin et al. 2005). However, several studies have demonstrated a neuroprotective function of CXCL1 against neuronal excitotoxicity (Deiva et al. 2004; Limatola et al. 2005; Mizuno et al. 2003). Thus, as for alpha and beta chemokines, CXCL3 could play a role in both neuroprotective and neurotoxic cascades induced by HIV replication in the CNS.

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2.6 Mechanisms of HIV-Induced Neurodegeneration: Roles for Excitotoxins and N-Methyl-d-Aspartate Receptors In addition to the enhanced expression of chemokines, enhanced expression of other potential neurotoxic factors such as excitatory amino acids, which include glutamate, quinolinic acid (QUIN), cysteine, and the amine N-Tox is associated with macrophage/microglia activation (Brew et al. 1995; Giulian et al. 1990; Giulian et al. 1993; Giulian et al. 1996; Yeh et al. 2000). Glutamate, which is the major excitatory neurotransmitter in the CNS, has been reported to be elevated in the CSF of HIVinfected individuals (Ferrarese et al. 2001), although this has been disputed (Espey et al. 2002; Espey et al. 1999). Because the concentration of glutamate in the synaptic cleft must be kept within a physiological range to avoid sustained toxic activation of neuronal glutamate receptors and excessive calcium influx (excitotoxicity) (Hyrc et al. 1997; Rothman 1984), altered glutamate homeostasis is thought to be a major pathway of neurodegneration in inflammatory brain diseases such as HIV infection (Kaul et al. 2001). Glutamate, QUIN, and N-Tox are all released (to varying levels) by HIV infected macrophages, and each of these has the potential to induce excitotoxicity through N-methyl-d-aspartate (NMDA) receptor activation (Giulian et  al. 1990; Jiang et al. 2001; O’Donnell et al. 2006). Therefore, the distribution and function of NMDAR within CNS neuronal populations is likely a major determinant of neuronal vulnerability to HIV-induced damage. The NMDAR, a subtype of glutamate receptor, is a voltage and ligand-gated calcium ion channel that generates excitatory postsynaptic currents through calcium influx into the neuron. Functional NMDAR are heteromeric assemblies of four subunits of at least 2 types: two NMDA-R1 (or NR1) subunits and two NMDA-R2 (or NR2) subunits. The subunit composition of NMDAR varies throughout neuronal development, and, to some degree, within different brain regions (Lynch and Guttmann 2001; Lynch and Guttmann 2002). The 8 variants of NR1 are derived from 1 gene via alternative splicing (Goebel et al. 2005) whereas 4 separate genes encode NR2 subunits (NR2A, NR2B, NR2C, and NR2D). Two variants of subtype NR3 also exist but their expression is not required for a functional NMDAR. NR1 subunits bind glycine, and NR2 subunits bind glutamate and quinolinic acid. The different NR2 subunits have different pharmacologic and biophysical properties and thus variations in the type of NR2 subunit can confer distinct properties to the receptor (Lynch and Guttmann 2001; Lynch and Guttmann 2002). For example, quinolinic acid activates NR2A- and NR2B-containing receptors but not those containing NR2C or NR2D. Furthermore, although all four NR2 subunits can bind glutamate with equal affinity, NR2A and NR2B trigger greater excitotoxicity than NR2C and NR2D. NR2 subtypes also have different specificities for pharmacologic inhibitors, which have been effectively used to distinguish which NR subunits are responsible for functional responses in NMDAR. The distribution of NMDAR subtypes offers one explanation for regional brain vulnerability to HIV-associated injury. Neonatal brain predominantly expresses

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NR2A, NR2B, and NR2D subunits and, in some regions, NR2C, over the course of development (Monyer et  al. 1994). In the adult rat brain, NR2A is ubiquitously expressed, whereas NR2B is restricted to the forebrain, and NR2C is largely restricted to the cerebellum (Kohr 2006). Notably, regions such as the hippocampus, striatum, and forebrain, which have high expression of NR2B, are often the areas demonstrating neuronal death in HIV infection whereas areas such as the cerebellum with NR2C expression are relatively spared (Archibald et  al. 2004; Conti et al. 1999; Everall et al. 1999). This suggests a role of specific NR2 subtypes in HIV-mediated neuronal excitotoxicity. Our group examined the role of NMDAR subtypes in determining susceptibility to HIV-induced neurotoxicity and found that neurons become vulnerable to injury from exposure to HIV-infected macrophages only after establishing functional NMDAR expression (O’Donnell et  al. 2006). We established an in  vitro model utilizing embryonic rat hippocampal neuronal cultures exposed to supernatants from HIV-infected macrophages and we found that neuronal death occurred only with the appearance of NR2A and NR2B subtypes as the neurons matured. As shown previously by others (Giulian et al. 1996; Jiang et al. 2001), we confirmed that the neurotoxic factor(s) released from the infected macrophages are of low molecular weight (<3 kD), and are heat- and protease-resistant excitotoxins that act through NMDAR. Furthermore, blockade of neurotoxicity at different neuronal developmental stages could be achieved using antagonists to specific NMDAR subunits (to either NR2A or NR2B) and this protection was consistent with the NR subtype expression profile of the cultured neurons. For example, inhibitors specific for NR2B/NR2B homodimers (Ifenprodil and Ro25-6981) were most effective earlier in the maturation process when NR2A was not heavily expressed. Neuronal protection in more mature cultures (with increased expression of NR2A and NR2B) required use of inhibitors that blocked both NR2B/NR2B homomeric receptors and NR2A/NR2B heteromeric receptors. In addition to glutamate, other amines released by activated macrophages that act at the NMDAR, such as quinolinic acid (QUIN), may also contribute to excitotoxity in HIV infection. Like glutamate, QUIN levels are shown to be elevated in CSF and brain parenchyma of HIV-infected patients and those with other CNS infections (Heyes et al. 1991; Heyes et al. 2001) (Achim et al. 1993). The accumulation of these excitatory amines points to malfunctioning glia cells since microglia are predominate producers of QUIN while both microglia and astrocytes regulate extracellular glutamate levels. QUIN and glutamate are metabolically processed by microglia and astrocytes and inflammatory mediators can alter the normal processing of these amines resulting in their accumulation in the extracellular space. QUIN is produced via the kynurenine pathway from the substrate l-tryptophan, and the key regulatory enzyme in this pathway, idoleamine 2, 3-dioxygenase (IDO), is upregulated in inflammatory states by cytokines such as IFN-gamma. In the brain, IDO is expressed by microglia, astrocytes, endothelial cells, and neurons. Macrophages/microglia are key QUIN producers because they express all enzymes of the pathway leading to QUIN production, whereas astrocytes predominantly have enzymes that shift production away from QUIN to other metabolites such as

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kynurenic acid, an antagonist of QUIN, and kynurenine (Guillemin et  al. 1999) (Heyes 1996). Bruce Brew and colleagues have proposed a model of QUIN metabolism in the brain where astrocytes play a neuroprotective role by minimizing production of QUIN (Guillemin et al. 2001; Guillemin et al. 2005). Thus, interactions between astrocytes and macrophages/microglia likely regulate extracellular QUIN concentrations, which, like glutamate, directly induce neuronal cell responses through NMDAR during HIV infection. Although it is as yet unclear whether chemokines have a direct effect on glutamate or QUIN metabolism, NMDAR expression, or NMDAR function, several studies have demonstrated that NMDAR-dependent excitotoxic neuronal injury results in a rapid and robust increase in CCL2 expression, in rat brain, and peripheral nerve (Galasso et al. 2000) (Kleinschnitz et al. 2004). A concomitant increase in CCR2 expression also occurs, suggesting a mechanism for recruitment of monocyte/macrophages to areas of exicitotoxic injury, such as that seen in HIV infection. Along these lines, QUIN also up-regulates chemokine (CCL2, CXCL12, CCR5, CXCL8) and chemokine receptor expression (CXCR3, CCR5, CCR3) in astrocytes (Croitoru-Lamoury et al. 2003). This also supports the hypothesis that excitotoxic injury induced by HIV promotes activation of multiple chemokine-mediated pathways that promote either further injury or that initiate protective responses to such injury. Further studies of the ability of chemokines and NMDAR ligands (glutamate, QUIN) expressed within the CNS to cross-modulate each other’s receptor expression and function could yield novel information about how chemokines influence the progression of excitotoxic injury in HAND and other neurodegenerative diseases. The potential for NMDAR antagonists to protect the CNS against HAND has recently been investigated in a multicenter therapeutic trial (ACTG) of Namenda (memantine), which is currently FDA-approved for use in Alzheimer’s disease (Schifitto et al. 2007). Although no clinically beneficial effect in neuropsychological test performance was observed during the 16-week treatment phase, there was a significant increase in the NAA/Cr ratio in the frontal white matter and parietal cortex in treated individuals, suggesting a potential neuroprotective effect. Further investigations of agents that block pathways (e.g., oxidative stress, glutamate and QUIN production) to neuronal excitotoxic injury and therapeutic trial designs that include longer duration trials (6 months or greater) are likely to follow (Bandaru et al. 2007; Brew et al. 2007; Clifford 2008; Evans et al. 2007).

2.7 Other Links Between Chemokines and Excitotoxic Injury: Glutamate Release Besides the clear role for chemokines in modulating recruitment of cells into the CNS in HIV infection, and the potential role for chemokines to directly modulate neuronal signaling, recent evidence has suggested a link between CNS chemokine expression and enhancement of excitotoxic injury through enhancement of glutamate

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release. Bezzi et al. (2001) demonstrated that CXCL12 rapidly (seconds) induces the release of glutamate from astrocytes in rat hippocampal brain slices in a calciumdependent manner. Detailed characterization of this release process revealed that it most likely occurs by inhibition of quantal-like glutamate exocytosis, which is independent of glutamate transporter (EAAT) reversal or osmotic damage, but which is dependent upon TNFa. Other studies have confirmed that CXCL12 can induce glutamate release in hypothalamic and substantia nigra neurons (Guyon and Nahon 2007; Guyon et  al. 2006), and modulate neuronal GABA release (Guyon et al. 2006). These interesting studies are the first reports of a direct link between neuronal chemokine production and enhancement of glutamate-mediated excitotoxicity, and they clearly extend previous observations of neuronal toxicity mediated by direct chemokine/neuronal signaling by CXCL10 (Sui et al. 2004; Sui et al. 2006) and CXCL12 (Kaul and Lipton 1999; Vergote et al. 2006; Zheng et al. 1999a; Zheng et al. 1999b). Additional studies are needed to more thoroughly define the abilities of chemokines to alter neurotransmitter metabolism in the CNS to better understand the mechanisms by which chemokines can modulate excitotoxic injury in HAND.

2.8 Therapeutic Considerations There is no doubt that HAART has changed the nature of HIV-infection and altered it from a uniformly fatal disease to a chronic, and often disabling, infection. Likewise, CNS manifestations of HIV-infection have also been modified by HAART. The severity of neurocognitive impairment has been lessened but not to a point where it has no impact on the quality of life, as even minor impairment can negatively affect survival. As systemic eradication of the virus is likely not possible in the near future, we are faced with addressing when and what types of therapies to initiate. The current guidelines for administering HAART recommend deferring therapy for asymptomatic patients until CD4 T + cell counts drop below 350. HAART is recommended for patients with symptoms or a history of an AIDSdefining illness (which includes HAD) and asymptomatic patients with CD4 T+ cell counts less than 200. These recommendations for deferred therapy are in contrast to early treatment recommendations to begin therapy soon after diagnosis. These newer recommendations take into consideration the increased likelihood of resistance with longer periods of unnecessary treatment, the negative side effects of HAART, many of which are not minor, offset with the longer life expectancy imparted by HAART. Because the virus enters into the CNS early in infection, this deferred therapy allows for virus replication in the CNS undoubtedly with concomitant neuronal damage that occurs with inflammation. It is unclear whether damage incurred during this period is a contributory reason/factor to why despite the HAART therapy, less severe HAND syndromes are still pervasive among those individuals receiving HAART. Perhaps there exists a threshold level of tolerable damage that may be reversible, and beyond this damage may result in neurological

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symptoms. If so, is there a role for adjunctive neuroprotective agents (NMDAR antagonists, chemokine modulators, antioxidants, others) before or after initiation of HAART?

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Chapter 3

HIV Co-receptors: The Brain Perspective Tracy Fischer-Smith and Jay Rappaport

3.1 Chemokines as Co-receptors for HIV Infection 3.1.1 CD4 The CD4 molecule, present on macrophages and helper T cells, had been recognized early in the history of HIV-1 research as the major receptor on cells susceptible to HIV-1 infection (Dalgleish et al. 1984; Klatzmann et al. 1984), as determined by monoclonal antibody blocking studies. While the expression of CD4 was sufficient to confer susceptibility of HIV-1 infection to other human cell types, there appeared to be a species-specific component that was required for infection. HIV-1 was unable to gain entry into mouse cells when human CD4 was either expressed ectopically (Maddon et  al. 1986) or in studies with human CD4 transgenic mice engineered to express CD4 molecules (Lores et  al. 1992). There was a clear requirement for additional molecules that could be provided in trans and studies with interspecies hybrid cells, demonstrating a necessity for specific human components, in addition to CD4, for infection (Weiner et al. 1991). It was proposed that other molecules (Maddon et al. 1986) or in fact a “second receptor” (Henderson and Qureshi 1993) was most likely needed for entry or fusion.

3.1.2 Discovery of Chemokine Receptors as “Second Receptors” for HIV-1 Infection A major clue leading to the identity of the second receptor was provided by the landmark studies aimed at identifying soluble inhibitory factors produced by CD8+ T cells (Cocchi et al. 1995). These studies identified the chemokines CCL5 (regulated T. Fischer-Smith and J. Rappaport (*) Temple University School of Medicine, Department of Neuroscience, 1900 N. 12th St., Biology Life Science Bldg., Rm. 246, Philadelphia, PA, 19122, USA e-mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_3, © Springer Science+Business Media, LLC 2010

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on activation, normal T-cell expressed and secreted, RANTES), CCL3 (macrophage inflammatory protein-1 alpha, MIP-1a), and CCL4 (macrophage inflammatory protein-1 beta, MIP-1b) as the major suppressive factors produced by CD8+ T cells. Furthermore, these recombinant chemokines could inhibit HIV-1 infection. At the time of this discovery, it was not yet appreciated that these C-C chemokines were indeed interfering with the binding of HIV to one of its coreceptors. CCR5, the receptor for these three chemokines was discovered the following year (Samson et al 1996). Several groups quickly determined that indeed, CCR5 was a coreceptor for HIV infection (D’Souza and Harden 1996). Meanwhile, a study by Berger’s group demonstrated that a 7-transmembrane protein, fusin, was the receptor for T-cell tropic HIV viruses (Feng et al. 1996). It is now evident that the major coreceptors for macrophage tropic and T-cell tropic HIV infection are CCR5 and CXCR4, respectively. Macrophage tropic virus replicates in both T cells and macrophages, whereas T-cell tropic virus is preferentially cytopathic for T cells, leading to cell fusion or syncytia. T-cell tropic virus appeared later in the course of disease in some patients and was associated with disease progression and the development of AIDS (Tersmette et al. 1988). The macrophage versus T-cell tropism of HIV-1 and, in fact, its preference for CCR5 versus CXCR4 are determined by sequences within the HIV-1 envelope (Cordonnier et  al. 1989; Shioda et  al. 1991), specifically the V1, V2, and V3 regions. While there seems to be a correlation between macrophage tropic virus with nonsyncytia-inducing phenotype (NSI), CCR5 utilization, and neutralization by antibodies to V3, such correlations are not absolute (Dittmar et  al. 1997; Stamatatos et al. 1997). In fact, certain T cell tropic variants can infect macrophages via CXCR4 and furthermore, not all viruses that can infect T cells or coreceptor transfected cell lines via CCR5 can actually infect macrophages. It appears, therefore, that coreceptor utilization is a major determinant of tropism; however, other factor(s) are clearly involved, particularly in macrophage infection.

3.2 Chemokines and Chemokine Receptors as Determinants of HIV-1 Infection and Disease 3.2.1 Chemokines and Chemokine Receptors in HIV-1 Transmission During HIV-1 infection, the initial transmission of virus occurs via the CCR5 receptor. The major evidence in support of this is that Caucasians with a homozygous 32 base pair deletion in CCR5 (CCR5∆32) are virtually uninfectable by HIV-1. This genotype appears to account for the resistance to HIV-1 infection exhibited by some individuals who were exposed several times, but not infected (Dean et al. 1996; Liu et al. 1996). While persons homozygous for this mutation are protected from HIV-1 infection, this mutation does not appear to confer reduced risk of infection in heterozygous individuals (Huang et al. 1996).

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Like the chemokine receptors, chemokine ligands also appear to play a role in protecting against HIV-1 transmission. Higher secretion of CCL3, CCL4, and CCL5 have been observed in peripheral blood mononuclear cells (PBMC) from hemophiliac patients who have experienced multiple HIV-1 exposure, but not been infected (Zagury et al. 1998). In fact, the reduced production of CCL3 has been associated with perinatal HIV-1 transmission in newborns, suggesting a role in innate immunity (Meddows-Taylor et  al. 2006). In sexual transmission, the chemokine CCL28 may also play a role in the resistance to HIV-1 infection via recruitment of HIV-1 specific IgA producing plasma cells to the mucosa (Castelletti et al. 2007). Genetic polymorphisms of chemokine ligands are also important in HIV-1 transmission. Certain CCL5 haplotypes appear to confer an increased risk for HIV-1 transmission and disease progression (Rathore et  al. 2008). In addition, the -403 G/A polymorphism is associated with a non-determined progressive multifocal leukoencephalopathy, (PML)-like leukoencephalopathy (NDLE) in HIV-1 infected individuals (Guerini et  al. 2008). Chemokine ligand genes CCL2 (monocyte chemoattractant protein-1, MCP-1), MCP-30, and Eotaxin, existing as a cluster on the long arm of chromosome 17, although not directly binding to the major chemokine receptor for HIV-1 infection (CCR5), appear to influence virus transmission. As such, chemokine–chemokine receptor interactions likely have additional effects on the immune response to viral infection capable of impairing HIV-1 transmission (Modi et al. 2003).

3.2.2 Chemokines and Chemokine Receptors and HIV-1 Disease Progression While the CCR5∆32 mutation in CCR5 protects against infection in homozygous individuals, heterozygous individuals do not appear to have this protection. Heterozygosity for CCR5∆32, however, does appear to have an effect in slowing disease progression (Eugen-Olsen et al. 1997), and the frequency of this mutation is enriched among slow progressors with HIV-1 infection (Rappaport et al. 1997; Stewart et al. 1997; Winkler et al. 2004). In addition to CCR5∆32, additional polymorphisms exist within the coding and 5¢untranslated regions of the CCR5 gene that may also influence the rate of HIV-1 disease progression. Within the CCR5 regulatory region, and to some extent the coding region, the considerable genetic diversity may suggest evolutionary pressure at this locus (Bamshad et al. 2002; Mummidi et al. 2000). One such haplogroup (HHE) is associated with a more rapid CD4+ T cell decline (Li et  al. 2005). Homozygosity for the 59356-T polymorphism, prevalent among African–Americans, is found to be associated with perinatal HIV-1 transmission (Kostrikis et al. 1999). Other regulatory region polymorphisms, including CCR5 59029A and 59353C, may promote disease progression; however, the CCCR5 59029G mutation appears to be protective (Clegg et al. 2000; McDermott et  al. 1998). In addition, a polymorphism in the CCR5 ligand CCL5 -28G is

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associated with non-progression, whereas the DC-Sign-139 139C mutation is associated with more rapid progression in a cohort of Japanese hemophiliacs (Koizumi et al. 2007). While initial transmission occurs via CCR5, it is interesting to note that a polymorphism in the CXCR4 receptor ligand CXCL12 (stromal cell– derived factor-1, SDF-1) SDF-1 3¢A is associated with an increased risk of infection in an African population (Petersen et al. 2005). Mutations in other coreceptors including CCR2 and CXCR4 also impact HIV-1 disease progression. The CCR2 -64I allele is reported to be associated with more delayed disease progression in some studies (Lee et al. 1998; Munerato et al. 2003; Smith et al. 1997; Winkler et al. 2004). In pediatric infection, the CCR2 -64I polymorphism is associated with delayed HIV-1 progression as well as a decreased risk of mother to child transmission (Mangano et  al. 2000). This mutation is surprisingly associated with increased rate of progression to neuropsychological impairment, suggesting that CCR2 or its ligand, CCL2 (monocyte chemoattractant protein-1, MCP-1), may have a specific role with respect to HIV-1 in the CNS (Singh et al. 2004). This hypothesis is supported by a similar role for the CCL2 -2578G mutation in conferring a reduced risk of acquiring HIV-1 infection, yet accelerating the development of HIV-1 associated dementia (HIVD) (Gonzalez et al. 2002). This mutation is associated with elevated CCL2 levels in cerebrospinal fluid (CSF), providing a likely mechanism for the observed affects on the development of central nervous system (CNS) disease in AIDS (Letendre et al. 2004). It is interesting that plasma CCL2 levels correlate with levels of plasma viremia and subclinical atherosclerosis (Joven et  al. 2006). Additionally, a correlation between CCL2 plasma levels and the CCL2 -2578G polymorphism is associated with atherosclerosis (Alonso-Villaverde et al. 2004). It is possible then that the effects of CCL2 in the CNS may result from its role in the periphery in HIV-1 infection. The role of chemokines and chemokine receptors in the development of HIVD is discussed further in Sect. 3.

3.2.3 Chemokines and Response to HAART Therapy Since chemokine and chemokine receptor mutations impact HIV-1 infection and disease progression, it has been of interest to determine the impact of certain protective polymorphisms in the response to anti-viral treatment. The CCR2 -64I and CXCL12 3¢A alleles are associated with more a favorable prognosis and virologic response to highly active anti-retroviral (HAART) therapy (Puissant et al. 2006). The fractalkine receptor, CX3CR1, that can also act as a coreceptor for HIV-1 infection, exhibits a polymorphism predicting HIV-1 disease progression in children (Singh et al. 2005). One such polymorphism, CX3CR1 -I249 M280, leads to decreased binding of its natural ligand CX3CL1 (fractalkine). Homozygosity for this allele is associated with more rapid disease progression (Faure et  al. 2000). Heterozygosity for M280, however, is associated with higher T cell counts in HAART treated patients (Passam et al. 2007).

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3.2.4 Chemokines as a Double-Edged Sword in HIV-1 Infection While chemokines are clearly not the only mechanisms for protection, they are probably among the multiple mechanisms providing resistance to HIV-1 infection (Piacentini et  al. 2008). Although chemokines that bind HIV-1 co-receptors can inhibit HIV-1 infection, viral infection has been shown to upregulate the production of several chemokines important to disease progression (Ansari et al. 2006; Choe et al. 2001; Poluektova et al. 2001) and most likely plays a role in the migration of uninfected cells toward foci of infection in  vivo. Modulation of chemokine– chemokine receptor interactions could influence a variety of immunologic processes with unintended consequences. It is interesting to note that while initial reports of CCR5∆32 homozygotes did not identify deficient phenotypes, there may be more subtle effects resulting in increased risk of certain diseases. For example, CCR5 genotype and the gene copy number of the cognate ligand CCL3-L1 can be used to determine the risk for the development of systemic lupus erythematosis (Mamtani et al. 2008). Since a defective receptor, caused by the CCR5D32 mutation, and a high copy number ligand appear predisposing, it is likely that interactions with other receptors/molecules are involved. In juvenile idiopathic arthritis the CCR5∆32 mutation is also predisposing (Scheibel et  al. 2008) and a similar association is observed with the development of Kawasaki disease (Burns et al. 2005). Additionally, the CXCL12 3¢A polymorphism has been linked to thrombotic events and myeloproliferative disorders (Gerli et al. 2005). In addition to the role of chemokines/chemokine receptors in viral infection, transmission, and pathogenesis, chemokines also play important roles in cellular trafficking and survival pathways. The role of chemokines and their receptors are of particular importance in the development of neurologic complications of AIDS, involving mechanisms discussed in the following sections.

3.3 Chemokines and Chemokine Receptors in HIV-1-Associated CNS Disease Among HIV-1-infected persons, approximately 5–10% develop a progressive subcortical dementia, termed HIV-1-associated dementia (HIVD). The introduction of HAART has reduced the incidence of HIVD dramatically, which was previously seen in as many as 30% of new AIDS cases during the pre-HAART era. The prevalence of CNS disease at autopsy, however, has risen and is most likely a reflection of HIV-1 infected persons now living longer with AIDS (Neuenburg et al. 2002; Sacktor et  al. 2001) yet ultimately failing HAART regimens. Consistent with a subcortical dementia, HIVD is a syndrome of motor, cognitive, and behavioral dysfunction. The neuropathology of HIVD, HIV encephalopathy (HIVE), is characterized by a generalized reduction of white matter with some grey matter loss,

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where the greatest reduction is seen in the basal ganglia and posterior cortex (Aylward et al. 1995; Aylward et  al. 1993). Other pathological features of HIVE include widespread reactive astrocytes and significant accumulation of mononuclear phagocytes (MPs), which include macrophages and microglia, in the CNS compartment. Microglial nodules and multinucleated giant cells are also observed and are commonly associated with areas of white matter degeneration and regions of necrosis (Budka et al. 1987). The neuropathogenesis of HIVD is not fully understood. In vitro and tissue based studies suggest that neuronal injury and apoptosis occurs both directly, through interaction of neurons with virus or viral proteins, and indirectly, through stimulation by neurotoxic factors produced by neighboring cells and/or through impaired function of supportive cells in the CNS. In HIVE, several chemokines and chemokine receptors are upregulated in the CNS and CSF and are believed to play a prominent role in promoting CNS disease through both mechanisms: directly, through interaction of chemokine receptors with virus or viral proteins and indirectly, through stimulation of secreted factors that are themselves directly or indirectly neurotoxic and recruitment of infected or uninfected monocytes/macrophages into the CNS compartment.

3.3.1 Direct Mechanisms of Neuronal Injury and Apoptosis Chemokine receptor expression has been detected by all cells of the CNS in normal brain tissue. Together with their ligands, these receptors play an important role in CNS development, neuronal survival and inflammation. Significant to HIV-1 infection of the CNS, microglia, astrocytes, and some neurons express major coreceptors for virus entry into cells, specifically CCR3 and CCR5, used by macrophage (M)-tropic virus and CXCR4, used by T-cell (T)-tropic virus. Dual tropic virus, seen at a relatively high frequency in the CNS of patients with HIVE (Mefford et al. 2008), is able to bind one or more of these receptors. In addition to key coreceptors for HIV-1 infection, other minor co-receptors, also believed to interact with intact virus or shed gp120, are expressed by cells of the CNS, including CX3CR1, CCR1, CCR2, and CCR4. Productive HIV-1 infection of the CNS is seen primarily in macrophages/microglia, with the major reservoir of productive virus found in the perivascular macrophage (Fischer-Smith et al. 2001). In conjunction with CD4, infection of macrophages/ microglia by different viral quasispecies occurs predominantly through CCR3 or CCR5 and may also act through other chemokine receptors, including CXCR4. Infection of astrocytes has been demonstrated and is believed to occur through a CD4-independent mechanism (Liu et al. 2004). In vivo and in vitro studies, however, indicate that astrocyte infection is non-productive (Di Rienzo et  al. 1998; Ranki et  al. 1995) or produces only minimal virus (Brengel-Pesce et  al. 1997; Gorry et  al. 1999; Neumann et  al. 1995). Similarly, productive viral infection of neurons has not been demonstrated in  vivo; however, HIV-1 nef and gag gene

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sequences have been detected in microdissected pyramidal neurons from the hippocampus (Torres-Munoz et  al. 2001). Regardless of the scarcity of evidence suggesting productive infection, the presence of viral proteins and/or cell stimulation through interaction of the virus with chemokines receptors expressed by astrocytes and neurons may contribute significantly to the disease process. Neuronal injury and/or loss in HIVE is believed to occur in part through direct interaction of HIV-1 envelope proteins with chemokine receptors present on some populations of nerve cells (Brandimarti et  al. 2004; Ryan et  al. 2002; Yi et  al. 2004). The HIV-1 envelope protein, gp120, which facilitates cellular entry of the virus through chemokine receptor interaction, can also be shed by infected cells and interact with these receptors apart from the complete virion, and activate, without infecting, the bound cell. Both M- and T-tropic virus and gp120 have been shown by several groups to induce neuronal cell death. This is inhibited by the addition of CCL5, CXCL12, CCL22 (macrophage-derived chemokine, MDC) or soluble CX3CL1 to the culture, suggesting interaction of gp120 with their recognized receptors, CCR1/CCR5, CXCR4, CCR4, and CX3CR1, respectively (Meucci et al. 1998). Indeed, HIVD patients with greater CSF levels of CCL5 and CCL4, scored higher on neuropsychological testing than those with low or undetectable levels (Letendre et  al. 1999). These natural ligands of CCR5 have also been shown to suppress HIV-1 infection of MPs (Cocchi et al. 1995), suggesting that the neuroprotective properties of CCL5 and CCL4 against the effects of gp120 may be both direct and indirect through competition with the virus for the CCR5 receptors on neurons, as well as macrophages/microglia. The contribution of gp120 and/or CXCL12 stimulation of neuronal CXCR4 to the development HIVD is not fully understood. Both, gp120 from a CXCR4utilizing virus strain (HIV-1 IIIB) and CXCL12 have been shown to have a direct neurotoxic effect on pure neuronal cell cultures in vitro (Hesselgesser et al. 1998). In a different system, however, that uses a glial feeder layer to support neuronal growth and differentiation, CXCL12 was shown to protect neurons against gp120 induced apoptosis (Meucci et al. 1998), potentially through regulation of retinoblastoma protein (pRb) in post-mitotic neurons by CXCL12 (Khan et  al. 2008). In contrast, a separate in vitro study that utilized a mixed culture system designed to recapitulate the CNS cellular complement showed that gp120 associated neurotoxicity occurred only in the context of macrophage/microglia activation (Kaul and Lipton 1999). Further, in this system, CXCL12 not only failed to protect neurons from gp120 induced apoptosis, but promoted apoptosis itself (Kaul and Lipton 1999). These seemingly incongruent findings may be the result of the fact that both gp120 and CXCL12 do not act solely on neurons but also act on other cells of the CNS. The interaction with other cells may then cause the production of multiple factors that may be neurotoxic and/or neuroprotective. For example, neuronal injury in HIVD is believed to be largely mediated by over-stimulation of the glutamate receptor, N-methyl-d-aspartate (NMDA), -coupled ion channels that allow excessive influx of Ca2+ ions. This influx triggers a variety of potentially harmful enzymes, free-radical formation and additional release of glutamate, an excitatory amino acid. Excess glutamate subsequently over-stimulates NMDA receptors on neighboring

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neurons and initiates further injury. CXCL12 has been shown to induce a rapid release of tumor necrosis factor-a (TNF-a) from astrocytes (Bezzi et  al. 2001) which inhibits the ability of astrocytes to take up excess glutamate (Fine et al. 1996). The role of chemokines in normal and diseased brain is still not fully understood and is the subject of continued investigations; however, these findings may demonstrate a dual nature of chemokines in both protecting neurons from apoptosis, as well as promoting neurotoxicity under specific pathological conditions. For example, HIV-1 infected macrophages secrete a highly neurotoxic form of CXCL12 (Zhang et  al. 2003) that results from N-terminal processing of CXCL12 by active matrix metalloproteinase-2 (MMP-2) (McQuibban et al. 2001). This may suggest a neurotoxic role for CXCL12 in the setting of HIV-1 infection of the CNS.

3.3.2 Indirect Mechanisms of Neuronal Injury and Apoptosis Apoptotic neurons are seen in HIVE autopsy tissue; however, their number does not correlate with the degree of CNS inflammation or severity of dementia (AdleBiassette et al. 1999). Interestingly, neuronal apoptosis is most often observed in areas associated with macrophage/microglial activation rather than the vicinity of infected cells, suggesting a significant contribution of soluble factors by activated macrophages/microglia, including chemokines, in promoting CNS disease (AdleBiassette et al. 1999). In support of this hypothesis, numerous in vitro studies have demonstrated the ability of infected macrophages or macrophages exposed to HIV-1 proteins to secrete chemokines found to be elevated in the CSF of patients with HIVD. The upregulation and action of these chemokines with their cognate receptor contribute to HIVE pathogenesis through several mechanisms, including recruitment of monocytes/macrophages into the CNS compartment, impairing astrocyte function and inducing microglia or astrocyte expression of factors that may promote CNS disease, including chemokines. 3.3.2.1 Monocyte/Macrophage Recruitment The recruitment and accumulation of macrophages in the CNS is significant to the development of HIVE. Once they are in the CNS compartment, infected and/or activated macrophages/microglia promote pathogenesis through viral spread, as well as secrete viral proteins and other factors, including chemokines, cytokines, reactive oxygen species (ROS) and EAAs that may stimulate and/or alter the action of other glia and/or promote neuronal injury and apoptosis. Over expression of chemokines, which function largely to recruit leukocytes to areas of inflammation, would most likely promote excess monocyte/macrophage infiltration into the CNS. Chemokines involved in monocyte/macrophage chemotaxis have been demonstrated in the CSF of patients with HIVD, including CCL2, CCL3, and CCL5; however, these chemokines appear to have different roles in disease pathogenesis.

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Brain macrophages, including perivascular macrophages and microglia, as well as astrocytes, produce CCL2, a potent monocyte/macrophage chemoattractant that appears to have a significant role in the development of HIVD. Patients with HIVD have increased CCL2 in the CSF, which correlates with the degree of dementia. In simian immunodeficiency virus (SIV)–infected rhesus macaques, an animal model for HIV-1 infection and HIVE, elevated CCL2 levels in the CSF preceded and predicted which animal would develop encephalitis (SIVE) (Zink et  al. 2001). The significance of CCL2 to the development of HIVD is further underscored by the finding that HIV-1-infected individuals homozygous for the CCL2 -2578G allele, a polymorphism that results in greater CCL2 expression in response to inflammatory stimulus (Rovin et  al. 1999), have a 4.5-fold increased risk of developing HIVD (Gonzalez et al. 2002). In contrast to CCL2 CSF levels, which are shown to correlate with the degree of dementia, greater CSF levels of CCL3 and CCL5 are associated with better neurocognitive function (Letendre et al. 1999). All three chemokines are involved in chemotaxis of monocytes/macrophages; however, CCL2 acts through the CCR2 receptor, whereas CCL3 and CCL5 act through the CCR5 receptor, a major coreceptor for HIV-1 infection of macrophages/microglia in the CNS, which is also expressed by neurons and astrocytes. The seemingly protective role of CCL3 and CCL5 may occur through competition with the virus for the CCR5 receptor, thus upsetting virus entry or cell stimulation/activation through gp120 interactions with CCR5. 3.3.2.2 Impaired Astrocyte Function and Chemokine Expression Astrocytes are believed to contribute significantly to HIVE neuropathogenesis through the release of glutamate and/or the failure to take up excess glutamate in the environment. This may occur, at least in part, through stimulation of astrocytes by chemokines released by neighboring macrophages/microglia, neurons or other astrocytes. Specifically, CXCL12 can impair glutamate release and uptake by astrocytes through upregulation of TNF-a and is significantly increased by astrocytes and neurons in HIVD (Rostasy et al. 2003). Astrocytes may also contribute to neuropathogenesis through upregulation and expression of factors that promote the development and progression of HIVD, including several chemokines. Recently, CXCL12, which is upregulated by astrocytes and significant to HIVE pathogenesis, has been implicated in monocyte/ macrophage recruitment in the CNS in HIVD through CXCR4 stimulation and decreased ICAM-1 adherence on microvascular endothelial cells (Malik et  al. 2008), suggesting an additional role for CXCL12 in the development of HIVD. CXCL10/(interferon-g inducible protein (IP)-10), which is found to be increased in the CNS of patients with HIVD and induced by HIV-1 gp120 stimulation of astrocytes, may also function to recruit additional leukocytes into the CNS (Asensio et  al. 2001). Further, CXCL10/IP-10 may contribute directly to neuronal injury through increased membrane permeability and subsequent apoptosis by means of caspase-3 activation (Sui et al. 2004).

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The HIV-1 protein, tat, has been shown by stereotaxic injection to induce macrophage invasion of the CNS and astrocytosis, with alterations in cytokine and chemokine production (Rappaport et al. 1999). Tat has also been shown to induce astrocytic expression of the CCL2 (Conant et al. 1998; Kutsch et al. 2000; Weiss et al. 1999) as well as interleukin (IL)-8 and CXCL10 by astrocytes (Kutsch et  al. 2000), presumably contributing to blood–brain barrier instability and leukocyte infiltration into the CNS. Moreover, tat, itself, exhibits CCL-2 (also CCL7, or MCP-3)-like chemotactic activity and contains a C-C region homology to beta chemokines (Albini et  al. 1998). It is interesting that Clade C tat proteins do not exhibit this activity due to a substitution at cysteine residue 31 (C to S) (Ranga et al. 2004). This alteration has been suggested to account for the reduced incidence of HIVD reported in Clade C infection in India (Ranga et al. 2004). Recent in vivo studies introducing infected human macrophages into SCID mice confirmed these cladespecific differences in the ability to induce encephalitis and furthermore, the tat and CCL2 dependence of CNS invasion in this model (Rao et al. 2008). IL-1b and TNF-a production by HIV-1 infected macrophages has also been shown to regulate IL-8 production by astrocytes. Like CCL2, IL-8 is up-regulated in the CSF of patients with HIVD and can promote monocyte migration. Moreover, IL-8 has been shown to synergistically enhance CCL2 mediated monocyte migration (Zheng et al. 2008) and may play an important role in macrophage recruitment and accumulation and in the CNS in HIVE.

3.4 Chemokines/Chemokine Receptor Pathways as Survival Pathways for Monocyte/Macrophage Reservoirs of HIV Infection While HAART has increased the life-expectancy and quality of life of individuals living with HIV-1 infection, the drugs that make up HAART only target virus replication and assembly without targeting the infected cell itself. This allows for the emergence of anti-retroviral “escape mutants” from already infected cells and subsequent development of AIDS. As such, cellular viral reservoirs are an important consideration in the fight against HIV-1 infection. Long-lived tissue macrophages have been implicated in maintaining reservoirs of virus in various compartments, including the CNS. Macrophage CCL5–CCR5 interactions are believed to contribute to macrophage longevity by promoting survival through anti-apoptotic signaling. The anti-apoptotic effect of this interaction occurs through bilateral activation of the phosphatidylinositol 3-kinase (PI3-kinase)- AKT and MEK-ERK signaling pathways (Tyner et al. 2005). Significant to HIV-1 infection, virus inducible levels of CCL5 are necessary for the anti-apoptotic events induced by CCL5-CCR5 interaction to occur (Tyner et al. 2005). Interestingly, whole virus or shed gp120 stimulation of CCR5 also induces PI3-kinase activity and is necessary for productive

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HIV-1 infection (Noble et al. 2003). Together, these findings suggest a significant role for the virus in maintaining a long-lived viral reservoir by inducing CCL5 production and/or through gp120 interactions with CCR5.

3.5 Chemokines/Chemokine Receptor Based Therapeutics: Trials and Tribulations While there are clear considerations regarding predisposition to certain diseases at the CCR5 locus, the lack of obvious phenotypic consequences in individuals homozygous for the CCR5∆32 deletion suggested that targeting CCR5 could have therapeutic utility in HIV-1 infection. Several approaches, including antibodies against CCR5, binding proteins, chemokine ligands, and small molecules have been used in studies designed to target HIV-1 and other diseases involving chemokine/ chemokine receptor interaction (Tsibris and Kuritzkes 2007; Wells et  al. 2006). Indeed, modified or variant forms of CCL5 have demonstrated effectiveness in preclinical in  vitro and animal studies as microbicides (Kawamura et  al. 2004; Kish-Catalone et al. 2007; Lederman et al. 2004). At the same time, small molecule approaches designed to block “Receptor Occupancy” have also resulted in a new class of “….roc” compounds. Several compounds targeting CCR5, including aplaviroc (GlaxoSmithKline), maraviroc (Pfizer) and vicriviroc (Schering Plough), have been developed and other companies, such as Merck, Millenium and Takeda, have developed additional compounds (Ribeiro and Horuk 2005). While apaviroc administration was halted due to toxicity findings in clinical studies, vicriviroc is in phase III clinical trials and maraviroc has been demonstrated to increase the percentage of treated individuals achieving undetectable plasma viremia in combination with other drugs and is now an approved treatment option. A major concern for this therapeutic strategy, however, is that a treatment interfering with CCR5 will foster the selection of CXCR4tropic HIV-1 variants that may be more pathogenic. Indeed, the emergence of such X4-using viruses during maraviroc treatment has been demonstrated and these variants appear to be selected from a pool of virus existing prior to treatment (Westby et al. 2006). For this reason, tropism analysis of patient virus is necessary prior to initiation of maraviroc therapy and maraviroc is not recommended as a first line therapy (Hammer et al. 2008). CCR5 expression likely plays a role in T-cell recruitment and may be involved in the development of autoimmune diseases. There is a negative association between the CCR5∆32 mutation and rheumatoid arthritis (Prahalad 2006). Furthermore, additional studies reviewed elsewhere suggest the involvement of CCR5 in multiple sclerosis, diabetes, and transplant rejection (Ribeiro and Horuk 2005). As such, it is likely that CCR5 antagonists developed for the treatment of HIV-1 infection can also be used for other diseases.

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Chapter 4

HIV Infection and the PNS Kevin Tan, Avindra Nath, and Ahmet Hoke

4.1 Introduction According to data from the Joint United Nations Program of HIV/AIDS (UNAIDS) and the World Health Organization (WHO), as of the end of 2005, there were 38.6 million adults and children living with HIV infection. There were an estimated 4.1 million new infections with HIV and an estimated 2.8 million deaths to AIDS. Changes in incidence along with rising AIDS mortality have caused global HIV prevalence to level off. However, the numbers of people living with HIV have continued to rise, due to population growth and, more recently, the introduction of highly active antiretroviral therapy (HAART). The availability of HAART, at least in the developed world, has a dramatic effect on suppressing HIV viral load (Sacktor 2002), decreasing the incidence of systemic opportunistic infections, reducing morbidity and mortality (Brodt et al. 1997; Palella et al. 1998; Moore and Chaisson 1999), decreasing inpatient hospitalizations (Fleishman and Hellinger 2003) and prolonging lifespan, and improving the prognosis of disease to the extent that the proportion of deaths from non-AIDS defining illnesses has superceded deaths caused by AIDS (Palella et al. 2006). Because of the improved survival, HIV infection is now considered a chronic disease rather than an acute fatal disease.

4.2 General Principles in HIV Neurological Disease HIV is neuroinvasive, neurovirulent, but is not especially neurotrophic (Manji and Miller 2004). It can potentially affect all areas of the neuroaxis, either directly, producing distinct neurological syndromes, or indirectly, by causing immunodeficiency with resultant susceptibility to opportunistic infections. General principles

K. Tan, A. Nath, and A. Höke () John Hopkins University School of Medicine Department of Neurology e-mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_4, © Springer Science+Business Media, LLC 2010

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used in management of patients with neurological complications of HIV include “time locking,” “parallel tracking,” and “layering” (Brew 2001). Prior to the introduction of HAART, the degree of HIV disease was related to its duration (“time locked”), which in turn was thought to be related to the CD4 cell count. In the post-HAART era, this is no longer true as many HAART-treated patients who have had HIV for long durations are well with normal CD4 cell counts. This principle should therefore be retermed “T cell locking” as the CD4 cell count becomes a better measure of degree of advancement of HIV disease (Brew 2003). The second principle is that of “parallel tracking,” which means that different complications affect different parts of the neuroaxis at the same time. An example is HIV spinal cord disease and peripheral neuropathy occurring simultaneously and distorting the symptoms and signs to varying extents depending on the severity of each condition. The third principle is that of “layering,” which means that several complications are superimposed one upon the other within one part of the neuroaxis. An example is a peripheral neuropathy related to HIV layered upon a previous neuropathy secondary to antiretroviral drugs.

4.3 Recognizing Peripheral Nervous System Involvement in HIV At the start of the HIV epidemic in the 1980s, the peripheral nervous system involvement in HIV infection was not widely appreciated. However, as the number of cases grew, it became obvious that not only were peripheral neuropathies common in HIV infection, but were also present in all stages of the disease, from seroconversion through end-stage immunodeficiency. The neuropathic complications occurred as a result of a variety of pathological processes in HIV infection (Verma 2001). The earliest reports of neurological complications of AIDS described distal symmetrical, painful sensory neuropathy occurring in HIV patients (Snider et al. 1983). Dysimmune inflammatory polyneuropathy was subsequently recognized as a complication of AIDS (Lipkin et al. 1985). Progressive polyneuropathy associated with cytomegalovirus (CMV) infection was documented as the first truly opportunistic infection of the peripheral nerve (Eidelberg et al. 1986). Of the neurological disorders associated with HIV infection, peripheral neuropathy has emerged as the commonest and is present at every stage of the disease (Wulff et al. 2000). HIV-associated neuropathy can be classified according to the timing of its appearance during HIV infection, its etiology, and whether it is primarily axonal or demyelinating. Some represent a consequence of HIV infection producing neuropathological damage, while others are related to opportunistic pathogens (Keswani et al. 2002). The spectrum of peripheral involvement in HIV infection includes distal symmetric polyneuropathy (DSP), toxic neuropathy from antiretroviral drugs, inflammatory demyelinating neuropathies (IDP), multifocal

AIDS

Progressive polyradiculopathy CMV polyradiculopathy

AIDS

AIDS

Opportunistic infection, eg. CMV

VZV radiculopathy or myeloradiculopathy

Stepwise progression

Early

Acute

Acute

Acute, subacute

Subacute or chronic

Early; pre-AIDS

Acute

Chronic inflammatory demyelinating polyradiculopathy Mononeuritis multiplex Vasculitic neuropathy

Early; pre-AIDS

Early; pre-AIDS

Seroconversion neuropathy

Inflammatory demyelinating neuropathy Acute inflammatory demyelinating polyradiculopathy

Any stage

Antiretroviral toxic neuropathy

Immune dysfunction: complement/macrophagemediated demyelinating neuropathy Immune dysfunction: demyelinating neuropathy

Mechanism Immune dysfunction: macrophage-mediated axonal injury Toxic neuropathy; mitochondrial damage Immune dysfunction

Lumbosacral pain; saddle anesthesia; rapidly progressive flaccid paraparesis Lumbosacral pain; saddle anesthesia; rapidly progressive flaccid paraparesis

(continued)

VZV infection: Schwann cellendothelial cells infection

CMV infection; necrotizing neuropathy

Multiple, asymmetric Dysimmune or vasculitic mononeuropathies, usually painful mechanisms Multiple, asymmetric CMV infection, Schwann cell mononeuropathies, usually painful infection: demyelinating neuropathy

Sensorimotor neuropathy; NCS show demyelinating features

Motor to sensory signs; NCS show demyelinating features

Clinical features Distal sensory loss and neuropathic pain; depressed or absent ankle reflexes Subacute; rarely acute Distal sensory loss and neuropathic with lactic acidosis pain Acute Generalized systemic illness, mononeuropathies

Table  4.1  Summary of peripheral nervous system involvement in HIV infection HIV-associated PNS disorder HIV disease stage Course Distal sensory neuropathy AIDS Subacute or chronic

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AIDS

AIDS, sometimes early

AIDS on HAART

AIDS

Autonomic neuropathy

Immune reconstitution inflammatory syndrome

Motor neuron disease

HIV disease stage

Diffuse infiltrative lymphocytosis syndrome

Table  4.1  (continued) HIV-associated PNS disorder Course

Subacute

Acute or subacute

Subacute or chronic

Subacute

Sjogren’s-like; symmetric or asymmetric sensorimotor, painful, multiple mononeuritis or distal sensory neuropathy Orthostatic hypotension; papillary abnormalities; sweating dysfunction; resting tachycardia Worsening or new neurological symptoms in the setting of immune reconstitution Motor dysfunction, muscle wasting with upper motor neuron signs; bulbar dysfunction

Clinical features

Possible immune mediated

Angiocentric CD8 hyperlymphocytosis in peripheral nerves; vascular mural necrosis Immune dysfunction: macrophage-mediated sympathetic ganglia injury Immune mediated

Mechanism

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mononeuropathies, progressive polyradiculopathies, and diffuse infiltrative lymphocytosis syndrome (DILS) (Ferrari et al. 2006) (Table 4.1).

4.4 Prevalence and Risk Factors of HIV-Associated Peripheral Neuropathy DSP is the most prevalent form of peripheral neuropathy in HIV in both the preHAART (Cornblath and McArthur 1988; So et al. 1988; Barohn et al. 1993; Fuller et al. 1993; Schifitto et al. 2002) and HAART eras (Morgello et al. 2004; Skopelitis et al. 2006; Smyth et al. 2007). These studies report the prevalence of DSP ranging from 11.5 to 55% of patients with AIDS, depending on the method of detection. The later studies done in the HAART era, which include patients who had toxic neuropathies related to the use of nucleoside reverse transcriptase inhibitors (NRTIs), show increased prevalence of peripheral neuropathy (Moore et al. 2000; Cherry et al. 2006). As toxic neuropathy from antiretroviral drugs cannot be distinguished from DSP clinically or electrophysiologically, they are often considered together as HIV-associated sensory neuropathies. There is a similar high prevalence of peripheral neuropathy (34%) in the pediatric population infected with HIV (Araujo et al. 2000). The frequency of IDP in the HIV-infected population is unknown but is thought to be rare (Wulff et al. 2000). In an outpatient population of HIV positive patients, mononeuritis multiplex and lumbosacral polyradiculopathy were found in less than 1% of patients with AIDS (Fuller et al. 1993). HIV-associated autonomic nervous system dysfunction is also not uncommon as up to 66% of patients have papillary involvement and 15% have sympathetic and parasympathetic involvement causing orthostatic hypotension and respiratory sinus arrhythmia (Gluck et al. 2000). While the prevalence of DSP continues to rise as patients with HIV infection live longer, the incidence of HIV-associated neuropathy may be on the decline. Schifitto et  al in the Northeast AIDS Dementia (NEAD) Consortium estimated the 1-year incidence of symptomatic neuropathy in a cohort of patients on HAART at 21% (Schifitto et  al. 2005), compared to an incidence of 36% in a prior cohort from the pre-HAART era (Schifitto et al. 2002). This suggests that HAART may change the natural history of HIV-associated DSP (Cornblath and Hoke 2006). The reported risk factors for HIV-associated sensory neuropathy are varied and may have changed since the availability of HAART. In the pre-HAART era, age, nutritional deficiencies, alcohol exposure, higher HIV viral load, and low CD4 counts (Moyle and Sadler 1998; Childs et al. 1999), as well as mood, other neurologic disorders and functional abnormalities (Schifitto et al. 2002) were neuropathy risk factors. In the HAART era, the use of NRTI (Cherry et al. 2006; Pettersen et  al. 2006) and exposure to protease inhibitor (PI) medication (Pettersen et  al. 2006; Smyth et al. 2007) are considered additional risk factors. Although hepatitis C mono-infection has been associated with peripheral nerve disease, and there is

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considerable overlap in the symptoms and signs of HIV and hepatitis C neuropathy, coinfection has not been shown to additively or synergistically increase the prevalence of neuropathy (Estanislao et al. 2005; Cherry et al. 2006).

4.5 Distal Symmetric Polyneuropathy DSP occurs mainly in the late phase of HIV infection, when there is severe immunosuppression. The initial symptoms are distal, painful and burning dysesthesias, and allodynia, which start in the toes and slowly progress up the legs over weeks to months. It is usually most severe on the soles of the feet and is typically worse at night. Involvement of the hands is much less common, but may begin at around the same time as symptoms reach the mid-leg level. Although patients may complain of weakness, it is rarely found on examination except late in the disease when there may be intrinsic foot muscle weakness (Cornblath and McArthur 1988). Examination usually reveals distal loss of sensory function to pain, temperature, and vibration modalities, whereas proprioception is usually normal or minimally impaired. The ankle reflexes are almost universally depressed or absent. The knee reflexes are often normal, but may be hyperactive, in which case a concomitant myelopathy should be considered (Dal Pan et  al. 1994). Antalgic gait may result from pain or the heightened sensitivity at the soles of the feet. There is no single diagnostic test for DSP. Investigations have an exclusionary and confirmatory role (Brew 2003). Spinal fluid analysis may show slightly elevated protein in a small number of patients and normal cell counts. Spinal fluid pleocytosis should raise the possibility of concurrent opportunistic infection (Hoke and Cornblath 2004). Electrophysiological studies, which predominantly test the function of large, myelinated fibers, usually show an axonal, length-dependent, predominantly sensory polyneuropathy (Cornblath and McArthur 1988; Tagliati et  al. 1999). However, there are caveats: 28% of asymptomatic HIV-infected patients have electrophysiologic abnormalities, and 19% have clinical evidence of DSP but normal conventional nerve conduction studies, presumably because of small-fiber involvement (Tagliati et  al. 1999). While absent sural sensory nerve action potentials are the commonest abnormality, there are motor conduction abnormalities that include reduced distal compound muscle action potential amplitudes, minimally reduced conduction velocities in the legs, and prolonged F-wave latencies in both arms and legs. Needle electromyography demonstrates denervation potentials and evidence of chronic partial denervation and reinnervation that is usually confined to leg and foot muscles (Hoke and Cornblath 2004). DSP is characterized by distal degeneration of long axons, in a “dying back” pattern. The density of unmyelinated fibers, and less commonly small and large myelinated fibers, is reduced (Araujo et al. 2000). Demyelination and remyelination are seen but are not prominent features, and are results of axonal degeneration. Punch skin biopsies reveal evidence of reduced intraepidermal nerve fiber density in the distal leg (Holland et  al. 1997), which is associated with the degree of neuropathic pain severity (Polydefkis et  al. 2002). Immunopathological studies

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have shown prominent macrophage activation (in some cases, subtle to prominent lymphocytic infiltration) with release of proinflammatory cytokines in the endoneurium of dorsal root ganglions and peripheral nerves (de la Monte et  al. 1988; Rizzuto et al. 1995; Pardo et al. 2001). HIV DNA or viral particles are rarely found in biopsy or autopsy nerve samples (Rizzuto et al. 1995).

4.6 Antiretroviral Toxic Neuropathy With the introduction of combination antiretroviral therapy in the mid 1990s, the incidence of most neurological complications of HIV has fallen dramatically. Although the incidence of HIV-associated DSP has decreased, the overall prevalence of peripheral neuropathy continued to increase (Sacktor 2002). This increase occurred at around the same time as NRTIs (particularly zalcitabine, ddC) were introduced into clinical practice (Berger et al. 1993; Blum et al. 1996). Longitudinal assessment of HIV patients in the Multicenter AIDS Cohort Study (MACS) showed that the incidence of sensory neuropathy increased in the face of declining incidence in most other neurological complications of HIV infection, and that this increase might have been attributable to the toxicity of HIV treatment (Bacellar et al. 1994). As additional NRTIs became available, it was found that didanosine (ddI) and stavudine (d4T) also caused a predominantly sensory neuropathy in a dose dependent fashion (Simpson and Tagliati 1995; Moyle and Sadler 1998). Furthermore, the risk of neuropathy may be additive or even synergistic when combinations of NRTIs are used (Moore et al. 2000). Because these drugs are an essential component of HAART, the NRTI-associated painful sensory neuropathy not only affects quality of life, but also severely limits viral suppression strategies. The phenotype and clinical presentation of antiretroviral toxic neuropathy (ATN) are similar to those of HIV-associated DSP. However, ATN is more likely to be painful, and has an abrupt onset and rapid progression. The main diagnostic clue is the temporal relationship of peripheral neuropathy to the start of NRTI therapy and stabilization, or at least the partial resolution when therapy is interrupted (Moyle and Sadler 1998). ATN most often develops after a mean of 16 to 20 weeks of treatment, unless there are other conditions that lower the threshold. Symptomatic improvement over weeks to months has been reported in two thirds of patients after discontinuation of the offending drug, but may be preceded by an initial period of worsening symptoms, also known as “coasting” (Berger et al. 1993). Despite the improvement, most patients do not return to a completely asymptomatic state (Hoke and Cornblath 2004). The electrophysiological and neuropathological changes are similar to the axonal neuropathy and prominent loss of unmyelinated fibers seen in DSP (Simpson and Tagliati 1995). Although the specific pathological mechanisms of ATN are not fully known, there is abundant indirect evidence of mitochondrial dysfunction as a principal mechanism. Prominent mitochondrial disruption and cristae abnormalities

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have been described with the use of some NRTIs and have been linked to interference with mitochondrial DNA synthesis. Myelin changes such as myelin splitting and edema have been observed in animal models of ddI peripheral neurotoxicity (Pardo et al. 2001). Dalakas and colleagues provided direct, pathological evidence of mitochondrial abnormalities in sensory nerves of patients with ddC-induced neuropathy. This mitochondrial dysfunction is attributed to the inhibition of DNA polymerase g, the enzyme responsible for mitochondrial DNA replication (Dalakas et al. 2001).

4.7 Neuropathies Associated With HIV Seroconversion A generalized systemic illness may accompany HIV seroconversion (Cooper et  al. 1985). Guillain-Barre syndrome (GBS) (Piette et  al. 1986), unilateral (Wiselka et al. 1987) or bilateral facial palsies (Wechsler and Ho 1989), bibrachial palsy (Calabrese et al. 1987) and sensory neuropathy (Denning 1988) have been reported to occur during this process, usually within 1–2 weeks of the acute febrile illness. Spinal fluid analysis may show a mild to moderate mononuclear pleocytosis and a mild increase in protein levels. The precise relationship to HIV viral load in the cerebrospinal fluid (CSF) or plasma is unknown (Brew 2003). There is no proven therapy, but most patients recover spontaneously without any treatment.

4.8 Inflammatory Demyelinating Polyradiculoneuropathies The inflammatory demyelinating polyradiculoneuropathies can been divided into two forms: acute inflammatory demyelinating polyneuropathy (AIDP form of GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP). They typically occur during early HIV infection in patients who are otherwise asymptomatic and probably represent autoimmune phenomena (Cornblath et al. 1987). The clinical and electrophysiological features and the response to immunomodulating therapies are indistinguishable from those seen in non-HIV associated AIDP and CIDP (Cornblath 1988). AIDP is clinically characterized by rapidly progressive ascending weakness associated with generalized areflexia, with progression to maximum illness in less than 4 weeks. It may occur with seroconversion (Piette et al. 1986) or as the first manifestation of illness that leads to the identification of concurrent HIV infection (Cornblath et al. 1987). CIDP is distinguished by its slower progression over several months and may be monophasic or relapsing. Motor findings usually predominate, and sensory symptoms and signs are usually minor. The cranial nerves may be involved, and respiratory paralysis occurs in acute cases (Hoke and Cornblath 2004).

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The presence of HIV infection, inversion of CD4/CD8 T-cell ratios, CSF lymphocytic pleocytosis (10 to 50 cells/mm3) in addition to elevated CSF protein and polyclonal elevation of serum immunoglobulins may help distinguish HIVseropositive from HIV-seronegative individuals with IDP. The primary electrophysiological finding is demyelination with variable degrees of axonal loss on serial follow up studies. This is manifest by some combination of prolonged distal and F-wave latencies, reduced conduction velocities, and compound motor action potential (CMAP) amplitudes, abnormal temporal dispersion and partial conduction block (Hoke and Cornblath 2004). In HIV-associated AIDP, the demyelinating form occurs more frequently than the axonal form. An immune attack on Schwann cells and myelin structure occurs and appears to be mediated by macrophages. A CD8 lymphocytic inflammatory infiltration of the peripheral nerve and roots is also seen (Pardo et al. 2001). In a few cases of AIDP, CMV inclusions in Schwann cells have also been found (Eidelberg et  al. 1986). The peripheral nerve pathology observed in sural nerve biopsies of HIV associated CIDP is similar to that in non-HIV-infected patients (Griffin et al. 1990). In early CIDP, pathological changes are characterized by lymphocytic and macrophage infiltration and demyelination (Cornblath et al. 1987; de la Monte et al. 1988). At later stages, remyelination, formation of onion bulbs and reduction in lymphocytic infiltration, small myelinated and unmyelinated fibers predominate (Pardo et al. 2001).

4.9 Mononeuropathy Multiplex Mononeuropathy multiplex (MM) is an infrequent complication that can occur in early HIV infection, because of dysimmune or vasculitic mechanisms (Gherardi et al. 1989; Chamouard et al. 1993; Bradley and Verma 1996; Schifitto et  al. 1997; Mahadevan et  al. 2001). Seroconversion-related MM has been described in a case report (Sugimoto et al. 2006). Most of these patients have a good prognosis as symptoms resolve spontaneously and treatment may not be necessary. In others, treatment has focused on intravenous immunoglobulin, judicious short-term use of steroids, and a combination of zidovudine and plasmapheresis (Chamouard et  al. 1993; Cohen et  al. 1993; Bradley and Verma 1996; Schifitto et al. 1997). In advanced AIDS, MM is usually associated with opportunistic infections such as CMV (Said et al. 1991; Roullet et al. 1994; Kolson and Gonzalez-Scarano 2001) or is secondary to lymphoma (Fuller et al. 1993). Despite a role for other herpes viruses in AIDS-associated myelitis, no substantive evidence has been published in support of a role for other viruses in the development of HIV-associated MM, including herpes simplex 1 or 2, varicella zoster, or Epstein Barr virus (Kolson and Gonzalez-Scarano 2001). MM can occur secondary to hepatitis B and C viruses, which are common co-infections of HIV-infected patients, particularly when there is an associated cryoglobulinemia (Taillan et al. 1993; Caniatti et al. 1996). Rarely

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has MM in HIV-infected patients with negative hepatitis B and C serologies been associated with cryoglobulinemia (Stricker et al. 1992; Le Lostec et al. 1994). The clinical presentation of MM in HIV-infected patients is similar to that in other patients with vasculitic neuropathy (Hoke and Cornblath 2004). It is characterized by symptoms and signs of sensory involvement, with numbness and tingling in the distribution of one peripheral nerve trunk. Sequential involvement of other noncontiguous peripheral or cranial nerves progresses over days to weeks. The initial multifocal and random neurologic features may evolve to symmetrical neuropathy (Ferrari et al. 2006). Electrophysiological studies often demonstrate multiple, asymmetric mononeuropathies, usually axonal in type, that cannot be localized to typical sites of entrapment (Keswani et al. 2002). CSF analysis reveals nonspecific abnormalities, such as elevated protein and mild mononuclear pleocytosis. Polymerase chain reaction (PCR) for CMV DNA and nerve or muscle biopsy may provide more specific diagnostic data (Roullet et al. 1994). Pathological studies of sural nerves in MM associated with the early stages of HIV infection show evidence of necrotizing vasculitis. The potential role of immune complex attack associated with hepatitis B and C or cryoglobulins as the main immunopathological mechanism has been proposed. In the later stages of AIDS, the CMV-associated MM is characterized pathologically by CMV inclusions in endothelial cells, focal demyelination, and Wallerian degeneration (Pardo et al. 2001).

4.10 Progressive Polyradiculopathy Progressive polyradiculopathy (PP) is an uncommon but well-described complication of HIV infection, occurring in advanced immunosuppression. It was detected in less than 2% of HIV positive patients referred for neurological evaluation (de Gans et al. 1990; Fuller 1992). The incidence of HIV-associated PP is thought to have declined in the era of HAART. It is usually attributed to CMV infection (Eidelberg et al. 1986; Behar et al. 1987; Miller et al. 1990; Fuller 1992; So and Olney 1994). However, it can be caused by other conditions, including lymphoma (Leger et  al. 1992; So and Olney 1994), syphilis (Lanska, Lanska et  al. 1988), mycobacterial infections (Corral et al. 1997; Masjuan et al. 1997), herpes simplex virus (Miguelez et al. 1999), and cryptococcus (Dromer et al. 1995). PP characteristically presents with subacute lumbosacral pain, “saddle anesthesia”, a rapidly progressive flaccid paraparesis and urinary retention over a few days to weeks (Eidelberg et al. 1986; So and Olney 1994). The motor involvement may be asymmetric (So and Olney 1994). A thoracic sensory level may be found in some patients (Eidelberg et al. 1986), and upper extremities may be involved late in the course of disease (Said et al. 1991). The majority of untreated individuals die within a few weeks. Many patients with CMV-related PP have co-existing systemic CMV infection such as retinitis (Behar et al. 1987; Miller et al. 1990).

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CSF findings in CMV-related PP are characterized by a marked polymorphonuclear pleocytosis, elevated protein level, and hypoglycorrhachia (de Gans et al. 1990; Miller et al. 1996). CMV can be recovered from CSF by culture, or more rapidly by PCR amplification of the viral genomic sequences. Lymphomatous meningitis is characterized by a lymphocytic pleocytosis. Electrodiagnostic study reveals primary evidence of axonal loss in lumbosacral roots with later denervation potentials in leg muscles and little or no evidence of demyelination (Hoke and Cornblath 2004). Radiological studies of the spinal cord should be performed to exclude focal compressive lesions of the cauda equina (Wulff et  al. 2000). Some patients will have thickened and sometimes enhancing nerve roots on imaging (Brew 2003). Autopsy studies in CMV-related PP have demonstrated marked inflammation and extensive necrosis of ventral and dorsal nerve roots (Eidelberg et al. 1986; Miller et al. 1990; Said et al. 1991). In severe cases, vascular congestion, edema, and infiltrates of polymorphonuclear and mononuclear cells have been observed around the inflamed nerve roots (Said et al. 1991). CMV has been described in neural tissue, based on both cytomegalic inclusions and immunocytochemical studies (Hoke and Cornblath 2004). Sural nerve biopsies in this syndrome have been relatively unrevealing, with only minimal degrees of inflammation (Cornblath 1988). In PP caused by lymphoma, infiltration of the ventral and dorsal nerve roots, including cauda equina by lymphomatous cells, has been observed (Leger et al. 1992).

4.11 Diffuse Infiltrative Lymphocytosis Syndrome Diffuse infiltrative lymphocytosis syndrome (DILS) is a rare systemic condition characterized by persistent peripheral CD8 lymphocytosis (CD8 cell count greater than 1,000 cells/mm3) and visceral CD8 T-cell infiltration of the salivary glands, lungs, kidneys, gastrointestinal tract, and peripheral nerves (Guillon et  al. 1987; Moulignier et al. 1997; Gherardi et al. 1998). Infiltration of the salivary glands and other visceral sites produces a Sjogren-like disorder (Itescu et al. 1989; Itescu et al. 1990; Kazi et al. 1996). Patients with DILS tend to have higher CD4 cell counts, fewer opportunistic infections, and longer survival times than typical HIV-infected patients (Ferrari et al. 2006). DILS-associated neuropathy has a variety of clinical presentations, including painful symmetric or asymmetric sensorimotor neuropathy, distal sensory neuropathy, mononeuritis multiplex, and demyelinating polyneuropathy (Gherardi et  al. 1998). Cranial neuropathy without evidence of a more generalized neuropathy may occur, typically as a facial nerve palsy in association with parotidomegaly (Itescu et al. 1990; Brew 2003). The neuropathy develops subacutely over days to weeks. In some cases, muscle weakness may be a result of an inflammatory myositis (Kazi et al. 1996). Electrophysiologic studies usually show axonal neuropathy, but in 15%, it is demyelinating (Moulignier et al. 1997). CSF is remarkable for a mild nonspecific lymphocytic pleocytosis, but with markedly raised protein up to 2 g/L (Brew 2003). Nerve biopsy shows marked angiocentric CD8 infiltrates without mural necrosis

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and abundant expression of HIV p24 protein in macrophages infiltrating the nerves (Moulignier et al. 1997; Gherardi et al. 1998; Price 1998). The lack of monoclonality in the T-cell infiltrates and the massive HIV proviral load in nerves distinguishes it as a HIV-related neuropathy rather than a malignant lymphomatous process (Gherardi et al. 1998; Authier and Gheradi 2003).

4.12 Autonomic Neuropathy There is conflicting data on whether or not autonomic nervous system dysfunction occurs in HIV infection (Hoke and Cornblath 2004). Early reports of small numbers of subjects suggested that dysautonomia may occur in the early (Villa et al. 1987; Welby et al. 1991) or advanced stages of infection (Craddock et al. 1987; Lin-Greenberg and Taneja-Uppal 1987; Miller and Semple 1987; Cohen and Laudenslager 1989; Ruttimann et al. 1991). However, one study that corrected for age and resting heart rate could find no increase in the number of abnormal autonomic function tests in individuals with AIDS, suggesting that dysautonomia may be nonspecific or falsely positive (Lohmoller et al. 1989). Later studies have reported subclinical autonomic neuropathy in otherwise neurologically asymptomatic individuals (Gluck et  al. 2000; Mittal et  al. 2004; Correia et  al. 2006). Severe autonomic neuropathy that occurs in patients with AIDS is generally accompanied by other forms of HIVassociated neuropathy (Freeman et al. 1990; Ruttimann et al. 1991). Failure of the parasympathetic autonomic system is manifested clinically by resting tachycardia, impotence, and urinary dysfunction. Sympathetic system abnormalities include orthostatic hypotension, syncope, diarrhoea, and anhydrosis. A variety of systemic and metabolic factors such as fluid depletion, electrolyte imbalance, malnutrition, peripheral, and central nervous system lesions, and medications may contribute to autonomic dysfunction (Wulff et  al. 2000; Verma 2001). The mechanism of autonomic dysfunction in HIV largely remains speculative. There may be both central and peripheral nervous system alterations (Gluck et al. 2000). The presence of T lymphocytes, macrophages with detectable HIV antigens and nerve cell degeneration in sympathetic ganglia of AIDS patients provides morphological evidence of autonomic nervous system damage (Chimelli and Martins 2002).

4.13 Neuropathies Associated with Immune Reconstitution HAART has been associated with numerous neurological immune reconstitution illnesses, which generally occur within 6 months after its introduction and are thought to constitute an aberrant immune response to opportunistic pathogens (Shelburne and Hamill 2003; Riedel et  al. 2006; Venkataramana et  al. 2006).

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Overall, it appears that immune reconstitution is only rarely associated with peripheral neuropathies in HIV-infected individuals. Makela and colleagues described the occurrence of a probable recurrent GBS six weeks after initiation of HAART and after a striking increase in CD4 cell count in an HIV-infected individual (Makela et al. 2002). Piliero and colleagues described an HIV-infected patient with AIDS who developed GBS, 26 days after initiation of a 6-drug HAART regimen, which had led to an impressive immune reconstitution (a rise in CD4 cell count from 31 to 602 cells/mL) (Piliero et al. 2003). Puthanakit and colleagues identified a child who developed GBS, 3 weeks after initiation of efavirenz-based HAART in a cohort of HIV-infected Thai children (Puthanakit et al. 2006).

4.14 Motor Neuron Disease There have been several reports of HIV-infected patients developing an illness very similar to motor neuron disease, with the clinical patterns mirroring those of amyotrophic lateral sclerosis (ALS) (Hoffman et al. 1985; MacGowan et al. 2001; Moulignier et al. 2001; Sinha et al. 2004; Verma and Berger 2006), progressive spinal muscular atrophy (PSMA) (Galassi et al. 1998), brachial amyotrophic diplegia (Berger et  al. 2005) and primary lateral sclerosis (PLS) (Verma and Berger 2008). These HIV-associated ALS syndromes differ from classical ALS in some respects. First, they occurred in younger patients unlike what is typical of classic ALS. Second, they were usually rapidly progressive. Third, the disorder did not progress inexorably, but improved in some patients after institution of antiretroviral therapy (Jubelt and Berger 2001). There is a suggestion that the relationship may be attributable to a quasispecies of HIV (von Giesen et  al. 2002). There was no consistent relationship of the ALS syndrome to the duration of HIV infection or stage of HIV disease. Further data are needed to clarify whether the association is truly causal (Verma and Berger 2006).

4.15 Pathology of HIV-Associated Peripheral Neuropathy The neuropathological analysis of HIV-associated neuropathies should include the different central and peripheral nervous system structures associated with sensory pathways, including spinal cord, dorsal root ganglia (DRG), peripheral nerve, and cutaneous nerve fibers (Pardo et al. 2001) (Fig. 4.1). The majority of studies have focused on the evaluation of the peripheral nerve, often from sural nerve biopsies and the DRG. Few studies have examined the pathology of sensory pathways in the

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Fig. 4.1  Hypothetical model of pathogenesis of pain in DSP. (1) Injury of peripheral nerve fibers due to multifocal inflammation and secreted macrophage activation products results in abnormal spontaneous activity of neighboring uninjured nociceptive fibers (“peripheral sensitization”). (2) Furthermore, the aberrant inflammatory response in DRG leads to alterations in neuronal sodium and calcium channel expression and ectopic impulse generation. (3) This results in central remodeling within the dorsal horn due to A-fiber sprouting and synaptic formation with pain fibers in lamina II, and maintenance of neuropathic pain (‘central sensitization’). Reproduced with permission from (Keswani et al. 2002)

spinal cord or skin nerve fibers. Most of the studies on the pathogenic mechanisms of HIV neuropathy have been focused on DSP and ATN, the commonest causes of peripheral nerve damage. The mechanisms of HIV-associated DSP are incompletely understood. Over the years, various hypotheses have been put forward, but recent data suggest that multiple mechanisms are likely to play a role in neuronal or axonal injury in DSP. These processes may be mediated by direct neurotoxicity of HIV or secreted viral proteins such as the envelope glycoprotein gp120, or by indirect mechanisms

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Fig. 4.2  Dorsal root ganglia pathology in HIV neuropathy is characterized by foci of macrophagelymphocytic infiltration (a) and Nagoette nodules (b). Infiltration by activated macrophages is demonstrated by immunostaining with anti-CD68 antibodies (c) (scale bar, 50 mm). Reproduced with permission of Wiley-Blackwell Publishing (Pardo et al. 2001)

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through neurotoxic cytokines released by infected or activated glial cells (Hoke and Cornblath 2004). The earliest recognized characteristic pathological feature is the axonal degeneration of long axons in distal regions (Cornblath and McArthur 1988; de la Monte et al. 1988; Fuller et al. 1993). The axonal damage follows a “dying back” pattern of degeneration, resembling the profile of other neuropathies that have prominent small sensory fiber involvement, such as amyloidosis and diabetic neuropathy. In many cases, there are epineural and endoneural perivascular inflammatory cells, such as T lymphocytes and activated macrophages (de la Monte et al. 1988). Other early neuropathological studies in populations of HIV patients have also shown variable degrees of involvement of the DRG with changes associated mostly with inflammation (Yoshioka et al. 1994; Rizzuto et al. 1995; Nagano et al. 1996) (Fig. 4.2). The inflammatory cells are comprised mostly of lymphocytes and activated macrophages or microglia cells. In many of these patients, there are reduced numbers of DRG neurons and increased frequency of Nageotte nodules (Yoshioka et al. 1994; Rizzuto et al. 1995; Nagano et al. 1996). The presence of infection by HIV of DRG neurons remains controversial (Yoshioka et al. 1994; Nagano et al. 1996). The most consistent pathology in the DRG appears to be activation of inflammatory mechanisms that lead to injury of DRG neurons and the subsequent loss of inputs to central sensory pathways (Rance et al. 1988). Few studies have focused on the pathological changes in the spinal cord associated with HIV neuropathy. Selective degeneration of the gracile tract in patients with sensory neuropathy, characterized by loss of axons and myelin sheaths in the

Fig. 4.3  Epidermal nerve fiber illustrated in a 50-mm vertical skin section, immunostained with the panaxonal marker anti–protein gene product 9.5. Skin section showing epidermal nerve fiber density (arrows) in the distal leg of a healthy adult (a) and reduced epidermal nerve fiber density and degenerating fibers (arrows) in the distal leg of a HIV-associated sensory neuropathy patient (b) (scale bar, 50 mm). Courtesy of Drs Gigi Ebenezer and Justin McArthur, Johns Hopkins University, Baltimore, Maryland, USA

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cervical and upper thoracic cord, suggested a “dying back” degeneration process of DRG neurons (Rance et al. 1988; Dal Pan et al. 1994). Other studies have shown the frequent coexistence of peripheral neuropathy in patients with vacuolar myelopathy (Dal Pan et al. 1994). Epidermal nerve fiber analysis by immunocytochemical techniques using the panaxonal marker protein gene product 9.5 (PGP 9.5) allows the study of epidermal innervation by small fiber C and Ad nerve fibers (McCarthy et  al. 1995; Holland et al. 1997). Studies of skin biopsies of HIV infected patients with DSP or ATN showed reduction in the number of epidermal fibers in distal areas of the lower extremities with an inverse correlation between neuropathic pain intensity and epidermal nerve fiber density (Polydefkis et al. 2002) (Fig. 4.3). There were also fewer epidermal fibers in HIV seropositive patients without clinical evidence of neuropathy, suggesting that HIV infection may be associated with the loss of cutaneous innervation even before the onset of sensory symptomatology (McCarthy et al. 1995).

4.16 The Role of Chemokine Receptors in HIV Neuropathogenesis HIV produces profound CD4 depletion, possibly through initial massive depletion of gut-associated memory T-cells, followed by chronic immune activation, leading to fatigue of homeostatic T-cell responses and progressive immunodeficiency (McArthur et al. 2005). Chemokines are small chemotactic cytokines that act as important messenger molecules between cells of the immune system. Chemokines produce their effects by activating a family of G-protein-coupled receptors. Chemokine receptors are all seven-transmembrane glycoproteins that are structurally related. They may be characterized into those that bind to specific ligands, or those that bind several chemokine ligands. There are also virally encoded (viral) chemokine receptors that represent shared receptors that have been transduced into the viral genome during evolutionary history (Premack and Schall 1996). Binding of HIV envelope glycoprotein gp120 to CD4 receptor solely is not sufficient for viral fusion and entry. An additional cell-surface cofactor is required for HIV infection. Chemokine receptors were identified to function as the cofactors for fusion and entry of HIV. Gp120 mediates binding of virus to the cell surface through high affinity interaction with CD4 receptor. However, it is the subsequent interaction with the appropriate chemokine receptor that is thought to trigger the final conformational changes, leading to fusion between the viral and cellular membranes (He et al. 1997; Horuk 1999). HIV strains are grouped according to the preferred site of replication. T-tropic viruses prefer replication in T lymphocytes and M-tropic viruses in macrophages. Use of chemokine receptors differs for each subgroup: CXCR4 (or fusin, the receptor for stromal cell-derived factor [SDF-1]) for T-tropic viruses and CCR5 (the receptor

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for Regulated on Activation, Normal T Expressed and Secreted [RANTES], also known as CCL5) for M-tropic viruses. M-tropic HIV strains are responsible for viral transmission and are the prevalent viral type isolated from asymptomatic individuals. With time, typically over years, T-tropic viral strains that are better able to infect T lymphocytes emerge. The emergence of T-tropic viral strains in infected individuals correlates with accelerated disease progression. Some strains of HIV exhibit dual tropisms and others can also use other chemokine receptors in addition to CXCR4 and CCR5. The precise determinants of HIV tropism in vivo are very complex and continue to be defined (Horuk 1999; Miller and Meucci 1999). In the brain, CCR5 and CCR3 have been postulated as coreceptors on microglia (He et al. 1997). After the HIV provirus has been integrated into the host-cell genome, it can be latent for years. During latency, while cellular function does not seem to be affected, viral replication continues actively (Ho et al. 1995). Some virally infected cells eventually die through apoptotic mechanisms (Banki et  al. 1998), although microglia are relatively resistant. Microglia and macrophages are able to sustain a productive infection without cellular activation. Chronic immune activation with HIV disease progression leads to dysregulation of macrophages, with the overproduction of various proinflammatory cytokines and chemokines within both the central and peripheral nervous system. This progression is crucial for the onset of HIVassociated sensory neuropathy (Tyor et al. 1995). In the central nervous system (CNS), chemokine receptors participate in HIVinduced neuropathology. Many types of cells in the brain possess a wide variety of chemokine receptors, including microglia, glia, and neurons. The existence of chemokine receptors on neurons suggests that chemokines might regulate neuronal function physiologically. These receptors might mediate some of the neurotoxic effects of HIV on the brain through their interactions with gp120. Neuronal death may be caused by direct mechanisms of gp120 on the same neuronal chemokine receptors, or indirectly through the release of neurotoxins (Miller and Meucci 1999).

4.17 Direct and Indirect Mechanisms of Neurotoxicity While direct infection of the neurons by HIV is not likely to be an important mechanism of neurotoxicity (Pardo et al. 2001), there is a complex interplay of HIV envelope glycoprotein gp120 neurotoxicity and other immunopathogenic factors in the mechanisms of axonal or neuronal injury (Hoke and Cornblath 2004). HIV infection most likely causes DRG cell body and axonal damage via different mechanisms of injury in each structure (Hahn et al. 2008). Gp120 directly invades the peripheral nerve and the DRG, resulting in neurotoxicity (Apostolski et al. 1993; Herzberg and Sagen 2001). The binding of gp120 to rat DRG neurons was initially found to be mediated by a glycoprotein (Apostolski et al. 1993). Herzberg and Sagen showed that limited peripheral nerve exposure to gp120 induces persistent painful sensory neuropathy in an in vivo rat model, which may be mediated by varying mechanisms in different parts of the sensory pathway (Herzberg and Sagen 2001). The gp120-exposed sciatic nerve exhibited transient

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Fig. 4.4  Simplified hypothesis of the mechanism of gp120-induced dorsal root ganglion (DRG) neurotoxicity. CXCR4 binding on Schwann cells by SDF-1a or gp120 results in the release of RANTES, which induces tumor necrosis factor (TNF)–a production by DRG neurons, and subsequent TNFR1-mediated neurotoxicity in an autocrine/paracrine fashion. Reproduced with permission of John Wiley & Sons, Inc. (Keswani et al. 2003b)

pathology, notably axonal swelling, and increased TNF-a within the nerve trunk. In addition, an intense astrocytic and microglial activation was observed in the spinal cord, which persisted and paralleled neuropathic pain behavior. Gp120 can also induce indirect neuronal injury through the Schwann cells (Keswani et  al. 2003b). When DRG sensory neurons and Schwann cells in a coculture paradigm were exposed to chemokine receptor CXCR4 and gp120, there was an upregulation of RANTES by the Schwann cells through the chemokine receptor CXCR4. RANTES bound to the chemokine receptor CCR5 on the neurons and induced upregulation of TNF-a, leading to classical apoptotic neuronal death in sensory neurons and axonal degeneration (Fig.  4.4). Axonal degeneration was partially blocked by a specific caspase inhibitor, but it was not clear if this effect was a direct action on the mechanism underlying axonal degeneration or it was an indirect effect due to the apoptotic death of the neuronal body. Melli and colleagues using a compartmentalized culture system, studied the mechanisms specific to axonal and neuronal toxicity (Melli et  al. 2006). In this in vitro cell culture method, the neuronal cell body was isolated from the axonal compartment, and each manipulated individually. The role of perineuronal and periaxonal Schwann cells was also examined individually. Their results showed that gp120 caused neuronal apoptosis and axonal degeneration through two, independent and spatially separate mechanisms of action: (1) an indirect insult to cell bodies, requiring perineuronal Schwann cells, resulting in neuronal apoptotic death and secondary axonal degeneration; (2) a direct, local toxicity exerted on axons through activation of mitochondrial caspase pathway that is independent of the cell body. This local axonal toxicity was mediated through binding of gp120 to axonal chemokine receptors, CCR5 and CXCR4 and was preventable by chemokine receptor

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a

b 120

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Normalized axon length

100

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80 60 40

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gp120

gp120 + anti-CXCR4

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Fig. 4.5  Gp120-induced local axonal degeneration is mediated directly by chemokine receptors on axons. Distal axons express both chemokine receptors CCR5 and CXCR4 as shown by triple immunostaining with anti-bIII tubulin, anti-CCR5, and anti-CXCR4 antibodies (63% of axons express CCR5 receptor and 94% express CXCR4) (a). Monoclonal antibodies anti-CXCR4 and anti-CCR5 prevented gp120 axonal toxicity when applied to the distal axons without Schwann cells (b) (scale bar, 200  mm). Reproduced with permission of Oxford University Press (Melli, Keswani et al. 2006)

blockers (Fig.  4.5). Periaxonal Schwann cells did not mediate this direct axonal toxicity, but rather played a neuroprotective role. The indirect mechanism of HIV-associated neurotoxicity involves immunopathogenic factors that cause peripheral nerve dysfunction (Verma et al. 2005). HIV may cause indirect damage by predominantly promoting macrophage infiltration in peripheral nerves and DRG. The increased number of reactive macrophages within peripheral nerves causes local release of proinflammatory neurotoxic cytokines, such as TNF-a, IFN-g, IL1, and IL6 (Breen 2002; Power et al. 2002), which cause axonal degeneration. An analysis of spinal cord and peripheral nerve cytokine profile in AIDS patients with vacuolar myelopathy and DSP showed increased TNF-a and decreased IL4 when compared to AIDS patients without neurological disease. The increased levels of TNF-a expression are predominantly due to macrophage or

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microglial production (Wesselingh et al. 1994). The presence of proinflammatory cytokines such as TNF-a, IFN-g, and IL6, as well as other mediators of inflammation such as nitric oxide, has been consistently demonstrated in the DRG of HIVinfected patients (Yoshioka et  al. 1994; Nagano et  al. 1996). The infiltrating infected macrophages may also serve as a reservoir of secreted viral proteins gp120 (Hoke and Cornblath 2004). At this stage, it is difficult to determine what role direct and indirect neurotoxicity plays in DSP. Further in vivo studies are needed to corroborate the in vitro studies. What is needed is a reliable animal model of HIV infection that also shows PNS neurotoxicity (Hoke and Cornblath 2004).

4.18 Pathology of Antiretroviral Toxic Neuropathy NRTIs are structural analogues of the natural nucleotides that form the building blocks of RNA and DNA in human cells. Their use as part of HAART has dramatically modified the natural history of HIV infection. They, however, cause a range of drug- or tissue-specific toxicities: zidovudine (AZT) causes myopathy; zalcitabine (ddC), didanosine (ddI), and lamivudine (3TC) cause neuropathy; stavudine (d4T) causes neuropathy or myopathy and lactic acidosis (Dalakas 2001). During phase I and II trials, the dose-limiting toxicity of didanosine, zalcitabine, and stavudine was identified as peripheral neuropathy (Dalakas 2001). The mechanisms by which NRTIs cause toxicity are not clearly established, but are hypothesized due to mitochondrial toxicity based on the “DNA polymerase g hypothesis” (Lewis and Dalakas 1995; Moyle 2000a, 2000b; Nolan and Mallal 2004). The clinical features of inherited mitochondrial disorders, such as peripheral neuropathy, myopathy, pancreatic dysfunction, and metabolic abnormalities including diabetes mellitus and lactic acidosis are similar to some of the observed side effects of NRTIs. Peripheral neuropathy is one of the most common features of the known mitochondrial disorders, and the NRTIs that have been associated with the development of ATN most potently inhibit mitochondrial DNA in vitro by interfering with mitochondrial DNA polymerase g, resulting in impairment of mitochondrial DNA synthesis and function (Cherry et al. 2003). As mitochondrial function is impaired to a degree at which a threshold of energy depletion is reached, symptoms become manifest. Studies on nerve biopsy specimens of patients treated with zalcitabine have shown structurally abnormal mitochondria in axons and Schwann cells (Dalakas et al. 2001). Reversal of established neuropathy appears to be a slow process that is dependent on the cessation of the offending NRTI drug. However, not all effects of NRTIs on mitochondria can be explained by the DNA polymerase g hypothesis. Other mechanisms, either secondary to or independent of inhibition of DNA polymerase g are involved in NRTI toxicity (Moyle 2000a, 2000b; Lewis et al. 2003). AZT is a potent inhibitor of mitochondrial DNA polymerase g but does not cause neuropathy in HIV patients (Dalakas 2001). Keswani and colleagues showed that NRTIs caused direct mitochondrial toxicity through

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inhibition of the mitochondrial transmembrane potential differential, leading to nonapoptotic DRG neuronal death. This effect was not preventable by specific caspase inhibitor, suggesting that the classical apoptotic pathway is not involved in NRTI neurotoxicity (Keswani et al. 2003a). In vivo evidence to suggest that patients who develop ATN are the same patients who have the greatest degree of mitochondrial dysfunction is also conflicting. Brew and colleagues found evidence of increased serum lactate levels in patients with ATN (Brew et al. 2003), but results from a cross-sectional study did not confirm this (Cherry et al. 2002). While there are many similarities between the clinical manifestations of inherited mitochondrial disorders and NRTI toxicity, the striking difference is the absence of CNS and renal manifestations among the effects of NRTIs, despite these agents being known to penetrate the CNS at therapeutic concentrations and many of them being predominantly renally excreted (Moyle 2000a, 2000b). L-acetyl carnitine depletion may be an additional mechanism for the neurotoxicity of NRTIs and a potential therapeutic target. The main function of L-acetyl carnitine is in mitochondrial b-oxidation of fatty acids and membrane energy balance. In the short term, depletion of acetyl carnitine disrupts mitochondrial metabolism and causes a toxic accumulation of fatty acids (Bremer 1990). L-acetyl carnitine potentiates nerve growth factor actions, promotes peripheral nerve regeneration and is neuroprotective in vitro, in vivo and in animal models of diabetic neuropathy. L-acetyl carnitine has analgesic properties, possibly mediated by increasing adrenocorticotrophic hormone and b-endorphin level, while L-carnitine also has favorable immunological benefits (Youle 2007). Famularo and colleagues found reduced serum levels of acetyl carnitine in patients with peripheral neuropathy on zalcitabine, stavudine or didanosine therapy compared with those on the same drug but without peripheral neuropathy (Famularo et al. 1997). These findings were, however, not confirmed in a study that found normal levels of acetyl carnitine in HIV-associated neuropathy patients (Simpson et al. 2001). Short term L-acetyl carnitine treatment has shown symptomatic benefits in ATN, although it is unclear if this effect is long lasting because of neuronal regeneration or is merely an analgesic effect (Scarpini et al. 1997; Osio et al. 2006). Longer term studies showed improvements in epidermal nerve fiber density (Hart et al. 2004) as well as the symptoms of neuropathy when patients were treated with L-acetyl carnitine (Hart et  al. 2004; Herzmann et  al. 2005). A short term randomized, double-blind, placebo-controlled study showed reduction in pain over a 2 week period in HIV patients with TNA (Youle and Osio 2007). In vivo, patients treated with AZT develop a mitochondrial myopathy with mitochondrial DNA depletion, deficiency of cytochrome c oxidase (complex IV), intracellular fat accumulation, high lactate production and marked phosphocreatine depletion (Lewis and Dalakas 1995; Dalakas 2001). Clinically, the patient presents with fatigue, myalgia, muscle weakness, wasting and elevated serum creatine kinase. Muscle biopsy shows “ragged red fibers”, the characteristic histopathologic changes of mitochondrial myopathy, caused by subsarcolemmal accumulation of mitochondria (Lewis and Dalakas 1995).

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4.19 The Role of Chemokine Receptors in Neuropathic Pain Neuropathic pain is a prominent feature of DSP and ATN. Symptoms include allodynia (pain evoked by a normal innocuous stimulus) and hyperalgesia (enhanced pain evoked by a noxious stimulus). All levels of the neuro-axis are involved in the pathogenesis of neuropathic pain states. Neuroinflammation and neuroimmune activation play a role in the generation and maintenance of persistent pain states (DeLeo and Yezierski 2001). The complex interactions of the immune cascade, including breakdown of the blood-brain barrier, production of reactive oxygen species, eicosanoids, cellular adhesion molecule upregulation, migration of antigen-specific B and T lymphocytes, infiltration of neutrophils and upregulation of cytokines, growth factors, and other signaling molecules such as substance P lead to cellular dysfunction and expression of chronic pain (DeLeo and Yezierski 2001). Neuropathic pain following peripheral nerve injury is a consequence of enhanced excitability associated with the chronic sensitization of nociceptive neurons in the peripheral and central nervous system (White et al. 2007). Cytokines and growth factors have been strongly implicated in the generation of pathological pain. Chemokines, along with cell adhesion molecules, play a major role in leukocyte orientation and migration in response to chemical stimuli, leading to recruitment of cells into areas of active inflammation. This chemokine response is critical in the CNS neuroinflammatory response to peripheral nerve injury that results in painful neuropathy (Sweitzer et al. 2002). Immune cell infiltration at the nerve site injury and increased endoneural levels of proinflammatory cytokines have been demonstrated in a rodent model of neuropathy (Wagner and Myers 1996). In addition, chemokines and their receptors can influence both the acute and chronic phases of pain by sensitizing nociceptive neurons. Neurons, glia, and microglia are able to synthesize and respond to chemokines, something that is independent of their role in the regulation of leukocyte chemotaxis and function (White et al. 2005a; White et al. 2007). An in vitro model study of embryonal rat DRG cells and intradermal inoculation into the rat hind paw demonstrated that chemokines and gp120 produce pain hypersensitivity by directly acting on chemokine receptors of the nociceptive neurons (Oh et al. 2001). White and colleagues showed that MCP-1 strongly excited injured DRG nociceptive and nonnociceptive neurons in culture (White et  al. 2005b). DRG chronic compression, a neuropathic pain model, caused upregulation of MCP-1/CCR2 signaling in both injured and adjacent uninjured DRGs (White et al. 2005b). Upregulated expression of CXCR4, CCR2, and CCR5 receptors, and their chemokine ligands has also been observed in populations of DRG neurons in chronic pain models (Bhangoo et al. 2007a, 2007b). The interactions of chemokine signaling between glia and DRG neurons appear to be important in mediating the chronic pain state associated with HIV infection (White et  al. 2005a; White et  al. 2007). Gp120 binds to both neuronal and nonneuronal CXCR4 and CCR5 chemokine receptors. In addition to the effects of inflammatory mediators released by virally infected leukocytes, there are at least two ways in which HIV-induced DSP may involve the direct effects of gp120 on chemokine receptors in the DRG: (1) viral protein shedding in the peripheral nervous system might enable

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gp120 to produce painful neuropathy via glial to neuronal signaling in the DRG and/ or spinal cord (Milligan et al. 2001; Keswani et al. 2003b) or (2) the direct activation of CCR5/CXCR4-bearing sensory neurons by gp120 (Herzberg and Sagen 2001; Oh et al. 2001). Keswani and colleagues have presented a model in which gp120 can act in both these ways (Keswani et al. 2003b; Keswani et al. 2006). The painful sensory neuropathy that is produced by NRTIs is indistinguishable from that resulting from HIV infection. Although it is widely believed that NRTIs initiate their effects on peripheral neurons through interactions with mitochondria (Dalakas 2001), exactly how this produces pain is unknown. Bhangoo and colleagues showed that ddC specifically upregulated expression of both SDF-1 and CXCR4 chemokine receptor in DRG glial cells and some neurons. This suggests that SDF-1 release from DRG glia might be involved in the autologous regulation of excitatory substances from these same cells and that released SDF-1 might also directly excite DRG neurons. Neuropathic pain induced by ddC was completely blocked by the CXCR4 antagonist AMD3100, illustrating the key role of CXCR4 signaling in this pathway (Bhangoo et al. 2007a, 2007b).

4.20 Models of HIV-Associated Peripheral Neuropathy In vitro and in vivo models of HIV-associated neuropathy are essential in the study of HIV peripheral neuropathogenesis and evaluation of potential therapeutic strategies. Development of therapeutic drugs has been hampered by the lack of reproducible models till recently. Early in  vitro models used either pharmacologically nonrelevant doses of NRTIs or a rat pheochromocytoma cell line (PC-12), which may not reflect the in  vivo phenotype of DRG sensory neurons (Chen et  al. 1991; Keilbaugh et  al. 1991; Cui et  al. 1997). Other cell culture models using T-lymphoblastoid cell culture, tissue-specific cell cultures, neuronal, and pancreatic cell cultures have been used to investigate mitochondrial toxicity of NRTIs but have their limitations for the prediction of clinical toxicity (Hoschele 2006). A more clinically relevant human DRG mixed neural cell culture model was developed by Hahn and colleagues to study the role of HIV-infected human macrophages in the pathogenesis of DSP (Hahn et al. 2008). Early attempts to find an animal model of HIV-associated sensory neuropathy met with little success. One group described a rabbit model of ddC-induced sensory neuropathy (Anderson et al. 1992). Keswani and colleagues established an in  vitro model of gp120 peripheral neurotoxicity using primary DRG cultures to elucidate novel pathogenic mechanisms and test neuroprotective strategies (Keswani et al. 2003b). By modifying the model with the addition of NRTIs, the group showed that the immunophilin ligand, FK506 but not cyclosporine A, prevented the development of neurotoxicity by ddC via a calcineurin-independent mechanism of neuroprotection (Keswani et  al. 2003a). These models of gp120 and NRTI neurotoxicity also

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established erythropoietin (EPO) derived from periaxonal Schwann cells of injured neurons, as a promising neuroprotectant of sensory neurons (Keswani et al. 2004b). In an animal model of distal axonopathy, the rat acrylamide toxicity model, systemic EPO administration prevented axonal degeneration and was associated with reduction in limb weakness and neuropathic pain behavior (Keswani et al. 2004a). The establishment of a robust in vivo rodent model of HIV-associated sensory neuropathy by administering ddI orally to gp120 transgenic mice will be useful in studying neuropathogenic mechanisms and future evaluation of other potential therapeutic strategies for HIV-associated neuropathy (Keswani et al. 2006). Rodent models of gp120 and antiretroviral-associated neuropathy have been developed by other groups. Wallace and colleagues demonstrated a persistent mechanical hypersensitivity in rats, with pathological changes in the peripheral and central nervous system following application of gp120 to the sciatic nerve (Wallace et al. 2007a, 2007b). The mechanical hypersensitivity was responsive to systemic treatment with gabapentin, morphine, and cannabinoid, but not with amitriptyline. By further systemic treatment with ddC with or without the concomitant delivery of perineural gp120, the group developed a neuropathic pain model to study the mechanisms underlying drug-induced neuropathy in the context of HIV infection (Wallace et al. 2007a, 2007b). Joseph and colleagues developed a rat model of NRTI-induced painful neuropathy using ddC, ddI, and d4T, producing dose-dependent mechanical hypersensitivity and allodynia (Joseph et al. 2004). They showed evidence of a calcium-dependent mechanism for mediating neuropathic pain. The lentivirus, feline immunodeficiency virus (FIV) causes immune suppression and neurological disease in its natural host, the domestic cat. Many common features of FIV and HIV have prompted its use as an animal model for HIV infection. Kennedy and colleagues reported development of FIV-induced neuropathy in cats (Kennedy et  al. 2004). Infection of neonatal cats with FIV resulted in macrophage infiltration of the DRG and peripheral nerves associated with reduction in epidermal nerve fiber density. Studies by this group suggest that both macrophages and CD8 lymphocytes play important roles in mediating an indirect neurotoxicity (Zhu et al. 2005; Zhu et al. 2006). Exposure of cat DRG neurons to FIV-infected macrophages resulted in axonal degeneration and neuronal atrophy and death, mediated through the activation of the inducible nitric oxide synthase in the infected macrophages (Zhu et al. 2005). FIV-infected cats also exhibited an increase in the number of CD8 lymphocytes in the DRG and peripheral nerves, and these FIV-infected lymphocytes caused axonal degeneration and neuronal death in an in vitro coculture system (Zhu et al. 2006). Using this model, Zhu and colleagues showed that ddI treatment during FIV infection resulted in additive pathogenic effects contributing to the development of ATN with correlated neurobehavioral changes, mitochondrial injury on neurons, and reduced brainderived neurotrophic factor (BDNF) production by Schwann cells in DRG, highlighting the convergent pathogenic effects that antiviral drugs might have in HIV infection (Zhu et al. 2007).

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4.21 Treatment of HIV-Associated Peripheral Neuropathy Currently, treatment of DSP and ATN is similar to many other neuropathies that have predominantly painful sensory involvement (Mendell and Sahenk 2003; Gonzalez-Duarte et  al. 2007). It is purely symptomatic as there are no proven regenerative therapies to reverse the underlying process. An 8-month prospective pilot study reported an improvement in subjective quantitative sensory testing (QST) in HIV-infected patients who responded to HAART (Martin et al. 2000). The patients who did not respond to HAART did not show any improvements in QST. It is possible that suppression of viral load will slow the progression of DSP. Some studies have found a correlation between viral load and incidence (Childs et  al. 1999), or severity (Simpson et al. 2002) of sensory neuropathy. Others, however, did not find any correlation between plasma viral loads and incidence of DSP or ATN (Brew et al. 2003). For ATN, it is reasonable to decrease the dose or discontinue the offending NRTI and change the HAART regimen if available. Failing this, the patient may need to continue the regimen with the addition of pain-modifying drugs. After discontinuation of a toxic NRTI, symptomatic improvement can be expected in most individuals within several months (Blum et al. 1996). Pain-modifying drug treatment has been based on studies in other painful neuropathies, especially diabetic neuropathy. However, there are several studies conducted in HIV-infected patients. The medications studied include gabapentin (Newshan 1998; La Spina et  al. 2001; Hahn et  al. 2004), amitriptyline (Kieburtz et al. 1998; Shlay et al. 1998), acupuncture (Shlay et al. 1998), mexilitine (Kemper et al. 1998; Kieburtz et al. 1998), memantine (Schifitto et al. 2006), prosaptide (Evans et al. 2007) and topical lidocaine (Dorfman et al. 1999; Estanislao et al. 2004), and capsaicin (Paice et al. 2000; Simpson et al. 2008b). Results of most of these studies have been disappointing as the placebo-controlled trials have generally not shown the agents to be more effective than placebo in relieving pain, and the degrees of pain relief reported in the smaller placebo-controlled or open-label trials have yet to be replicated in larger placebo-controlled trials. To date, those symptomatic therapies shown to be effective in relieving the pain of DSP in randomized, placebo-controlled trials are lamotrigine (Simpson et  al. 2000; Simpson et  al. 2003), recombinant human nerve growth factor (NGF) (McArthur et al. 2000), a high dose capsaicin patch (Simpson et  al. 2008a), and cannabinoids (Abrams et al. 2007). Narcotic analgesics are effective in controlling the chronic pain associated with DSP and ATN, but should be used judiciously because of the high incidence of side effects and abuse potential. The principle is to use a long-acting narcotic with regular dosing and restrict short-acting analgesics for break-through pain. L-acetyl carnitine, a compound with a known safety profile, has shown symptomatic benefits (Scarpini et al. 1997; Hart et al. 2004; Herzmann et al. 2005; Osio et al. 2006) and improvements in epidermal nerve fiber density (Hart et al. 2004) in HIV patients with ATN. A short term randomized, double-blind, placebo-controlled

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study showed reduction in pain (Youle and Osio 2007). A prospective, 48-week, placebo-controlled study is now completed and is under analysis, and results will be published in due course (Youle and Osio 2007).

4.22 Conclusion In summary, the neuropathogenesis of HIV peripheral neuropathy comprises several contributing factors. These include advanced immunosuppression and chronic inflammation involving activated and HIV-infected macrophages within peripheral nerves and the DRG neuron, leading to a pathogenic cascade mediated by a host of factors including proinflammatory cytokines, chemokines, and perhaps proteases and matrix metalloproteinases, causing nerve dysfunction and generating neuropathic pain. Direct and indirect mechanisms of secreted HIV envelope protein gp120 mediate neurotoxicity through its effects on neurons and glial components such as microglia and Schwann cells. NRTIs exacerbate peripheral neurotoxicity by impairing mitochondrial DNA synthesis and function. These result in a failure to elaborate and export structural proteins in nerves, including neurofilament and tubulin that are required to maintain axon caliber and sensory terminal integrity. Chemokines and their cognate receptors play a central role in mediating the neuroinflammatory response, HIV transmission and infection, neurotoxic effects of gp120, neuronal and glial signaling, and nociceptive neurons sensitization.

References Abrams DI, Jay CA et al (2007) Cannabis in painful HIV-associated sensory neuropathy: a randomized placebo-controlled trial. Neurology 68(7):515–521 Anderson TD, Davidovich A et al (1992) Peripheral neuropathy induced by 2’,3’-dideoxycytidine. A rabbit model of 2¢,3¢-dideoxycytidine neurotoxicity. Lab Invest 66(1):63–74 Apostolski S, McAlarney T et  al (1993) The gp120 glycoprotein of human immunodeficiency virus type 1 binds to sensory ganglion neurons. Ann Neurol 34(6):855–863 Araujo AP, Nascimento OJ et al (2000) Distal sensory polyneuropathy in a cohort of HIV-infected children over five years of age. Pediatrics 106(3):E35 Authier FJ, Gheradi RK (2003) Peripheral neuropathies in HIV-infected patients in the era of HAART. Brain Pathol 13(2):223–228 Bacellar H, Munoz A et al (1994) Temporal trends in the incidence of HIV-1-related neurologic diseases: Multicenter AIDS Cohort Study, 1985–1992. Neurology 44(10):1892–1900 Banki K, Hutter E et al (1998) “Molecular ordering in HIV-induced apoptosis. Oxidative stress, activation of caspases, and cell survival are regulated by transaldolase.”. J Biol Chem 273(19):11944–11953 Barohn RJ, Gronseth GS et al (1993) Peripheral nervous system involvement in a large cohort of human immunodeficiency virus-infected individuals. Arch Neurol 50(2):167–171 Behar R, Wiley C et  al (1987) Cytomegalovirus polyradiculoneuropathy in acquired immune deficiency syndrome. Neurology 37(4):557–561

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von Giesen HJ, Kaiser R et al (2002) Reversible ALS-like disorder in HIV infection. An ALS-like syndrome with new HIV infection and complete response to antiretroviral therapy. Neurology 59(3):474 author reply 474–5 Wagner R, Myers RR (1996) Endoneurial injection of TNF-alpha produces neuropathic pain behaviors. Neuroreport 7(18):2897–2901 Wallace VC, Blackbeard J et al (2007a) Pharmacological, behavioural and mechanistic analysis of HIV-1 gp120 induced painful neuropathy. Pain 133(1–3):47–63 Wallace VC, Blackbeard J et  al (2007b) Characterization of rodent models of HIV-gp120 and anti-retroviral-associated neuropathic pain. Brain 130(Pt 10):2688–2702 Wechsler AF, Ho DD (1989) Bilateral Bell’s palsy at the time of HIV seroconversion. Neurology 39(5):747–748 Welby SB, Rogerson SJ et al (1991) Autonomic neuropathy is common in human immunodeficiency virus infection. J Infect 23(2):123–128 Wesselingh SL, Glass J et al (1994) Cytokine dysregulation in HIV-associated neurological disease. Adv Neuroimmunol 4(3):199–206 White FA, Bhangoo SK et al (2005a) Chemokines: integrators of pain and inflammation. Nat Rev Drug Discov 4(10):834–844 White FA, Sun J et  al (2005b) Excitatory monocyte chemoattractant protein-1 signaling is upregulated in sensory neurons after chronic compression of the dorsal root ganglion. Proc Natl Acad Sci USA 102(39):14092–14097 White FA, Jung H et al (2007) Chemokines and the pathophysiology of neuropathic pain. Proc Natl Acad Sci USA 104(51):20151–20158 Wiselka MJ, Nicholson KG et  al (1987) Acute infection with human immunodeficiency virus associated with facial nerve palsy and neuralgia. J Infect 15(2):189–190 Wulff EA, Wang AK et  al (2000) HIV-associated peripheral neuropathy: epidemiology, pathophysiology and treatment. Drugs 59(6):1251–1260 Yoshioka M, Shapshak P et al (1994) Expression of HIV-1 and interleukin-6 in lumbosacral dorsal root ganglia of patients with AIDS. Neurology 44(6):1120–1130 Youle M (2007) Acetyl-L-carnitine in HIV-associated antiretroviral toxic neuropathy. CNS Drugs 21(Suppl 1):25–30 discussion 45–6 Youle M, Osio M (2007) A double-blind, parallel-group, placebo-controlled, multicentre study of acetyl L-carnitine in the symptomatic treatment of antiretroviral toxic neuropathy in patients with HIV-1 infection. HIV Med 8(4):241–250 Zhu Y, Jones G et al (2005) Lentivirus infection causes neuroinflammation and neuronal injury in dorsal root ganglia: pathogenic effects of STAT-1 and inducible nitric oxide synthase. J Immunol 175(2):1118–1126 Zhu Y, Antony J et al (2006) CD8+ lymphocyte-mediated injury of dorsal root ganglion neurons during lentivirus infection: CD154-dependent cell contact neurotoxicity. J Neurosci 26(13):3396–3403 Zhu Y, Antony JM et al (2007) Didanosine causes sensory neuropathy in an HIV/AIDS animal model: impaired mitochondrial and neurotrophic factor gene expression. Brain 130(Pt 8):2011–2023

Chapter 5

HIV Latency and Reactivation: Role in Neuropathogenesis Anupam Banerjee, Michael R. Nonnemacher, and Brian Wigdahl

5.1 Introduction A hallmark of human immunodeficiency virus type 1 (HIV-1) and other members of the retroviridae family is reverse transcription of their RNA genome and concomitant integration of the resultant DNA copy into the genome of target cells, thereby allowing them to utilize the host cell transcriptional machinery to synthesize components necessary for viral replication. HIV-1-infected patients have benefited enormously from the use of the combinatorial drug regimen introduced more than a decade ago, known as highly active antiretroviral therapy (HAART). As a result of the use of HAART, viral burden is often lowered beyond detectable limits, thereby facilitating the rejuvenation of the host immune system allowing patients to lead longer and more productive lives. Although effective, HAART is unable to completely cure infection due to the persistence of a long lived, drug-insensitive reservoir of integrated, transcriptionally silent proviruses in subpopulations of susceptible cells in the peripheral blood as well as solid tissues and organs. As a result, upon discontinuation of therapy and/or under certain stimulatory conditions, rebound of viremia is observed resulting in virus reseeding the periphery from these “latent” cellular reservoirs.

5.2 Types of Latency and Major Cellular Reservoirs In 1995, HIV-1 latency was first documented in HIV-1-infected patients when ex vivo T cell cultures were found to contain a subpopulation of cells that produced infectious virions when stimulated with T cell activators (Chun et al. 1995; Finzi et al. 1997). Latently infected T cells are rare, to the order of one in a million resting A. Banerjee, M.R. Nonnemacher, and B. Wigdahl () Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA, 19129, USA e-mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_5, © Springer Science+Business Media, LLC 2010

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CD4+ T cells (Chun et al. 1997). Accurate estimates of the frequency of other possible cellular reservoirs have not yet been documented. Another defining feature of latently infected cells is their longevity. This is due to the inherent biological property of these specific cellular subsets. In the case of resting memory CD4+ T cells, the slow decay rate (half life = 44 months in HAART patients) (Finzi et al. 1999) of these cells helps the virus to stay in a state of reversible genomic activation. “Latency” is defined as a state of dormancy which is accompanied by very low levels of HIV-1 mRNA production and the absence of infectious virus production (Brooks et  al. 2003; Hermankova et  al. 2003). Often, the RNA that is produced appears to be truncated (Adams et al. 1994; Kao et al. 1987; Lassen et al. 2004b), thereby leading to inefficient production of viral proteins and infectious virus. This makes it a difficult task to reliably distinguish latently infected cells within a pool of uninfected cells. To complicate the therapeutic control of HIV-1 infection, it has been estimated that the latent pool of infected cells is established very early in the course of HIV-1 pathogenesis and disease. In such a scenario, the normal window of time associated with the introduction of HAART will fail to disrupt the onset of viral latency. Consequently, current treatment strategies are more focused on the control of viremia rather than eradication of endogenous proviral DNA associated with latent infection (Chun et al. 1998; Finzi et al. 1997).

5.2.1 Pre-integration Latency Previous studies have proposed that latent infection involves two forms that are defined by the integration state of the proviral genome and the phenotype of the infected cell. Pre-integration latency is observed following fusion of the viral membrane with the cytoplasmic membrane of the susceptible host cell and arises as a result of failure of the proviral DNA genome to integrate into the host cell chromatin. In the case of unactivated, resting CD4+ T cells, certain inherent biological characteristics are the most common limiting factors for HIV-1 infection. Reduced nucleotide levels in these cells are often the restricting factor for successful reverse transcription (Fig. 5.1). It has been estimated that it may take up to 3 days for this process (Meyerhans et  al. 1994). Additionally, insufficient pools of ATP pose an energy barrier to transport of the pre-integration complex across the nuclear membrane (Fig. 5.1) (Bukrinsky et al. 1991, 1992; Zack et al. 1990). The pre-integration latent form of proviral DNA is extremely labile and the efficiency of complete reverse transcription before the onset of decay processes is only about 50% (Zhou et  al. 2005). Even after reverse transcription, the viral cDNA has a narrow window of time (t1/2 = 1 day) before degradation begins (Fig.  5.1) (Pierson et  al. 2002). Although pre-integration latency is more frequent than post-integration latency, its contribution to long-term persistence of the virus and the disease process itself is of less clinical importance. Other host factors, which form integral components of the host innate immune arsenal, also serve to specifically inhibit HIV-1 replication in susceptible cells by

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Fig. 5.1  Regulators of pre- and post-integration latency. Pre-integration latency is regulated as the viral RNA is reverse transcribed into the proviral DNA (A). This is controlled by the availability of the nucleotide pool, half life of the forming proviral cDNA copy, and the interaction of the viral protein Vif with the cellular antiviral protein APOBEC, especially family members 3G and 3F. It is also regulated at the step of transport across the nuclear membrane through the availability of ATP as the process requires energy (B). Post-integration, the proviral DNA copy of the viral genome, is regulated mainly by the availability of host transcription factors, especially NF-kB and NFAT (C)

acting at the level of viral cDNA synthesis and/or viral cDNA nuclear import. One such group of cellular antiviral response factors belongs to the APOBEC (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like) family of polynucleotide–cytidine deaminases. The 23-kD HIV-1 accessory protein “viral infectivity factor” or vif plays a critical role in HIV-1 pathogenesis in  vivo. Experiments performed with HIV-1 strains deficient in vif (Dvif) identified APOBEC3G as a specific inhibitor of viral infection in non-permissive cells like primary human CD4+ T cells. Additionally, resistance of peripheral blood monocytes and immature dendritic cells to HIV-1 has been attributed, in part, to high levels of APOBEC3G; whereas establishment of macrophages as receptive hosts is consistent with low levels of this enzyme (Peng et al. 2007). In turn, HIV-1 vif counteracts the action of APOBEC3G (Sheehy et  al. 2002). This is also consistent with the fact that Dvif HIV-1 viruses are able to replicate in permissive cells having very low levels of APOBEC3G. Among the human APOBEC proteins, APOBEC3G and APOBEC3F

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are the most potent antagonists of HIV-1 infectivity (Fig.  5.1) (Alce and Popik 2004; Fan and Peden 1992; Madani and Kabat 2000; Simon et  al. 1998). Specifically, these enzymes are incorporated into the virions as they are being assembled in an infected cell. In subsequent rounds of infection in susceptible cell populations, they mediate cytidine-to-uridine (dC-to-dU) changes in the nascent reverse transcripts, which get translated into guanosine-to-adenosine (G-to-A) hypermutations in the plus-strand viral cDNA (Suspene et  al. 2004; Yu et  al. 2004a). Apart from direct viral gene inactivation upon integration (Mangeat et al. 2003), the high content of deoxy-uracils may also trigger host DNA repair enzymes known as uracil-DNA-glycosidases, thereby leading to degradation of minus-strand viral DNA (Harris et  al. 2003). Additionally, barriers to tRNA3Lys annealing to viral RNA during reverse transcription, plus-strand DNA transfer as well as proviral integration have also been proposed (Guo et al. 2007; Li et al. 2007; Luo et al. 2007; Mbisa et  al. 2007). Vif opposes APOBEC3G-restriction by sequestering it into an E3 ubiquitin ligase complex comprised of Elongin B/C, Cullin5, and ring box-1 (Fig.  5.1). This results in polyubiquitylation and subsequent proteosomal degradation of cellular APOBEC3G (Mehle et al. 2004; Sheehy et al. 2003; Stopak et al. 2003; Yu et al. 2003, 2004b).

5.2.2 Post-integration Latency and the Resting CD4+ T Cell Post-integration latency arises after the viral genome has been reverse transcribed and the mature reverse transcript has translocated into the nucleus, ultimately getting stably integrated into the host chromosome. The major cell type, which is implicated in the maintenance of this type of latency, is the resting memory CD4+ T cell (Table 5.1). In the early stages of infection, the number of activated CD4+ T cells harboring integrated provirus outweighs the number of resting CD4+ T cells (Bukrinsky et al. 1991; Chun et al. 1997; Stevenson et al. 1990; Zack et al. 1990). This may be explained by the fact that CCR5, the preferred HIV-1 co-receptor in early infection is present at very low levels on naïve T cells and gets upregulated upon antigen encounter and subsequent T cell activation, thereby facilitating infection. Antigen stimulation also alleviates blocks to reverse transcription (Chiu et al. 2005; Zhou et  al. 2005), nuclear entry (Bukrinsky et  al. 1992), initiation (TongStarksen et  al. 1987), and concomitant transcriptional elongation (Adams et  al. 1994; Lassen et al. 2004b) as well as export of nascent viral mRNAs (Lassen et al. 2006) commonly encountered in naïve resting CD4+ T cells. However, within the resting CD4+ T cell population, the memory CD4+ T cells are quantitatively more predominant over naive CD4+ T cells (Chun et  al. 1997) for post-integration latency. The most commonly accepted model suggests that this type of latency is primarily established when an activated T cell retreats back to a resting memory phenotype. During the reversion to a quiescent state, although cells express adequate levels of CCR5 for infection, the cellular microenvironment is not conducive for post-integration steps in the viral life cycle (Persaud et  al. 2003).

Astrocytes

Cells of the CNS Microglial cells

Takahashi et al. (1996), Tornatore (1994), Trillo-Pazos et al. (2003), Wang et al. (2004)

Albright et al. (2004), Cosenza et al. (2002), Watkins et al. (1990)

Table 5.1  Summary of HIV-I cellular reservoirs Reservoir tissue Inclusive cells Reference(s) Peripheral blood Monocyte/ Bailey et al. (2006), Carr et al. (1999), macrophages Chun et al. (1998), Crowe et al. (1990, 1992), Diamond et al. (2004), Groot et al. (2008), Ho et al. (1986), Kicffer (2004), Lambotte et al. (2000), McElrath et al. (1991), Nicholson et al. (1986), O’Brien (1994), Sharova et al. (2005), Zhu et al. (2002) Dendritic cells Geijtenbeek et al. (2000), Otero et al. (2003) CD4+ T cells Chun et al. (1997), Duh et al. (1989), Kinoshita et al. (1997), Persaud et al. (2003)

(continued)

Microglia might serve as a potential reservoir for latent HIV-1, capable of infectious virus production under activation conditions. In initial stages of HIV-1 disease, the number of productively infected microglial cells is low; however, during late stages of infection their numbers rise significantly There is detection of viral DNA and RNA in postmortem brain tissues of AIDS patients, but rarely viral antigens, suggesting that astrocytes may harbor latent virus

During reversion from an activated to a quiescent state, although cells express adequate levels of CCR5, the cellular microenvironment is not conducive for post-integration steps. Since endogenous levels of inducible transcription factors is low, LTR activation is inefficient resulting in non-expression of viral gene products allowing these cells to escape immune surveillance and act as passive carriers of HIV-1 for their natural lifespan

This cell type is thought to be a viral reservoir due to identification of PPC resulting from an infection event culminating in propagation and entrenchment of viral sequence in deep tissues as these cells proliferate and differentiate into tissue macrophages. These tissue macrophages are refractory to the cytopathic effects of HIV-1 gene products. They can support a continuous, low-level of virus production throughout their lifetime. Monocytes have been found to harbor latent proviral DNA throughout the course of disease. MDMs are capable of reseeding infectious virus to the periphery, especially in late stage disease when CD4+ T cells are depleted DCs might not be a significant source of latent provirus in vivo

Rationale

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Neural progenitor cells

Bone marrow Hematopoietic progenitor cells Minor reservoirs Mast cells

Table 5.1  (continued) Reservoir tissue Inclusive cells

Toll-like receptor signaling can reactivate HIV-1 replication, raising the possibility that these cells can serve as an inducible reservoir When nestin+ multi-potential cells are differentiated towards a neuronal lineage post-infection, there is a negligible increase in virus replication. When these cells were transfected with a pNL4–3 molecular clone and stimulated with TNP-a, there is an upregulation in virus production, suggestive of reactivation from latency

Sundstrom et al. (2004)

Lawrence et al. (2004)

Reseeding of a viral variant from a rare infection event of a few progenitor cells raises the possibility that the bone marrow compartment may be a source of latent provirus

Rationale

Bailey et al. (2006)

Reference(s)

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Since the endogenous levels of inducible transcription factors like NF-kB and NFAT (Duh et al. 1989; Kinoshita et al. 1997) in these cells have been reported to be very low, HIV-1 promoter activation is inefficient resulting in non-expression of viral gene products (Figs. 5.1 and 5.2). Therefore, these cells can escape immune surveillance and act as passive carriers of HIV-1 for their natural lifespan.

5.2.3 Monocyte–Macrophage Latency In addition to memory CD4+ T cells, other as yet undefined, physiologically important, long-term cellular reservoirs for HIV-1 are likely to exist. Support for this hypothesis has come from experiments which have revealed that the virus which re-emerges after cessation of HAART does not always appear identical to the resting CD4+ T cell virus pool (Chun et al. 2000). Another key piece of evidence stems from observations wherein comparisons were made between length polymorphisms in variable segments of the HIV-1 env gene of viruses isolated from both the resting CD4+ T cell compartment in patients on HAART as well as rebound virus from patients who were discontinuous with their treatment (Zhang et  al. 2000). Surprisingly, two out of a total of five patients exhibited differences in the lengths of env sequences between the two samples. Based on these and other observations, it is now generally accepted that inspite of a strict HAART regimen where viremia drops down below <50 copies/ml (Dornadula et al. 1999), some residual free virus can still be detected in the plasma of patients. This “residual viremia,” amounting to as low as 1 copy/ml (Maldarelli et al. 2007; Palmer et al. 2003), might represent recrudescence of virus from steady-state viral reservoirs (Shen and Siliciano 2008). More recent elegant studies have provided additional support for this hypothesis. Detailed phylogenetic analyses of residual viremia from HAART-responsive patients revealed that in about half of these patients, there was a preponderance of a single sequence in the plasma, which was infrequently detected in the resting CD4+ T cell pool (Bailey et al. 2006). Equally intriguing was the recovery of this “predominant plasma clone” (PPC) in repeated samplings up to 3 years. It has been suggested that the PPC might be the result of an infection event in the monocyte– macrophage lineage, culminating in propagation and entrenchment of a viral sequence in deep tissues as these cells proliferate and differentiate into tissue macrophages (Bailey et al. 2006). This residual viremia also represents a nonevolving viral entity with no novel drug resistance mutations in patients who switched from an initial non-suppressive HAART regimen to a subsequent successful one (Bailey et al. 2006; Hermankova et al. 2001; Kieffer et al. 2004). This further strengthened the argument that latency was established during the early stages of primary disease (Chun et al. 1998) and that in addition to resting CD4+ T cells, cells of the monocyte–macrophage lineage may also provide a sanctuary for latent provirus (Table 5.1 and Fig. 5.3). These cells possess a diverse array of important biological characteristics, which make them a prime candidate for such a phenomenon.

Fig. 5.2  Models of post-integration latency modulated through transcriptional regulation. (a) The LTR of the integrated proviral genome remains transcriptional silent due to recruitment of histone deacetylase-1 (HDAC-1) by NF-kB homodimers p50 and/or by LSF and YY1, which maintains the nucleosomes, nuc-0 and nuc-1, in a deacetylated state (red ovals) keeping the chromatin in a condensed state and preventing the recruitment of RNA polymerase. (b) Basal transcription driven by the LTR occurs in the absence of the viral protein Tat. It is driven primarily through the upregulation of cellular transcription factors such as NFAT and the p65 subunit of NF-kB, which help recruit the RNApol II complex to the transcriptional start site. However in the absence of Tat, transcription from the HIV-1 LTR produces predominantly short RNA as a result of the hypophosphorylated state of RNApol II (arrows on CTD) and the activity of 5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) and negative elongation factor complex (NELF), which bind to hypophosphorylated RNA pol II and inhibit transcriptional elongation. However, there is a low level accumulation of longer transcripts that results in a build up of viral regulatory proteins, including Tat, which has been shown to eventually lead to the next stage of viral transcription, Tat-mediated transcription (c). As the concentration of Tat increases, increased Tat binding to the trans-activation response region (TAR) structure on the viral RNA results in the increased recruitment of the positive transcription elongation factor b (P-TEFb), the cyclin dependent kinase 9 (Cdk9), and cyclin T1 (CycT1) . Recruitment of PTEFb to TAR has been shown to induce hyperphosphorylation of CTD by Cdk9 resulting in the dissociation of DSIF and NELF. Acetylation of Tat at Lys 50 has been shown to create a binding site for p300/CREB binding protein-associated factor (PCAF) promoting the formation of a ternary complex of Tat-PCAF and P-TEFb. The interaction of PCAF with acetylated Tat has been shown to compete against TAR RNA binding of Lys50-acetylated Tat, and causes its dissociation from TAR RNA, thereby enhancing the transcriptional elongation of HIV-1

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Fig. 5.3  Interplay of cellular and tissue reservoirs during neuropathogenesis. The central dogma of HIV-1 infection of the central nervous system (CNS) centers on the peripheral blood monocyte becoming infected and immune-activated, transversing the blood–brain barrier, and delivering HIV-1 to the cells of the central nervous system. Once in the CNS, this perivascular macrophage productively releases infectious progeny HIV-1 that may lead to the infection of microglial and astrocytic cell populations within the CNS. These two main cell populations of the CNS are thought to be the main CNS cellular reservoirs (*CR). During active viral replication within the CNS, it is also likely that HIV-1 can reseed the periphery either as free virus or by infecting immune cells that are involved in the immune surveillance of this tissue. In addition, the bone marrow is thought to play a key role in the development of neuropathogenesis, especially in the context of bone marrowderived monocyte-CNS macrophage turnover, as a viral reservoir that may seed the peripheral blood and CNS with HIV-1-infected cells of the monocyte–macrophage lineage

First, peripheral blood monocytes traffic to various tissues where they differentiate into macrophages, which are long-lived cells. Second, tissue macrophages are relatively refractory to the cytopathic effects of HIV-1 gene products (Ho et al. 1986; Nicholson et al. 1986). They can also support a continuous albeit low-level of virus production throughout their lifetime due to several host-cell replication blocks such as barriers in reverse transcription (O’Brien 1994) and nuclear import (Diamond et  al. 2004). Inefficient expression of viral antigens therefore assists the virus in inadvertently escaping host immune surveillance mechanisms. Monocytic cells also exhibit low levels of infection, which is largely attributed to reduced surface levels of the HIV-1 receptor CD4, thereby diminishing the possibility of viral attachment. Consequently, the size of the infected monocyte–macrophage pool is estimated to be rather small compared to the infected T cell pool with successful integration events detected in less than 1% of monocytes. As with resting CD4+ T cells, monocytes have been found to harbor latent proviral DNA throughout the

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course of the disease (McElrath et al. 1991) and it is possible to recover infectious virus from this reservoir in HAART-responsive patients ex vivo, under stimulatory conditions (Lambotte et al. 2000; Zhu et al. 2002). In fact, the viral DNA decay rate in CD14+ monocytes was found to be much slower (~41.3 months) when compared to that in activated (~19.8 months) or resting CD4+ T cells (~23.5 months) (Zhu et  al. 2002). HIV-1-infected monocyte-derived macrophages (MDMs) have been shown to fuse with and transmit virus to autologous peripheral blood CD4+ leukocytes (Carr et  al. 1999; Crowe et  al. 1990, 1992; Sharova et  al. 2005). More recently, it has been discovered that the underlying mechanism involves formation of a transient multi-molecular complex at the interface between HIV+ MDMs and uninfected CD4+ T cells, characterized by the presence of HIV-1 env-CD4 receptor colocalization (Groot et al. 2008). This infectious “virological synapse” is reminiscent of stable junctions formed between T cells and dendritic cell-T cells, a hallmark of direct viral spread in  vivo. MDMs also exhibit enhanced expression of P-glycoprotein transporters, which impart resistance to protease inhibitors (PIs) that are often key components of HAART cocktails. Exclusion of this class of drugs from infected cells allows increased post-translational processing of gag-pol polyproteins by the viral aspartyl protease, thereby facilitating late stages of the HIV-1 replication cycle. The aforementioned observations outline the central role of MDMs as an in  vivo latent reservoir capable of reseeding infectious virus to the periphery, especially in late stages of disease when CD4+ T cells are depleted.

5.2.4 Cells of the CNS It is now widely believed that HIV-1 enters the central nervous system (CNS) early in the course of the disease (An et al. 1999; Davis et al. 1992), soon after infection of peripheral T cells and monocytes. Perivascular macrophages and microglial cells are the cell populations which are the main HIV-1 producing cells in the CNS and are implicated in the development and manifestation of HIV-1-associated dementia (HAD) and a range of other HIV-1-associated neurocognitive disorders (HAND) most commonly observed in advanced stages of disease (Garden 2002; Williams and Hickey 2002). These cells possess surface CD4 as well as CCR5, much like their counterpart tissue macrophages (He et al. 1997; Lavi et al. 1997) and can be infected by HIV-1. Their involvement as prime targets for HIV-1 is also dictated by their respective locations in brain tissue. Since perivascular macrophages are found on the parenchymal side of the blood–brain barrier (BBB) adjacent to the microvasculature, they are much more likely to come into direct contact with HIV-1-infected cells of the monocyte–macrophage lineage traversing the BBB into the brain. The “Trojan Horse” hypothesis suggests that HIV-1-infected monocytes in the periphery continuously replenish the pool of perivascular macrophages, thereby acting as carriers of HIV-1 into the brain (Fig. 5.3) (Haase 1986; Peluso et al. 1985). In comparison, microglia are found dispersed throughout the brain and have slower turnover rates

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(Hickey et al. 1992; Lassmann et al. 1993). Although proximity to the periphery makes perivascular macrophages more amenable to infection and subsequent virion production (Cosenza et al. 2002; Fischer-Smith et al. 2004; Takahashi et al. 1996; Wiley et al. 1986; Williams et al. 2001b), this does not minimize the role played by long-lived microglia in amplifying the neuropathological cascade. Studies have also established that microglia might serve as a potential reservoir for latent HIV-1 (Table  5.1 and Fig.  5.3), capable of infectious virus production under activation conditions (Albright et  al. 2004; Watkins et  al. 1990). This is supported by the observation that in the initial stages of HIV-1 disease, the number of productively infected microglial cells is low; however, during late stages of the infection their numbers significantly increase (Cosenza et al. 2002). With regard to identifying the molecular mechanism behind post-integration latency in microglial cells, it was discovered that unlike the restrictive mechanism operating in resting CD4+ T cells, reverse transcription, integration, and subsequent viral mRNA production events were not significantly altered in unactivated microglia. However, a post-translational block was identified with aberrant p55Gag processing by the viral protease. The inefficient release of infectious viral particles was attributed to this hindrance in Gag processing. However, in the presence of serum, cytokines, and growth factors, this block was alleviated (Albright et al. 2004). Astrocytes are the most abundant cell type in the CNS and are critical regulators of normal brain homeostasis. They serve to maintain the BBB integrity, secrete neurotransmitters and other compounds of the neuronal circuitry, provide nourishment to diverse cell types including neurons, and even act as sentinels of the CNS, secreting immuno-modulatory molecules. The extent of their susceptibility to HIV-1 in  vivo is controversial as a result of the trace amounts of surface CD4. However, persistent infection of astrocytes has been documented in in  vitro cell culture models with production of low levels of infectious virus (Brack-Werner et al. 1992; Kramer-Hammerle et al. 2005; Wang et al. 2004). Detection of viral DNA and RNA in postmortem brain tissues of acquired immunodeficiency syndrome (AIDS) patients (Takahashi et al. 1996; Trillo-Pazos et al. 2003; Wang et al. 2004) but rarely viral antigens, suggests that astrocytes may harbor latent virus (Table 5.1 and Fig. 5.3). Under proinflammatory stimuli, treatment of astrocytes with cytokines like tumor necrosis factor (TNF)-a and IL-1b that are abundant in late stage disease could activate virus from latency and initiate new rounds of infection (Tornatore et al. 1994).

5.2.5 Dendritic Cells Dendritic cells (DCs) are functionally classified into myeloid (MDCs) and plasmacytoid DCs (PDCs), both of which are potent professional antigen presenting cells (APC) but vary in the expression of a number of surface molecules. They also exhibit differences in their susceptibility to various strains of HIV-1 (Lore et  al. 2005; Smed-Sorensen et al. 2005). Their role in the dissemination of HIV-1 is of

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utmost importance as they are touted to be the first cell type to be infected during vaginal transmission (Spira et al. 1996). The mode of DC-mediated transmission of HIV-1 to other susceptible host cells can either be cis or trans, depending on whether DCs are directly infected or not, respectively (Dong et al. 2007). During infection in trans, DCs capture virions at the primary site of infection and subsequently migrate into the lymph nodes to transfer infectious virus to autologous CD4+ T cells. In this mode of infection, the surface receptor “Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin” (DC-SIGN) binds and internalizes virions in non-degradative compartments within DCs where they can retain infectivity for several days (Geijtenbeek et al. 2000), thus contributing to the chronic persistence of the virus for a limited time in  vivo. However, studies involving individuals undergoing HAART failed to detect HIV-1 proviral DNA in peripheral blood DCs from patients with undetectable levels of viral RNA (Otero et al. 2003), suggesting that DCs might not be a significant source of latent provirus in vivo (Table 5.1).

5.2.6 Bone Marrow Cell Populations In humans, the HIV-1 viral load in the BM has been found to be very similar to the viral load in the blood, both when cell-free virus was examined (Regez et al. 2005), and when cell-associated virus was assessed (McElrath et  al. 1989), suggesting active viral replication in the BM. Bone marrow abnormalities in HIV-1-infected patients most commonly include dysplasias as well as cytopenias of multiple hematopoietic lineages (Jenkins et al. 1998; Koka et al. 1998). Several mechanisms may contribute to the HIV-1-induced suppression of hematopoiesis, including but not limited to direct infection of hematopoietic progenitor cells (HPCs) (Harbol et al. 1994). Studies have unanimously showed that HPCs express the HIV-1 receptor CD4 and the co-receptors CXCR4 and CCR5 at the level of mRNA and protein. Despite the detection of the HIV-1 coreceptors on HPCs, attempts to detect in vitro and in vivo infection of these cells have generated inconsistent observations. The reason for these confounding results has often been the purity of the CD34+ cell population and their differentiation status (presence of mixed population of CD34+ stem cells as well as multipotent progenitor cells). Also, the partial resistance despite the expression of CD4, CCR5, and CXCR4 may be explained by the ability of these cells to secrete endogenous CCR5 ligands MIP-1a, MIP-1b, and RANTES (Majka et al. 1999, 2000) as well as the CXCR4 ligand SDF-1 (Aiuti et al. 1999), which may compete with the virus and block infection. In vitro, CD34+ progenitors have been reported to be susceptible to HIV-1 infection, albeit with varying efficiency (Furlini et al. 1996; Ruiz et al. 1998). It has been reported that concurrent human herpesvirus-6 (HHV-6) infection renders two human hematopoietic progenitor (TF-1 and KG-1) cell lines susceptible to HIV-1, indicating that under certain conditions, the refractility to infection can be bypassed (Furlini et al. 1996). Additional

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studies with the TF-1 cell line reinforce this possibility and demonstrate that as the cells are induced to differentiate along the myeloid lineage, cell surface co-receptors are upregulated, which in turn correlates with the increased susceptibility of these cells to HIV-1 (Alexaki et al. 2007; Quiterio et al. 2003). In the light of these observations, the hypothesis put forward by Bailey et al. proposing reseeding of a viral variant from a rare infection event of a few progenitor cells raises interesting possibilities about the bone marrow compartment being a source of latent provirus (Table 5.1 and Fig. 5.3) (Bailey et al. 2006).

5.2.7 Other Potential Minor Reservoirs Although several other cellular subpopulations in the bone marrow, peripheral blood, and brain express CD4 as well as CXCR4 and CCR5 and can be infected in culture to varying degrees, there is paucity of information regarding the recovery of infectious virions from these cells. Importantly, it has been shown that Toll-like receptor signaling can reactivate HIV-1 replication in latently infected mast cells (Sundstrom et  al. 2004), thereby raising the possibility of these cells serving as an inducible HIV-1 reservoir. More recently, elegant work by Lawrence et al. has provided direct evidence for the infection of neural progenitor cells in vitro with CXCR4-utilizing strains IIIB and NL4–3. When these nestin+ multi-potential cells were differentiated towards an astrocytic phenotype several days post-infection, there was a burst in virus production. However, there was negligible increase in viral replication when they were differentiated towards a neuronal lineage. When these undifferentiated progenitor cells were transfected with a pNL4–3 molecular clone followed by stimulation with TNF-a, there was a marked upregulation in virus production, suggestive of reactivation from latency (Lawrence et al. 2004). This is in line with results obtained from other in vitro cell culture models of latency and raises the possibility that brain-derived progenitor cells could harbor HIV-1 in a latent state in vivo.

5.3 Maintenance of Latency The HIV-1 genome is about 9.8 kb in length, with two identical long terminal repeats (LTRs) flanking the genes encoding the structural proteins (gag, pol, and env), regulatory proteins (Tat and Rev), and accessory proteins (Vpu, Vpr, Vif, and Nef  ) at the 5¢ and 3¢ ends. The gag gene encodes for the matrix p17 (MA), capsid p24 (CA), and viral nucleocapsid p7 and p6 (NC) proteins. The pol gene encodes three enzymes: protease (PR), reverse transcriptase (RT), and integrase (IN). The env gene encodes the precursor gp160, which is proteolytically processed into gp120 and gp41, components of the viral envelope. Soon after reverse transcription and nuclear import of the pre-integration complex (PIC), IN catalyzes the integration of double-stranded viral cDNA with the host cell genome. Within the integrated

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DNA (provirus), the 5¢ LTR acts as the viral promoter and serves as the template for initiation of host RNA-polymerase II-directed transcription (Goff 2001). The LTR is approximately 640 base pairs in length and is divided into three regions: unique 3 (U3), repeat (R) and unique 5 (U5) (Fig. 5.2). The LTR U3 region is subdivided into the core region, enhancer region, and modulatory region (Fig. 5.2). The core region includes the TATAA element upstream of the transcriptional start site and the GC box array consisting of three GC-rich Sp1 transcription factor binding sites (Fig. 5.2). The core region is essential for HIV-1 basal transcription. The core region is preceded by the enhancer region constituting the two tandem NF-kB binding sites (Fig.  5.2). The modulatory region is located further upstream from the NF-kB sites and includes binding sites for various transcription factors such as CCAAT/enhancer binding proteins (C/EBP), activating transcription factor/cyclic AMP response element binding (ATF/CREB) factors, lymphocyte enhancer factor (LEF-1), and nuclear factor of activated T cells (NF-AT) (Fig. 5.2) (Pereira et al. 2000; Rohr et  al. 2003; Wu and Marsh 2003). Maintenance of post-integration latency is intimately intertwined with parameters governing availability of host transcription factors, viral regulatory proteins, chromatin reorganization, and other stochastic events related to efficiency of transcription.

5.3.1 Chromatin Determinants Since successful integration is a prerequisite for concomitant HIV-1 gene expression (Mitchell et  al. 2004), it raises interesting possibilities about the transcriptional status of the virus being dictated by the immediate chromatin environment. A related question is whether HIV-1 has a predilection to integrate into certain regions of the genome. Elegant work by a number of investigators has demonstrated that different retroviruses exhibit unique target site preferences. Murine leukemia virus (MLV) was shown to favor sequences encoding 5¢ ends of mRNAs (Mitchell et al. 2004; Wu et al. 2003) in HeLa cells. In contrast, HIV seems to have a strong bias towards transcriptionally active genes, as demonstrated by in vitro studies conducted with the human lymphoid SupT1 cell line (Schroder et  al. 2002) as well as human peripheral blood mononuclear cells (PBMCs) and primary lung fibroblasts (Mitchell et  al. 2004). This is also consistent with in  vivo analyses of PBMCs obtained from untreated HIV-1-infected patients (Liu et al. 2006). Using ligationmediated polymerase chain reaction (PCR), studies have estimated that the frequency of integration in transcription units hovered from 75 to 80% (Mitchell et al. 2004). Interspersed within these 100–250 kb “open conformation” regions were CpG islands that excluded HIV-1 integration events. Paradoxically, integration intensity was reduced in genes that were highly expressed indicating that very high levels of transcription were not ideal for integration events. This has suggested that a certain level of accessibility of the chromatin might be optimum for integration. Consistent with this observation, introns of actively transcribed genes have been found to select T cells from HAART-compliant patients (Han et  al. 2004) for HIV-1 integration in

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CD4+ memory T cells. Furthermore, the nature of local, site-specific chromatinassociated proteins might act in concert to allow integration in only certain topographical regions. Last, the viral integration machinery should be compatible for tethering with these host factors. Indeed, CpG islands are known to be regulatory regions that bind unique sets of transcription factors, which may preclude any interactions with the HIV-1 integration machinery, yet be more attuned to the MLV machinery. On a global scale, integration frequencies showed a positive correlation with gene-dense chromosomes (Mitchell et  al. 2004). It is to be noted that this targeting on a chromosomal level might be a consequence of as yet undefined factors that are associated with, for example, spatial positioning of chromosomes within the nucleus, thereby serving as critical factors regulating transcriptional silencing and reactivation of viral gene expression (Misteli 2004). Another interesting explanation for post-integration latency that deals with the nature of the integration site is known as promoter interference (Lassen et al. 2004a). Random integration into transcriptionally active regions of the host genome might result in close proximity of the HIV-1 LTR to a strong upstream cellular promoter, resulting in suppression of the HIV-1 promoter resulting in “transcriptional read-through.” Integration of the proviral DNA interfaces it with the normal molecular architecture of the host chromatin, which is assembled into nucleosomes. Each nucleosome contains a protein core made of eight histone molecules (H2A, H2B, H3, and H4) and 146 nucleotide-long double-stranded DNA wrapped around it (Alberts et  al. 2002). Since nucleosome structure is thought to limit the accessibility of host transcription factors, one might presume that their real time conformation would determine transcriptional activity of the HIV-1 promoter. Independent of the integration site, two nucleosomes (designated nuc-0 and nuc-1) are precisely organized at the viral promoter DNA (Verdin 1991; Verdin et al. 1993). In a transcriptionally quiescent state, nuc-0 (positioned at nucleotide (nt) −405 to −245 relative to the transcriptional start site) and the nuc-1 (positioned at nt +20 to +165 relative to the transcriptional start site) define two open nucleosome-free regions in the viral DNA, extending from −244 to +19 and from +166 to +256 relative to the transcription start site (Fig. 5.2). These open regions include the HIV-1 LTR modulatory, enhancer/core region, transcription factor binding sites for AP3-L, Sp1 (Verdin et al. 1993) and USF (Jones and Peterlin 1994) and a region overlapping the primer-binding site immediately downstream of the 5¢LTR (Van Lint et  al. 1997). Promoter bending by these proteins (d’Adda di Fagagna et al. 1995; Ikeda et al. 1993) has been thought to keep this stretch of DNA nucleosome free by hindering histone assembly. Genomic footprinting studies have confirmed that independent of the activation state, most of these cis-acting elements are always occupied by cognate cellular transcription factors, indicating that transcriptional activation is more likely to be dependent on the nucleosomal conformation rather than binding site accessibility (Demarchi et al. 1993). In fact, it has been proposed that displacement of nuc-1 is a prerequisite for HIV-1 transcription (Verdin et al. 1993) as observed in response to T cell activation stimuli (Verdin et al. 1993). There are two basic mechanisms that modulate the conformation of a nucleosome, thus affecting the regulation of transcription: (1) Post-translational modifications of N-terminal tails of histones, namely

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acetylation, phosphorylation, and methylation. These factors include histone acetyl transferases (HATs) like p300/CREB binding protein (p300/CBP) and p300/CBPassociating factor (PCAF) (Ogryzko et al. 1996, 1998). (2) ATP-dependent chromatin remodeling complexes such as the SWI/SNF family. These processes are regulated by acetylation of specific lysine residues within the N-termini of selective core histones, with HATs neutralizing positive charges on these amino acids; thereby weakening histone–DNA interactions. This is necessary for trans-activation as this makes the DNA more “open” or accessible to the transcriptional machinery (Berger 2002; Cosma 2002). In contrast, recruitment of histone-deacetylases (HDACs) results in transcriptional repression. In representative cellular models of HIV-1 latency like the J-Lat system, recruitment of histone deacetylase-1 (HDAC1) by NF-kB p50 was found to constitutively maintain the nucleosomes nuc-0 and nuc-1 in a deacetylated state, thus keeping the chromatin in a condensed state and consequently impairing RNA polymerase II recruitment and transcriptional initiation from the HIV-1 LTR (Fig.  5.2) (Williams et  al. 2006). Knockdown of p50, which itself lacks a trans-activation domain, has not only been shown to induce an increased basal promoter activity but has also been shown to increase the responsiveness to the HIV-1 trans-activator protein Tat, suggesting a role for constitutive p50 binding to the HIV-1 LTR as a mechanism for post-integration latency in CD4+ T cells (Williams et al. 2006). The importance for HDAC1 in virologic latency is reiterated by studies that have proposed a dynamic model for LTR regulation in T cells by two cellular transcriptional regulators YY1 and LSF (Coull et al. 2000). They form a trimeric complex with HDAC1 at a region spanning nucleotides −10 to +27 of the HIV-1 LTR. While LSF-1 binds to DNA, YY1 serves as an intermolecular bridge to anchor HDAC1 to this region (Fig. 5.2) (Coull et al. 2000). Histone-methylation can have diverse effects depending on the tissue or the amino acid residue under consideration. For example, methylation of lysine-9 (K9) on histone-3 (H3) by histone methyl transferases (HMTs) has been shown to be linked with transcriptional silencing, just as methylation of K4 is associated with activation (Fischle et al. 1999). Accordingly, it has been demonstrated that the participants involved in maintenance of the heterochromatic state at the integrated HIV-1 promoter consisted of the methyltransferase Suv39H1 which specifically mediates H3–K9 trimethylation; and the heterochromatin protein-1g (HP1g), which has been shown to bind the amino acid residue on one hand and recruit the HMT on the other (Bannister et al. 2001; Cheutin et al. 2003; Grewal and Moazed 2003). The role of HP1g in HIV-1 post-integration latency was further confirmed in the HIV-1-infected U1-monocytic model, where its knockdown resulted in reactivation of viral replication (du Chene et al. 2007). Apart from histone-methylation, hypermethylation of CpG sites found within the HIV-1 LTR itself is also responsible for repression of basal and activation-induced promoter activity in the ACH-2 cell line (Ishida et  al. 2006), which serves as a representative CD4+ T cell HIV-1 latency model. Interestingly, post integration latency in microglial cells seems to be the result of the concerted action of both HDACs as well as HMTs on core histone H3 in nuc-1 (Marban et  al. 2007). COUP-TF interacting protein 2 (CTIP2), a transcriptional repressor interacts with HDAC1 and HDAC2 via its N-terminus to repress

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transcription off the HIV-1 promoter in TZMbl cells, a HeLa cell line derivative that carries two integrated copies of the HIV-1 LTR; one driving luciferase and the other driving beta-galactosidase. This was corroborated in experiments with a microglial cell line transfected with either an episomal HIV-1 LTR-luc plasmid or a HIV-1 pNL4–3 vector, wherein knockdown of HDAC1/HDAC2 led to a dramatic increase in CTIP2 knockdown-mediated increase in viral promoter activity and viral replication. Immunoprecipitation and deletion experiments also revealed the interaction of CTIP2 with the HMT Suv39H1 via its central 145–434 residues, with subsequent HP1 binding. This sequential hypoacetylation and methylation of H3 provides the framework for an attractive model for in vivo HIV-1 persistence in the CNS in a latent state.

5.3.2 Availability of Cellular Transcription Factors Availability of host cell transcription factors and viral proteins regulate HIV-1 gene expression in the context of specific cell types, cell cycle regulation, cellular differentiation, and cellular activation (Krebs et al. 2002). Sequestration of two critical transcription factors NF-kB and NFAT in the cytoplasm of resting CD4+ T cells contributes to the repressive state of the HIV-1 LTR in these cells (Lassen et  al. 2004a). The paucity of these factors within the nucleus is reversed in response to activation signals. T cell receptor (TCR) crosslinking or cytokine stimulation (e.g., TNF-a, IL-7) or mitogens (e.g., protein kinase C activators like the phorbol ester PMA and prostratin) lead to nuclear translocation of these molecules and subsequent binding to overlapping cognate sites in the HIV-1 LTR, thereby upregulating basal and Tat-mediated promoter activity (Fig.  5.2). TCR ligation also induces transcription and heterodimerization of the c-jun/c-fos complex AP-1, which is absent in resting T-cells (Liu 2005) and synergizes with NFAT and NF-kB to promote HIV-1 gene expression. Murr-1 has been identified as an innate inhibitor of HIV-1 replication in resting CD4+ T lymphocytes (Ganesh et al. 2003). It has been postulated that such an effect might be a direct consequence of its physical interaction with the ubiquitin-complex protein E3 ligase, preventing this enzyme from targeting phospho-IkB for degradation, possibly playing a role in nuclear exclusion of activated NF-kB in vivo. This, in turn, might play an inhibitory role in basal and TNF-a-stimulated NF-kB activation.

5.3.3 Availability of Viral Proteins The transactivation-responsive region (TAR) is located immediately downstream of the transcriptional start site in the HIV-1 LTR, encompassing nucleotides +1 to +59 (Berkhout et al. 1989; Muesing et al. 1987), and is required for the function of the viral trans-activator protein Tat. In the absence of Tat (Fig. 5.2b), short transcripts

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(30–50 nucleotides) predominate (Cujec et al. 1997; Garcia-Martinez et al. 1997; Parada and Roeder 1996). In the presence of Tat however (Fig. 5.2c), transcription is increased several hundred-fold with the level of long transcripts increasing significantly with a consequent increase in viral gene expression (Dayton et al. 1986; Fisher et al. 1986; Ghosh et al. 1993; Laspia et al. 1990). In a mature transcript, TAR adopts a hairpin structure including a six-nucleotide loop, a trinucleotide pyrimidine bulge and extensive duplex structure (Berkhout et al. 1989). U23, in the bulge, is critical for Tat binding (Churcher et al. 1993; Rana and Jeang 1999; Weeks and Crothers 1991). Tat has specifically been shown to associate with Tat-associated kinase (TAK), equivalent to the positive-transcription elongation factor b (p-TEFb) found in Drosophila, and composed of cyclin T1 and cyclin dependent kinase 9 (CDK9) (Mancebo et  al. 1997; Wei et  al. 1998; Zhu et  al. 1997) in a sequential manner. CDK9 then has been shown to phosphorylate the C-terminal domain (CTD) of RNA polymerase II and promote transcription elongation (Mancebo et al. 1997; Zhou et al. 1998; Zhu et al. 1997). Therefore, the lack of HIV-1 gene expression in latently infected cells might not only arise due to absence of Tat, but also as a result of extremely low levels of CDK9 and cyclin T1, as observed in resting CD4+ T cells (Ghose et al. 2001). In addition, mutations in the Tat gene, the Tat responsive element itself might also contribute to the latent phenotype, as evident from experiments performed in the U1 (Emiliani et al. 1998) and ACH-2 (Emiliani et al. 1996) cell lines, respectively. Tat might also function as a co-activator to recruit histone acetyltransferases, including CBP/p300 and PCAF to the LTR (Marzio et  al. 1998). Tat-recruited HATs presumably acetylate histones in LTR-proximal nucleosomes, remodeling nuc-1 and potentiating transcription (el Kharroubi and Martin 1996; Verdin, Paras, and Van Lint, 1993). Besides initiation and elongation of transcription, Tat might also play a role in capping and stabilization of nascent viral transcripts by interacting with mRNA capping proteins Mce1 and Hcm1 (Chiu et  al. 2001, 2002). Furthermore, Tat itself has been shown to be a substrate for the HAT enzyme activity associated with CBP/p300 and PCAF (Benkirane et al. 1998; Bres et al. 2002a, b; Deng et al. 2000; Van Lint et al. 1996). The HAT activity of CBP/p300 can also influence the activity of the NF-kB p50 subunit. The increased acetylation of p50 leads to an increase in p50 DNA binding and a concomitant transcriptional activation of the HIV-1 promoter (Furia et al. 2002). The SWI/SNF family of proteins is another family of proteins that interact with Tat and play a role in the regulation of the HIV-1 promoter. The SWI/SNF proteins are integral components of the yeast RNA pol II holoenzyme (Wilson et al. 1996) and have the ability to utilize the energy from ATP hydrolysis to disrupt DNA–histone contacts and allow access to transcriptional activators (Peterson and Tamkun 1995; Wilson et al. 1996). In the context of HIV-1 transcription, SWI/SNF proteins are required for Tat’s transactivation ability and generation of mature full-length transcripts. Physical interaction between Tat and the remodeling subunits INI1 and Brm has been observed (Mahmoudi et al. 2006; Treand et al. 2006). In fact, T cell activation by mitogenic stimuli induces recruitment of SWI/SNF complex subunits Jun-3, BRG-1, and ATF-3 (Henderson et al. 2004). In particular, BRG-1 has been

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shown to be recruited to the Ap1-III site located at the 3¢ boundary of nuc-1 (Henderson et al. 2004). T cell activation also enhances the endogenous pools of inositol phosphate, increasing the activity of SWI/SNF by an as yet unexplained mechanism (Shen et al. 2003; Steger et al. 2003; Zhao et al. 1998). The viral protein Rev may also play a role in HIV-1 latency. Expression of the viral Rev protein is essential for the nuclear export of genomic RNA as well as unspliced and/or singly spliced transcripts (Cullen 2003), which are ultimately translated into structural, regulatory, and enzymatic viral proteins. Retention of Rev and Tat (viral transactivator proteins) transcripts in the nucleus of resting CD4+ T cells from HAART patients (Lassen et al. 2006) might be involved in the maintenance of post-integration latency in these cells. Importantly, this phenomenon is non-existent in activated T cells.

5.4 RNA Interference RNA interference, a recently discovered biological process involved in the regulation of gene expression at a post-transcriptional level, has generated immense excitement due to its potential for targeting viral as well as cellular transcripts. Recently, it has been found that cellular miRNAs inhibit HIV-1 expression in primary resting CD4+ T cells by interactions with the 3¢-termini of HIV-1 transcripts, thereby leading to translational inhibition of key HIV-1 encoded proteins, including Tat and Rev (Huang et al. 2007). Inefficient Tat and Rev could exacerbate viral latency in these cells. A more indirect mechanism has been predicted, wherein cellular miRNAs miR-17-5p and miR-20 have been demonstrated to silence PCAF, a critical player in Tat-mediated trans-activation of the HIV-1 LTR (Triboulet et  al. 2007). Interestingly, there have been reports supporting (Omoto et al. 2004) and refuting (Lin and Cullen 2007) the existence of HIV-1 encoded miRNAs (viRNA), involved in modulating either viral or cellular gene expression. Equally controversial is the origin of these viRNAs, with TAR being suggested as a candidate (Klase et  al. 2007). A putative role for TAR in sequestering the pre-miRNA processing enzyme Dicer’s interacting partner TAR-RNA binding protein (TRBP) has been put forward (Bennasser et al. 2006). Tat itself has also been touted to inhibit the endogenous RNAi pathway by its ability to directly inhibit Dicer activity. In order to shed light on the importance of the RNAi-HIV interface, the relevant studies need to be carefully scrutinized in the light of infection and the ensuing kinetics of viral replication in physiologically relevant susceptible host cell populations in vivo.

5.5 Neuropathogenesis and Reseeding from Reservoirs HIV-1 infection results in dissemination of the virus to different cellular and tissue compartments. Some of these anatomical sanctuaries, for example the genital tract (Byrn and Kiessling 1998) and the CNS (Kravcik et al. 1999; Rolinski et al. 1997),

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are poorly penetrable to HAART. This suboptimal exposure to antiviral drugs might therefore lead to a preferential, step-wise selection and accumulation of drug resistant strains in these reservoirs. Along with the resistant viral variants, specific viral quasispecies might also get archived in their latent form (Bailey et al. 2006; Ruff et al. 2002; Verhofstede et al. 2004). The notion of independent viral evolution in these distinct anatomical sites is supported by sequencing and phylogenetic analyses of protease and V3–V5 envelope regions that reveal a differential distribution of HIV quasispecies in PBMCs, plasma, spleen, brain, lymph nodes and lung (Clarke et al. 2000). Variations in drug penetration efficacies within a compartment might give rise to localized resistance patterns as evident from the isolation of distinct HIV genotypes from different regions of the brain (Chang et  al. 1998). Compartmentalization of genetic variants may represent an adaptation on part of the virus to utilize specific micro-environmental cues within the CNS, either for maintaining an active or a passive transcriptional state. In support of this hypothesis are findings that consensus B (conB) HIV-1 viral variants having mutations in C/ EBP site I, denoted as 3T (C-to-T change at nucleotide position 3) and C/EBP site II, denoted as 6G (T-to-G change at nucleotide position 6) and 4C (T-to-C change at nucleotide position 4), were found to be more prevalent in HIV-1-associated dementia (HAD) patients (25%, 10%, and 7%, respectively) and absent from nondemented patients (Hogan et  al. 2003). Notably, the 6G variant accumulated in mid-frontal gyrus (Burdo et al. 2004), a neuroanatomical region known to be permissive for high-level HIV-1 replication (Glass et  al. 1995) and the 4C variant localized to the cerebellum (Burdo et al. 2004), known to support low level HIV-1 gene expression. Since 6G and 4C configurations represent high and low affinity C/ EBP binding sites in the HIV-1 LTR (Burdo et al. 2004), it is intuitive to hypothesize that these variants evolved to persist in localized niches to maintain active replication or latency, contributing to the development of neurologic disease. In addition to the brain, the above studies also revealed that all of these variants were only found in the peripheral blood of patients early in disease, strengthening the argument that viral egress from the peripheral blood compartment into the brain occurs early in the natural course of HIV-1 pathogenesis. The bone marrow might serve a key role in the development of neuropathogenesis, especially in the context of bone marrow-derived monocyte-CNS macrophage turnover (Fig. 5.3). It is now well established that repopulation of macrophages in the brain by peripheral blood monocytes is considerably accelerated in cases of inflammation (Fig.  5.3) (Williams et  al. 2001a). In HIV-1-infected patients, the CD14low/CD16+ monocytic sub-population may increase to as high as 40% of the total circulating monocytes (Thieblemont et al. 1995). These cells are believed to migrate more effectively in the CNS than the classical CD14hi/CD16– monocytes and have been shown to accumulate in perivascular regions of the brain, co-localizing with HIV-1-specific proteins (Fischer-Smith et al. 2001, 2004). Furthermore, these cells have been construed to make a vital contribution towards development of HAD, as studies have shown that they have a more activated, pro-inflammatory phenotype (Pulliam et al. 2004), and that their population is expanded in patients with HAD rather than non-demented HIV-1-infected patients (Pulliam et al. 1997).

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More recently, the CD14low/CD16+ monocytes were also shown to be more susceptible to productive HIV-1 infection (Ellery et al. 2007). However, there is still considerable debate whether they were infected in the BM, potentially as developing monocyte progenitors, or whether infection occurred while in peripheral blood. Interestingly, HIV-1-specific sequences from the BM resemble sequences from the deep white matter of the brain, more than any other tissue, suggesting direct HIV-1 trafficking from the BM and diffusion into the CNS (Liu et al. 2000). The observation that anemia has a striking correlation with HAD further implies that HIV-1induced pathogenesis of the BM may impact HAD progression (McArthur et  al. 1993). Similarly, a recent report showing that BM diffusion rates correlate with dementia severity (Ragin et al. 2006) provides additional support for the hypothesis that events in the BM could be associated with the development of HAD (Gartner 2000).

5.6 Concluding Remarks Although the current HAART regimen against HIV-1 is extremely effective in eliminating plasma viral burden by impeding ongoing viral replication in infected cells, complete eradication remains an elusive therapeutic goal due to the persistence of the latent virus pool. Identification of latently infected cells within a greater pool of cells with a “resting” phenotype is hindered by the absence of any distinguishable characteristics. Although it is widely believed that reactivation from latency in  vivo is prompted by hyper-activation of infected immune cell populations, factors determining the threshold of activation of these latent viral genomes are ill-defined. In the case of suppressive HAART, virus release from this reservoir is a stochastic event and often proceeds at undetectable levels. Another confounding factor is the extremely slow decay rate of these integrated viral entities. In the light of these practical challenges, novel treatment strategies have started focusing towards purging latent virus. Although still in its infancy, structured or supervised treatment interruption (STI) strategies have generated a lot of interest and involve cycles of treatment withdrawal followed by re-initiation. Besides allowing temporary respite from the side effects of continual drug use, the rationale behind such intermittent breaks in treatment is that it will allow rapid replication of HAARTsuppressed viral quasispecies as well as the re-emergence of latent viral strains, which can then be targeted by resumption of therapy. In the absence of HAART, cells harboring archived latent drug-resistant strains may also get transiently activated with subsequent production, processing, and presentation of viral antigens. As a result, they might become more susceptible to cytolytic T cell-mediated lysis. However, the efficacy of STI has been debatable till date, especially in managing acute infection and highlights the need to conceive optimal strategies on an individual basis depending on the clinical correlates of HIV-1 disease severity. Along with devising novel treatment approaches using existing antiretrovirals, a greater emphasis has to be placed on developing compounds with better pharmacodynamic and

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pharmacokinetic parameters, including but not limited to reduced toxicity and improved penetration capability into CNS and testes. Acknowledgments  These studies were funded in part by the Public Health Service, National Institutes of Health through grants (B. Wigdahl, Principal Investigator) from the National Institute of Neurological Disorders and Stroke, NS32092 and NS46263, and the National Institute of Drug Abuse, DA19807

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Chapter 6

HIV Coreceptors and Their Roles in Leukocyte Trafficking During Neuroinflammatory Diseases Robyn S. Klein and Erin E. McCandless

6.1 Overview Due to the increasing resistance of HIV-1 to antiretroviral therapies, there has been much emphasis on the discovery and development of alternative therapeutics for HIV-1-infected individuals. The chemokine receptors CXCR4 (Bleul et al. 1996a; Feng et al. 1996; Nagasawa et al. 1996; Oberlin et al. 1996) and CCR5 (Alkhatib et al. 1996; Deng et al. 1996; Dragic et al. 1996) were identified as target molecules from the time their role as coreceptors for HIV-1 entry into leukocytes was first discovered 10  years ago. Initial studies focused on the use of the chemokine ligands, or altered derivatives, of CXCR4 and CCR5 to prevent the entrance of HIV-1 into immune cells (Schols 2006). While these studies showed some initial promise, there was evidence of significant caveats to their use, including selection of alternative coreceptor utilizing strains (Marechal et al. 1999; Mosier et al. 1999) and the potential to cause inflammatory side effects. These data prompted the development and study of small molecule inhibitors of CXCR4 and CCR5, which have also been used to examine the roles of these molecules in a variety of inflammatory and infectious diseases. Since their discovery as HIV-1 coreceptors, expression of CXCR4 and CCR5 has been detected on diverse leukocyte populations and has been shown to influence the promotion, maintenance, and regulation of inflammation (Bleul et  al. 1996b; D’Apuzzo et al. 1997; Granelli-Piperno et al. 1996; Klein and Rubin 2004; Mohle et al. 1998; Sozzani et al. 1997; Wu et al. 1997). The trafficking of CXCR4and CCR5-expressing leukocytes occurs in a wide range of diseases with diverse etiologies that affect a variety of tissue sites, including the central nervous system (CNS). Because most antiretroviral therapies are unable to efficiently cross the blood–brain barrier (Boffito et al. 2006), the CNS retains special status as a potential viral reservoir during HIV-1 infection. It is therefore imperative to consider how R.S. Klein (*) and E.E. McCandless Department of Internal Medicine, Division of Infectious Diseases, Washington University School of Medicine, 660 South Euclid Ave, St. Louis, MO, 63110, USA e-mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_6, © Springer Science+Business Media, LLC 2010

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targeting HIV-1 coreceptors impacts on leukocyte trafficking into the CNS, as it could influence the incidence, progression and severity of advanced HIV-1 infection, including the development of HIV-1-associated neurological diseases. For example, as HIV-1-infected immune cells are believed to bring virus in to the CNS (FischerSmith and Rappaport 2005), enhancement of leukocyte trafficking into the CNS could lead to increases in CNS viral replication and dissemination. Alternatively, increased parenchymal entry of immune cells might promote the induction of demyelinating disease via enhanced entry of myelin specific T cells into the CNS (Hellings et al. 2002; Holz et al. 2000; Lunemann et al. 2004; Muraro et al. 2002). Additionally, the prevention of leukocyte trafficking by receptor antagonism could lead to increased susceptibility to lethal opportunistic infection, as was observed in multiple sclerosis (MS) patients treated with natalizumab, a humanized monoclonal antibody against a4-integrin, an adhesion molecule shown to be essential for the migration of leukocytes into the CNS (Adelman et al. 2005). In either scenario, the effects on CNS leukocyte trafficking resulting from CXCR4 and CCR5 antagonism could be especially detrimental when applied to an immunocompromised patient population such as one infected with HIV-1. The following chapter will discuss the current data relating to the role of CXCR4 and CCR5 in leukocyte trafficking in the CNS. Included is a discussion of the contributions of CXCR4 and CCR5 to neuroinflammatory diseases caused by infection with either HIV-1 or WNV and by autoimmune mechanisms such as in MS. To gain preliminary insight into how altering leukocyte trafficking patterns in the CNS could affect disease outcome, studies utilizing animal models of autoimmune and virologic CNS diseases and receptor antagonism or deficiency will be covered. Finally, the potential benefits and/or hazards of targeting CXCR4 and CCR5 in the context of HIV infection will be addressed.

6.2 Leukocyte Trafficking into the CNS The movement of leukocytes out of the blood and into diseased tissue is the hallmark of inflammation. The coordination of this movement begins in the secondary lymphoid tissue where leukocytes are primed with information regarding antigen specificity, become activated, and up-regulate molecules that can direct their infiltration into specific target tissues. The general sequence of events leading to leukocyte entrance into inflamed tissue is a well-defined process involving selectins, integrins and chemokines (Butcher and Picker 1996). The first step in this sequence involves a family of molecules that bind sialylated carbohydrates known as selectins, which are expressed on the activated endothelium and mediate the “capture and rolling” of lymphocytes along the vascular wall. The rolling motion of a lymphocyte is converted to firm adhesion by the combined action of integrins and chemokines. Integrins are expressed by lymphocytes, and endothelial cells express their carbohydrate ligands. Integrins undergo conformational changes in response to activation of G-protein-coupled chemokine receptors, which are also expressed by trafficking

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lymphocytes (Johnston and Butcher 2002; Springer 1994). Chemokine ligands, which comprise a large family of proteins that generally direct lymphocyte extravasation into tissues, thus accomplish directed homing of lymphocytes. Although the molecular patterns required to enable trafficking from the circulation into the CNS are incompletely understood, three important molecules, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 (Laschinger and Engelhardt 2000), and P-selectin (Piccio et al. 2002) appear to play essential roles in this process. ICAM-1, VCAM-1 and P-selectin are all expressed by activated endothelial cells during induction of disease or by treatment with the inflammatory mediators tumor necrosis factor alpha (TNF-a), CD40 ligand (CD40L), lipopolysaccharide (LPS) and IL-1b (Ubogu et  al. 2006). After the CNS endothelium is activated, the expression of P-selection and ICAM-1 mediate the capture of circulating T cells through interactions with P-selectin glycolipid (PSGL)-1, and lymphocytefunction-associated antigen (LFA)-1 (integrin aLb2), respectively (Biernacki et al. 2001; Bullard et al. 2007; Piccio et al. 2002). Firm adhesion of trafficking T cells depends upon ICAM-1, while monocytes utilize VCAM-1, although both share a common receptor, very-late antigen (VLA)-4 (Floris et al. 2002). The CNS is protected from immune cell intrusion by a highly specialized system of microvasculature known as the blood–brain barrier (BBB) (Lucas et al. 2006). The BBB is comprised of a network of various cell types and modifications that contribute to maintaining its immune-privilege status including endothelial cells joined by tight junctions, their encasement by pericyte-embedded basement membranes and an additional barrier comprised of glial cell foot-processes. These modifications create an additional area through which leukocytes must exit to gain parenchymal entry known as the perivascular space, which is unique to the CNS (Ballabh et al. 2004). Under normal conditions, this formidable barrier is effective at limiting the trafficking of leukocytes from the blood into the CNS parenchyma. However, when the BBB is compromised, immune cells are able to gain access to the CNS. The extent of BBB penetration and leukocyte entrance depends upon disease etiology (Frohman et al. 2006). Despite differences in the extent of parenchymal entry during infectious or autoimmune diseases, infiltrating leukocytes first accumulate in the perivascular spaces. The perivascular infiltrate is therefore the cardinal lesion associated with all neuroinflammatory diseases. Control of immune infiltration of the CNS poses a unique dichotomy in which its limitation is greatly desired for the treatment of MS, but might also be detrimental during infectious disease where leukocytes are required to clear pathogens. Understanding the specific mechanisms regulating the trafficking of leukocytes across the blood–brain barrier is therefore paramount in developing therapies that prevent or promote inflammation within the CNS. The HIV-1 coreceptors CXCR4 and CCR5 bind to ligand members of a family of molecules known as chemokines, or chemotactic cytokines. While the hallmark function of these small proteins is the direction of leukocyte trafficking, they can also participate in cellular events such as activation and costimulation (Bajetto et al. 2001a). Members of the chemokine family can be classified as either homeostatic or inflammatory based on their temporal expression (Charo and Ransohoff 2006; Kim 2005). Although traditionally the CNS had been thought to be protected from immune acti-

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vation, it is now known that a variety of chemokines and their receptors are expressed in the CNS, both constitutively and during inflammatory diseases, including CXCR4, CCR5 and their ligands (Bajetto et al. 2001b; Cartier et al. 2005). Classically categorized as a homeostatic chemokine, the ligand for CXCR4, known as CXCL12 or SDF-1, can be detected in the quiescent CNS at the microvasculature and by subpopulations of neurons (Krumbholz et  al. 2006; Stumm et  al. 2002). CXCR4 is also expressed constitutively throughout the CNS and can be found on endothelial cells, oligodendrocytes, microglia, and neurons (Klein and Rubin 2004). In the absence of inflammation, CXCL12 and CXCR4 direct the movement and proliferation of and recognition between a variety of resident neural cells types during CNS development (Lu et  al. 2002; Pujol et  al. 2005; Stumm et  al. 2003). Neurophysiologic roles for these molecules in the adult CNS have not been established. During inflammatory conditions, however, expression of CXCL12 is altered (McCandless et al. 2006; McCandless et al. 2008a), suggesting that its role in the CNS is not limited to a developmental one. Because CXCR4 is ubiquitously expressed on leukocytes, it is likely that BBB expression of CXCL12 affects the trafficking of leukocytes, including CD4+, CD8+ T cells and macrophages (Bleul et al. 1996b; Klein and Rubin 2004). Much of the work studying inflammatory-induced CXCL12 has been done in the context of autoimmune neuroinflammation, such as MS and its murine model experimental autoimmune encephalomyelitis (EAE), during which endothelial cells, neurons, microglia, and astrocytes have all been shown to increase their expression of CXCL12 (Ambrosini et al. 2005; Calderon et al. 2006; Krumbholz et al. 2006; McCandless et al. 2006; McCandless et al. 2008a). In compliment, increased numbers of CXCR4-expressing T cells and macrophages traffic to and infiltrate the CNS during MS, leading to demyelination and neuronal injury (Frohman et al. 2006; Prat and Antel 2005). More recent studies have examined the roles of CXCL12 and CXCR4 during encephalitis due to neurotropic viruses, such as the West Nile virus (WNV) and in the context of neuroAIDS (Langford et al. 2002; McCandless et al. 2008b; Peng et al. 2006). These studies have focused on CXCL12 expression by various BBB constituents including endothelial and glial cells and collectively support the notion that CXCR4 plays an important role in regulating the trafficking of leukocytes into the CNS parenchyma. Unlike CXCR4, expression of CCR5 and its four ligands, CCL3, -4, -5 and -8, only occurs during inflammatory states. CCR5 may be expressed by activated T cells, macrophages, microglia and astrocytes (Bleul et al. 1997; Granelli-Piperno et al. 1998; Qin et al. 1998; Wahl et al. 1999), depending on the disease context. In most neuroinflammatory diseases, including neuroAIDS and MS, CCL3, -4, and -5 are expressed within inflammatory lesions (McManus et al. 1998; Simpson et al. 1998; Van Der Voorn et al. 1999) while CCR5 is expressed by infiltrating mononuclear cells (Simpson et al. 2000; Sorensen et al. 1999), suggesting a role for these molecules in the recruitment of immune cells during inflammation. While most of this data is correlative, several recent studies utilizing mice with targeted deletion of CCR5 or examining cohorts of patients with natural mutations of CCR5 or its ligands have begun to shed light on the differential roles of CCR5 in the trafficking mononuclear cell subsets in to the CNS (Table  6.1). While initial interpretations

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Table 6.1  Role of CCR5 in neuroinflammatory disorders Effect of CCR5 deletion/ Disease polymorphism

Reference

Murine models

Tran et al. (2000) Glass et al. (2001)

EAE Chronic MHV

Acute MHV

LCMV MS Human diseases

Flavivirus infection

No effect Decreased macrophage infiltration and demyelination Decreased CD4+ T cell trafficking into the CNS, impaired viral clearance; No effect on CD8+ T cell trafficking into the CNS Enhanced fatality with delayed CD8+ T cell infiltration No effect; CCR5D32 not protective Delayed onset of disease

Glass and Lane (2003)

Bennetts et al. (1997) Barcellos et al. (2000)

Decreased severity of disease Schreiber et al. (2002) Associated with early death Gade-Andavolu et al. (2004) No effect on disease Kantarci et al. (2005) No association with MS Ristic et al. (2006) Associated with MS Favorova et al. (2006) Protective role Otaegui et al. (2007) Decreased severity of disease Van Veen et al. (2007) Increased risk of symptomatic Glass et al. (2006) WNV infection Increased risk of symptomatic Lim et al. (2008) WNV infection Associated with tickborne Kindberg et al. (2008) encephalitis (TBEV) EAE experimental autoimmune encephalomyelitis, MHV murine hepatitis virus, LCMV lymphocytic choriomeningitis virus, MS multiple sclerosis, WNV West Nile virus, TBEV tick-borne encephalitis virus

suggested a simple “summon and response” relationship, it has now become apparent that CCR5-expressing leukocytes may be involved in both clearance of infection and in the counter-regulation of T cell responses that might lead to postinfectious inflammatory sequelae.

6.3 NeuroAIDS Neurological disease associated with HIV-1 infection results from primary replication within the CNS, which generally occurs during advanced stages of the disease when viral isolates reportedly expand their coreceptor usage from CCR5 to

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CXCR4. However, viruses that replicate within the CNS primarily infect cells via CCR5, suggesting a distinctive role for this receptor in the biology of neuroAIDS. Consistent with this, CCR5 has been detected on HIV-infected macrophages within both the CNS and the peripheral blood in individuals with advanced disease. Additionally, T cell-tropic viruses that traffic in and out of the brain during progressive HIV-1 disease may play a greater role in HIV-1-associated neuropathogenesis than macrophage-tropic viruses, which have been shown to induce less neurotoxicity (Zheng et al. 1999). As the accumulation of macrophages within the CNS has been correlated with encephalitis and dementia (Marcondes et  al. 2008), it is possible that CXCR4 and/or CCR5 play roles in the physiologic turnover of macrophages within the CNS. In this section, we will discuss the evidence implicating CXCR4 and CCR5 in the trafficking of leukocytes into the CNS during HIV-induced neuroinflammatory diseases. As infection and replication of HIV-1 and other viruses in brain macrophages and microglia represent the principal reservoir and vehicle for viral dissemination in nonlymphoid tissues, understanding the mechanisms that promote the infiltration of these cells is essential for developing targeted therapies aimed at depleting this reservoir.

6.3.1 Human Studies The first insights into the essential role of macrophages in the neurodissemination of HIV came from studies examining chemokine receptor expression on postmortem specimens from patients with HIV encephalitis. In a majority of these specimens, both CXCR4 and CCR5 were detected on brain macrophages and microglia within inflammatory lesions, which also exhibited staining for HIV-1 antigen (Bonwetsch et  al. 1999; Sanders et  al. 1998; Vallat et  al. 1998). The overall frequency of CCR5-expressing perivascular macrophages was positively correlated with severity of HIV-1-induced neurologic disease, whereas the frequency of CXCR4-expressing macrophages did not correlate with disease severity (Vallat et  al. 1998). CCR5 expression was also significantly enhanced in HIV-1-specific CD8 T cells taken from cerebrospinal fluid versus peripheral blood of HIV-infected patients without neurologic disease (Shacklett et al. 2004). Ligands for these receptors (CXCL12, CCL2, -3, -5) were found to be expressed by activated astrocytes (Peng et al. 2006; Sanders et al. 1998), suggesting that turnover of macrophages may increase during neuroinflammatory states. Characterization of chemokine receptors found on T lymphocytes and monocytes in brain sections from subjects with various neuroinflammatory diseases, however, revealed that CCR1 and CCR5 are present on perivascular and parenchymal monocytic cells whereas only CCR5 was present on parenchymal macrophages (Trebst et al. 2003). These findings suggest that CCR5-expressing mononuclear cells, macrophages, and microglia contribute to progression of neurologic disease of individuals with AIDS by promoting virus entry and replication within the CNS. As CCR5 was also found to be expressed by normal microglia in nonencephalitis brain specimens, this receptor may play a role in the physiologic turnover of these

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cells, although this remains a topic of much debate (Ajami et al. 2007; Mildner et al. 2007). Using a coculture of endothelial cells and astrocytes that models several aspects of the human blood–brain barrier, Weiss et al. (1999) examined the mechanism whereby the HIV-derived factor Tat, a protein that activates viral gene expression (Deng et al. 2002), may facilitate monocyte transmigration. HIV-1 Tat induced significant expression of CCL2 by astrocytes and upregulated expression of CCR5 on human monocytes (Weiss et al. 1999). Although they demonstrated that transmigration across an in vitro BBB model could be inhibited by antiCCL2 antibodies, others have shown that antiCCR5 and antiCCR1 also both abrogate monocyte migration in similar models suggesting that a variety of inflammatory processes could augment monocyte migration into the CNS (Ubogu et al. 2006).

6.3.2 Macaque Model The macaque model of neuroAIDS using simian immunodeficiency virus (SIV) has confirmed that viruses utilizing CCR5, such as SIV(mac)251, can cause primary disease in the CNS via the infiltration of SIV-infected mononuclear cells. Similar to patients with HIV-encephalitis, macaques with SIV encephalitis exhibit elevated CNS levels of CCL3–5 and perivascular infiltrates expressing CXCR4 and CCR5. Within these infiltrates, CCR5 localized specifically to cells within microglial nodules (McManus et  al. 2000; Westmoreland et  al. 1998). This model has further demonstrated that CCR5 expression on blood monocytes and brain microglia and/ or macrophages distinguishes animals that develop encephalitis from those that do not (Marcondes et  al. 2008). Recently, IL-15 treatment of SIVmac251-infected macaques was associated with decreased percentages of CCR5-expressing CD4+ T cells within the peripheral blood (Mueller et al. 2008). IL-15 had previously been reported to improve the survival and effector function of HIV- and SIV-specific CD8+ T cells and to up-regulate CCR5 on human CD4+ T cells. Because IL-15 treatment of acute SIV infection of rhesus macaques also led to an increase in the viral set point and acceleration of disease, the authors speculated that the decreased percentages of peripheral CCR5-expressing CD4+ T cells might indicate that the trafficking of these cells into tissue increases infection. Interestingly, one of the IL-15-treated animals developed neuropathological signs of early SIV encephalitis with increased mononuclear cell infiltrates and parenchymal microglial nodules, suggesting that increased expression of CCR5 on virally infected mononuclear cells promotes viral entry and neuropathology (Mueller et al. 2008).

6.4 CNS Autoimmunity MS, a chronic demyelinating disease of the CNS, is the most common cause of nontraumatic disability among young adults (Frohman et al. 2006). At the cellular level, MS is mediated by myelin-specific CD4+ T cells that destroy oligodendrocytes

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and trigger a cascade of inflammatory events leading to recruitment of additional immune cells that induce progressive demyelination and, ultimately, axon destruction. Clinically, MS is characterized by variations in the progression of disease over time, which has led to three clinical classifications of the disease. Relapsing-remitting MS (RRMS), the most common presenting form of MS, is a stable condition interrupted by recurrent attacks of temporary neurological disability. RRMS often evolves into secondary-progressive MS (SPMS), in which patients no longer remit but rather progressively deteriorate and accrue neurological disability. Primary-progressive MS (PPMS) follows a continuously declining course beginning at disease onset. In MS, it is generally accepted that acute inflammatory lesions begin with breakdown of the BBB (McFarland and Martin 2007), leading to the formation of perivascular infiltrates of mononuclear cells, parenchymal penetration by myelin-specific T cells, and subsequent recruitment and activation of monocytes/microglia that cause demyelination and axonal damage (Hauser et al. 1986; Huseby et al. 2001; Prat and Antel 2005; Ransohoff et al. 2003). A variety of proinflammatory chemokines, including ligands of both CXCR4 and CCR5, are detected within the CNS of individuals with autoimmune neuroinflammatory diseases. Thus, in patients with MS and in mice with EAE, CCL3–5 are elevated within the CNS and CSF and treatments that decrease the levels of these ligands lead to decreased infiltration of mononuclear cells and diminished disease (Eltayeb et al. 2007; Fischer et al. 2000; Glabinski et al. 2002, 2000; IronyTur-Sinai et al. 2006; McCandless et al. 2006). CXCR4 and CCR5 ligands, however, exhibit differential roles in the pathophysiology of CNS autoimmunity, which can be traced to their cellular sources and location. CXCL12, which is expressed by the CNS microvasculature, regulates BBB immune privilege while CCL3–5 is expressed by glial cells and promotes the migration of leukocytes into the CNS parenchyma. In this section, we will discuss the roles of CXCR4 and CCR5 in CNS autoimmune diseases, drawing from studies utilizing the EAE model as well as tissue samples derived from MS patients.

6.4.1 CXCL12 and CXCR4 At many tissue sites, CXCL12 expression increases during autoimmune disease and CXCR4 participates in the localization, proliferation and activation of effector leukocytes at inflamed tissues sites (Garcia-Vicuna et al. 2004; Nagase et al. 2001; Nanki and Lipsky 2000). AMD3100, a bicyclam specific antagonist of CXCR4 signaling (De Clercq 2003; Hatse et al. 2002), has been employed to analyze the role of this receptor in a variety of biological processes, as targeted deletion of either CXCL12 or CXCR4 leads to embryonic lethality due to defects in the development of multiple organ systems (Ma et al. 1998; Zou et al. 1998). Amelioration of disease in a variety of murine models of autoimmunity has been accomplished via chronic treatment with AMD3100 (Lukacs et  al. 2002; Matthys et  al. 2001), suggesting that CXCR4 activation is required during the expression of certain autoimmune diseases. During CNS autoimmunity, activation of CXCR4 may be necessary

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for the development of myelin-specific T cells as use of mutant chemokine ligands that antagonize CXCR4 decreased EAE by inhibiting the sensitization phase of the disease, leading to decreased activation of encephalitogenic T cells (Kohler et al. 2008). The role of CXCL12 and CXCR4 within the CNS during autoimmunity, however, is more complicated. CSF derived from patients with neuroinflammatory diseases contains elevated levels of CXCL12 (Giunti et al. 2003). In studies using CNS tissues derived from both mice and humans with and without neuroinflammatory diseases, BBB expression of the CXCL12b isoform has been observed to be dynamic, exhibiting alterations in its level and location depending on the disease context (McCandless et al. 2008a, b, 2006). In normal CNS tissues CXCL12b expression occurs along the abluminal surfaces of CNS endothelial cells where leukocytes, which ubiquitously express the receptor CXCR4, engage it when attempting to enter the CNS. The polarized expression of CXCL12b acts to localize mononuclear cells to perivascular spaces during neuroinflammation, thereby limiting their entry into the CNS parenchyma. This subcompartment retention, which is analogous to the role of CXCL12b in lymphoid compartments, could be an integral component of CNS protection from the pathologic consequences of lymphocyte-induced glial cell activation. Consistent with this, in both humans and mice with CNS autoimmune disease, BBB expression of CXCL12b increases and relocates across the inflamed venules, allowing the egress of leukocytes from CNS perivascular spaces into the parenchyma, leading to glial activation and demyelination. This altered pattern of CXCL12 expression at the BBB was shown to be highly specific for MS, occurring in 10–100% of venules within inflammatory lesions in postmortem CNS specimens. Other diseases affecting the CNS such as viral encephalitis, CNS lymphoma and Alzheimer’s disease did not show altered CXCL12 expression at the BBB (McCandless et al. 2008a). Alteration in homeostatic CXCL12 expression also correlated with increased astrocyte expression of CXCL12 within the glial limitans (Calderon et  al. 2006; McCandless et al. 2008a). Recent studies have implicated the cytokine interleukin (IL)-1b in the regulation of CXCL12 expression and location within the CNS. Exposure to IL-1b, as well as myelin basic protein (MBP) induces CXCL12 expression by astrocytes in vitro (Calderon et al. 2006). Administration of IL-1b, but not TNF-a, to naïve mice induced CXCL12 relocation in approximately 90% of vessels, and mice with targeted deletion of the IL-1R do not relocate CXCL12 at the microvasculature after immunization with MOG (McCandless et al., 2009). Consistent with this, mice with targeted deletion of IL-1R are resistant to EAE (Matsuki et al. 2006). Further studies will determine the mechanisms of this effect with regard to the infiltration of CXCR4-expressing mononuclear cells. Use of a phospho-specific antibody against the ligand-activated form of CXCR4 also revealed an association between relocation of CXCL12 and activation of CXCR4 within lumenal leukocytes (McCandless et al. 2008a). These data suggest that aberrant expression of CXCL12 at the BBB could contribute both to leukocyte entry into and egress from perivascular spaces. Consistent with this, administration of AMD3100 to mice with EAE leads to widening of inflammatory lesions, increased demyelination and worsened clinical disease. These results demonstrate

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a critical role for CXCL12b in regulating the trafficking of lymphocytes through the perivascular space during CNS autoimmunity. CXCL12 has also been implicated in the initial myelination of the CNS. During development, the migration, proliferation and differentiation of oligodendrocyte precursor cells (OPCs) that populate the spinal cord, hindbrain and basal forebrain with mature oligodendrocytes is accomplished via the synergistic effects of growth factors and chemokines, including CXCL12 (Benveniste and Merrill 1986; Dziembowska et  al. 2005; Franklin 2002; Frost et  al. 2003; Hinks and Franklin 1999; Kadi et al. 2006; Redwine and Armstrong 1998; Tsai et al. 2002; Wu et al. 2000). Studies examining spinal cord myelination in embryonic mice with targeted deletion of CXCR4 suggest that it may control the survival and migration of OPCs in this CNS region (Dziembowska et al. 2005). Interestingly, studies of a variety of CXCR4-expressing cell types have indicated that CXCL12 may synergize with platelet-derived growth factor (PDGF), transforming growth factor (TGF)-b1 or insulin-like growth factor (IGF)-1 in promoting CXCR4-mediated localization, proliferation, survival and maturation (Akekawatchai et  al. 2005; Avecilla et  al. 2004; Basu and Broxmeyer 2005; Kadi et al. 2006; Kortesidis et al. 2005; Lataillade et al. 2000; Sanders et al. 2000). Thus the presence of growth factor receptors on OPCs may enable migration and proliferation during the initial myelination of the CNS according to the precise location and timing cues provided by other factors. During inflammation, however, new cues in the form of Th1 cytokines and chemokines may be required to trigger these developmental attributes. CXC chemokines may also play a role in remyelination during CNS autoimmunity. Recently, subpopulations of cells expressing markers of OPCs (NG2, O4) have been identified within the adult mammalian brain (Dawson et  al. 2003). Studies examining the expression of chemokines within MS lesions, however, have been controversial with some investigators detecting the in situ expression of CXC receptors 1–3 on proliferating oligodendrocytes while others have not (Filipovic et  al. 2003; Omari et al. 2006, 2005). These investigations did not include antiCXCR4 in the panel of chemokine receptor antibodies used in their immunohistochemical analyzes. Studies inducing EAE in mice with targeted deletion of CXCR2 did not appear to uncover any enhancement in disease susceptibility or progression in these mice. However this single study focused on the role of CXCR2-expressing neutrophils in EAE, so extensive analyzes of demyelination and recovery in comparison with wild-type mice were not performed (Abromson-Leeman et al. 2004). Further studies on the roles of CXCR2 and CXCR4 are clearly warranted as these chemokines may play differential roles in various aspects of the remyelination process.

6.4.2 CCR5 and Its Ligands The role of CCR5 in the trafficking of leukocytes during CNS autoimmune diseases is poorly understood. Studies indicate that under noninflamed, physiologic states, few T cells enter the CNS and there is minimal CNS engraftment of blood-derived

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monocyte precursors of microglia (Ajami et  al. 2007; Mildner et  al. 2007). However, in disease states where the BBB is disrupted, expression of a variety of chemokines, including CCL5, is increased, as is the trafficking of antigen-specific lymphocytes and a subpopulation of monocytes that differentiate into ramified parenchymal microglia (Mildner et  al. 2007). Thus, CCR5 may be important for both the trafficking of autoreactive lymphocytes and the demyelinating process believed to be initiated and maintained by activated monocytes and microglia within the CNS parenchyma. In support of this, active MS lesions contain CCR5 expressing infiltrating T cells comprised of members of both the adaptive (CD4+, CD8+) and innate (gdTCR+) arms of immunity (Rinaldi et al. 2006). While autoreactive CD4+ and CD8+ T cells have been shown to play roles in CNS demyelinating diseases in murine models, gd T cells have recently come to the forefront as important participants in the initial induction of EAE (Lees et al. 2008; Odyniec et al. 2004; Smith and Barnum 2008; Szalai and Barnum 2004; Szalai et  al. 2005). Interestingly, gd TCR+T cell lines derived from MS patients, compared to lines derived from healthy control subjects, expressed lower levels of CCR5 but higher levels of ligand (CCL5), suggesting the presence of an autoregulatory loop (Murzenok et al. 2002). Additional CCL3 and CCR5 expressing cells present within MS lesions are foamy macrophages and activated microglia (Balashov et  al. 1999; Simpson et  al. 2000; Sorensen et  al. 1999). In a study by Trebst et al. (2003), 70% of CSF CD14+ monocytes derived from MS patients during exacerbations were found to express CCR1 and CCR5, regardless of the stage of disease, versus <20% of circulating monocytes. CCR1/ CCR5 expressing monocytes were found in perivascular infiltrates and at demyelinating edges of lesions. While early lesions contained CCR1/CCR5-expressing monocytes and CCR1/CCR5 negative microglia, those examined at later stages contained macrophages that expressed only CCR5. The authors suggest that a subset of CCR1+/CCR5+ blood monocytes traffic into the CNS where, in the presence of ligands they are retained and, which upon further activation, down-regulates CCR1 and upregulates CCR5. A recent study identified four patterns of demyelination in active MS lesions. In all four of these patterns, infiltrating monocytes coexpress CCR1 and CCR5. The characteristics of pattern II lesions suggested a primary inflammatory mechanism of myelin injury, while pattern III lesions showed features consistent with oligodendrocyte degeneration. In pattern II lesions, the number of cells expressing CCR1 significantly decreased while CCR5 increased in late active compared with early active demyelinating regions. In striking contrast, numbers of cells expressing CCR1 and CCR5 were equal in all regions of pattern III lesions (Mahad et al. 2004). Support for the role of CCR5 in the trafficking of CD4 T cells and macrophages during CNS autoimmunity also comes from studies using viral models of demyelination. Intracranial infection of the coronavirus mice with mouse hepatitis virus (MHV) results in an immune response-mediated demyelinating disease that serves as another model MS. During MHV-induced demyelination, CD4+ T cells amplify demyelination by attracting macrophages into the CNS following viral infection by mechanisms that are not yet understood. In studies using mice with targeted deletion

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of CCR5, virus-specific CD4+ T cells were unable to traffic into the CNS which led to higher CNS viral titers early during infection (Glass and Lane 2003b). CCR5 is not, however, required for the trafficking of virus-specific CD8+ T cells in this model as there were no differences in trafficking patterns of CCR5−/− CD8+ T cells. On the contrary, loss of CCR5 led to increased activity of virus-specific, cytotoxic CD8 T cells and eventual viral clearance that was no different from wildtype mice (Glass and Lane 2003a). However, the authors did observe a significant decrease in the extent of macrophage recruitment and demyelination during the chronic demyelinating phase of MHV CNS disease. Analysis of chemokine receptor expression on infiltrating macrophages and microglia revealed that most of them expressed CCR5. This was one of the first studies implicating CCR5 in the trafficking of macrophages into the CNS during a neuroinflammatory disease. CCR5 is among a group of chemokine receptors expressed by T cells that are essential for their effector functions during Th1 inflammatory responses. Studies indicate that cytokines that contribute to the development of autoreactive T cells upregulate their expression of CCR5 and enhance their encephalitogenic properties (Bagaeva et al. 2003) and that DT390-RANTES-SRalpha, a recombinant immunotoxin, prevents EAE via decreasing the numbers of CCR5+-infiltrating cells within the CNS (Jia et  al. 2006). Consistent with this, increased percentages of CCR5 peripheral blood mononuclear cells (PBMCs) can be found in the blood and CSF of MS patients versus healthy controls or patients with other types of neurological diseases (Martinez-Caceres et al. 2002b). PBMCs that express CCR5 include lymphocytes, monocytes and myeloid-derived dendritic cells (mDCs), which are major stimulators of T cells. Comparisons of CCR5 mRNA on PBMCs derived from patients with different forms of MS revealed significantly increased expression in those derived from PPMS versus SPMS, RRMS and control patients (Jalonen et al. 2002), suggesting that synthesis of CCR5 within leukocytes increases with severity of MS. CD209+ CCR5+ dendritic cells are abundant in nonlesional gray matter in multiple sclerosis and may thus play a role in the activation of autoreactive T cells within the CNS parenchyma, leading to exacerbations (Cudrici et al. 2007). Both memory T cells and mDCs have been shown to exhibit increased expression of CCR5 and CCL5 in the blood and CSF of MS patients (Pashenkov et al. 2002). CSF memory T cells, which could potentially differentiate into effector cells via antigen encounter derived from MS patients, express disproportionately high levels of CCR5 when compared with peripheral blood mononuclear cells (Sorensen et al. 1999, 2002; Zang et al. 2000). During relapse, CD4 and CD8 cells within the CSF are enriched for CCR5 while during remission, CCR5 alone is reduced in CSF CD4 T cells suggesting that CCR5 expression on those particular cells is a marker of disease activity (Misu et al. 2001). Treatment with immunosuppressive drugs alters the numbers of CCR5expressing T cells in the periphery. Both cyclophosphamide and glatiramer therapy, for example, reverse the increase in the percentages of IFN-g-producing CCR5 expressing T cells in MS patients (Allie et al. 2005; Karni et al. 2004). In addition, treatment with methylprednisolone can decrease the percentages of CCR5-expressing CD4 T cells in the peripheral blood (Martinez-Caceres et al. 2002a). IL-12 stimulates myelin-reactive T cells to up-regulate the beta-chemokine receptor, CCR5, in correlation with the

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acquisition of central nervous system-infiltrating and encephalitogenic properties (Bagaeva et  al. 2003). In contrast, in  vitro treatment of T cells with IFN-b inhibits expression of CCL3 and CCL5 mRNA, and surface expression of their receptor CCR5, suggesting that the mechanism of IFN-beta treatment of MS lies in impairment in T cell trafficking (Zang et al. 2000). Consistent with this, treatment with IFN-b is associated with a decrease in CCR5-expressing mononuclear cells in the peripheral blood (Teleshova et al. 2002). Similarly, Sorensen and Sellebjerg (2001) found that T cells in the peripheral blood of SPMS patients exhibited lower levels of CCR5, which may have left this compartment and trafficked into the CNS (Sorensen and Sellebjerg 2001). These studies suggest that targeting CCR5 might be useful for the treatment of CNS autoimmune diseases and, in support of this, peripheral administration of antiCCL3 antibodies has been shown to prevent recurrence of autoimmune anterior uveitis in Lewis rats (Manczak et al. 2002). However, genetic approaches have not been as conclusive in that CCL3- and CCR5-deficient mice are both fully susceptible to EAE (Tran et al. 2000) and studies on patients with the CCR5D32 allele, which encodes a truncated, nonfunctional protein, reveal an unclear relationship between CCR5 activity and MS severity. Approximately a dozen genome screenings of MS cohorts and multiplex MS families have evaluated the relationship between the presence of the CCR5D32 allele and disease onset, severity and outcome of MS. The majority of these studies suggest that while decreased activation of CCR5 does not confer protection from MS (Bennetts et al. 1997), it may delay disease onset and attenuate recurrent disease activity (Kantor et  al. 2003; Sellebjerg et  al. 2000). In addition, patients with the CCR5D32 allele have been observed to have more benign clinical courses with smaller lesion volumes, lower black hole ratio on MRI and higher percentages of lesions with signs of remyelination (Kaimen-Maciel et  al. 2007; Schreiber et  al. 2002). Similar findings have been reported for other CCL5 polymorphisms including the lowproducer alleles CCL5−403*G and CCR5+303*G, which were associated with reduced risk of severe axonal loss and reduced T2 hyperintense and T1 hypointense lesion volumes on MRI, respectively (van Veen et al. 2007). In contrast, high-producer alleles CCL5−403*A and CCR5+303*A were associated with a worse clinical disease course and early age at onset. Similarly, a study of CCR5D32 allele carriage in a Spanish population revealed a statistically significant difference between the study group and the control group for the carriers of at least one deleted allele in which the allele was more frequent in the control group, suggesting a possible protective effect of this deletion against MS (Otaegui et al. 2007). Most of these authors concluded that CCR5 antagonism may attenuate disease activity in MS or may provide a therapeutic target to modulate inflammatory demyelination (Sellebjerg et al. 2000). Several studies have refuted these findings, suggesting instead that the CCR5D32 polymorphism is either not a major determinant of susceptibility for MS (Kantarci et al. 2005; Ristic et al. 2006; Sellebjerg et al. 2007; Silversides et al. 2004) or is positively associated with MS (Favorova et al. 2002), especially in patients with worsened outcome including primary progressive disease and early death (Gade-Andavolu et al. 2004; Pulkkinen et al. 2004). The use of novel CCR5 antagonists in various animal models of MS may be necessary to address the role of CCR5 in the neuropathogenesis of MS.

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6.5 West Nile Virus Encephalitis WNV is a neurotropic flavivirus now endemic within the Northern hemisphere that cycles between ornithophilic mosquitoes and natural bird reservoirs but may also incidentally infect humans and other vertebrate animals (Campbell et  al. 2002; Glaser 2004; Williams 2004). While most WNV infections are asymptomatic or manifest as a mild, flu-like illness, potentially fatal neuroinvasive infections, including meningitis, encephalitis and anterior myelitis, occur in the elderly or immunocompromised. In the CNS, WNV targets cortical, midbrain, cerebellar and spinal cord neurons leading to their injury or death (Fratkin et al. 2004; Hunsperger and Roehrig 2006; Samuel et al. 2006; Shrestha et al. 2003). The high incidence of WNV neuroinvasive disease in patients on antiT cell therapies (Katz and Bianco 2003; Kleinschmidt-DeMasters et al. 2004) and in mice with T cell deficiencies (Shrestha et al. 2006; Wang et al. 2006, 2003a, b) indicate that, similar to other neurotropic viruses, the clearance of WNV within the CNS relies heavily on cell-mediated immune responses that promote the migration and effector functions of T cells into the CNS parenchyma. Experiments in mice have established that some chemokines and their receptors have essential roles in directing leukocytes to the CNS to clear WNV from infected neuronal cells. CCL3–5, chemokines that all bind the chemokine receptor CCR5, are strongly induced in the CNS after WNV infection (Glass et al. 2005, 2006; Klein et al. 2005; Shirato et al. 2004), and targeted deletion of CCR5 is associated with depressed leukocyte trafficking, increased viral burden and enhanced mortality (Glass et al. 2005). WNV encephalitis is associated with the early expression of CXCL10 by virally infected neurons that proceeds in a caudal to rostral direction within the CNS with significantly higher levels detected in the cerebellum by day 5 postinfection (Klein et al. 2005). Loss of CXCL10 was associated with decreased recruitment of WNV-specific CD8 T cells into the CNS, high CNS viral loads and enhanced mortality. The identification of CXCL12 as a key regulator of leukocyte trafficking at the BBB led to recent studies evaluating the role of this chemokine in the migration of virus-specific T cells during WNV encephalitis.

6.5.1 CXCL12 and CXCR4 The discovery that CXCL12 serves to prevent excessive immune cell entry by localizing infiltrating immune cells to perivascular spaces led to the hypothesis that BBB expression of CXCL12 might also prevent infiltrating virus-specific T cells from entering the CNS during infections with neurotropic viruses. Indeed, analysis of human postmortem specimens from patients who have succumbed to WNV revealed that CXCL12 maintains its polarity at the BBB (McCandless et al. 2008a, b) and that infiltrating T cells remained primarily sequestered within perivascular spaces with little parenchymal entry in most CNS regions (McCandless et al. 2008a, b). Using a murine model of lethal WNV encephalitis, the authors demonstrated that

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CXCL12 mRNA and protein at the BBB are down-regulated during the course of WNV encephalitis and that the decreased levels of CXCL12 are associated with a concomitant decrease in the numbers of perivascular T cells and an increase in the numbers of parenchymal T cells. In addition, administration of a CXCR4 antagonist early in the course of CNS infection led to increased parenchymal penetration of WNV-specific CD8+ T cells, enhanced viral clearance and improved survival from 10 to 50%. Interestingly, the augmented numbers of infiltrating CD8+ T cells was associated with decreased glial cell activation, suggesting that T cell entry into the CNS for the purpose of viral clearance does not necessarily lead to inappropriate immune activation and pathology. It is also probable that virus-specific T cells that can efficiently eliminate a pathogen trigger mechanisms for efficient T cell egress from or elimination within the CNS versus T cells that gain entry into the CNS but do not encounter their antigens. Further studies are necessary to identify the molecules that regulate pathways of T cell exit. The demonstration that CXCR4 inactivation leads to increased leukocyte entry poses a dilemma for the use of CXCR4 antagonists in patients infected with HIV-1. While it suggests that CXCR4 antagonism might promote the entry of virus-specific T cells, it also raises the issue of whether the entry of HIV-infected monocyte might also be enhanced, promoting the CNS as a reservoir of virus. Because HIV-specific CD8 T cell responses are defective in chronic HIV infection (Trabattoni et al. 2004), CXCR4 antagonism in the setting of HIV infection may therefore tip the balance in favor of extensive HIV neuroinvasion and acceleration of HIV encephalitis. Further preclinical evaluations that focus on the CNS may be warranted before novel CXCR4 antagonists are approved as antiinfectives for patients with HIV-1.

6.5.2 CCR5 The role of CCR5 in viral infections of the CNS has been studied using a variety of viral models including MHV, lymphocytic choriomeningitis (LCMV) and WNV (de Lemos et  al. 2005; Glass and Lane 2003b; Glass et  al. 2005; Nansen et  al. 2000). As observed in CNS autoimmune diseases, CCR5 expression is essential for the trafficking of CD4+ T cells in all of these viral models. In addition, impaired trafficking of CD8+ T cells and macrophages was observed in LCMV- and WNVinfected mice with targeted deletion of CCR5. In all circumstances, CCR5-deficient mice had increased viral burdens and, in the cases of LCMV and WNV, adoptive transfer of splenocytes from virally-infected CCR5+/+ mice into infected CCR5−/− mice increased leukocyte accumulation in the CNS and increased survival rates to approximate those observed in infected CCR5+/+ control mice. These studies indicate a critical role for CCR5 in antiviral immune responses within the CNS and suggest that targeting CCR5 to prevent infection with HIV-1 could have dire consequences if patients become infected with neurotropic viruses. In support of this, several recent studies have evaluated the clinical outcomes observed in flavivirus-infected patients that carry the CCR5D32 allele. One group

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has now published two reports that associate deficiency of CCR5 with symptomatic WNV infection. In one report, which examined WNV infection and carriage of CCR5D32 in cohorts of patients from Colorado and Arizona, loss of CCR5 was associated with increased neuroinvasive disease (Glass et  al. 2006). In a metaanalysis of the Colorado and Arizone cohorts plus two additional cohorts from Illinois and California, homozygosity for the CCR5D32 allele was found to be higher in patients with symptomatic WNV infection (Lim et al. 2008). The discrepancy in the severity of WNV infection associated with CCR5D32 homozygosity between the two studies was blamed on underpowering as the small sample sizes were unable to show such an association for a gene found in populations of European descent with allelic frequencies ranging from 0 to 0.29 (McNicholl et al. 1997). In addition, neither study demonstrated any impact of CCR5D32 homozygosity on age incidence in symptomatic, WNV-infected patients.

Autoimmunity tight junction

IL-1R

Viral encephalitis

IL-1R

CXCL12 CXCL12 CXCL12 CXCR4

IL-1b

T cell

T cell

IL-1R IL-1b

T cell

CXCL12 CXCL12 CXCL12

IL-1b T cell

T cell

T cell

glial limitans

CXCL12 IL-1R

CXCL12 IL-17R

CXCL12 CXCL12 T cell

T cell

T cell

T cell

T cell

T cell

Fig.  6.1  Role of CXCL12 in T cell trafficking at the BBB. Depicted are proposed models for CXCL12-mediated regulation of T cell egress from perivascular spaces in the context of autoimmune and WNV encephalitides. In autoimmune disease, CXCR4-expressing T cells express the cytokine IL-1b (McCandless et al., 2009), which binds its receptor, IL-1R, on endothelial cells. This induces the intracellular uptake of CXCL12 via unknown mechanisms, allowing T cells to exit from the perivascular space. In the case of WNV encephalitis, IL-1b is not expressed within the CNS (Cheeran et  al. 2005; Kong et  al. 2008) and T cells remain localized to perivascular spaces until late in the course of disease when CXCL12 levels are down-regulated and T cells begin to enter the parenchyma (McCandless et al. 2008b)

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Carriage of the CCR5D32 allele, however, may be a risk factor for severe infections with flaviviruses, in general. Kindberg et  al. (2008) performed CCR5D32 genotyping among Lithuanian patients with tick-borne encephalitis (TBE), an often fatal infection caused by the TBE flavivirus (TBEV). As with WNV, TBEV in most individuals induces a self-limited, febrile illness with influenza-like symptoms while certain individuals, for unknown reasons, develop severe meningoencephalitis. In the Kindberg study, a significant increase in CCR5D32 allele prevalence was observed in patients with TBE compared with nonTBE aseptic meningoencephalitis subjects and healthy subjects were seronegative for TBE. Carriage of the CCR5D32 allele was also associated with increased clinical severity of disease, although individuals with CCR5D32 were not members of the group with the most severe symptoms (Kindberg et al. 2008). Thus, there are likely to be additional risk factors for severe TBE neuroinvasive disease that are currently unknown.

6.6 Conclusions Coreceptor antagonism for the prevention of widespread cellular entry has been considered an attractive approach to halt the progression of HIV-1 ever since the discovery that carriage of CCR5D32 confers resistance to initial infection with HIV-1. However, the multifunctional role of HIV-1 coreceptors suggests that additional insights are required to identify unforeseen hazards of pharmacological chemokine receptor inactivation. This appears to be particularly important in the case of CXCR4 antagonism, which leads to increased trafficking of mononuclear cells into the CNS, potentially impacting on the CNS viral reservoir. While few studies have directly addressed how HIV-1 infection itself impacts on leukocyte CNS entry and no studies have implicated CXCR4 in this process; the studies outlined in this chapter strongly suggest that HIV-1 enters the CNS within mononuclear cells and that this migration is enhanced during CXCR4 antagonism. Because HIV-infected macrophages and microglia have also been implicated in the inflammatory-mediated destruction of neurons (Adamson et al. 1996; Dawson et al. 1993; Giulian et al. 1996; Nottet et al. 1995; Persidsky et al. 2000), CXCR4 antagonists could potentially increase both the incidence and severity of neuroAIDS. Thus, studies examining the impact of CXCR4 antagonism on CNS viral loads and neuronal injury are warranted prior to the approval and implementation of this new class of antiHIV drugs. Use of CCR5 antagonists, on the other hand, might limit HIV-1 neuroinvasion and/or alter the immunopathology ascribed to CCR5-expressing mononuclear cells that enter the CNS. As outlined above, CCR5 is involved in the CNS trafficking of HIV-infected mononuclear cells and plays a role in glial cell activation and amplification of inflammatory responses. Thus, inactivation of CCR5 could prove to be efficacious in slowing the progression of HIV-1 infection. These speculations, however, have not undergone rigorous evaluation in animal models of neuroAIDS. Thus, the effect of newly approved CCR5 antagonists on HIV infection with the CNS is currently unknown and will therefore be determined empirically. If CCR5

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antagonism does prove to be efficacious for the prevention of neuroAIDS, based on the virologic studies in animal models described above, its use would not be recommended if alternative immunotherapeutic agents that promote CD8 T cell-mediated clearance of HIV-1 were developed and implemented. Therefore, use of CCR5 antagonists will need to be continually reevaluated through animal model experimentation as novel therapeutic targets emerge and are translated into new drug treatments for HIV-infected patients. Acknowledgments  This work was supported by NIH/NINDS NS052632 and by grants from the National Multiple Sclerosis Society and the Midwest Regional Center for Excellence in Emerging Infectious Diseases (all to R.S.K.). The authors would also like to thank Drs. Eric Lyng and Jigisha Patel for their critical readings of the manuscript.

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Oberlin E, Amara A, Bachelerie F, Bessia C, Virelizier JL, Arenzana-Seisdedos F, Schwartz O, Heard JM, Clark-Lewis I, Legler DF, Loetscher M, Baggiolini M, Moser B (1996) The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1. Nature 382:833–835 Odyniec A, Szczepanik M, Mycko MP, Stasiolek M, Raine CS, Selmaj KW (2004) Gammadelta T cells enhance the expression of experimental autoimmune encephalomyelitis by promoting antigen presentation and IL-12 production. J Immunol 173:682–694 Omari KM, John GR, Sealfon SC, Raine CS (2005) CXC chemokine receptors on human oligodendrocytes: implications for multiple sclerosis. Brain 128:1003–1015 Omari KM, John G, Lango R, Raine CS (2006) Role for CXCR2 and CXCL1 on glia in multiple sclerosis. Glia 53:24–31 Otaegui D, Ruiz-Martinez J, Olaskoaga J, Emparanza JI, de Munain AL (2007) Influence of CCR5Delta32 genotype in Spanish population with multiple sclerosis. Neurogenetics 8:201–205 Pashenkov M, Teleshova N, Kouwenhoven M, Smirnova T, Jin YP, Kostulas V, Huang YM, Pinegin B, Boiko A, Link H (2002) Recruitment of dendritic cells to the cerebrospinal fluid in bacterial neuroinfections. J Neuroimmunol 122:106–116 Peng H, Erdmann N, Whitney N, Dou H, Gorantla S, Gendelman HE, Ghorpade A, Zheng J (2006) HIV-1-infected and/or immune activated macrophages regulate astrocyte SDF-1 production through IL-1beta. Glia 54:619–629 Persidsky Y, Zheng J, Miller D, Gendelman HE (2000) Mononuclear phagocytes mediate blood– brain barrier compromise and neuronal injury during HIV-1-associated dementia. J Leukoc Biol 68:413–422 Piccio L, Rossi B, Scarpini E, Laudanna C, Giagulli C, Issekutz AC, Vestweber D, Butcher EC, Constantin G (2002) Molecular mechanisms involved in lymphocyte recruitment in inflamed brain microvessels: critical roles for P-selectin glycoprotein ligand-1 and heterotrimeric G(i)linked receptors. J Immunol 168:1940–1949 Prat A, Antel J (2005) Pathogenesis of multiple sclerosis. Curr Opin Neurol 18:225–230 Pujol F, Kitabgi P, Boudin H (2005) The chemokine SDF-1 differentially regulates axonal elongation and branching in hippocampal neurons. J Cell Sci 118:1071–1080 Pulkkinen K, Luomala M, Kuusisto H, Lehtimaki T, Saarela M, Jalonen TO, Elovaara I (2004) Increase in CCR5 Delta32/Delta32 genotype in multiple sclerosis. Acta Neurol Scand 109:342–347 Qin S, Rottman JB, Myers P, Kassam N, Weinblatt M, Loetscher M, Koch AE, Moser B, Mackay CR (1998) The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J Clin Invest 101:746–754 Ransohoff RM, Kivisakk P, Kidd G (2003) Three or more routes for leukocyte migration into the central nervous system. Nat Rev Immunol 3:569–581 Redwine JM, Armstrong RC (1998) In vivo proliferation of oligodendrocyte progenitors expressing PDGFalphaR during early remyelination. J Neurobiol 37:413–428 Rinaldi L, Gallo P, Calabrese M, Ranzato F, Luise D, Colavito D, Motta M, Guglielmo A, Del Giudice E, Romualdi C, Ragazzi E, D’Arrigo A, Dalle Carbonare M, Leontino B, Leon A (2006) Longitudinal analysis of immune cell phenotypes in early stage multiple sclerosis: distinctive patterns characterize MRI-active patients. Brain 129:1993–2007 Ristic S, Lovrecic L, Starcevic-Cizmarevic N, Brajenovic-Milic B, Jazbec SS, Barac-Latas V, Vejnovic D, Sepcic J, Kapovic M, Peterlin B (2006) No association of CCR5delta32 gene mutation with multiple sclerosis in Croatian and Slovenian patients. Mult Scler 12:360–362 Samuel MA, Morrey JD, Diamond MS (2006) Caspase-3 dependent cell death of neurons contributes to the pathogenesis of West Nile virus encephalitis. J Virol 81(6):2614–2623 Sanders VJ, Pittman CA, White MG, Wang G, Wiley CA, Achim CL (1998) Chemokines and receptors in HIV encephalitis. AIDS 12:1021–1026 Sanders VJ, Everall IP, Johnson RW, Masliah E (2000) Fibroblast growth factor modulates HIV coreceptor CXCR4 expression by neural cells. HNRC Group. J Neurosci Res 59:671–679 Schols D (2006) HIV co-receptor inhibitors as novel class of anti-HIV drugs. Antiviral Res 71:216–226 Schreiber K, Otura AB, Ryder LP, Madsen HO, Jorgensen OS, Svejgaard A, Sorensen PS (2002) Disease severity in Danish multiple sclerosis patients evaluated by MRI and three genetic

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Teleshova N, Pashenkov M, Huang YM, Soderstrom M, Kivisakk P, Kostulas V, Haglund M, Link H (2002) Multiple sclerosis and optic neuritis: CCR5 and CXCR3 expressing T cells are augmented in blood and cerebrospinal fluid. J Neurol 249:723–729 Trabattoni D, Piconi S, Biasin M, Rizzardini G, Migliorino M, Seminari E, Boasso A, Piacentini L, Villa ML, Maserati R, Clerici M (2004) Granule-dependent mechanisms of lysis are defective in CD8 T cells of HIV-infected, antiretroviral therapy-treated individuals. Aids 18:859–869 Tran EH, Kuziel WA, Owens T (2000) Induction of experimental autoimmune encephalomyelitis in C57BL/6 mice deficient in either the chemokine macrophage inflammatory protein-1alpha or its CCR5 receptor. Eur J Immunol 30:1410–1415 Trebst C, Staugaitis SM, Tucky B, Wei T, Suzuki K, Aldape KD, Pardo CA, Troncoso J, Lassmann H, Ransohoff RM (2003) Chemokine receptors on infiltrating leucocytes in inflammatory pathologies of the central nervous system (CNS). Neuropathol Appl Neurobiol 29:584–595 Tsai HH, Frost E, To V, Robinson S, Ffrench-Constant C, Geertman R, Ransohoff RM, Miller RH (2002) The chemokine receptor CXCR2 controls positioning of oligodendrocyte precursors in developing spinal cord by arresting their migration. Cell 110:373–383 Ubogu EE, Cossoy MB, Ransohoff RM (2006) The expression and function of chemokines involved in CNS inflammation. Trends Pharmacol Sci 27:48–55 Vallat AV, De Girolami U, He J, Mhashilkar A, Marasco W, Shi B, Gray F, Bell J, Keohane C, Smith TW, Gabuzda D (1998) Localization of HIV-1 co-receptors CCR5 and CXCR4 in the brain of children with AIDS. Am J Pathol 152:167–178 Van Der Voorn P, Tekstra J, Beelen RH, Tensen CP, Van Der Valk P, De Groot CJ (1999) Expression of MCP-1 by reactive astrocytes in demyelinating multiple sclerosis lesions. Am J Pathol 154:45–51 van Veen T, Nielsen J, Berkhof J, Barkhof F, Kamphorst W, Bo L, Ravid R, Verweij CL, Huitinga I, Polman CH, Uitdehaag BM (2007) CCL5 and CCR5 genotypes modify clinical, radiological and pathological features of multiple sclerosis. J Neuroimmunol 190:157–164 Wahl SM, Greenwell-Wild T, Peng G, Hale-Donze H, Orenstein JM (1999) Co-infection with opportunistic pathogens promotes human immunodeficiency virus type 1 infection in macrophages. J Infect Dis 179(Suppl. 3):S457–S460 Wang T, Scully E, Yin Z, Kim JH, Wang S, Yan J, Mamula M, Anderson JF, Craft J, Fikrig E (2003a) IFN-gamma-producing gamma delta T cells help control murine West Nile virus infection. J Immunol 171:2524–2531 Wang Y, Lobigs M, Lee E, Mullbacher A (2003b) CD8+ T cells mediate recovery and immunopathology in West Nile virus encephalitis. J Virol 77:13323–13334 Wang T, Gao Y, Scully E, Davis CT, Anderson JF, Welte T, Ledizet M, Koski R, Madri JA, Barrett A, Yin Z, Craft J, Fikrig E (2006) Gamma delta T cells facilitate adaptive immunity against West Nile virus infection in mice. J Immunol 177:1825–1832 Weiss JM, Nath A, Major EO, Berman JW (1999) HIV-1 Tat induces monocyte chemoattractant protein-1-mediated monocyte transmigration across a model of the human blood-brain barrier and up-regulates CCR5 expression on human monocytes. J Immunol 163:2953–2959 Westmoreland SV, Rottman JB, Williams KC, Lackner AA, Sasseville VG (1998) Chemokine receptor expression on resident and inflammatory cells in the brain of macaques with simian immunodeficiency virus encephalitis. Am J Pathol 152:659–665 Williams K (2004) Modes of transmission for West Nile virus. Clin Lab Sci 17:56 Wu L, Paxton WA, Kassam N, Ruffing N, Rottman JB, Sullivan N, Choe H, Sodroski J, Newman W, Koup RA, Mackay CR (1997) CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro. J Exp Med 185:1681–1691 Wu DT, Woodman SE, Weiss JM, McManus CM, D’Aversa TG, Hesselgesser J, Major EO, Nath A, Berman JW (2000) Mechanisms of leukocyte trafficking into the CNS. J Neurovirol 6(Suppl. 1):S82–S85 Zang YC, Samanta AK, Halder JB, Hong J, Tejada-Simon MV, Rivera VM, Zhang JZ (2000) Aberrant T cell migration toward RANTES and MIP-1 alpha in patients with multiple sclerosis. Overexpression of chemokine receptor CCR5. Brain 123(Pt 9):1874–1882

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Section II

Chapter 7

Chemokine Proteolytic Processing in HIV Infection: Neurotoxic and Neuroimmune Consequences David Vergote, Christopher M. Overall, and Christopher Power

7.1 Introduction Together with transcriptional and translational regulation, posttranslational modification is a pivotal mechanism regulating protein abundance and function. Proteolysis has been suggested to be the most important posttranslational modification of proteins – it affects every protein and can result in marked changes in activity and eventual clearance (Doucet et al. 2008). Not only do protein degradation and processing modulate protein stability, permitting extra- or misfolded protein recycling, but they are also important evolutionary strategies for generating bioactive molecules that affect cell function and survival. Two major enzymatic mechanisms are known to be involved in protein degradation and processing: (1) ubiquitin-dependent degradation of proteins by the proteasome and (2) protease-dependent maturation and processing of proteins with ensuing effects on their biological functions. Indeed, the maturation of numerous neuropeptides involves sequential proteolytic cleavages of a precursor protein by different proteases leading to peptide products with pleiotropic effects (see (Hallberg and Nyberg 2003) for review). Requisite maturation by proteolysis has also been reported for molecules involved in immune response including inflammatory proteins (pro-IL-1b by caspase-1/ICE and MMP-9 (Cerretti et  al. 1992; Schonbeck et al. 1998), pro-TNF-a by ADAM17/TACE (Moss et al. 1997), TGFb by plasmin (Yee et al. 1993), receptors (protease-activated receptors by their ligands) (Noorbakhsh et al. 2003), or elements of the complement cascade (Gasque 2004) to reveal their full activity. Several proteins acquire neuropathogenic properties following a proteolytic processing; one of the best examples occurs in

D. Vergote and C. Power () Department of Medicine (Neurology), 6-11 Heritage Medical Research Centre, University of Alberta, Edmonton, AB T6G 2S2, Canada e-mail: [email protected] C.M. Overall Molecular Biology and Biochemistry and Oral Biological and Medical Sciences, University of British Columbia, V6T 1Z3, Vancouver, BC, Canada O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_7, © Springer Science+Business Media, LLC 2010

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Alzheimer’s disease in which the pathogenicity of amyloid peptides depends on proteases, namely secretases, involved in amyloid precursor protein (APP) maturation. This chapter will describe how the proteolysis of chemokines might participate in the neuropathogenesis of HIV infection, thus contributing to the development of the central nervous system disorder termed HIV-associated dementia (HAD).

7.2 Chemokine Proteolysis Overview Numerous proteases have been described to process specific chemokines, cleaving either their amino- or carboxy-terminal ends with ensuing altered structural and biological properties, leading to the highly topical area of chemokine degradomics (Davis et al. 2005; McQuibban et al. 2000, 2002; Overall et al. 2002; Van Damme et al. 1999; Cox and Overall 2008). The NH2-terminal end preceding the first two conserved cysteinyl residues of a chemokine is generally critical for the activation of its cognate receptor as it is the principal region of the molecule responsible for the triggering of the receptor (Gong and Clark-Lewis 1995; Nufer et  al. 1999; Valenzuela-Fernandez et  al. 2002). Hence, proteolytic cleavage of this region has been shown to specifically change the biological properties of a chemokine. Table  7.1 summarizes the available data regarding the proteolytic cleavage of chemokines that might bind to non-orphan chemokine receptors described as having HIV co-receptor functions (CCR1, CCR2, CCR3, CCR8, and CXCR6 besides CCR5 and CXCR4). Importantly, HIV-1 sequences and strains derived from the CNS rely chiefly on CCR5 (and not CXCR4) for productive infection of target cells including microglia and macrophages (Gorry et  al. 2001; Power et  al. 1998). Antagonists of these receptors could interfere with HIV attachment and entry but chemokine truncation might also impact on its cognate biological properties. The full effect of truncation depends on the cleavage site for each specific chemokine and therefore on both the cleavage site specificity of the protease and the nature of the chemokine used as a substrate. Indeed, the proteolysis of CCL8 by CD26 and MMP-8 (Tester et al. 2007) removing the first two and five residues, respectively, strongly decreases the chemotactic properties of the chemokine. However, cleavage of CCL3 by the same proteases removing also the two aminoterminal residues actually increases the chemotactic properties of CCL3 (Proost et al. 2000; Van Coillie et al. 1998). Several chemokines are completely degraded by specific proteases, e.g. CCL2 by neutrophil elastase (Witherden et al. 2004) or CCL3 and CCL4 by cathepsin D (Wolf et al. 2003). Of note, while the cleavage of CCL14 by plasmin leads to a degradation of the chemokine, it transiently activates the chemokine before further degrading the chemokine resulting in a short half-life chemotactic activity (Vakili et al. 2001). Nevertheless, the inability to detect a product of proteolysis should be interpreted with care as it might reflect either a real and complete degradation initiated by the protease or an inability to detect the cleavage product by the specific methods used. A shift from agonistic to antagonistic properties toward its cognate receptor may occur upon proteolytic cleavage independently of any affinity modulation and examples are numerous. Indeed, while aminoterminal proteolysis of CCL2,

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Table 7.1  Proteolytic processing of chemokines interacting with described HIV coreceptor Biological Chemokine Protease Product consequences References CCL2 Neutrophil Degradation Witherden et al. (2004) elastase EhCP2 Before a R Modulation CK Pertuz Belloso et al. (2004) MMP-1, -3, -8 5–76 Dec. CK/antiMcQuibban et al. (2000, inflam in vivo 2002) MMP-12 Antag. Dean et al. (2008) CCL3 Cathepsin D Degradation from Wolf et al. (2003) Cterm Elastase17–70; 32–70 Loss CK Ryu et al. (2005) proteinase 3 Cathepsin G 24–70; 28–70 Loss CK Ryu et al. (2005) CD26/DPPIV 3–70 Inc. CK on CCR1 Proost et al. (2000) CCR5/keep anti-HIV activity CCL4 Cathepsin D Degradation Wolf et al. (2003) from Cterm Iwata et al. (1999), Noso CCL5 sCD26/mCD26 3–68 Dec. CK on et al. (1996), Oravecz CCR1 but not et al. (1997), Proost et al. on CCR3 (1998c), Schols et al. (1998) Oravecz et al. (1997), sCD26/mCD26 3–68 Inc. antiHIV Proost et al. (1998c), activity/shift Schols et al. (1998) CCR1 to CCR5 Lim et al. (2005, 2006) Cathepsin G 4–68 Dec. CK and antiHIV activity Proost et al. (1998a), Loss CK/antag. CCL7 MMP-1, 2, 3, 13, 5–76 Struyf et al. (1998), CCR1/2/3/ 14, MT1McQuibban et al. antiinflam. MMP (2000, 2002) in vivo MMP-12 5–76 Antag. Dean et al. (2008) CCL8 CD26/DPPIV 3–76 Dec. CK and Oravecz et al. (1997), Van signaling Coillie et al. (1998) 6–76 Chemokine Proost et al. (1998a) inhibitor MMP-3/MMP-12 5–76 CCR2 antag. McQuibban et al. (2002), Dean et al. (2008) CCL11 N. americanus Inactivation Culley et al. (2000) MMP CD26/DPP IV 3–74 Partial CCR3 Oravecz et al. (1997), antag. Struyf et al. (1999) Ellyard et al. (2007) Elastase/ cathepsin G/ plasmin (continued)

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Table 7.1  (continued) Chemokine Protease

Product

CCL13

MMP-1,-3

4–75; 5–75; 8–75

CCL14

MMP-12 EhCP2 Plasmin

uPA CCL15

CCL23

CXCL12

Cathepsin G Chymase Neutrophil elastase Cathepsin G/ chymase Elastase CD26/DPP IV/ DPP8

Leukocyte elastase

Biological consequences

References

Dec. CK/CCR2/3 McQuibban et al. (2002) antag./antiinflam. in vivo 5–75 Dean et al. (2008) Before a R Modulation CK Pertuz Belloso et al. (2004) Vakili et al. (2001) HCC(9–74) + Inc. activity degradation but further degradation HCC(9–74) CCR1/3/5 agonist/ Detheux et al. (2000), antiHIV Vakili et al. (2001) 24–92; 27–92; Inc. CK and Berahovich et al. (2005), 29–92 signaling Richter et al. (2005) 29–92 Inc. CK activity Berahovich et al. (2005) 22–92 Inc. CK and Berahovich et al. (2005), signaling Richter et al. (2005) 27–99 Inc. CCR1 activity Berahovich et al. (2005) 30–99 3–67

4–67

MMP-1,-2,-3,-9,- 5–67 13,-14 Cathepsin G 6–67 antag antagonist; CK chemotactism; properties

Inc. CCR2 activity Berahovich et al. (2005) Christopherson et al. Inactivation at (2002), Ohtsuki et al. CXCR4/Dec. (1998), Proost et al. antiHIV-1 (1998b), Shioda et al. (1998), Ajami et al. (2008) Valenzuela-Fernandez et al. No CXCR4 (2002) binding/ function/loss anti-HIV McQuibban et al. (2001), Shift receptor Vergote et al. (2006), to CXCR3/ Zhang et al. (2003) neurotox./inflam. Inactivation Delgado et al. (2001) Dec decrease; inc increase; antiinflam anti-inflammatory

CCL7, CCL8, and CCL13 by metalloproteases (MMPs) does not alter the chemokine ability to bind their receptor, their agonistic activities are eliminated and the cleaved products become receptor antagonists (McQuibban et al. 2002). However, this does not necessarily prevent antagonists from activating the receptor, by an antagonistic mechanism involving a mandatory MAPK activation, which has been recently described for CCL11 engagement of CCR2 (Ogilvie et al. 2004). Proteolytic cleavage may also activate the chemokine. For example, CCL14 naturally displays a very weak affinity for CCR1 and is unable to bind to CCR3 and CCR5 but shows an increase in CCR1 affinity and gain of CCR3 and CCR5 affinity associated with chemotactic activity on lymphoblasts, monocytes, and eosinophils upon

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urokinase-type plasminogen activator cleavage, thereby becoming a potent CCR1, CCR3, and CCR5 agonist (Detheux et al. 2000; Vakili et al. 2001). Similarly, CD26processing of CCL4 increases the chemokine’s ability to signal through CCR1 and CCR2 without altering its affinity for CCR5 (Guan et al. 2002, 2004). Interestingly, the specific proteolysis of a few chemokines leading to unexpected effects has also been described. CCL20 and CXCL7 need to be processed by cathepsins to display antimicrobial activity (Hasan et  al. 2006; Krijgsveld et  al. 2000). Effects of proteolysis on CCL5 and CXCL12 (also termed Stromal cell-Derived Factor (SDF)-1a) generating products with pathogenic properties will be discussed below. Aminoterminal processing of chemokines by proteases may result in the decrease of the chemokine’s potency in terms of its biological activities or complete inactivation of the chemokine at its cognate receptor. Alternatively, a switch from receptor agonism to antagonism (or to new properties acquired by the cleaved molecule) may emerge following cleavage. It is unlikely that aminoterminal truncation of a chemokine alters binding to glycosaminoglycans (GAG) as the main determinants involved in GAG binding are located more centrally within the chemokine sequence or near the C-terminus (Allen et al. 2007). Compared to the amino-terminal end, the carboxy-terminus of chemokines is usually not involved in receptor binding but may modulate chemokine-receptor affinity, GAG binding, other chemokine activities, or be the seeding point of complete degradation of the molecule (Ehlert et al. 1995; Wolf et al. 2003). A recent study showed that CXCL11 presents a carboxyterminal binding site for CXCR3 and is involved in GAG binding. The proteolytic cleavage of CXCL11 by MMPs releases the chemokine from GAGs, thereby destroying tissue-fixed haptotactic gradients of CXCL11 and potentially disrupting Th1 cell migration (Cox et al. 2008). Chemokine properties are regulated transcriptionally and translationally by glycosylation, by the presence of decoy receptors like GAG, or by their targeting to the ubiquitin–proteasome system. Chemokine proteolysis involves another mechanism by which chemokine activity is regulated either finely (“subtle” changes in affinity) or completely (agonist to antagonist transition or, as will be discussed later, changes in receptor specificity). This regulatory mechanism is modulated by specific proteases but occurs in constitutive or inducible manners.

7.3 Chemokine Proteolysis Altering HIV Binding to Its Coreceptors 7.3.1 HIV-Induced Proteases Proteases are crucial enzymes induced by HIV to alter the physiology of the central nervous system. Indeed, proteases participate in brain infection, helping infected peripheral cells to cross the blood–brain barrier, as well as in the viral neuropathogenesis as will be later discussed. We will first describe examples of

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three families of host proteases regulated by HIV-1 and their impact on its lifecycle by processing chemokines: MMPs, CD26/DPP IV, and cathepsins. 7.3.1.1 MMPs MMPs are zinc-dependent metalloproteases classically described in extracellular matrix remodeling, allowing tissue healing or cell migration. The proinflammatory potential of MMPs is broadly acknowledged, especially within the nervous system where they have been described to participate in the neuropathogenesis of multiple sclerosis, Alzheimer’s disease, malignant gliomas, stroke, or neuroviral infections (see (Yong et  al. 2001) for review). However, more recent data also have revealed potent anti-inflammatory roles for MMPs with more MMPs exhibiting previously unrecognized beneficial roles in inflammation (Cox and Overall 2008; Dean et al. 2008; McQuibban et al. 2000). The ability of HIV to induce the production and/or the secretion of proteases by its cellular targets has been reported especially for MMP-2 and -9. This property of the virus was initially shown in 1992, as observed for HIV-infected monocytes that secreted metalloproteases necessary for the infected cells to cross epithelial barriers (Dhawan et al. 1992). Since then numerous studies have linked HIV infection with increased MMP activity, particularly with the viral proteins Tat, gp120, and gp41 (see (Mastroianni and Liuzzi 2007; Webster and Crowe 2006) for review) and with a downregulation of tissue inhibitors of MMPs (TIMPs) (Suryadevara et al. 2003). One of the beneficial effects of antiretroviral therapies is to abolish the increase of MMP-9 occurring in HIV infected mononuclear cells as recently shown (Latronico et al. 2007). These observations were confirmed in animal models of lentivirus-induced immunodeficiency. Indeed, SIV-infected macaques develop cognitive and motor deficits, alterations in evoked potentials, and rapid disease progression associated with the expression of MMP-9 in the brain (Berman et al. 1999). Moreover, infection with feline immunodeficiency virus (FIV) induces expression of MMP-2 (but not MMP-9) and is associated with neurotoxicity and neurobehavioral deficits (Johnston et al. 2002). These in vivo results corroborate the detection of MMP-2, -7, and -9 in the cerebrospinal fluid of HIV-infected patients and with the demonstration that level of MMP-2 activity is associated with neurodegeneration (Conant et al. 1999; Sporer et al. 1998; Zhang et al. 2003). Of particular interest, an alternate HIV co-receptor, mannose receptor, appears to be involved in the upregulation of MMP-2 in astrocytes (Lopez-Herrera et  al. 2005). Moreover, these effects were specific to neurovirulent FIV strains or to non-neurovirulent chimeric virus containing the cerebrospinal fluid-derived FIV env gene as blood-derived viral strains were not as neuropathogenic (Johnston et al. 2002). Diversity in neurovirulence among brain-derived HIV-1 strains also exists and Johnston et al. demonstrated that infection of primary macrophages containing brain-derived HIV envelope sequences associated with the predetermined

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diagnosis of HIV-associated dementia (but not with brain-derived envelope containing viruses from non-demented AIDS patients) induced MMP-2 and -9, an effect perhaps also mediated by the viral protein Tat (Johnston et  al. 2000, 2001). Thus, the protease-inducing properties appear to be closely related to the neurovirulence of the individual lentiviral strain. Other neurotropic retroviruses such as HTLV-I (Giraudon et al. 1997) have been shown to induce MMP expression, but the occurrence of the specific cleavage of chemokines in these infections remains unclear.

7.3.1.2 CD26/DPP IV CD26, also named dipeptidyl peptidase (DPP) IV because of its enzymatic function, is a membrane bound protease specifically cleaving the two first aminoterminal residues from its substrates providing the P1 residue is a prolyl. Modulation of its dipeptidyl peptidase activity was proposed to be a promising therapeutic target for T cell dysregulation disorders including diabetes and malignancy (reviewed in (Aytac and Dang 2004). Several years before the description of the chemokine receptors CCR5 and CXCR4 as the principal HIV coreceptors (Dragic et al. 1996; Oberlin et  al. 1996), CD26 was posited as a membrane coreceptors for HIV (Callebaut et al. 1993). Indeed, both viral proteins, gp120 and Tat, are able to interact with mCD26. Interestingly, a general decrease in CD26 expression is observed in T cells from HIV-infected patients (De Pasquale et al. 1989). This phenomenon might be explained by the increased susceptibility to HIV infection of CD26expressing T cells compared to their CD26-negative counterparts (Callebaut et al. 1998) but is prevented by immune reconstitution through HAART (Keane et  al. 2001). CD26 appears to interfere with the viral binding to the T cells although this effect depends on its level of expression. Indeed, moderate expression of CD26 sensitizes T cells to infection while a high level of expression prevents HIV fusion (Callebaut et al. 1998). Moreover, CD26 appeared to be necessary for gp120/gp41induced apoptosis in T cells (Jacotot et al. 1996), but a high viremia correlates with a decrease in CD26 enzymatic activity (Hosono et al. 1999). Interestingly, the viral protein Tat is considered as a “natural” inhibitor of CD26 (Ohtsuki et  al. 2000). Indeed, the binding of Tat to sialylated CD26 inhibits the dipeptidyl peptidase enzymatic activity of CD26 (Smith et al. 1998). This might be an important mechanism for the virus to create viral reservoirs throughout the body, particularly in lymphoid tissue. As CD26 can either generate or eliminate anti-HIV molecules through the processing of chemokines (for example CCL5 (Schols et al. 1998) and CXCL12 (McQuibban et al. 2001), thus modulating the availability of HIV coreceptors and CD26 that regulates gp120/gp41-mediated cell death, this protease appears to be important in different stages of viral replication in T cells. While CD26 has been detected on glial cells (Shimizu et al. 1995), its role in HIV infection within the central nervous system is, however, still unclear.

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7.3.1.3 Cathepsins Proteases of the cathepsin family exhibit their activity principally within the lysosomes as they are optimally active at low pH, although catheptic activity can occur, albeit less efficiently, at neutral pH but for limited time periods. Beside their established role in innate immunity (Pham 2006), cathepsins have been implicated in the pathogenesis of cancer, stroke, and Alzheimer’s disease (Nixon and Cataldo 2006; Palermo and Joyce 2008; Qin et al. 2008). The enzymatic activity of cathepsins also appears to be critical for Ebola virus entry within cells through proteolysis of the virus glycoproteins (Chandran et al. 2005). Upon HIV infection, cathepsin D, an aspartic cathepsin, activity together with collagenase activity is decreased in circulating monocytes. In contrast, polymorphonuclear cells show an increase in cathepsin D and elastase activities although lymphocytes show only a slight increase in elastase activity (Prin-Mathieu et al. 2001). Cathepsins D and G (a serine protease) have been reported to increase the ability of HIV to grow in lymphocytic cultures and to increase the macrophage susceptibility to HIV, respectively (El Messaoudi et al. 1999; Moriuchi et al. 2000). In the brain, cathepsins are broadly expressed by astrocytes, microglia, and neurons. In the frontal lobe of HIV-infected human brains, cathepsins B and C activities are increased in the white matter while only cathepsin C activity is increased in the cortex. This finding is associated with an increase in cathepsin D immunoreactivity in cortical activated microglial cells, hypertrophic astrocytes, perivascular macrophages, and multinucleated cells (Gelman et al. 1997). Hence, cathepsins are implicated in the neuropathogenesis of HIV with potential effects on chemokine proteolysis but direct interactions have yet to be demonstrated to date.

7.3.2 Regulation of Anti-HIV Properties of Chemokines by Limited Proteolysis Most aminoterminal processing by proteases inactivates chemokines’ anti-HIV properties (Table  7.1), such as those of CXCL12 upon its processing by CD26, leukocyte elastase, or MMPs (Christopherson et al. 2002; McQuibban et al. 2001; Ohtsuki et al. 1998; Proost et al. 1998b; Shioda et al. 1998; Valenzuela-Fernandez et al. 2002). However, there are examples in which chemokines increase or acquire anti-HIV properties upon aminoterminal proteolysis. Indeed, CD26-processed CCL5 possesses antiviral properties while the full-length CCL5 does not. The acquisition of antiviral properties by CCL5(3–68) is associated with the shift of receptor preference from CCR1/CCR3 to CCR5 (Schols et  al. 1998). Similarly, associated with acquisition of CCR1/CCR3/CCR5 affinity, urokinase-type plasminogen activator-processed CCL14 acquires anti-HIV properties (Detheux et  al. 2000; Vakili et al. 2001). Moreover, other proteolytic products from a chemokine have been proposed to modulate HIV infection. Both CCL14 processing by trypsin and CCL5 processing

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by CD26 generate the anti-HIV products CCL14(9–74) and CCL5(3–78) through the acquisition of or an increase in CCR5 affinity (Detheux et al. 2000; Schols et al. 1998) suggesting that chemokine truncation in the context of a viral infection might be considered as either a pathogenic mechanism benefiting the pathogen or a defense response to the infection increasing host protection. Nevertheless, the occurrence and implications of CCL5 and CCL14 processing in CNS remain unknown although trysinogen IV is known to be expressed in the CNS and likely exerts its actions on proteinase-activated receptor-2 contributing to neuroprotection during HIV infection (Noorbakhsh et al. 2005).

7.4 Role of Chemokine Proteolysis in HIV Neuropathogenesis: CXCL12 and MMPs The role of proteases, particularly metalloproteases, is widely recognized in neurodegeneration (Yong et al. 2001). Regarding HIV neuropathogenesis, neurotoxicity of HIV Tat expressing macrophages conditioned media was shown to be prevented by MMP-2 and MMP-7 antibodies. In vivo experiments confirmed the ability of MMP inhibitors to prevent Tat-mediated neurotoxicity (Johnston et  al. 2001). Moreover, FIV-mediated neurotoxicity was also attenuated by the small molecule MMP inhibitor prinomastat and by MMP-2 blocking antibodies (Johnston et al. 2002). Our group recently described a mechanism underlying the neuronal cell death occurring during HIV-infection of CNS and associated with the proteolytic cleavage of CXCL12, which is a natural antagonist for HIV-1. We showed that numerous lentivirus-induced MMPs can cleave the aminoterminus of the chemokine CXCL12 (Johnston et  al. 2000; McQuibban et  al. 2001), generating a neurotoxic product whose abundance is increased in the cortex of HIV-infected patients, particularly those diagnosed with HIV-associated dementia (Vergote et al. 2006; Zhang et al. 2003). CXCL12 processing is associated with a loss of affinity of the chemokine for its cognate receptor, CXCR4, while acquiring affinity for CXCR3 whose ligands have been shown to exert neurotoxicity properties (Vergote et al. 2006).

7.4.1 MMP-Mediated CXCL12 Processing in the Brain Associated with HIV Infection 7.4.1.1 CXCL12 Cleavage by MMPs The ability of CD26/DPPIV, leukocyte elastase, and cathepsin G to process CXCL12 giving rise to CXCL12(3–67), CXCL12(4–67), and CXCL12 (6–67), respectively, was previously demonstrated. These proteolytic products failed to activate CXCR4 or induce chemotaxis, intracellular calcium signaling, or any anti-HIV properties (Christopherson et  al. 2002; Delgado et  al. 2001; Ohtsuki et  al. 1998;

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Fig.  7.1  CXCL12 sequence showing the reported processing by proteases. Highlighted are the sequences involved in CXCR4 binding and activation, and the two peptides, CXCL12(5–19) and CXCL12(20–33), evaluated for their ability to compete with CXCL12(5–67) in binding to its receptor

Proost et al. 1998b; Shioda et al. 1998; Valenzuela-Fernandez et al. 2002). In 2001, McQuibban et  al. also demonstrated in  vitro that CXCL12 was a substrate for MMPs, with MMP-2 and also MMPs-1, -3, -9, -13, and -14 able to process CXCL12 resulting in a four amino acids truncated form of CXCL12(5–67) at the amino terminus of both alpha and beta isoforms of CXCL12 (Fig. 7.1) (McQuibban et al. 2001). This processing was specific as MMPs-7 and -8 were unable to process CXCL12(McQuibban et al. 2001). The highly potent chemotactic (and HIV antagonistic) CXCL12 was thus converted into CXCL12(5–67) exerting neither chemotactic nor HIV antagonistic activities. Few chemokine processing products observed in  vitro have been confirmed in vivo in large part because of the difficulty in generating specific detection tools such as antibodies. Nonetheless, the CXCL12 truncation product, CXCL12(5–67), was detected in brains of HIV/AIDS patients but not in brains of patients without HIV infection despite the presence of other neurological diseases. Cells exhibiting CXCL12(5–67) immunoreactivity were shown to be CD45-positive suggesting that the main source of CXCL12(5–67) in HIV-infected brains were monocytoid (macrophage/microglia) cells. The relevance of this localization was confirmed by the co-localization in these cells of MMP-2, also upregulated in HIV-positive brains as mentioned above suggesting that CXCL12 may be processed intracellularly by MMP-2, possibly in the secretory pathway. Oxidative stress has been shown to promote activation of MMPs resulting in intracellular degradation of the metalloprotease substrates thus supporting the above hypothesis (Okamoto et al. 2001; Wang et al. 2002). It warrants bearing in mind that proteases and their substrates may have different or supplementary effects if present intracellularly opposed to extracellularly as they may be exposed to different substrates/targets. Interestingly, the abundance of CXCL12(5–67) in HIV-infected brains was linked to the neurological status. Indeed, CXCL12(5–67) appeared to be more abundant in brains of patients diagnosed with HIV-associated dementia compared to the brains of non-demented HIV/AIDS patients (Vergote et al. 2006). This finding demonstrated that CXCL12(5–67)

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was an in vivo product of CXCL12 cleavage by MMPs driven by HIV infection. The putative beneficial effect of this processing for the virus will be discussed later in this chapter. 7.4.1.2 MMP Processing of CXCL12 is Associated with a Shift of Receptor Affinity from CXCR4 to CXCR3 In keeping with the examples discussed above, the various properties of chemokines are clearly linked to the integrity of their aminoterminal domain preceding the two first conserved cysteines (Gong and Clark-Lewis 1995; Nufer et  al. 1999; Valenzuela-Fernandez et al. 2002). Indeed, the truncation of the aminoterminal end of CCL2, CCL5, CCL7, or CCL8 by MMPs, dipeptidyl peptidases, or cathepsins results in a decrease, or even in a complete loss, of chemoattractivity (Table 7.1). On the other hand, aminoterminal processing of CCL14, CCL15, or CCL23 increases or reveals the chemotactic properties of the chemokines (Table 7.1). As a specific example, the truncation of the two first residues from CCL3 by DPPIV increases the chemotaxis induced by activation of CCR1 and CCR5 but a further aminoterminal processing by cathepsin G or elastase finally results in a loss of chemotactic activity (Proost et al. 2000; Ryu et al. 2005). Antiviral potency might be associated with or independent of the chemotactic potency as receptor activation might be dissociable from the ability of its ligand to bind. In the case where the ligand loses its ability to activate the receptor (in general, to induce chemotaxis) without losing its affinity for the receptor, the ligand is considered as an antagonist like CCL13 after its truncation by MMPs (McQuibban et al. 2002). If the chemokine is a ligand for several receptors, its chemotaxis might be differentially regulated by truncation. CCL5 truncation by DPPIV, while decreasing its chemotactic properties on monocytes, does not change its chemoattractivity for eosinophils reflecting a loss of CCR1 affinity but no change in CCR3 affinity (Noso et al. 1996; Oravecz et al. 1997, Proost et al. 1998c, Schols et al. 1998). Hence, CXCL12 processing by MMPs is not an exception as it selectively alters the chemokine properties. Indeed, while CXCL12 is a potent chemoattractant for CXCR4-expressing pre-B lymphoma cells and primary CD34+ stem cells, MMPprocessed CXCL12 is unable to attract these cells in transwell experiments (McQuibban et al. 2001). Moreover, CXCL12 is a potent inhibitor of infection by T cell-tropic (CXCR4-dependent) HIV strains through competition with the virus for CXCR4 binding, but its aminoterminal cleavage completely abolishes its antiviral properties. These data corroborate the observations obtained with the CD26-, the leukocyte elastase- and the cathepsin G-processed CXCL12, CXCL12(3–67), CXCL12(4–67), and CXCL12(6–67), also presenting a loss in chemotactic and anti-HIV properties (Christopherson et al. 2002; Delgado et al. 2001; Ohtsuki et al. 1998; Proost et al. 1998b; Shioda et al. 1998; Valenzuela-Fernandez et al. 2002) highlighting the crucial role of CXCL12 aminoterminus integrity to confer antiviral properties to the chemokine through CXCR4 binding.

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Keeping in mind that aminoterminal processing of numerous chemokines was reported to modify their affinity for their receptors, and that it was observed that CXCL12 loses its chemotactic and antiviral properties following MMP processing, we speculate that the limited CXCL12 proteolysis by MMPs altered the ability of the chemokine to bind its receptor, namely CXCR4. Binding experiments showing that CXCL12(5–67) was altered in its ability to compete with CXCL12 in binding to CXCR4 together with the prevention of CXCL12(5–67) neurotoxic properties (further described later) by the pertussis toxin suggested a shift of receptor from CXCR4 to another G protein-coupled receptor (McQuibban et  al. 2001; Zhang et al. 2003). Of interest, unexpected properties of CXCL12(5–67) were prevented by CXCR3 antagonists; it will be discussed later. Further binding studies confirmed the affinity of CXCL12(5–67) for CXCR3 as the CXCR3 antagonist, CXCL11(5– 73), but not the full-length CXCL12, efficiently competed with CXCL12(5–67) in binding on monocytoid cells (Vergote et  al. 2006). Moreover, the aminoterminal sequence of CXCL12(5–67) seemed to be crucial for the binding as the synthetic peptide CXCL12(5–19) containing the RFFRESH motif, being the primary docking site of the chemokine to CXCR4 (Crump et  al. 1997), mimicked the CXCL12(5–67) in binding to CXCR3 (Fig. 7.1). Although CXCL12 is considered to be a constitutively expressed chemokine, CXCR3 ligands, CXCL9–11 and CXCL4, are inducible/inflammatory chemokines. Nevertheless, like CXCR3 ligands, CXCL12 does not carry the ELR (Glu-LeuArg) motif correlating with angiogenic properties besides the CXC motif. In this regard, CXCL12 constitutes an exception as, despite the absence of the ELR motif, it does display angiogenic properties (Deshane et  al. 2007). CXCR3’s ability to bind the MMP-processed CXCL12 is not completely surprising because relationships between CXCL12 and CXCR3 ligands have already been reported. Indeed, CXCR3 and CXCR4 together with their respective ligands have been involved in established immunological phenomena such as memory immune response or chemoattraction of specific NK cells or T cells subsets (Campbell et  al. 2001; Hauser et al. 2002; Poggi et al. 2004). As will be discussed later, CXCL10 shares common properties with CXCL12(5–67) (Sui et al. 2006; Vergote et al. 2006; van Marle et al. 2004). On one hand, CXCL12 was shown to possess a modest affinity for CXCR3 (Weng et al. 1998) but, on the other hand, Soejima and Rollins, after describing the ability of CXCL10 to bind to a receptor different from CXCR3 and glycosaminoglycans, were unable to completely rule out the possibility of a CXCR4 affinity of CXCL10 (Soejima and Rollins 2001). Furthermore, the CXCR3 ligand, CXCL11, and CXCL12 have recently been shown to share some high affinity for an alternate receptor CXCR7 (Balabanian et al. 2005; Burns et al. 2006). All these homologies between CXCR3 and CXCR4 systems are probably due to the structural similarity between their ligands, particularly in their aminoterminus responsible for the ligand-receptor specificity (Booth et al. 2002). Of importance, a receptor preference change after a proteolytic cleavage of a chemokine has been described for CCL5, in which its cleavage by CD26 alters its receptor preference from CCR1 to CCR5 thus increasing its antiviral activity (Proudfoot et al. 1999; Schols et al. 1998), but conversely MMP-2 cleavage of CXCL12 abolished its antiviral properties (McQuibban et al. 2001).

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7.4.2 Pathogenic Effects of MMP-Processed CXCL12 on Neurons Besides the intrinsic neurotoxicity of some HIV-1 proteins (Vpr, Nef, Tat, gp120) (Jones et al. 2007; van Marle et al. 2004), HIV-mediated innate immune activation in the central nervous system can generate pathogenic molecules, as reviewed previously (Gonzalez-Scarano and Martin-Garcia 2005; Jones and Power 2006; Kaul et al. 2001). Among these innate immune effects, chemokines play incompletely defined roles and the CXCL12 truncation by MMPs alters the chemokine biological effects and reveals numerous novel actions toward neuronal and/or glial cells. 7.4.2.1 Perturbation of Neuronal Membrane Physiology Current influxes regulated by ionic transport through the axonal membrane are the basis of neuronal physiology allowing interneuronal communication and thus conferring its function to the central nervous system. In a comparative study, CXCL12(5–67) was shown to depress whole cell current at low concentrations (10 nM) in primary rat neurons by evoking a reduction of a calcium-activated potassium conductance, whereas CXCL12 had no effect on such currents at a similar concentration (Vergote et  al. 2006). Interestingly, this feature was mimicked by CXCL10 treatment and was prevented by a CXCR3 antagonist, thus confirming receptor shift to CXCR3 (Vergote et al. 2006). Neuronal excitability results from the alteration of this conductance (Jassar et al. 1999) suggesting that neurons might be more susceptible to excitotoxicity in presence of CXCL12(5–67) at low concentrations. Moreover, even if not shown in neuronal cells, in monocytoid cells, the CXCL12 ability to mobilize internal calcium is lost upon MMPs processing. A feature once again shared with CXCR3 ligands (Vergote et al. 2006) and confirmed by the loss of the ability to mobilize calcium of other aminoterminal truncated CXCL12 (Delgado et  al. 2001; Proost et  al. 1998b; Valenzuela-Fernandez et  al. 2002). If the loss of calcium mobilization is confirmed in neurons, the loss of affinity for CXCR4 might be the initial cause of the depression of whole-cell currents preceding an impairment of basal neuronal functions. 7.4.2.2 Neurotoxicity of the MMP-Processed CXCL12 MMP-induced neurotoxicity is mediated by several mechanisms. The mechanisms described appear to be associated with or without accompanying the proteolytic activity. Indeed, while MMP-mediated degradation of myelin is clearly associated with the enzymatic activity of the implicated proteases (Liuzzi et  al. 1994), interactions of MMPs with integrins and the resulting neurotoxicity are independent of the proteolytic activity (Conant et al. 2004), suggesting that MMPs can be seen not only as proteases but also as molecules possessing pleiotropic activities. An unexpected property of CXCL12(5–67) was described by our group in which HIV-mediated

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neurotoxicity was prevented in vitro and in an in vivo model by prinomastat, a general MMP inhibitor. CXCL12(5–67) was identified as the major neurotoxin in this model (Zhang et al. 2003). Several lines of evidence suggested the apoptotic nature of the cell death-induced by CXCL12(5–67). Indeed, CXCL12(5–67)-exposed neurons showed initial cell condensation, became TUNEL-positive and showed an increase in both p53 and caspase-3 abundance together with a decrease in Bcl-2 abundance ((Vergote et al. 2006) and unpublished data). The death-inducing effect of CXCL12(5–67) was specific to neurons as both LAN-2 and NG108 neuronal cell lines and human fetal neurons (but not astrocytic, monocytoid, pre-B lymphoma, or primary CD34+ stem cells) displayed apoptosis at comparable concentrations of CXCL12(5–67). Interestingly, while the synthetic peptide, CXCL12(5–19), mimicking the CXCL12(5–67) aminoterminus, was able to bind CXCR3 and initiate the neuropathogenic effects of CXCL12(5–67) by reproducing CXCL12(5–67) effects on neurons regarding process retraction, it was not able to go on to cause neuronal death. This latter observation suggested that the complete activation of CXCR3 by CXCL12(5–67) required domains other than the CXCL12(5–19) necessary for the initial docking and the subsequent partial activation of CXCR3. The involvement of CXCR3 in the neurotoxicity was confirmed by preventing CXCL12(5–67) neurotoxicity with a CXCR3 blocking antibody, the CXCR3 antagonist CXCL11(5–73), and following CXCR3 knock-down (Vergote et al. 2006). CXCR3 activation by CXCL10 has previously been reported to induce neuronal cell death in the context of HIV. Indeed, we and other groups showed that the HIV accessory protein Nef-induced neurotoxicity and the neuronal cell death mediated by SIV were mediated by CXCL10 (Sui et al. 2006; van Marle et al. 2004). However, concurrent processes including the loss of CXCL12’s neuroprotective properties, mediated by CXCR4 through proteolysis, might also contribute to HIV neuropathogenesis (Khan et al. 2008).

7.4.3 Immunogenic Properties of MMP-Processed CXCL12 on Glial Cells It is widely recognized that chemokines released by activated glial cells or by neurons act as neuroimmunomodulatory factors together with cytokines, free radicals, and excitatory amino acids after injury (Biber et al. 2002; Gonzalez-Scarano and Martin-Garcia 2005). MMPs have been considered as markers of inflammation since the mid-1990s. Several studies implicated CXCL12 and MMPs in HIVinduced cerebral inflammation. As examples, HIV-infected macrophages-induced astrogliosis was shown to be dependent on both CXCL12 and MMPs (Okamoto et al. 2005) and gp120-induced IL-1b accumulation and neuronal apoptosis were also associated with an increase of MMP-2 and -9 expression and were prevented by MMP inhibitors (Russo et al. 2007). These data suggested that CXCL12(5–67) might regulate cerebral inflammation. While CXCL12 does not exert substantial inflammatory (including both pro- and anti-inflammatory) effects on monocytoid and astroglial cells in the nanomolar range, its aminoterminal truncation by MMPs

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exacerbates its inflammatory effects on these cells (Vergote et  al. 2006). CXCL12(5-67)’s immunogenic effects on glial cells were also prevented by CXCR3 antagonists thus linking the novel immunogenic properties of the chemokine with the change in receptor preference discussed above (Vergote et al. 2006). As mentioned earlier, aminoterminal processing of CXCL12 prevents intracellular calcium release in monocytoid cells. The difference of concentration range necessary to evoke immunogenic (1–10 nM) or neurotoxic (100–1,000 nM) effects probably reflects earlier/larger immunogenic consequences compared with later and more restricted neurotoxic consequences of CXCL12(5–67) production associated with HIV infection of the central nervous system.

7.4.4 MMP-Processed CXCL12 Effects in an In Vivo Model A feature shared by all lentiviruses, including HIV, is their ability to induce neuroinflammation and neuronal injury within the central nervous system. These effects are mediated by the virus properties of neuroinvasion, neurotropism, and neurovirulence (Patrick et  al. 2002). In the initial paper describing CXCL12(5–67) neurotoxicity, Zhang et al showed that the intrastriatal implantation of the truncated chemokine as a mouse model for neuroAIDS induced neurobehavioral deficits directly correlated with two cardinal features associated with chronic HIV infection (Zhang et al. 2003): (1) a widespread gliosis, represented by microglial and astrocytic cell hypertrophy, and (2) neuronal cell death to a similar extent compared to the implantation of conditioned media from macrophages infected with a pseudotyped virus encoding the envelope protein of a brain-derived virus. Interestingly, the neurotoxic features seem to be exacerbated for brain-derived viruses originated from patients showing clinical signs of dementia as blood-derived or non-demented brain-derived viral strains show only minimal neurotoxic properties suggesting a sequence specificity for neurotoxicity that remains to be fully elucidated (Johnston et al. 2000). The complete prevention of neurobehavioral deficits associated with the above conditioned media by coimplanting a blocking antibody targeting MMP-2 suggested that MMP-2 is the principal MMP responsible for the in vivo CXCL12 truncation while other MMPs can poorly compensate its absence. The in vivo neurotoxic properties of CXCL12(5–67) were later confirmed together with the additional description of the prevention of neuropathogenic effects of CXCL12(5–67) by the CXCR3 antagonist, CXCL11 (5–73), implying that CXCR3-mediated CXCL12(5–67) effects within the brain caused a neurological disease phenotype (Vergote et  al. 2006). Moreover, in  vitro immunogenic properties of the processed chemokine were conserved in this in vivo model as glial cell activation was accompanied by cytokine induction in the brain of CXCL12(5–67)-implanted mice. In an in vivo model of neuroAIDS, CXCL12(5–67) therefore recapitulates the acquired properties demonstrated in vitro (Fig. 7.2). Together with its detection in HIV-positive human brains, these data confirm the relevance of the mechanisms described above.

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Gliosis Neuroinflammation Neuronal loss Neurobehavioral deficit

Fig. 7.2  Additive neuropathogenic properties exerted by a proteolytically cleaved chemokine as a consequence of a change in receptor specificity, culminating in neurodegeneration. HIV-induced MMPs process the aminoterminal peptide of constitutively expressed CXCL12 generating in the brain the peptide CXCL12(5–67) with novel receptor specificities and ensuing novel bioactive properties. Indeed, this proteolytic product acting through CXCR3 instead of CXCR4 may perturb the membrane physiology, activate an apoptotic signaling pathway on neurons, and induce the expression of neuroinflammatory molecules in astrocytes and microglia. In vivo neuroinflammation, neuronal loss, and neurobehavioral abnormalities caused by CXCL12(5–67) were mediated by CXCR3

7.4.5 Evolutionary Advantages for the Virus The success of an infection by an infectious pathogen depends on the pathogen’s ability to circumvent the immune response developed by the host. Chemokines play a crucial role in the immune response manifested upon infection by attracting immune cells at the site of infection. Several examples of host–pathogen interactions at the chemokine level have been described so far. Entamoeba histolytica, a pathogenic intestinal amoeba responsible for dysentery, is able to produce more than 20 cysteine proteases. Among them, the EhCP2 (E. histolytica Cysteine Protease 2) has been shown to modulate chemotactic activity of CCL2, CCL13, and CXCL8 by aminoterminal cleavage (Pertuz Belloso et al. 2004). Another metalloprotease produced by the hookworm Necator americanus can process CCL11, thereby preventing its chemotactic properties (Culley et al. 2000). It is therefore legitimate to wonder if these parasites may influence HIV neuropathogenesis. Numerous studies reported the link between E. Histolytica and HIV but, in spite of the number of studies,

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conclusions are difficult to draw clearly as results depending on the different populations are contradictory (Chen et al. 2007; Moran et al. 2005). Cerebral infection by HIV was associated with the increase expression of CXCL12 in the brain (Langford et al. 2002). This increase of CXCL12 expression can probably be considered as a protective mechanism set up by the host to try circumventing the viral infection by producing a highly potent anti-HIV molecule. However, in a coevolutionary view of the host–pathogen interactions, the virus seems to have “learned” how to use this defense mechanism to its own advantage. Indeed, by processing CXCL12, the virus thus eliminates a competitor for the binding to cell surface receptor broadening the infection of neural cells within the brain together with increasing the neuropathogenicity of the virus. In this regard, the immunogenic properties acquired by CXCL12 upon MMPs truncation might increase the susceptibility of neural cells to viral infection as cerebral inflammation has been shown to facilitate HIV infection (Albright et al. 2000). Another advantage for the virus is the use of a host protease to generate a key element for its neurotoxicity without having to encode either the protease or the neurotoxin itself, thereby avoiding any extension of its extremely compact genome. No obvious advantage for the virus seems to be brought by the death of neurons. Thus, the neurotoxicity generated by HIV appears to be a non-adaptive consequence of the viral infection, detrimental for both viral spread out within human population and host survival. We speculate here that with the short common history between the virus and the host (likely less than 100 years), the virus and host still need to coevolve toward a more adaptive strategy for complementing their life cycles. Indeed, just as SIV infection in nonhuman primates has a much longer common history and has become benign for susceptible old world nonhumate primates, the interaction between HIV and humans might also achieve this state in the future.

7.5 Concluding Remarks Chemokines proteolytic maturation is a burgeoning field but the relevance of most in vitro observations needs to be demonstrated in more physiological and relevant models. The chief example we have developed herein is MMP-2 processing of CXCL12, indicating the importance of the understanding of the relevance together with the consequences of substrate processing. Indeed, chemokine processing, particularly by proteases with diverse substrate specificity, such as MMPs, may generate unsuspected ligands that participate in major physiological processes, as illustrated in neurodegeneration and neuroinflammation occurring upon HIV infection. Moreover, the roles of CXCR3 in HIV-induced neuronal death underline the involvement of several chemokine receptors in indirect neuropathogenic events together with their established roles as viral coreceptors. Studies of chemokine processing linked to HIV infection have provided several potential therapeutic targets. Treatment of HIV/AIDS patients with HAART includes

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protease inhibitors targeting the HIV protease. Including a MMP inhibitor might be beneficial in preventing the neurological disorders associated with HIV infection. Moreover, CXCR3, while being known as a weak HIV coreceptors, was not considered as an interesting enough target for anti-HIV drugs. However, we have emphasized throughout this chapter the major role of this chemokine receptor in HIV neuropathogenesis, thus reevaluating CXCR3 antagonism as an opportunity to treat HIV infection, and it may be a fruitful avenue for future research. Acknowledgments  The authors thank Leah DeBlock for assistance with manuscript preparation. D.V. was supported by a Toupin Chair Fellowship. C.P. and C.M.O. hold Canada Research Chairs (T1) in Neurological Infection and Immunity, and Metalloproteinase Proteomics and Systems Biology, respectively. This research was supported by the Canadian Institutes for Health Research (CIHR), the Canadian Foundation for AIDS Research (CANFAR), National Institutes of Health (NIMH), and an Infrastructure Grant from the Michael Smith Research Foundation (University of British Columbia Centre for Blood Research). The authors have no conflicting financial interests.

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Chapter 8

Chemokines and Chemokine Receptors in the Brain Stéphane Mélik Parsadaniantz, Ghazal Banisadr, Philippe Sarret, and William Rostène

8.1 Introduction As described in previous chapters, chemokines are involved in virtually all pathologies exhibiting an inflammatory component. This includes nervous system pathologies, such as neurodegenerative or neuroinflammatory diseases, where the expression of a number of chemokines and chemokine receptors in activated astrocytes and microglia increases. This suggests their involvement in the activation of the central nervous system (CNS) defense mechanisms (Bleul et al. 1996; Oberlin et al. 1996; Thibeault et al. 2001). However, a new paradigm has recently emerged from the current literature supporting the view that chemokines and their receptors are also expressed by neuronal cells within the CNS and that they are further implicated as modulators of CNS functions. This concept opens a new field of investigation in Neuroscience (Rostene et  al. 2007). The discovery of cells in CNS structures expressing and responding to chemokines raises the following questions: (1) Why do these neural cells express chemokines and chemokine receptors? (2) How do chemokines affect the activity of neurons under physiological and physiopathological circumstances?

8.2 Expression of Chemokines and Their Receptors in the CNS 8.2.1 Chemokine Receptor Expression in the CNS A large number of data, based on both in vitro and in vivo studies, have reported the expression of chemokine receptors in the CNS. Among chemokine receptors

S.M. Parsadaniantz, G. Banisadr, P. Sarret, and W. Rostène (*) UMRS 968 Inserm-Universite P et M Curie Paris 6, Institut de la Vision, 17 Rue Moreau 75012, Paris, France e-mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_8, © Springer Science+Business Media, LLC 2010

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which belong to the G-protein coupled receptor family, a constitutive expression of several chemokine receptors has been reported, notably for CCR1–5, CXCR2–4 and CX3CR1. Interestingly, under pathological conditions, the majority of CCR members (CCR1–CCR6) and CX3CR1 are expressed by neurons, astrocytes and microglial cells. 8.2.1.1 CCR Family Neuronal expression of CCR1 was established in cultures of a human neuroblastoma cell line, NT2.N (hNT) neurons (Hesselgesser et al. 1997) as well as in hippocampal neurons (Meucci et al. 1998). Neuronal expression of CCR2 was initially described in primary cultures of human neurons and in hNT neurons (Coughlan et  al. 2000). Recently, our group established that CCR2 is constitutively expressed by neurons in rat brain through autoradiographic and histochemical studies (Banisadr et al. 2002a). Neuronal expression of CCR2 was mainly identified in the cerebral cortex, caudate putamen, globus pallidus, substantia innominata, supraoptic and paraventricular hypothalamic nuclei, ventromedial thalamic nucleus and substantia nigra. Furthermore, this work demonstrated the coexistence of CCR2 in neuronal cell bodies expressing markers for classical neurotransmitters such as choline acetyltransferase-immunoreactivity (cholinergic neurons) in the caudate putamen and substantia innominata, as well as in tyrosine hydroxylase-positive neurons (dopaminergic neurons) in the substantia nigra pars compacta (Banisadr et al. 2005, 2002b). Neuronal CCR3 and CCR5 were detected in both normal and HIV/SIV-infected brains (Sanders et al. 1998; Westmoreland et al. 1998; Vallat et al. 1998; Klein et al. 1999). CCR4 was detected in dorsal root ganglia (DRG) neurons (Oh et al. 2001), in hippocampal pyramidal neurons (Meucci et al. 1998) and more recently in cerebellar Purkinje neuron cultures (Gillard et al. 2002). Expression of CCR5 was reported in pyramidal neurons of the cortex and the hippocampus of rats (Meucci et al. 1998) as well as macaque brains (Westmoreland et al. 2002). Moreover, our group carried out an autoradiographic and immunohistological study showing that neurons constitutively express CCR5 in discrete neuroanatomical regions (i.e. the motor cortex, caude putamen, hypothalamus, hippocampus, substantia nigra, periaqueductal gray, cerebellum) (Figs. 8.1 and 8.2). CCR9 and CCR10 mRNA were also amplified in rat hippocampal pyramidal neurons (Meucci et al. 1998). 8.2.1.2 CXCR Family It was shown by immunohistochemistry on post-mortem sections, that CXCR2 was expressed in neurons of various regions of the human adult nervous system including the hippocampus, cerebellum, pontine nuclei and spinal cord. The labeling Fig.  8.1 (continued) nucleus, (k, l) Hi hippocampus, (m, n) So supraoptic nucleus, (o, p) Arc ­hypothalamic arcuate nucleus, (q, r) PH posterior hypothalamic area, (s, t) SN Substantia nigra. All sections were observed with an Olympus fluorescent microscope (BX 61). Scale bars = 50 mm. (Unpublished data)

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Fig. 8.1  Rostrocaudal neuroanatomical distribution of CCR5-immunoreactivity in the telencephalon, diencephalon and mesencephalon using a CCR5 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Regions corresponding to pictures are depicted in coronal diagrams taken from the Paxinos and Watson (1998). (a, b) M Motor cortex, (c, d) CPu caudate putamen (striatum), (e, f) SID substantia innominata dorsal part, (g, h) GP globus pallidus, (i, j) Me medial amygdaloid

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Fig. 8.2  Autoradiographic distribution of CCR5 binding site in coronal sections of the adult rat brain using [125I]MIP-1b/CCL4. Sections were incubated with 33 pM [125I]MIP-1b/CCL4 in the absence or in the presence of 20 nM unlabeled MIP-1b/CCL4 (nonspecific binding) at 4°C for 18 h. Photographs of autoradiogram films were obtained using a computer-based image analysis system (Biocom, Les Ulis, France). Acb nucleus accumbens, CA1 field CA1 of hippocampus, Cerebellum, Cg cingulate cortex, CPu caudate putamen, DM dorsomedial hypothalamic nucleus, DR dorsal raphe nucleus, GP globus pallidus, M motor cortex, MM medial mammillary nucleus, PAG periaqueductal gray, Pf precommissural fornix, PH posterior hypothalamic area, Pn pontine nucleus, Po posterior thalamic nuclear group, Py pyramidal tract, S subiculum, SN substantia nigra, V visual cortex according to (Paxinos and Watson 1998) nomenclature. The section in the bottom right corresponds to the nonspecific binding obtained with an excess 20 nM of unlabeled MIP-1b/CCL4. (Unpublished data)

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was mainly localized on cell bodies, dendrites and axons (Horuk et al. 1997; Xia et  al. 1997). The constitutive neuronal expression of CXCR3 was detected by immunohistochemistry in the cerebral cortex, hippocampus, striatum, cerebellum and spinal cord of the human brain (Xia et al. 2000). In addition, in vitro studies showed that in hNT cells as well as in primary cultures of neurons, functional CXC chemokine receptors including CXCR2, CXCR3 and CXCR4 are expressed (Hesselgesser et al. 1997; Coughlan et al. 2000; Meucci et al. 1998; Lavi et al. 1997; Xia et al. 2000; Bajetto et al. 1999; Ohtani et al. 1998; Lazarini et  al. 2000; Zheng et  al. 1999). Indeed, CXCR4 is obviously the most studied CXC receptor in the nervous system. In addition to its in vitro expression mentioned above, Lavi et  al. (1997) and Sanders et  al. (1998) demonstrated the expression of CXCR4 protein in neurons of the dentate gyrus and pyramidal neurons of the hippocampus, in post-mortem tissue of HIV-negative patients and an over-expression in HIV-patients. Using the same methodology, it was shown that CXCR4 was expressed by neurons in the hippocampus, dentate gyrus and cerebral cortex of the macaque brain (Westmoreland et al. 2002, 1998). Indeed, they reported a main localization for CXCR4 at the somatodendritic and axonal level. It was also demonstrated that CXCR4 was not exclusively located directly at the synapse but in its vicinity, in agreement with what was reported for G-protein coupled receptors of several neuropeptides (Pujol et al. 2005). Further, a rodent orientated study focusing at in situ hybridization and immunohistochemistry showed the constitutive neuronal expression of CXCR4 in brain (Stumm et  al. 2002). The same year, our group established by an immunohistochemical method that CXCR4 is constitutively expressed by neurons in rat brains (Banisadr et  al. 2002a). Neuronal expression of CXCR4 was mainly found in the cerebral cortex, caudate putamen, globus pallidus, substantia innominata, supraoptic and paraventricular hypothalamic nuclei, ventromedial thalamic nucleus and substantia nigra. Furthermore, this work demonstrates the coexistence of CXCR4 in neuronal cell bodies expressing choline acetyltransferase-immunoreactivity in the caudate putamen and substantia innominata, as well as in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. CXCR5 mRNA was, for itself, amplified in the granular and Purkinje cell layers of the mouse cerebellum (Kaiser et al. 1993).

8.2.1.3 CX3CR1 The expression of the fractalkine receptor (CX3CR1) was investigated in the rodent CNS. Some immunohistological studies revealed that some neuronal cells express CX3CR1 in rat hippocampal neurons (Meucci et al. 2000), mouse cortical neurons (Mizuno et  al. 2003), human neurons (Hatori et  al. 2002), as well as in patients experiencing AIDS-associated encephalitis (Tong et al. 2000). However, CX3CR1 is mainly present on resident microglia throughout the brain, in the parenchyma, choroid plexus and meninges.

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8.2.2 Chemokine Expression in the CNS 8.2.2.1 CC Chemokines The preferential ligand of CCR2, CCL2, is expressed by NT2.N neurons (Coughlan et al. 2000) and during human CNS development (Meng et al. 1999). In addition, the induction of neuronal CCL2 expression is also described in a model of facial nerve lesion (Flugel et al. 2001) and upon brain ischemia (Che et al. 2001). More recently, we established by immunohistochemistry that CCL2 is constitutively expressed in rat neurons, in discrete neuroanatomical regions (Banisadr et al. 2003). Neuronal expression of CCL2 was mainly pinpointed in several brain areas such as the cerebral cortex, globus pallidus, hippocampus, hypothalamic nuclei, substantia nigra and facial and trigeminal nuclei and in Purkinje cells of cerbellum. Moreover, we provided evidence that CCL2, like CXCL12 colocalizes with classical neurotransmitters such as acetylcholine in the substantia innominata and in dopaminergic neurons in the substantia nigra pars compacta and the ventral tegmental area. On the other hand, CCL3 is expressed by neurons of the dentate gyrus, hippocampus and cerebral cortex, either in normal brain or in the CNS of patients coping with Alzheimer’s disease (Xia et  al. 1998). CCL5 neuronal expression was observed ex vivo after viral infection (Patterson et  al. 2003). In a middle cerebral artery occlusion (MCAO) mouse model of brain ischemia, it was observed that cortical neurons rapidly expressed the chemokine CCL21 in the penumbra of the ischemic core. Because healthy brain tissue did not express CCL21, the expression is suggested to be specifically linked to endangered neurons (Biber et al. 2001).

8.2.2.2 CXC Chemokines Among CXC chemokines, neuronal expression of CXCL1 has been detected in the rat spinal cord where its expression is developmentally regulated (Robinson et al. 1998). CXCL10 over-expression is described in neurons after MCAO-induced brain ischemia in rat (Wang et al. 1998) and viral infection by human immunodeficiency virus encephalitis (HIVE) (Sui et  al. 2004). Another CXC chemokine present in neurons is CXCL12. This chemokine was detected in vitro in rat neurons and neuronal progenitors, where it induced a dose-dependent chemotactic response. This chemotaxism is inhibited by Pertussis toxin, which uncouples Gi proteins, by the bicyclam AMD3100, a highly selective CXCR4 antagonist, as well as by an inhibitor of the MAP kinase pathway (Lazarini et al. 2000). Analysis of the brain patterns of CXCL12 expression of HIVE-positive patients revealed intense somato-dendritic neuronal CXCL12 immunoreactivity (Langford et  al. 2002). Moreover, Stumm et  al. (2002) demonstrated the presence of CXCL12 transcripts in the rat cortex and hippocampal neurons. Interestingly, neurons syn-

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thesize a-isoform of CXCL12 mRNA transcripts while endothelial cells of cerebral microvessels selectively express CXCL12 b-isoforms. Under LPS stimulation, a dramatic decrease in endothelial CXCL12 mRNA expression throughout the forebrain occurs but with no impact on neuronal CXCL12. More recently, we established by immunohistochemistry that CXCL12 is constitutively expressed in rat neurons in discrete neuroanatomical regions (Banisadr et  al. 2003). Indeed, neuronal expression of CXCL12 was mainly identified in the cerebral cortex, substantia innominata, globus pallidus, hippocampus, paraventricular and supraoptic hypothalamic nuclei, lateral hypothalamus, substantia nigra and oculomotor nuclei. Moreover, we provided evidence that CXCL12 is constitutively expressed in cholinergic neurons in the medial septum and substantia innominata and in dopaminergic neurons in substantia nigra pars compacta and the ventral tegmental area.

8.2.2.3 CX3CL1 Several in  vivo and in  vitro studies demonstrate that neurons are the principal source of CX3CL1 production in human, macaque and rat brain (Harrison et  al. 1998; Schwaeble et al. 1998; Nishiyori et al. 1998; Chapman et al. 2000; Boddeke et al. 1999). Its expression is mainly observed in the cerebral cortex, hippocampus, caudate putamen, thalamus and olfactory bulb. It was noted that neuroinflammatory processes or in vitro neuron damaging left CX3CL1 mRNA expression at a stable level (Chapman et al. 2000; Hatori et al. 2002; Mizuno et al. 2003). Because microglia were shown to express the CX3CR1 receptor, a role for CX3CL1/CX3CR1 signaling through a neuron-microglia interaction was strongly suggested (Harrison et  al. 1998; Schwaeble et  al. 1998; Nishiyori et  al. 1998; Chapman et  al. 2000; Boddeke et al. 1999).

8.3 Role of Chemokines in Regulating CNS Activity Since chemokines are often associated with the pathogenesis of neuroinflammatory diseases, the neuronal expression of chemokines and their cognate receptors strongly suggests their involvement in brain function regulation. Indeed, chemokines fulfill all requirements which have been associated with a neurotransmitter/neuromodulatory action (Rostene et al. 2007). As reported above, chemokines can be synthesized not only in glial cells but also in neurons where they are coexpressed with classical neurotransmitters. They are located in nerve endings and can be released by these nerve terminals under membrane depolarization. Chemokine receptors are found both on pre- and postsynaptic neurons where they influence electrophysiological potentials, intracellular signaling and physiological effects (Fig. 8.3).

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Fig.  8.3  Chemokines are neuromodulators. Some chemokines can be synthesized, as their own receptor, by the same neuron (a). A chemokine released by exocytosis can modulate the electrical activity of neurons after binding to its own presynaptic receptor (autoreceptor). Chemokines can also act on other neurons and glial cells (astrocytes and microglia) which express chemokine receptors (b). (Adapted from Rostene and Melik-Parsadaniantz, Pour la Science 2008, 369 ; 66–72)

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8.4 Neuromodulatory Action of Chemokines Chemokines play an important role in the regulation of synaptic activity in the brain. Such properties of chemokines have been described in several neuronal systems (For review: Ragozzino 2002). In this chapter we will exclusively focus on two examples to illustrate the neuromodulatory effects of chemokines.

8.4.1 Effect on Neurotransmission By means of unilateral intranigral injection, we recently demonstrated that CXCL12 decreased dopamine (DA) content and increased extracellular DA and its metabolite concentrations in the ipsilateral striatum. A similar CXCR4-mediated effect was reported on DA release and identified by a contralateral rotatory behavior (Skrzydelski et al. 2007). Our data demonstrate that CXCL12 may act as a stimulatory modulator of DA neurotransmission mediating locomotor behavior. On the other hand, CXCL12 induces changes in the action potential frequencies of DA neurons (Guyon et al. 2006, 2005) as well as an increase of the evoked postsynaptic potentials recorded extracellularly in the CA1 field of hippocampal slices (Zheng et al. 1999). In cerebellar slices, CXCL12 induces a Tetrodotoxin-sensitive increase in the frequency of g-aminobutyric acid (GABA)-ergic and glutamatergic synaptic currents in Purkinje neurons (Limatola et al. 2000; Ragozzino et al. 2002). In addition, CXCL12 elicits a slow inward current and an increase in intracellular Ca2+ in Purkinje neurons (Limatola et al. 2000). These effects are considerably reduced by ionotropic glutamate receptor antagonists, but fully active in a medium in which synaptic transmission is inhibited. This fact indicates the currents might be mediated by extrasynaptic glutamate, possibly released from the surrounding glial and/ or nerve cells. This is further supported by a study in which a CXCR4-dependent glutamate release from glial cells was reported and a functional role for CXCR4 in the cross-talk between glial cells and neurons was suggested (Bezzi et al. 2001). These authors demonstrated the CXCL12/CXCR4 binding to stimulate the elevation of intracellular Ca2+ and the extracellular release of TNFa in astrocytes. Interaction of TNFa with its receptors initiated a second sequence of intracellular signaling leading to generation of PGE2, which controls glutamate release and astrocyte communication (Bezzi et al. 2001). A study from Giovannelli et  al. (1998) also provides evidence that Purkinje neurons of mouse cerebellar sections respond to human CXCL8 and CXCL1 by (1) a cytosolic Ca2+ increase compatible with IP3 formation; (2) an enhancement of neurotransmitter release; and (3) an impairment of long-term depression of synaptic strength (LTD). The authors suggest that CXCL8 and CXCL1 could play a neuromodulatory role in mouse cerebellum activity, more precisely under an infectious aggression. Likewise, stimulatory effects on neuronal transmission are observed with CXCL2 in cultured cerebellar granule cells, where this CXC chemokine

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enhances both evoked and spontaneous postsynaptic currents in patch clamped Purkinje neurons from rat cerebellar slices (Ragozzino et al. 1998). It was also reported that some chemokines including CCL22, CCL5 and CX3CL1 could regulate neuronal signaling through the inhibition of Ca2+ channels (Oh et al. 2002). In hippocampal neurons, chemokines like CCL22 and CX3CL1 blocked the frequency of spontaneous glutamatergic excitatory postsynaptic currents recorded from these neurons and reduced voltage-dependent Ca2+ currents in the same neurons (Meucci et al. 1998). In addition, CX3CL1 can negatively modulate EPSCs at Schaffer collateral-CA1 active synapses acting at postsynaptic sites through mechanisms involving CX3CR1 by reducing postsynaptic AMPA-evoked currents (Ragozzino et al. 2006). Another report describes that the stimulation of CXCR1 and CXCR2 by CXCL8 in freshly isolated cholinergic septal neurons induces the fast and repetitive inhibition of the voltage-dependent Ca2+ currents (Puma et al. 2001). All these data illustrate the action of chemokines on the electrical activity of brain neurons.

8.4.2 Involvement of Chemokines in Nociception Chemokines and their receptors were described as modulators of the nociceptive pathways in rodents either peripherally or centrally. Indeed, subcutaneous administration of various chemokines induces a local hyperalgesia (Cunha et  al. 1991; Cunha et al. 2000; Oh et al. 2001; Zhang et al. 2005). Chemokine receptors were detected in sensory neurons both in situ (Zhang et al. 2005) and in cultured cells (Oh et  al. 2001), where they co-localize with nociceptor markers such as the peptides substance P and CGRP (Qin et al. 2005; Jung et al. 2008). In the spinal cord, chemokines and their receptors were detected in neurons as well as in glial cells. Intrathecal administration of chemokines produces hyperalgesia, suggesting constitutive expression of functional receptors in the spinal cord (Milligan et  al. 2004). Interestingly, spinal chemokines appear to serve as mediators of neuroneuronal, neuro-glial and/or glial-glial communication depending on the different ligand-receptor interactions. Furthermore, chemokines and their receptors are strongly regulated in the nociceptive pathway during the course of pathological pain in animal models. Thus, chemokines are now regarded as important mediators in the field of nociception and are therefore promising pharmacological targets for the treatment of pain (White et al. 2007). Of particular importance throughout this book, HIV-1 appears to interact indirectly with neurons via binding of the external envelope protein, gp120, to the chemokine receptors CXCR4. It was shown that gp120 can directly influence the activity (Oh et al. 2001) and survival (Keswani et al. 2003) of sensory neurons and interact with axons in  vitro leading to toxicity (Melli et  al. 2006). Furthermore, peripheral administration of HIV-1 gp120 is associated with the development of pain-related behavior in rats (Herzberg and Sagen 2001) suggesting that HIV-1 gp120 interaction with peripheral nerves is responsible for the generation of

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neuropathic pain in humans. Antiretroviral approaches also result in increased nociception. Indeed, neuropathic pain was reported to be associated with administration of Nucleoside Reverse Transcriptase Inhibitors (NRTIs) used in the treatment of HIV-1 infection (Joseph et al. 2004). Moreover, it was reported that NRTIs up-regulate signaling via CXCR4 in the dorsal root ganglia (DRG) and that CXCL12/CXCR4 signaling is fundamental to anti-retroviral drug 2¢,3¢-dideoxycytidine (ddC)-induced tactile allodynia (Bhangoo et  al. 2007). This suggests that CXCL12 and CXCR4 mediate HIV associated painful neuropathy both as a consequence of viral infection and as an antiviral treatment side effect. It was shown that subsets of cultured rat small and medium DRG neurons expressed CXCR4 (Oh et al. 2001) and that gp120 as well as CXCL12 were able to excite sensory cells and trigger the release of substance P. A more detailed analysis of the signaling cascades induced by perineural gp120 injection was recently published (Wallace et  al. 2007). Administration of gp120 followed by mechanical but not thermal hypersensitivity, results in profound phenotypic modifications including changes in DRG neuropeptide expression pattern and spinal glia activation. In addition to these peripheral and spinal effects of gp120, CXCR4 exerts pronociceptive actions by cross-desensitizing µ-opiate receptors in the periaqueducal gray matter (Szabo et al. 2002). Another heteromerization mechanism between CXCR4 and d-opioid receptor was recently reported, resulting in a suppression of both receptors signaling without interfering with ligand binding or receptor desensitization (Pello et al. 2008). Indeed, this study hypothesizes that CXCR4/Delta opioid receptor heterodimers are silent complexes since CXCR4 does not activate JAK 2 and Delta opioid receptor is unable to trigger Gai respectively. Further insight into the mode of action of CXCR4 in nociception was provided recently by the demonstration that CXCL12 injection into the periaqueducal grey matter prevents the antinociceptive effect of cannabinoid agonists (Benamar et  al. 2008). Together, these results indicate that the CXCR4 system might modulate all the nociceptive signals from the peripheral nervous system to the brain, acting on various neuronal systems and making it a putative pharmacological target in the treatment of pain but more particularly in HIV associated neuropathy. Another chemokine, CCL2, is closely linked to nociception (Sun et al. 2006). Indeed, it was demonstrated that the chemokine CCL2 and its cognate receptor CCR2 are expressed in DRG neurons and the spinal cord under physiological and pathological conditions (Bhangoo et  al. 2007; Dansereau et  al. 2008; Gosselin et al. 2005), suggesting a possible autocrine/paracrine modulatory action. In addition to its direct link in nociceptor physiology, a significant line of evidence shows that CCL2 is implicated in central pain signaling (White et  al. 2007). Indeed, intracisternal or intrathecal injection of this chemokine activates nociceptive behaviors in the rat (Ahn et al. 2005; Tanaka et al. 2004; Dansereau et al. 2008). Notably, a rapidly increasing number of publications report an up-regulation of CCL2 signaling in pathological pain paradigms. First, in rodent neuropathic pain models, CCL2 and CCR2 expression are increased in sensory neurons (Tanaka et al. 2004). In addition, CCL2 shows sensitizing actions in freshly isolated sensory neurons from chronically compressed DRG (Sun et  al. 2006). Moreover, in a

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model of cancer pain in the rat, CCR2 is up-regulated in the cervical dorsal spinal cord in response to inflammatory tumor progression in forepaws (Vit et al. 2006). Finally, the idea of CCL2/CCR2 contribution in pathological pain is strongly supported by the initial observation that CCR2 deficient mice are resistant to the induction of allodynia following sciatic nerve ligation (Abbadie et al. 2003). The modes of action of CCL2 on sensory transmission are multiple and yet many remain to be discovered. Recent data have emphasized the importance of glial activation in various types of pathological pain (for review, see Tsuda et al. 2005). Like other chemokines, there is a growing body of evidence suggesting that CCL2 plays a role for microglial and astrocyte activation (for review, see Abbadie 2005). Indeed, it was recently demonstrated that spatial and temporal relationships exist between CCL2 expression and spinal glia activation following peripheral nerve injury (Zhang and De Koninck 2006). Mice overexpressing CCL2 in astrocytes also exhibited enhanced nociceptive behavior in response to both thermal and chemical stimuli (Menetski et al. 2007). The reduced number of activated astrocytes and microglia in CCR2-null mice further supports this hypothesis (Abbadie et al. 2003; Zhang and An 2007). A fundamental finding was that upon stimulation, neurons from sensory pathways induce the cleavage of the membranous CX3CL1 and release the soluble chemokine (Milligan et al. 2004). Interestingly, expression of the CX3CR1 receptor is present on spinal microglia suggesting a role in neuroglial crosstalk in nociception (Verge et  al. 2004). In this context, intrathecal administration of CX3CL1 dose-dependently causes mechanical allodynia and thermal hyperalgesia in the rat, confirming constitutive CX3CR1 expression but also its involvement in nociceptive signaling (Milligan et  al. 2004). It was shown that expression of CX3CL1 and CX3CR1 are sustained in persistent neuropathic or inflammatory animal pain models (Verge et al. 2004; Lindia et al. 2005). The mechanism by which CX3CL1 and CX3CR1 modify nociception threshold is not completely elucidated. Because CX3CL1 is conveyed at the extracellular surface of spinal neurons and spinal sensory afferents and is released upon strong activation, CX3CR1 receptors are primarily expressed by microglia in the dorsal spinal, it has previously been suggested to be a neuron-to-glial signal that induces astroglia and microglia activation (Milligan et al. 2004, 2005). Another mode of action of CX3CL1 is the inhibition of opioid anti-nociceptive effect in the periaqueductal grey (PAG). Indeed, a recent study showed that intra-PAG injection of CX3CL1 before administration of m, d and k opioid agonists significantly attenuates the anti-nociception induced by each of these peptides (Chen et al. 2007). In contrast, at the peripheral level, it was recently observed that CX3CL1 and CX3CR1 exert anti-nociceptive actions in the course of neuropathic pain in mice. Indeed, intraneural injection of CX3CL1 into the sciatic nerve significantly delays the development of allodynia in the spared nerve injury model and CX3CR1 knockout mice show a pronounced allodynia in this same pain model (Holmes et  al. 2008). Taken together, these results suggest an anti-allodynic role for CX3CL1 and its receptor at the periphery while having the opposite effect in the CNS of mice.

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8.5 Conclusions and Perspectives As we broaden our knowledge about chemokines, their pivotal roles in many biological functions are becoming clear, especially in neurobiology. Because the chemokinergic system has been well preserved during evolution, it suggests that it is essential for the coordination, regulation and fine-tuning of several types of biological responses including neuromodulation. The recent chemokinergic insights open a new avenue in neurophysiology while increasing the mechanistic complexity of neurotransmission and neuro-glia interactions. Thus, these advances are a first step towards the development and use of chemokine agonists or antagonists as pharmacological treatments in CNS pathologies. However, a better structure-­activity study for several of these chemokines requires to be ­carried out. Acknowledgments  The authors were supported by the Institut national de la recherche médicale (INSERM), Université Pierre et Marie Curie (UPMC), Agence nationale pour la recherche (ANR), Centre national de la recherche scientifique (CNRS) and the Canadian Institutes of Health Research (CIHR).

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Szabo I, Chen XH, Xin L, Adler MW, Howard OM, Oppenheim JJ, Rogers TJ (2002) Heterologous desensitization of opioid receptors by chemokines inhibits chemotaxis and enhances the perception of pain. Proc Natl Acad Sci U S A 99:10276–10281 Tanaka T, Minami M, Nakagawa T, Satoh M (2004) Enhanced production of monocyte chemoattractant protein-1 in the dorsal root ganglia in a rat model of neuropathic pain: possible involvement in the development of neuropathic pain. Neurosci Res 48:463–469 Thibeault I, Laflamme N, Rivest S (2001) Regulation of the gene encoding the monocyte chemoattractant protein 1 (MCP-1) in the mouse and rat brain in response to circulating LPS and proinflammatory cytokines. J Comp Neurol 434:461–477 Tong N, Perry SW, Zhang Q et al (2000) Neuronal fractalkine expression in HIV-1 encephalitis: roles for macrophage recruitment and neuroprotection in the central nervous system. J Immunol 164:1333–1339 Tsuda M, Inoue K, Salter MW (2005) Neuropathic pain and spinal microglia: a big problem from molecules in “small” glia. Trends Neurosci 28:101–107 Vallat AV, De Girolami U, He J et  al (1998) Localization of HIV-1 co-receptors CCR5 and CXCR4 in the brain of children with AIDS. Am J Pathol 152:167–178 Verge GM, Milligan ED, Maier SF, Watkins LR, Naeve GS, Foster AC (2004) Fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) distribution in spinal cord and dorsal root ganglia under basal and neuropathic pain conditions. Eur J NeuroSci 20:1150–1160 Vit JP, Ohara PT, Tien DA, Fike JR, Eikmeier L, Beitz A, Wilcox GL, Jasmin L (2006) The analgesic effect of low dose focal irradiation in a mouse model of bone cancer is associated with spinal changes in neuro-mediators of nociception. Pain 120:188–201 Wallace VC, Blackbeard J, Pheby T, Segerdahl AR, Davies M, Hasnie F, Hall S, McMahon SB, Rice AS (2007) Pharmacological, behavioural and mechanistic analysis of HIV-1 gp120 induced painful neuropathy. Pain 133(1–3):47–63 Wang X, Ellison JA, Siren AL, Lysko PG, Yue TL, Barone FC, Shatzman A, Feuerstein GZ (1998) Prolonged expression of interferon-inducible protein-10 in ischemic cortex after permanent occlusion of the middle cerebral artery in rat. J Neurochem 71:1194–1204 Westmoreland SV, Alvarez X, deBakker C, Aye P, Wilson ML, Williams KC, Lackner AA (2002) Developmental expression patterns of CCR5 and CXCR4 in the rhesus macaque brain. J Neuroimmunol 122:146–158 Westmoreland SV, Rottman JB, Williams KC, Lackner AA, Sasseville VG (1998) Chemokine receptor expression on resident and inflammatory cells in the brain of macaques with simian immunodeficiency virus encephalitis. Am J Pathol 152:659–665 White FA, Jung H, Miller RJ (2007) Chemokines and the pathophysiology of neuropathic pain. Proc Natl Acad Sci U S A 104:20151–20158 Xia M, Qin S, McNamara M, Mackay C, Hyman BT (1997) Interleukin-8 receptor B immunoreactivity in brain and neuritic plaques of Alzheimer’s disease. Am J Pathol 150:1267–1274 Xia MQ, Bacskai BJ, Knowles RB, Qin SX, Hyman BT (2000) Expression of the chemokine receptor CXCR3 on neurons and the elevated expression of its ligand IP-10 in reactive astrocytes: in vitro ERK1/2 activation and role in Alzheimer’s disease. J Neuroimmunol 108:227–235 Xia MQ, Qin SX, Wu LJ, Mackay CR, Hyman BT (1998) Immunohistochemical study of the beta-chemokine receptors CCR3 and CCR5 and their ligands in normal and Alzheimer’s disease brains. Am J Pathol 153:31–37 Zhang J, De Koninck Y (2006) Spatial and temporal relationship between monocyte chemoattractant protein-1 expression and spinal glial activation following peripheral nerve injury. J Neurochem 97:772–783 Zhang JM, An J (2007) Cytokines, inflammation, and pain. Int Anesthesiol Clin 45:27–37 Zhang RX, Liu B, Wang L, Ren K, Qiao JT, Berman BM, Lao L (2005) Spinal glial activation in a new rat model of bone cancer pain produced by prostate cancer cell inoculation of the tibia. Pain 118:125–136 Zheng J, Thylin MR, Ghorpade A et al (1999) Intracellular CXCR4 signaling, neuronal apoptosis and neuropathogenic mechanisms of HIV-1-associated dementia. J Neuroimmunol 98:185–200

Chapter 9

Chemokine Signaling in the Nervous System and Its Role in Development and Neuropathology Richard J. Miller

9.1 Introduction In addition to its effects on the immune system, HIV-1 infection is associated with numerous deleterious effects on the nervous system. Infection of the central nervous system (CNS) can cause inflammatory disease in the brain (HIV-1 encephalitis, HIVE) characterized by reactive microglia and astrocytes, white matter abnormalities, microglial nodules, perivascular inflammation, multinucleated giant cells, and neuronal cell loss – in areas including the cortex, basal ganglia, and limbic system. These effects on the CNS produce cognitive and motor symptoms in adults and are associated with neurodevelopmental problems in children. HIV-1 can produce dementia (HAD) or milder forms of cognitive impairment known as minor cognitive motor disorder (MCMD). This occurs in about 10–20% of patients with acquired immunodeficiency syndrome (AIDS) (Gonzalez-Scarano and Martin-Garcia 2005; Kaul et  al. 2001; Sacktor et  al. 2002; Williams and Hickey 2002). Highly active antiretroviral therapy (HAART) reduces HIV-1 mRNA levels in plasma and cerebrospinal fluid (CSF), and improves neurocognitive function and CNS abnormalities as detected using neuroimaging studies (Sacktor et al. 2002). However, as most current antiviral drugs used in HAART have relatively poor CNS penetration, the CNS remains an important reservoir for the virus. Additionally, neurocognitive impairment continues to be a significant and increasingly frequent complication of HIV-1 infection (McArthur et al. 2003; Sacktor et al. 2002). In the peripheral nervous system (PNS), HIV-1 infection and its treatment using HAART are associated with the development of neuropathic pain syndromes characterized by severe lancinating pain as well as parathesias and burning pain in the extremities. Damage to peripheral nerves has been associated with these syndromes. HIV-1-associated polyneuropathy has become the most common neurological complication of HIV-1 infection (Pardo et al. 2001). More than half of individuals with

R.J. Miller  (*) Department of Molecular Pharmacology and Biological Chemistry, Northwestern University School of Medicine, 303 E Chicago Ave, Chicago, IL, 60637, USA e-mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_9, © Springer Science+Business Media, LLC 2010

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advanced HIV-1 infection display signs of a neuropathy (Morgello et  al. 2004; Schifitto et al. 2002). Distal-symmetric polyneuropathy (DSP) is the most frequent subtype, especially in the later stages of AIDS (Pardo et al. 2001). It is also the case that the use of antiretroviral agents can themselves cause a peripheral neuropathy that may be clinically indistinguishable from HIV-DSP(Cornblath and Hoke 2006). The pathogenesis of HIV-DSP- and HAART-related neuropathy remains unclear. The most characteristic pathological feature is the distal degeneration of long axons (Pardo et  al. 2001) accompanied by macrophage infiltration (Bradley et  al. 1998; Pardo et al. 2001) and sometimes a modest loss of neurons in the dorsal root ganglia (DRG) (Esiri et  al. 1993; Rance et  al. 1988). Furthermore, there is degeneration within the centrally-directed extension of sensory DRG neurons (Rance et al. 1988). It has been of great interest to try to understand the cellular and molecular basis of the effects of HIV-1 on the nervous system. The best starting point is to consider what is known about the effects of HIV-1 in the immune system. It has been well established that the infection of leukocytes by the virus is mediated by coreceptors: CD4 and a chemokine receptor. In most cases, the chemokine receptor employed is CCR5 or CXCR4. Different strains of the virus utilize one or the other receptor, or in rarer cases, both the types. The tropism of the virus results in its ability to primarily infect macrophages or lymphocytes, although there are many variations on this theme. HIV-1 variants in the brain are genetically and biologically distinct from those in lymphoid tissues and other organs. The molecular basis of HIV-1 neurotropism is not completely understood, and viruses that bind to CCR5, CXCR4, or both receptors have been isolated from patients with HAD (Dunfee et al. 2006; Mefford et al. 2008). Nevertheless, except in certain cases of low efficiency infection of cells such as astrocytes, chemokine receptors appear to be essential components of HIV-1 neuropathogenesis. This being the case, it is important to have a clear understanding of the normal roles played by chemokines and their receptors in the physiology of the nervous system in order to understand how these processes may be subverted by the virus to produce the observed neuropathology. Based on the extensive knowledge of the effects of HIV-1 on the immune system, one possible point of interaction for HIV-1 in the brain is with immune cells such as macrophages and microglia, a relationship that is likely primarily responsible for HIVE. However, over the last 10 years, there has been an increasing realization that chemokine signaling also plays a direct role in regulating neuronal function. Thus, in addition to microglia, chemokine receptors are expressed by neurons, microglia, and neural progenitors, so these cells may also be considered potential targets for the virus (Tran and Miller 2003). This article will discuss some of the newly appreciated functions for chemokines in the nervous system and how these help to explain the neuronal effects of HIV-1.

9.2 Chemokines and the Development of the Nervous System The properties and functions of chemokines were initially elucidated based on their effects on the hematopoietic system, and their functions in the nervous system were not originally appreciated. Interestingly, the very first observation indicating that

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chemokines might have independent effects on the brain came from investigations of HIV-1 neuropathogenesis. Thus, Brenneman et  al. (1988) published a paper demonstrating that gp120, the coat protein of HIV-1 that actually engages the coreceptors, CD4 and CCR5/CXCR4, on target cells, could produce apoptosis of cultured neurons. This has proved to be a very robust observation that has been repeated in numerous studies including studies carried out in  vivo (Toggas et  al. 1994). Such observations naturally raised questions about the mechanism of action of gp120 in this regard. Several suggestions were made as to the identity of the “receptor” for gp120 on neurons, which at the time were not known to express chemokine receptors. These possibilities included the HIV-1 coreceptor, CD4, and other molecular entities (Lipton et al. 1994). The first report of the action of a chemokine on neurons was published in 1993. The study demonstrated that IL-8 could increase the survival of cultured neurons (Araujo and Cotman, 1993). However, as can be appreciated from its name, IL-8 was not known to be a chemokine at that time and was instead classed as an “interleukin.” Indeed, the expression of chemokine receptors by neurons was not generally appreciated until around 1997/1998 when several reports suggested this. These reports included observations of the expression of chemokine receptors by neuronal cell lines (Hesselgesser et  al. 1997), primary cultures of neurons (Meucci et  al. 1998; Ohtani et al. 1998), and in brain sections from HIV-1, Alzheimer’s disease, and other patients (Horuk et al. 1997; Westmoreland et al. 1998; Xia et al. 1997). Furthermore, data were obtained, suggesting functions for chemokine signaling in the development of the nervous system (Zou et  al. 1998) as well as in neuronal survival and communication (Giovannelli et al. 1998; Meucci et al. 1998). In the light of this new information, studies on the mechanism of gp120 toxicity shifted to consider whether chemokine receptors, such as CXCR4, expressed by neurons were responsible for these toxic effects and whether gp120-induced apoptosis still occurred in the absence of CD4, which is not expressed by neurons in appreciable amounts (Hesselgesser et  al. 1997, 1998; Miller and Meucci 1999). Several excellent studies have subsequently demonstrated the widespread expression of CXCR4 by neurons and other cell types in different parts of the nervous system (McGrath et al. 1999; Stumm et al. 2002, 2003). On the basis of the expression patterns of CXCR4 (and also CCR5) there are several potential targets for gp120 in the brain including microglia and astrocytes in addition to neurons. Therefore, the effects of HIV-1 may result from direct actions on neurons and glia as well as indirect effects resulting from HIVE. Further, chemokine signaling might be an important participant at several points in these events. Aside from providing the targets for HIV-1-induced neuropathogenesis, researchers have also studied the normal functions served by chemokines in the nervous system. One answer to this question can be obtained by considering the evolution of chemokines and their receptors. There are a large number of chemokines that have been identified to date, more than 50 in most mammals. As far as we know, all the actions of chemokines are transduced through stimulation of a family of related, G protein-coupled receptors (Tran and Miller 2003). Research into the pattern of evolution of chemokines and their receptors has revealed that a large expansion of the chemokine family occurred in parallel with the development of a sophisticated

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immune system in higher vertebrates (Huising et  al. 2003). This is consistent because chemokines have been shown to be key regulators of leukocyte migration in higher organisms. In this context one chemokine – CXCL12 (also called stromal cell derived factor-1 or SDF-1) stands out as being ancient and highly conserved (Huising et al. 2003). Indeed, homologues of SDF-1 and its receptor, CXCR4, are present in animals so ancient that they do not possess an immune system (Huising et  al. 2003). Hence, it is believed that there are ancient functions for chemokine signaling that do not concern the regulation of leukocyte migration. The hypothesis that CXCR4 signaling is important in areas beyond immunology has now been widely documented. One of the first indications of this fundamental role for the SDF-1/CXCR4 signaling axis came from the analysis of phenotypes observed for CXCR4 (Tachibana et al. 1998; Zou et al. 1998) and SDF-1 knockout mice (Ma et  al. 1998; Nagasawa et  al. 1996) and mutant zebrafish (Knaut et  al. 2005). In general, the phenotypes of these two types of mice appear very similar, consistent with the long held view that SDF-1 and CXCR4 constitute a unique signaling combination. It should be noted however, that recently another chemokine receptor, CXCR7, has also been shown to bind SDF-1, and so it is possible that some of the effects of SDF-1 are not mediated by CXCR4. The full extent of the importance of CXCR7 signaling in development and in the adult remains to be determined. (Miao et al. 2007; Schönemeier et al. 2008; Sierro et al. 2007; Valentin et al. 2007). A role for CXCR4 in development is clearly of interest considering the profound effects of HIV-1 on children (Epstein and Gelbard 1999). There are a large and ever increasing number of phenotypes that have been associated with deletion of the SDF-1/CXCR4 genes. These include deficits in b-lymphopoiesis and myelopoiesis (Nagasawa 2007; Nagasawa et al. 1996; Nie et al. 2004; Zou et al. 1998), cardiogenesis (Miao et al. 2007; Nagasawa et al. 1996; Zou et al. 1998), angiogenesis (Tachibana et al. 1998; Zou et al. 1998), neurogenesis (Lu et al. 2002; Stumm et al. 2003; Tran et al. 2007), gastrulation (Nair and Schilling 2008), and germ cell migration and development (Dumstrei et  al. 2004). Collectively, these phenotypes can be explained by the observation that CXCR4 signaling is important in regulating the migration of different types of stem/progenitor cells. This then appears to be the original role of chemokine signaling, and CXCR4 signaling in particular. Seen in this light, the extensive role played by chemokine signaling in the control of leukocyte trafficking can be thought of as an evolutionary development of this original function. Consistent with this idea, it has been demonstrated that of all chemokines and chemokine receptors, SDF-1 and CXCR4 are the most widely expressed during the development of the embryo (Knaut et al. 2003, 2005; McGrath et al. 1999; Moepps et al. 2000; Rehimi et al. 2008; Tissir et al. 2004; Yusuf et al. 2005). Moreover, the expression patterns for both SDF-1 and CXCR4 are highly dynamic, consistent with the possibility that they have shifting developmental roles in the formation of many different tissues. SDF-1 and CXCR4 expression have been observed very early, as early as blastocyst formation, in frog, chick, zebrafish, and mouse embryos. CXCR4 and SDF-1 are both expressed by embryonic stem cells derived from the inner cell mass of the developing blastocyst and it has been observed that SDF-1 enhanced the survival and migration of these cells (Guo et al. 2005).

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In addition, SDF-1/CXCR4 signaling is important in gastrulation (Nair and Schilling 2008). Thus, it is likely that CXCR4 signaling plays a central role in stem cell function from very early in development. In addition to its role in stem cell migration, development, and organogenesis, CXCR4 signaling also seems to have tissue-specific developmental roles that are distinct and appropriate to each tissue. For example, in the development of the nervous system, CXCR4 signaling also functions as an axon guidance cue (Chalasani et  al. 2003, 2007; Lieberam et  al. 2005). Hence, the role of CXCR4 signaling in development is complex and stage specific, ranging from an initial role in regulating the migration and functions of stem cells to tissue specific effects on differentiated cells. Clearly, HIV-1 might interfere with any of these processes and so might have effects on neural systems by this mechanism. Examination of the phenotypes of SDF-1 and CXCR4 knockout mice (Ma et al. 1998; Zou et al. 1998) demonstrated that among other effects a clear abnormality was observed in the development of the cerebellum. During the development of this part of the brain, cerebellar granule cell progenitors are located and proliferate in a subpial region known as the external granule cell layer (EGL). Once the pool of progenitors has expanded sufficiently, postmitotic cells migrate inwardly to form the internal granule cell layer (IGL), which is the normal location of mature granule cells. However, in CXCR4- and SDF-1-deficient mice it was observed that granule cell progenitors migrated inward at an inappropriately early time resulting in ectopically located progenitors within the Purkinje cell layer. This phenotype can be explained by considering the normal distribution of CXCR4 and SDF-1 expression in the cerebellum during development. CXCR4 is expressed by proliferating progenitors in the EGL, and SDF-1 is synthesized and secreted by the overlying meninges. Therefore, SDF-1-mediated chemoattraction serves as a signal for maintaining progenitors within the EGL (Klein and Rubin 2004; Klein et  al. 2001). This is a proliferative environment in which factors like Sonic hedgehog (SHH) stimulate progenitor division. Indeed, SHH and SDF-1 have cooperative effects on progenitor proliferation. If CXCR4 signaling is interrupted, progenitors are not retained in the EGL and respond to other chemoattractant factors resulting in their early inward migration. Following these initial reports, numerous other neuronal phenotypes have also been observed in SDF-1/CXCR4 deficient mice. In the hippocampal dentate gyrus (DG), for example, deletion of CXCR4 resulted in a significant phenotype in which granule cells were observed to be ectopically placed along the normal route of granule cell progenitor migration (Bagri et al. 2002; Lu et al. 2002) (Fig. 9.1). The development of the DG takes place in the period immediately after birth. Granule cell progenitors migrate from the wall of the lateral ventricle to form a “germinal matrix” in which they proliferate further and form the two blades of the DG. The expression patterns for SDF-1 and CXCR4 in the developing DG, as well as in the phenotype of CXCR4 knockout mice, are consistent with a model in which SDF-1 is secreted by meningeal cells that line the route of migrating CXCR4-expressing progenitors, and progenitors are stalled in their migration when CXCR4 signaling is interrupted (Fig. 9.1). This is not the only role for CXCR4 signaling in the development

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Fig. 9.1  Left panels (a–g) Localization of CXCR4 receptors and SDF-1 by in situ hybridization in the developing dentate gyrus at different embryonic (e) and postnatal days. dg dentate gyrus; ncx neocortex; iz intermediate zone; ms migratory stream; mng meninges. Right panels (a–g) demonstrate the placement of dentate granule cells indentified using staining for Prox-1 in wild type (wt) and CXCR4 knockout (−/−) mice. The figure also shows the localization and reduction of BrdU incorporation in the wild type and CXCR4 knockout mice (From Lu et al. 2002)

of this part of the brain. For example, SDF-1 can also act as an axon guidance cue, and it plays a critical role in promoting the growth of perforant fibers from the entorhinal cortex to the DG. Additional neuronal phenotypes observed in CXCR4 knockout mice include defects in the placement of developmentally important Cajal–Retzius cells (Berger et al. 2007; Paredes et al. 2006; Stumm et al. 2003), cortical GABAergic interneurons (Stumm et al. 2003, 2007; Tiveron et al. 2006), and GnRH-secreting forebrain neurons (Schwarting et al. 2006). In all of these situations it appears that the progenitors of these neurons utilize SDF-1-mediated chemoattraction to attain their final positions, and that lack of CXCR4 signaling results in interrupted progenitor migration. Thus, these examples differ from the situation prevailing in the cerebellum

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where instead SDF-1 is used to maintain progenitors in a proliferative environment and where interruption of CXCR4 signaling results in abnormally enhanced early migration of progenitors. Nevertheless, the basis for each phenotype is the chemoattractive effect of SDF-1 on CXCR4-expressing neural progenitors. SDF-1 regulated stem cell migration is also a feature of the peripheral nervous system. Here CXCR4 is expressed by neural crest-derived DRG progenitors migrating from the dorsal aspect of the neural tube, and SDF-1 is expressed by mesenchymal cells that line their route of migration (Belmadani et al. 2005). Thus, the anatomical scenario is similar to that observed in the developing DG. Disruption of CXCR4 signaling again results in a phenotype in which DRG neurons exhibit interrupted migration, and this results in abnormally formed DRGs in mice, or trigeminal ganglia in zebrafish (Knaut et al. 2005). Zebrafish also exhibit a variety of other sensory cell phenotypes when CXCR4 signaling is disrupted (Li et al. 2004). The development of DRG neurons also highlights the fact that SDF-1 frequently has tissuespecific effects during development in addition to those on stem cell migration. As indicated above, SDF-1 has been observed to act as an axon guidance cue for developing axon growth throughout the nervous system. This includes the axons of developing DRG neurons, where the action of SDF-1 reduces the repellant effects of the factor semaphorin 3A (Chalasani et al. 2007). Hence the growth pattern of sensory neuron axons is also abnormal in CXCR4 knockout mice. After exposure to SDF-1 during axon outgrowth, embryonic DRG neurons express both CXCR4 receptors and SDF-1, suggesting some kind of autocrine effect of CXCR4 signaling on these cells (Belmadani et  al. 2005; Odemis et  al. 2005). Indeed, interference with CXCR4 signaling in vivo or in culture strongly reduces the survival of DRG neurons. Thus, depending on the stage of development, SDF-1 produces effects on progenitor migration, axon development, and survival of DRG neurons. In adult animals SDF-1 assumes yet another role, acting as a neurotransmitter that can stimulate DRG neuron excitability and produce pain (see below, Bhangoo et al. 2007a, b; Oh et al. 2001). Furthermore, the expression of CXCR4 receptors by DRG neurons or glia may act as a binding site where T-tropic strains of HIV-1 can produce neuronal excitation, pain, or even death (Melli et al. 2006; Oh et al. 2001). Therefore, it is clear that CXCR4 signaling serves important role during the entire lifetime of a DRG neuron. As in the example of DRG neurons, it has also been demonstrated that CXCR4 signaling has effects on the survival and proliferation of neural stem cells in other parts of the developing embryo and in the adult (see below). To put these observations in context, it should be understood that the role of CXCR4 in the development of stem cells is not unique to the nervous system. CXCR4 expression by tissue-specific stem cells has been reported for embryonic stem cells and germ cells, as well as for progenitors from skeletal muscle, heart, liver, endothelium, and renal and retinal epithelia (Ratajczak et al. 2006). Indeed, as is clear from the name, “stromal cell derived factor-1,” that SDF-1-activated signaling also plays a key role in the development of hematopoietic stem cells which give rise to blood and related tissues. In mammals, the first primitive hematopoietic stem cells (HSCs) are found in the yolk sac, and the first definitive HSCs are located a few days later in a structure termed the aorta–gonad–mesonephros (AGM). From the AGM, HSCs

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migrate to the fetal liver which, during the second trimester of gestation, becomes the major mammalian organ for hematopoiesis. By the end of the second trimester, HSCs leave the fetal liver and colonize the bone marrow (BM). In mice deficient in SDF-1 or CXCR4, HSCs migrate appropriately from the AGM to the fetal liver but not from the liver to the BM (Nagasawa 2007; Zou et al. 1998). This indicates that the latter migration is dependent on CXCR4 signaling, consistent with the extensive expression of SDF-1 by cells observed in the BM. In addition to the disposition of HSCs, CXCR4 signaling also has tissue-specific roles in the development of leukocytes, b-lymphopoiesis being deficient in CXCR4 knockout mice (Nagasawa 2007; Nie et al. 2004; Zou et al. 1998). Moreover, HSCs are still retained in the BM in adult mice through SDF-1-mediated chemoattraction, and disruption of this process allows efflux of HSCs into the blood (Chute 2006; Lapidot et al. 2005; Nagasawa 2007; Welner and Kincade 2007). Indeed, this observation has proven clinically useful. For example, CXCR4 antagonists such as the drug AMD3100 can be used to mobilize HSCs from the BM for therapeutic purposes such as collection of HSCs prior to transplantation (Cashen et al. 2007). Following transplantation, intravenously administered HSCs are observed to home to the BM in a CXCR4-dependent fashion and this phenomenon can be used to reconstitute a depleted hematopoietic system in diseases such as chronic myelogenous leukemia and aplastic anemia (Chute 2006; Dar et al. 2006; Lapidot et al. 2005). A role for CXCR4 signaling during development has been recognized in numerous other instances. For example, primordial germ cells (PGCs) give rise to gametes in the gonads and are often the earliest cell lineage to be specified. Analysis of PGC migration in mice and zebrafish in which genes for SDF-1 or CXCR4 have been deleted, or in which expression of these molecules has been suppressed, shows aberrant colonization of the gonads by PGCs (Dumstrei et  al. 2004; Raz 2003). Cardiac development is also aberrant in CXCR4 or SDF-1 knockout mice. During the development of the heart, a subpopulation of cardiac neural crest cells migrate to colonize the outflow tract endocardial cushions prior to septation, the process through which a single outflow vessel, the truncus arteriosus, becomes the ascending aorta and the pulmonary trunk (Snider et al. 2007). Migration occurs via the third, fourth, and sixth pharyngeal arches (Jiang et  al. 2000). However, in both CXCR4- and SDF-1-deficient mice the region of the ventricular septum is abnormal (Nagasawa et al. 1996; Zou et al. 1998). As SDF-1 is expressed in the developing heart tissue (McGrath et al. 1999) and CXCR4 is expressed in migrating cells of the neural crest (Belmadani et al. 2005), it is possible that interruption of this process is the basis for this phenotype. This would be consistent with other defects in neural crest development observed in CXCR4 knockout mice, including defects in formation of the DRG (see above, (Belmadani et al. 2005) and positioning of melanoblasts in hair follicles (Belmadani 2008). A further interesting phenotype identified in SDF-1/CXCR4 knockout mice is a deficiency in blood vessel development, initially observed in the gastrointestinal system (Tachibana et al. 1998). Consistent with such observations, CXCR4 receptors have been shown to be expressed by hemangioblasts, the earliest common precursor to hematopoietic and endothelial stem cells, found in yolk sac

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blood islands (McLeod et  al. 2006) and also by endothelial cells that can be derived from embryonic stem cells (ESCs) in culture (Chen et al. 2007). In the latter case, these endothelial cells expressed CXCR4 and migrated towards an SDF-1 gradient. SDF-1 enhanced the formation of blood vessels in a matrigelbased assay. Hence, there is good reason to believe that CXCR4 signaling in endothelial progenitors is of considerable significance in the development of vascularization in the embryo.

9.3 The Role of Chemokines in the Control of Adult Neurogenesis It has been clearly demonstrated that CXCR4 signaling is a widely used method for regulating the migration and development of stem cells occurring in many tissues during organogenesis. However, it is known that stem cells are retained in many adult tissues and can be expanded on demand to cope with stressful situations such as damage or infection when they are required for tissue repair and to maintain tissue homeostasis (Kucia et al. 2005b; Ratajczak et al. 2006). A good example of this is the production of blood cells from HSCs that reside in the adult BM. The turnover rate of most blood cells is relatively fast compared to other tissues and so their numbers must be continuously restored. Thus, HSCs are constantly called upon to manufacture different types of blood cells in order to maintain normal homeostasis or under conditions of stress when specific subsets of leukocytes must be rapidly expanded.   As discussed above, the retention of HSCs in the adult BM is under the control of CXCR4 signaling and HSCs can be mobilized into the blood when this signaling is disrupted (Nagasawa 2007). This is a basic model for many types of stem cell-mediated repair programs in the adult. Thus, stem cells used for repair of nonhematopoietic tissues may also be deposited in stem cell niches during development and are mobilized when required through CXCR4-mediated signaling. Indeed, in addition to HSCs, the adult BM contains other types of stem cells that can be used for repair of different tissues (Fox et al. 2007; Kucia et al. 2005a). It is likely that these cells express CXCR4 receptors and can follow SDF-1 gradients to areas of damage where they effect repairs. One example is the repair of endothelial tissue following damage to blood vessels. In this case, SDF-1 is released from platelets that become associated with the damaged region. CXCR4expressing endothelial progenitors in the BM and elsewhere then migrate to the damaged region (Hristov et  al. 2007). Following myocardial infarction, SDF-1 expression increases in the damaged portion of the heart, and mesenchymal stem cells, also located in the BM, can migrate to this region for cardiac repair purposes (Fox et al. 2007). It has even been demonstrated that CXCR4-expressing mesenchymal stem cells can enter the brain following stroke (Hill et al. 2004) or under other circumstances (Sano et  al. 2005). Indeed, a population of CXCR4-expressing cells that also

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express neuronal markers such as nestin have been observed in the BM and are mobilized into the blood in response to stroke (Kucia et al. 2006). In this situation, SDF-1 expression in the brain is upregulated primarily by perivascular astrocytes (Hill et  al. 2004; Miller et  al. 2005; Stumm et  al. 2002). It is possible that the migrating BM cells act as a source of new neurons under these or related circumstances. It appears counter-intuitive that neural progenitors are located in the BM, outside the blood-brain barrier. However, under injury conditions the barrier’s permeability changes to allow progenitors to cross. It was thought for a long time that new neurons were not produced from endogenous sources in the brains of higher mammals once they had attained maturity. However, it is now clear that populations of neural progenitors that actively produce new neurons, oligodendrocytes, and astrocytes exist primarily in the subventricular zone (SVZ) surrounding the lateral ventricle and the subgranular zone of the dentate gyrus (DG), and probably in other regions as well (Gould 2007). As in other tissues such as the BM discussed above, these cells are thought to exist in special stem cell niches where the influences important for stem cell development can be coordinated (Palmer et  al. 2000). In the brain, both blood borne and neuronal influences are thought to be important for the development of new neurons. It is thought that new neurons produced on an ongoing basis in the DG, for example, contribute to certain types of hippocampal plasticity, and that new neurons normally produced in the olfactory bulb (OB) appear to be of importance in maintaining odor detection in the context of constant neuronal turnover in this part of the brain (Conover and Notti 2008; Imayoshi et al. 2008; Gould 2007). Therefore, this ongoing neurogenesis is envisaged as having a homeostatic role analogous to that of HSCs in replenishing leukocytes under normal circumstances. However, as with the BM, new neurons and glia derived from neural stem cells may contribute to the brain’s efforts to repair itself in the face of injury. New neurons need to be produced to repair the chronic neurodegeneration that occurs in conditions such as amyotrophic lateral sclerosis (motor neurons), Parkinson’s Disease (dopaminergic neurons), or the rapid neurodegeneration associated with stroke (Imitola 2007; Steiner et al. 2006). Similarly, new oligodendrocytes need to be produced for repair of demyelinating diseases such as multiple sclerosis. The role of chemokine signaling in the development of adult neural stem cells under these various circumstances has been actively studied. It is clear that adult neural stem/progenitor cells located in the SVZ and DG express chemokine receptors, including high levels of CXCR4 receptors (Berger et  al. 2007; Tran et  al. 2007) (Fig. 9.2). Considering the DG as an example, CXCR4 is expressed by the most immature radial glia-like stem cells as well as their progeny including rapidly-amplifying cells, neuroblasts, and immature granule neurons. Furthermore, SDF-1 is also expressed by neuronal cells in the DG (Banisadr et  al. 2003; Stumm et al. 2002). The close juxtaposition of SDF-1 and CXCR4 in the adult DG suggests that CXCR4 may be an important regulator of adult neurogenesis in this part of the brain. Indeed, it now appears that SDF-1 may be important for several reasons.

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Fig. 9.2  Left panels (a–o), illustrate the expression pattern for CXCR4 receptors in the developing and postnatal dentate gyrus using CXCR4-EGFP BAC transgenic mice (d, arrow, Cajal–Retzius cells) (From Tran et  al. 2007). Right panels illustrate the distribution of SDF-1 protein in the dentate gyrus of SDF-1-mRFP fusion protein BAC transgenic mice. Note the punctate distribution in secretory vesicles. Some of these vesicles are localized to blood vessels (white arrow) and some are observed in the soma of large neurons (arrowhead). As can be observed in the bottom panels, these large neurons express parvalbumin (blue staining) indicating that they are GABAergic interneurons. mol molecular layer; GrDG granule cell layer; PoDG hilus (From Bhattacharrya et al. 2008)

In the adult DG it is thought that the ongoing level of neurogenesis must be regulated in part through a feedback mechanism by which the granule cells of the DG can increase or decrease the level of new neuron production according to ongoing need. This process has been called “excitation/neurogenesis coupling.” However, exactly how this is achieved has not been clear. One important observation concerns the role of the neurotransmitter gamma-aminobutyric acid (GABA). The actions of GABA on mature neurons usually produce inhibition and hyperpolarization due to activation of a ligand gated ion channel (GABA-A receptor) that is permeable to cloride ions. Owing to the distribution of Cl– across the cell membrane (low inside, high outside), the influx of Cl– produces an inhibitory hyperpolarizing current. On the other hand, in immature neurons, where cells have a high internal Cl– concentration, Cl– moves out of the cell and the action of GABA is excitatory. Usually, the first synapses to be formed with developing neurons are excitatory, GABAergic synapses, and this is true in the case of developing granule cells in the adult DG (Duan et al. 2008). Recent studies have demonstrated that SDF-1 is actually stored in neurotransmitter vesicles in DG neurons, including DG GABAergic interneurons such as basket cells (Bhattacharrya et al. 2008) (Fig. 9.2). Both GABA and SDF-1 can be released in the DG and may cooperate with one another to regulate the functions of neural stem cells such as their proliferation and differentiation (Bhattacharrya et al. 2008). It is interesting to note that for the synapses that at immature granule cell progenitor synapses both GABA and SDF-1 appear to be tonically released and produce a tonic level of progenitor activation. This is apparent when electrophysiological records from

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Fig. 9.3  Tonic activation of GABA-A and CXCR4 receptors in the adult dentate gyrus stem cell niche. Figure shows voltage clamp recordings from EGFP expressing cells in acutely isolated slices from the hippocampus of nestin-EGFP transgenic mice. Application of either a GABA-A antagonist (bicuculline) or a CXCR4 antagonist (AMD 3100) elicited an outward current when recording from these cells (From Bhattacharrya et al. 2008)

neural progenitors reveal an outward current observed upon the addition of either a GABA-A (bicuculline) or CXCR4 (AMD3100) antagonist (Fig.  9.3). In keeping with this result, both GABA and SDF-1 produce inward currents in DG progenitors that serve to depolarize the cells (Duan et al. 2008; Bhattacharrya et al. 2008). Tonic SDF-1 release is also supported by the fact that the CXCR4 receptors on these cells are substantially downregulated through ligand-induced endocytosis (Kolodziej et al. 2008). The tonic release of SDF-1 may normally help to integrate the level of neural activity in the DG with the ongoing level of neurogenesis. One major conclusion from these data is that chemokines can act as neurotransmitters in the DG, and this finding has had widespread influence.

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There are several other conditions under which chemokines can be expressed by neurons (Fryer et al. 2006; Schreiber et al. 2001). These include the expression of CCL21 in cortical neurons following injury and the expression of MCP-1 by sensory neurons in association with chronic pain syndromes (see below). As is the case for SDF-1 in the DG, chemokines expressed in these other neurons are also released and play a neuromodulatory role. Hence, CCL21 may be important to communication between injured neurons and microglia (de Jong et  al. 2005, 2008), and MCP-1 release from sensory neurons may be important to the regulation of excitability in these cells (see below). It is also possible that some of the actions of HIV-1 in the brain might be mediated through effects of the virus on adult neurogenesis through binding to CXCR4 receptors expressed by DG neural progenitors. Indeed, deficits in neurogenesis in AIDS patients have been reported (Krathwohl and Kaiser 2004a, b; Okamoto et  al. 2007). It has been shown that at least some T-tropic gp120s can activate signaling via the CXCR4 receptor. This can lead to activation of the p38 pathway and a reduction in stem cell proliferation, a mechanism that could explain the deficits observed in patients (Okamoto et al. 2007). Although the role of chemokine signaling in the adult DG seems clear, no such role has been established so far for neurogenesis in the SVZ and OB. It is clear that progenitor cells in the SVZ express several chemokine receptors including CXCR4 (Tran et al. 2007), but whether chemokine signaling is important to the migration of these cells to the OB is unclear. However, both SDF-1 and CXCR4 are also expressed in the OB (Stumm et al. 2002), and so it is possible that the migration of interneurons from the core of the OB to the outer layers of the structure may be under the control of chemokine signaling. It is also likely that chemokine signaling is important to neurogenesis that occurs in the context of brain pathology. For example, in response to neurodegeneration, such as that occurring following a cortical or subcortical stroke, there are attempts by the brain to repair itself. This response involves the migration of endogenous neural progenitors from the SVZ to the area surrounding the infarct (Imitola 2007; Ohab et al. 2006; Robin et al. 2006). It is likely that activated astrocytes and microglial cells proximal to the brain lesion and endothelial cells asso­ ciated with neovascularization secrete chemokines such as SDF-1 or MCP-1 which, in turn, act upon chemokine receptors expressed by endogenous neural progenitors and stimulate their directed migration towards the site of the lesion (Belmadani et al. 2006). Moreover, one strategy for treating diseases like stroke is to introduce exogenous neural stem cells into the brain in the hope that they will participate in brain repair. Such a strategy is analogous to the use of HSCs to reconstitute damaged or irradiated BM. These neural progenitors, which can be expanded in cell culture, also express chemokine receptors and thus can “home” to sites of chemokine production in the brain (Tran et al. 2004). In support of such possibilities it has been observed that both CCR2- (Liu et al. 2007) and CXCR4- (Ohab et al. 2006; Thored et al. 2006) expressing progenitors are found within stroke-induced lesions in the brain, and that interference with chemokine signaling blocks this recruitment (Belmadani et  al. 2006; Ohab et  al. 2006). The migration of neural progenitors is not only

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required for brain repair in the context of neurodegeneration, but is also important to brain repair as the result of demyelination that occurs in diseases such as Multiple Sclerosis. Here again it is thought that the inflammatory response associated with demyelinating lesions can act as a source of chemokines attracting progenitors that then develop into oligodendrocytes and remyelinate damaged neurons. In keeping with this possibility it has been demonstrated that oligodendrocyte progenitors (OPs) express chemokine receptors such as CXCR4 and that chemokines can produce (or sometimes inhibit) a chemotactic response by these OPs (Dziembowska et al. 2005; Kadi et al. 2006; Maysami et al. 2006). Injection of OPs into the lateral ventricle or intravenously has demonstrated the migration of these cells to sites of demyelinating lesions, their clear development into oligodendrocytes (Banisadr et al. unpublished observations), and an improvement in the clinical score associated with experimental autoimmune encephalomyelitis, the major rodent model for MS. Although the above discussion highlights the important role of chemokine signaling in development and repair in many tissues, these same receptors may also play a role in several pathological processes in which stem cells migrate and develop improperly. A particularly important example of this process concerns the development of tumors, including brain tumors. In this case, it is currently thought that the growth of many tumors may depend on the properties of cancer “stem cells,” which represent neoplastic versions of the stem cells that normally generate or repair most tissues. These cancer stem cells can give rise to tumor cells in primary tumors and can also metastasize to seed tumors in other areas of the body. It is clearly important to identify the factors that help to enhance the growth and “health” of tumors and also contribute to their further distribution. Considerable evidence now suggests that chemokine signaling, including CCR2 and CXCR4 signaling, can contribute to both of these phenomena (Kucia et al. 2005a; Orimo et al. 2005; Orimo and Weinberg 2006). First, SDF-1 is produced by many tumors where it is suspected to have autocrine, growth-promoting effects on the developing tumor. It also enhances the growth of blood vessels that are important for further tumor growth and development, indicating a key role for CXCR4 (or possibly CXCR7). Even more striking is the possibility that expression of CXCR4 or other chemokine receptors by cancer stem cells allows them to follow SDF-1 gradients and seed tumors at remote sites. It is clear that such sites are non-random and commonly involve the lungs, liver, bone marrow, or lymph nodes, areas of constitutively high SDF-1 expression. It is also clear that the hypoxic environment prevailing in parts of a tumor allows HIF-1a-induced upregulation of CXCR4 expression by different types of cancer stem cells. This process also helps them to “home” to sources of SDF-1 such as the bone marrow (Ceradini and Gurtner 2005). This aberrant role for SDF-1/CXCR4 signaling probably involves not only the growth and metastasis of tumors, but almost certainly other types of diseases which involve the abnormal migration and subsequent growth of cells (Xu et al. 2007). For example, in pulmonary fibrosis (PF) aberrant development of fibroblast-like cells in the lung produces fibroblastic foci, abnormal lung remodeling, and eventually fatal lung dysfunction. It is likely that the source of this aberrant fibroblast production

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is a type of circulating fibroblast-like progenitor cell called a fibrocyte-a type of circulating mesenchymal stem cell. These cells express CXCR4 receptors and can home to sources of SDF-1 (Agostini and Gurrieri 2006; Scotton and Chambers 2007; Snider et al. 2007). Although the reason is not clearly understood, it appears that in PF the lung produces increased amounts of SDF-1 leading to the attraction and development of fibrocytes. In animal models of the disease, on bleomycin administration to the lung, CXCR4 antagonists reduce fibrocyte influx and lung fibrosis. Thus, the potential role for CXCR4 signaling in diseases such as PF and cancer also suggests the possibility that CXCR4 antagonist drugs may constitute novel types of therapy in such disorders. Thus, in these examples of stem cell related pathology, as well as in the normal development of the organism and the normal repair response of the adult, the effects of SDF-1/CXCR4 signaling appear to be of key regulatory importance.

9.4 Chemokine Signaling in Pathological Pain States As discussed above, chemokine signaling is also important to the functioning of the peripheral nervous system. Chemokine signaling appears to be of particular importance in the phenomenon of chronic pain (White et al. 2007). In this situation, the upregulation of chemokine expression and release by neurons (in this case, sensory neurons) appear to be of central importance in the genesis of pain syndromes. It is therefore worth examining this relationship as an example in which chemokines can be used as neuromodulators in the context of pathology. It is also an excellent example of how chemokines can act at the interface of inflammation and neuronal function. As discussed this interface is of great importance in HIV-1 infection and HAART. Both infection by the virus or HAART can produce several types of chronic pain syndromes, most commonly distal symmetric polyneuropathy, as described above (Hahn et al. 2008). The sensation of acute pain (acute nociception) is clearly of great importance for the survival of all complex organisms. However, in a large number of clinical situations patients can develop chronic pain syndromes in which the sensation of pain is no longer associated with normal and physiologically appropriate aversive stimuli. Such “pathological” pain is generally observed to result from damage to the nervous system, the toxic effects of drugs (e.g., HAART), infection (e.g., HIV-1), and several other causes. Under these circumstances, patients may develop spontaneous sensations of pain or pain produced by stimuli that are not normally perceived as noxious, such as light touch or warm or cool temperatures. The chronic and intractable nature of these syndromes may have extremely negative effects on the quality of life. Furthermore, many chronic pain syndromes are difficult to manage pharmacologically. Although drugs that afford a degree of relief to some individuals are available, they are not universally effective. Powerful pain killing drugs such as opiates also produce some relief, but taking such drugs chronically

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may be associated with the development of serious opioid-related problems such as tolerance and dependence. It is widely believed that chronic pain behaviors involve fundamental changes in the properties of the neurons that signal pain. For example, changes may occur in the properties of sensory neurons in the dorsal root (DRG) and trigeminal ganglia as well as higher up the neuraxis. It appears that some DRG neurons change their complement of neurotransmitters, receptors, and ion channels resulting in hyperexcitability and ectopic firing (Devor 2006). In addition, changes in synaptic transmission at the level of the dorsal horn (interneurons) and higher up the neuraxis (descending controls) result in central sensitization to pain-related stimuli (D’Mello and Dickenson 2008). It seems reasonable to assume that a fuller understanding of the molecular and cellular mechanisms that underly these changes will provide new therapeutic targets allowing more effective treatment and, perhaps, the permanent reversal of chronic pain states including those associated with HIV-1. Chronic pain such as that occurring following HIV-1 infection or HAART is a widespread health problem, thus it has been intensively investigated by both academic and industry-based laboratories and many new insights have been forthcoming. However, putting these discoveries together into a logical framework for improving the treatment of chronic pain has yet to be achieved. Nevertheless, some important advances in our overall understanding of these syndromes have been achieved. It is now clear that the stimuli which initiate chronic pain syndromes initially provoke some aspect of the inflammatory response. Inflammation can be considered to be an adaptive response to all noxious stimuli. Inflammatory responses represent a continuum which starts with the activation of resident tissue macrophages and may subsequently require the influx of additional leukocytes depending on the seriousness of the situation (Medzhitov 2008). If such inflammatory responses are successful in resolving a problem, then the organism will once again return to a state of homeostasis. If the problem remains unresolved, then the chronic nature of some elements of the inflammatory response will often result in tissue dysfunction. One manifestation of this appears to be chronic pain, although why this should be the case is unclear from the evolutionary standpoint. Nevertheless, if the genesis of the problem is viewed as resulting from a dysfunctional inflammatory response it provides us with novel targets for therapeutic intervention in chronic pain syndromes. The relationship between initiation of the inflammatory response and the development of chronic pain is now well established. The activation of cells with immune competency such as Schwann cells, astrocytes, satellite glial cells, microglia, and macrophages, all of which exist in close proximity to primary afferent nerves, has been clearly demonstrated. Further, interference with the activation of these cells prevents or limits the development of pain (Romero-Sandoval et  al. 2008; Uçeyler and Sommer 2008; Watkins et al. 2007a, b). However, although this model of events provides us with a new way of looking at the genesis of chronic pain the precise molecular pathways that ultimately lead to pain are complex and incompletely understood. It is clear however, that following the initiation of the inflammatory response, the generation of inflammatory cytokines is a key event in the

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development of pain behavior. For example, the activation of Toll-like receptors on resident macrophages by a variety of cellular proteins expressed in connection with the cellular stress response, or by molecular components derived from bacteria and viruses, will produce upregulation of inflammatory cytokine expression (Guo and Schluesener 2007). Exactly what comes next is unclear, but upstream cytokines such as TNF-a and IL1-b direct the synthesis of numerous downstream mediators that orchestrate further aspects of the response (Uçeyler and Sommer 2008). Chemokines are widely employed as downstream effectors in the inflammatory cytokine cascade. When considering the genesis of chronic pain in particular, the chemokine MCP-1/CCL2 and its receptor, CCR2, have been identified as being of great importance (White et al. 2007). One result that clearly demonstrates this conclusion is the phenotype of CCR2 receptor ko mice. These mice show an almost complete lack of development of neuropathic pain in some injury models and reduced development in several others (Abbadie et  al. 2003). Moreover, CCR2 receptor antagonists have similar effects (Bhangoo et  al. 2007a, b). On the other hand, overexpression of MCP-1, or administration of the chemokine by several routes to rodents, produces manifestations of pain hypersensitivity (Menetski et al. 2007). Several studies have attempted to identify the molecular and cellular basis for this type of result, although a precise explanation is still far from clear. Following the initial observation that numerous chemokine receptors are expressed by neurons, Oh et al. (2001) demonstrated that neonatal DRG neurons in culture expressed many of these receptors. They also revealed that addition of chemokine agonists to these cells produced powerful excitation. Intraplanar injection of several chemokines produced acute pain behavior in adult rodents. In addition, Oh et al. demonstrated that the HIV-1 coat protein, gp120, also produced agonist-like effects on cultured DRG neurons, including excitation, and produced pain when injected by the intraplanar route. The authors therefore suggested that chemokines might have a direct role in mediating pain in the context of inflammation, a situation in which chemokine expression is strongly upregulated. Moreover, in the model envisaged by Oh et  al. the effects of chemokines are primarily produced through their direct effects on the excitation of sensory neurons. As mentioned above, this proposed role of chemokine signaling in pain received important support from a paper published in 2003 by Abbadie et  al. describing the impressive reduction in different pain behaviors observed in CCR2 ko mice. This study demonstrated that mechanical allodynia resulting from the administration of formalin or CFA was reduced and that long-term mechanical allodynia resulting from sciatic nerve ligation was almost completely absent in mutant mice. Furthermore, intraplanar administration of MCP-1 elicited mechanical allodynia. Although these data were generally consistent with those of Oh et  al, Abbadie et  al. suggested a different model to explain the apparent role of CCR2 signaling in pain which was based on the potential role of MCP-1/CCR2 signaling in Wallerian degeneration (WD). It had been previously demonstrated that, in response to WD-related injuries, MCP-1 was upregulated in nerve-associated Schwann cells and/or endoneurial fibroblasts (Toews et al. 1998). It was suggested that MCP-1 expression upregulated in these cells played a central role in the attraction of macrophages into the injured nerve. Indeed, according

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to Carroll and Frohnert (1998), MCP-1-expressing cells in the injured sciatic nerve are not present in Wlds mutant mice in which the WD is reduced or abolished under many circumstances. In keeping with these considerations, Abbadie et al. observed a sustained influx of CCR2-expressing macrophages into the DRG and sciatic nerve in response to nerve injury accompanied by upregulation of mRNA for CCR2 in the same locales. No expression of CCR2 by neurons was observed in either the DRG or spinal cord. Yet, the authors identified immunoreactivity for CCR2 in microglia in the spinal cord (SC) of injured animals. They therefore proposed a model in which MCP-1 had two major functions. The first of these was to attract macrophages into the DRG and nerve as part of the cellular mechanism of WD. It was proposed that these macrophages might then secrete proalgesic factors. Moreover, it was suggested that MCP-1 also activates CCR2-expressing microglia in the SC and that these cellular interactions subsequently result in the release of proalgesic factors which further participate in the cellular changes contribute to the development of chronic hypernociceptive states. In support of this latter proposal, Abbadie et  al. also observed that the number of activated microglia and astrocytes was reduced following nerve injury in the SC of CCR2 ko mice. However, a different model was suggested indicating that the sensory neurons themselves might be the site of chemokine/receptor interaction in the generation of chronic pain, something that would have been predicted from the studies of Oh et al. discussed above. Tanaka et al. (2004) demonstrated that in a neuropathic pain model (sciatic nerve ligation) MCP-1 expression (mRNA and protein) was actually upregulated by the DRG neurons themselves after 24/48 h ipsilateral, and to some extent contralateral, to the injury. These observations were consistent with other studies demonstrating that MCP-1 could be upregulated in different types of neurons in response to injury (Flugel et al. 2001; Schreiber et al. 2001). Tanaka et al. considered that the targets of MCP-1 action might include blood monocytes following its release from peripheral nerve terminals, the DRG by a process of cell autonomous chemokine release and mutual CCR2 receptor stimulation, and the spinal cord following transport and release by centrally terminating DRG axons. The authors also demonstrated that intrathecal injection of MCP-1 produced transient pain hypersensitivity lasting 2 h which supports the latter possibility. White et al. (2005) further examined the possibility that DRG neurons might be the site of MCP-1 action. Using a compressed ganglion model of neuropathic pain these authors observed that CCR2 receptors were expressed in the cell bodies of DRG neurons of all sizes in the injured and closely juxtaposed ganglia around 3/5 days following the initiation of injury. Similarly, as observed by Tanaka et  al., MCP-1 was expressed by DRG neurons but not by other cell types around the same time. CCR2 was also expressed by some non-neuronal cells which might be macrophages or satellite glial cells. Importantly, White et  al. also demonstrated that, consistent with the CCR2 expression data, MCP-1 excited neurons of all sizes in the DRG from injured but not control animals. Consistent with these excitatory effects, Qin et  al. (2005) also demonstrated that addition of MCP-1 to cultured DRG neurons elicited release of the nociceptive neurotransmitter, calcitonin gene-related peptide (CGRP), from these cells, presumably as a result of increased neuronal excitation.

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In summary, these data illustrate the fact that there are numerous sites at which MCP-1/CCR2 signaling might elicit proalgesic effects resulting from its release in the periphery, the DRG, and the spinal cord. Different types of data collected using different models have led to the suggestion of different theories highlighting the roles of neuronal or nonneuronal cells. Indeed, these theories are not necessarily mutually exclusive. However, in several instances these observations have included the expression of MCP-1 by sensory neurons indicating that it may play a neuromodulatory role. In order to further understand the role of neuronal MCP-1 expression, Zhang and De Koninck (2006) examined MCP-1 expression following sciatic nerve injury. In their experiments MCP-1 expression was upregulated by DRG neurons quite rapidly (<1 day). In addition to the DRG, the authors presented evidence that MCP-1 was also transported by neurons to the spinal cord where MCP-1-labeled terminals were colocalized with CGRP. MCP-1 was also observed to be expressed by a population of neurons in the ventral horn. No MCP-1 expression colocalized with microglial cells. However, microglial and subsequently astrocyte activation was clearly evident. The authors concluded that one role for MCP-1 may be to be released from the central terminals of sensory neurons and, as described above, to activate CCR2-expressing microglia. This interesting model, in which microglia are the prime targets for the actions of MCP-1 is reminiscent of the results discussed above for the CNS in which neurons upregulate CCL21 and use it as a neuromodulator for contacting microglia. Thus, this type of chemokine signaling might be a general method by which neurons can interact with immune cells like microglia in the nervous system. This would certainly also be true for the chemokine CXC3CL1 (fractalkine), which can clearly play a role in neuron to microglial communication. The hypothesis that MCP-1 expressed by DRG neurons plays a similar role has been further examined in several other publications. For example, Wallace and colleagues investigated the role of MCP-1/CCR2 signaling in a model of HIV-1associated neuropathic pain (Wallace et al. 2007). In this case nociceptive behavior was induced either by application of the HIV-1 coat protein gp120 alone to the sciatic nerve by itself or accompanied by the administration of the antiretroviral agent, Zalcitabine (ddC). These manipulations produce pain hypersensitivity and associated activation of spinal microglia. Inhibition of microglial activation with minocycline delayed the expression hypernociceptive behavior. Moreover, as observed by several others (see above) MCP-1 expression was upregulated in DRG neurons in association with gp120 treatment. The same group also used a chronic constriction injury of the sciatic nerve to demonstrate that MCP-1 was upregulated by both injured and uninjured DRG neurons that expressed CGRP and P2X3 purinergic receptors (Thacker et  al. 2008). Importantly, the authors showed that MCP-1 was both transported by primary afferent fibers in an anterograde fashion and released into the superfusate bathing an ex vivo preparation of the dorsal horn and attached dorsal roots following nerve stimulation. Consistent with this, intrathecal or intraspinal injections of MCP-1 into naïve rats produced mechanical pain hypersensitivity and associated activation of microglia. Finally, intrathecal administration of an antibody to MCP-1 reversed pain behavior

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in the sciatic nerve injury model as well as the associated microglial activation observed under these circumstances. Hence, these data provide strong evidence for the model suggested by Abbadie et  al. and Zhang and De Koninck, in which centrally released MCP-1 produces chronic pain via direct activation of CCR2expressing microglia located in the spinal cord. Indeed, in addition to these reports several other studies have demonstrated that direct administration of MCP-1 by a number of different routes to either naïve or injured animals can elicit pain. Further support for a role for MCP-1/CCR2 signaling in nociceptive behavior was provided by a completely different approach. In this case, Zhang et al. (2007) made use of chimeric mice to further examine the relative roles of resident spinal microglia and infiltrating blood-borne macrophages in the generation of chronic pain following injury to the sciatic nerve. As with several other investigators, Zhang et al. demonstrated that chronic pain failed to develop in CCR2 ko mice. However, when CCR2 was selectively knocked out in either the central (spinal cord) or peripheral (blood/bone marrow) compartment, pain still developed. The authors demonstrated that intrathecally administered MCP-1 increased microglial activation and this did not occur in CCR2 ko mice. Zhang et al. also demonstrated that circulating macrophages entered the spinal cord in association with injury where they proliferated and became microglia. This process was also dependent on CCR2 activation and did not occur when MCP-1 was neutralized. Considering all of the above data one may arrive at the following model for explaining the participation of MCP1/CCR2 signaling in the genesis of chronic pain. In addition to any role MCP-1 may have in the attraction of macrophages that are involved in WD, an early event in the generation of chronic pain is the upregulation of MCP-1 expression by DRG neurons. This has been a consistent observation. MCP-1 expressed by DRG neurons is stored in secretory vesicles and is transported to the periphery and spinal cord where it can be released from nerve terminals following nerve excitation (Thacker et al. 2008). In the SC, the targets of this MCP-1 are CCR2-expressing microglia (Abbadie et al. 2003). Activation of these microglia triggers a pathway which eventually results in nociceptive hypersensitivity. The pathway is not entirely clear but presumably activated microglia release further proalgesic mediators and substances that then produce astroglial activation and other events that contribute to the development of chronic pain (Menetski et  al. 2007). Influx of CCR2-expressing, circulating macrophages by centrally released MCP-1 can also contribute to the pool of activated microglia (Zhang et al. 2007). Hence, it may be concluded that in order to therapeutically target pain development, both central CCR2-expressing cells (microglia) and peripherally circulating cells (macrophages) must be targeted. Such a model clearly explains several key observations including the role of CCR2 signaling as demonstrated by the phenotype of CCR2 ko mice, neutralizing antibodies, and CCR2 antagonist drugs, as well as the role of MCP-1 when upregulated and released from primary afferents, the observed requirement for activated microglia in the cord (as well as influx of macrophages), and the observation that MCP-1 administration produces hypernociception. It is also worth considering alternative actions of MCP-1/CCR2 signaling in the generation of pain. As indicated above, another possible site of action is the DRG

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Fig. 9.4  Upregulation of MCP1 and CCR2 expression in neurons of the dorsal root ganglia in association with the development of neuropathic pain. Upper panels illustrate expression of CCR2 receptors by in situ hybridization in naïve rats (a) and animals subjected to Chronic Compression of the DRG (CCD) a model for neuropathic pain (b). CCR2 is expressed in small satellite cells and many neurons (white arrows). Bottom panels (c, d) illustrate the expression of MCP-1 (immunohistochemistry, red) under the same circumstances. Many neurons (red arrows) express the chemokine (From White et al. 2005)

itself. In this case it is clear that the expression of MCP-1 is upregulated in the cell bodies of DRG neurons where it is also packaged into secretory vesicles (Fig. 9.4). Furthermore, it is apparent that, at least in some studies, CCR2 receptors are expressed by DRG neurons in association with nociception (White et  al. 2005; Bhangoo et al. 2007a, b; Jung et al. 2008). Thus, a model based on cell autonomous or near-neighbor actions of MCP-1 released from the cell bodies of DRG neurons should be considered. It is possible that direct effects of MCP-1 on DRG neuronal excitability are of prime importance, as it is clear that MCP-1 strongly excites CCR2-expressing DRG neurons and such increased excitation is certainly an important feature of chronic pain behavior (White et al. 2005; Sun et al. 2006). The mechanism of MCP-1 induced excitation is not entirely clear and will certainly depend on the class of DRG neurons involved. However, regulation of TRP channels (Jung et al. 2008), K+ channels (Sun et al. 2006), and Na+ channels (Wang et al. 2008) have all been suggested and are probably involved. Given the fact that upregulation of

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MCP1/CCR2 by DRG neurons occurs some days following the initial injury, it is also likely that excitation produced in this fashion may be more important for the maintenance of chronic pain rather than its initiation. Thus, according to this view the major site of action of MCP-1 is within the DRG rather than in the SC. Whatever the precise site of action of MCP-1, it is clear that inhibition of this chemokine’s action or inhibition of its synthesis might be considered as a novel therapeutic approach in chronic pain conditions. The potential effectiveness of this approach is evident from the results of administration of antibodies against MCP-1 in several of the studies discussed above and from the effectiveness of CCR2 receptor blockers and the phenotype of CCR2 ko mice. In addition to inhibition of MCP-1 action, inhibition of MCP-1 synthesis by DRG neurons may be an alternative target for therapeutic involvement in chronic pain. It has been observed that MCP-1 synthesis in DRG neurons is under the control of nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) (Jung et al. 2008). Indeed, the role of MCP-1 in this situation is reminiscent of other “inflammatory” scenarios. For example, in obese mice, which can also be thought of as another form of tissue stress, adipocytes upregulate MCP-1 synthesis and this is important for the associated development of insulin resistance (Kanda et  al. 2006). Under such conditions it may also be possible to engage signaling pathways that antagonize the NFkB-induced upregulation of the inflammatory cytokine (e.g., MCP-1) response. For example, activation of the PPAR signaling pathway may have this type of effect (Bensinger and Tontonoz 2008). As far as the expression of CCR2 by DRG neurons is concerned, this has been shown to be directed by the Ca(2+)-dependent transcription factor, nuclear factor of activated T-cells (NFAT), suggesting that this might also be a target for intervention by drugs that inhibit calcineurin or other upstream NFAT regulators (Jung et al. 2008). Although MCP1/CCR2 signaling seems to have general relevance to the development of chronic pain states, other chemokines and their receptors may also be important under certain conditions. From the point of view of the present discussion, SDF-1/CXCR4 signaling also seems to be involved in the generation of HIV-1/ NRTI-related pain states. Administration of a single dose of the antiretroviral agent, ddC, to rodents elicited long-lasting bilateral pain hypersensitivity (Bhangoo et al. 2007a, b). Under normal conditions, the expression of CXCR4 in the DRG is limited to satellite glial cells and some neurons. However, following NRTI treatment the expression of SDF-1/CXCR4 was upregulated by DRG neurons of diverse sizes. Perhaps more relevant to the clinical situation, application of gp120 to the sciatic nerve produced upregulation of MCP-1 and CCR2 in DRG neurons together with associated, unilateral pain hypersensitivity. Appropriately, this behavior could be reversed by a CCR2 receptor antagonist. However, when gp120 and ddC were combined, both MCP1/CCR2 and SDF1/CXCR4 were upregulated (Bhangoo et al. unpublished observations). Under these circumstances, the upregulation of SDF-1/ CXCR4 is much greater than with just ddC treatment alone, and many neurons express this ligand and receptor. Interestingly, in this situation the pain hypersensitivity behavior is blocked by AMD 3100 but not by a CCR2 receptor blocker, indicating that CXCR4 signaling is the main component underlying this pain. These findings are interesting because with NRTI/gp120 treatment both MCP-1/CCR2 and SDF1/

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CXCR4 are upregulated. It is not clear why CXCR4 signaling is dominant. However, one interesting possibility is that if CCR2 and CXCR4 are upregulated in the same neurons, they may form heterodimers in which CXCR4 is dominant (Sohy et al. 2007). Indeed, the ability of chemokine receptors to homo or heterodimerize with other chemokine receptors or with other receptors such as opioid receptors raises a whole new set of possible considerations when investigating how chemokines may regulate neuronal functions.

9.5 Conclusions The above discussion highlights some of the many roles that chemokine signaling has now been shown to play in the nervous system. The important role of SDF-1/ CXCR4 in development is clear, as is the role of MCP-1/CCR2 in the genesis of neuropathology. One concept that has emerged is the idea that chemokines can act as neurotransmitters. Thus, in the examples discussed above, it can be seen that SDF-1 (in the DG) and MCP-1 (in the DRG) can both play such a role. It is likely that other chemokines that are expressed in neurons under different circumstances can also act as neuromodulators. Overall, it now appears that chemokines are extremely versatile neuromodulators that are important in virtually every aspect of brain development, function, and pathology.

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Kadi L, Selvaraju R, de Lys P, Proudfoot AE, Wells TN, Boschert U (2006) Differential effects of chemokines on oligodendrocyte precursor proliferation and myelin formation in  vitro. J Neuroimmunol 174:133–146 Kanda H, Tateya S, Tamori Y, Kotani K, Hiasa K, Kitazawa R, Kitazawa S, Miyachi H, Maeda S, Egashira K, Kasuga M (2006) MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity. J Clin Invest 116:1494–1505 Kaul M, Garden GA, Lipton SA (2001) Pathways to neuronal injury and apoptosis in HIVassociated dementia. Nature 410:988–994 Klein RS, Rubin JB (2004) Immune and nervous system CXCL12 and CXCR4: parallel roles in patterning and plasticity. Trends Immunol 25:306–314 Klein RS, Rubin JB, Gibson HD, DeHaan EN, Alvarez-Hernandez X, Segal RA, Luster AD (2001) SDF-1 alpha induces chemotaxis and enhances Sonic hedgehog-induced proliferation of cerebellar granule cells. Development 128:1971–1981 Knaut H, Werz C, Geisler R, Nusslein-Volhard C (2003) A zebrafish homologue of the chemokine receptor Cxcr4 is a germ-cell guidance receptor. Nature 421:279–282 Knaut H, Blader P, Strahle U, Schier AF (2005) Assembly of trigeminal sensory ganglia by chemokine signaling. Neuron 47:653–666 Kolodziej A, Schulz S, Guyon A, Wu DF, Pfeiffer M, Odemis V, Höllt V, Stumm R (2008) Tonic activation of CXC chemokine receptor 4 in immature granule cells supports neurogenesis in the adult dentate gyrus. J Neurosci 28:4488–4500 Krathwohl MD, Kaiser JL (2004a) Chemokines promote quiescence and survival of human neural progenitor cells. Stem Cells 22:109–118 Krathwohl MD, Kaiser JL (2004b) HIV-1 promotes quiescence in human neural progenitor cells. J Infect Dis 190:216–226 Kucia M, Reca R, Jala VR, Dawn B, Ratajczak J, Ratajczak MZ (2005a) Bone marrow as a home of heterogenous populations of nonhematopoietic stem cells. Leukemia 19:1118–1127 Kucia M, Reca R, Miekus K, Wanzeck J, Wojakowski W, Janowska-Wieczorek A, Ratajczak J, Ratajczak MZ (2005b) Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1-CXCR4 axis. Stem Cells 23:879–894 Kucia M, Wojakowski W, Reca R, Machalinski B, Gozdzik J, Majka M, Baran J, Ratajczak J, Ratajczak MZ (2006) The migration of bone marrow-derived non-hematopoietic tissue-committed stem cells is regulated in an SDF-1-, HGF-, and LIF-dependent manner. Arch Immunol Ther Exp (Warsz) 54:121–135 Lapidot T, Dar A, Kollet O (2005) How do stem cells find their way home? Blood 106:1901–1910 Li Q, Shirabe K, Kuwada JY (2004) Chemokine signaling regulates sensory cell migration in zebrafish. Dev Biol 269:123–136 Lieberam I, Agalliu D, Nagasawa T, Ericson J, Jessell TM (2005) A Cxcl12-CXCR4 chemokine signaling pathway defines the initial trajectory of mammalian motor axons. Neuron 47:667–679 Lipton SA, Yeh M, Dreyer EB (1994) Update on current models of HIV-related neuronal injury: platelet-activating factor, arachidonic acid and nitric oxide. Adv Neuroimmunol 4:181–188 Liu XS, Zhang ZG, Zhang RL, Gregg SR, Wang L, Yier T, Chopp M (2007) Chemokine ligand 2 (CCL2) induces migration and differentiation of subventricular zone cells after stroke. J Neurosci Res 85:2120–2125 Lu M, Grove EA, Miller RJ (2002) Abnormal development of the hippocampal dentate gyrus in mice lacking the CXCR4 chemokine receptor. Proc Natl Acad Sci USA 99:7090–7095 Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, Kishimoto T, Bronson RT, Springer TA (1998) Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc Natl Acad Sci USA 95:9448–9453 Maysami S, Nguyen D, Zobel F, Pitz C, Heine S, Hopfner M, Stangel M (2006) Modulation of rat oligodendrocyte precursor cells by the chemokine CXCL12. Neuroreport 17:1187–1190 McArthur JC, Haughey N, Gartner S, Conant K, Pardo C, Nath A, Sacktor N (2003) Human immunodeficiency virus-associated dementia: an evolving disease. J Neurovirol 9:205–221

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Okamoto S, Kang YJ, Brechtel CW, Siviglia E, Russo R, Clemente A, Harrop A, McKercher S, Kaul M, Lipton SA (2007) HIV/gp120 decreases adult neural progenitor cell proliferation via checkpoint kinase-mediated cell-cycle withdrawal and G1 arrest. Cell Stem Cell 1:230–236 Orimo A, Weinberg RA (2006) Stromal fibroblasts in cancer: a novel tumor-promoting cell type. Cell Cycle 5:1597–1601 Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem R, Carey VJ, Richardson AL, Weinberg RA (2005) Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121:335–348 Palmer TD, Willhoite AR, Gage FH (2000) Vascular niche for adult hippocampal neurogenesis. J Comp Neurol 425:479–494 Pardo CA, McArthur JC, Griffin JW (2001) HIV neuropathy: insights in the pathology of HIV peripheral nerve disease. J Peripher Nerv Syst 6:21–27 Paredes MF, Li G, Berger O, Baraban SC, Pleasure SJ (2006) Stromal-derived factor-1 (CXCL12) regulates laminar position of Cajal-Retzius cells in normal and dysplastic brains. J Neurosci 26:9404–9412 Qin X, Wan Y, Wang X (2005) CCL2 and CXCL1 trigger calcitonin gene-related peptide release by exciting primary nociceptive neurons. J. Neurosci Res 82(1):51–62 Rance NE, McArthur JC, Cornblath DR, Landstrom DL, Griffin JW, Price DL (1988) Gracile tract degeneration in patients with sensory neuropathy and AIDS. Neurology 38:265–271 Ratajczak MZ, Zuba-Surma E, Kucia M, Reca R, Wojakowski W, Ratajczak J (2006) The pleiotropic effects of the SDF-1-CXCR4 axis in organogenesis, regeneration and tumorigenesis. Leukemia 20:1915–1924 Raz E (2003) Primordial germ-cell development: the zebrafish perspective. Nat Rev Genet 4:690–700 Rehimi R, Khalida N, Yusuf F, Dai F, Morosan-Puopolo G, Brand-Saberi B (2008) Stromal-derived factor-1 (SDF-1) expression during early chick development. Int J Dev Biol 52:87–92 Robin AM, Zhang ZG, Wang L, Zhang RL, Katakowski M, Zhang L, Wang Y, Zhang C, Chopp M (2006) Stromal cell-derived factor 1alpha mediates neural progenitor cell motility after focal cerebral ischemia. J Cereb Blood Flow Metab 26:125–134 Romero-Sandoval EA, Horvath RJ, DeLeo JA (2008) Neuroimmune interactions and pain: focus on glial-modulating targets. Curr Opin Investig Drugs 9:726–734 Sacktor N, McDermott MP, Marder K, Schifitto G, Selnes OA, McArthur JC, Stern Y, Albert S, Palumbo D, Kieburtz K, De Marcaida JA, Cohen B, Epstein L (2002) HIV-associated cognitive impairment before and after the advent of combination therapy. J Neurovirol 8:136–142 Sano R, Tessitore A, Ingrassia A, d’Azzo A (2005) Chemokine-induced recruitment of genetically modified bone marrow cells into the CNS of GM1-gangliosidosis mice corrects neuronal pathology. Blood 106:2259–2268 Schifitto G, McDermott MP, McArthur JC, Marder K, Sacktor N, Epstein L, Kieburtz K (2002) Incidence of and risk factors for HIV-associated distal sensory polyneuropathy. Neurology 58:1764–1768 Schönemeier B, Kolodziej A, Schulz S, Jacobs S, Hoellt V, Stumm R (2008) Regional and cellular localization of the CXCl12/SDF-1 chemokine receptor CXCR7 in the developing and adult rat brain. J Comp Neurol 510:207–220 Schreiber RC, Krivacic K, Kirby B, Vaccariello SA, Wei T, Ransohoff RM, Zigmond RE (2001) Monocyte chemoattractant protein (MCP)-1 is rapidly expressed by sympathetic ganglion neurons following axonal injury. Neuroreport 12:601–606 Schwarting GA, Henion TR, Nugent JD, Caplan B, Tobet S (2006) Stromal cell-derived factor-1 (chemokine C-X-C motif ligand 12) and chemokine C-X-C motif receptor 4 are required for migration of gonadotropin-releasing hormone neurons to the forebrain. J Neurosci 26:6834–6840 Scotton CJ, Chambers RC (2007) Molecular targets in pulmonary fibrosis: the myofibroblast in focus. Chest 132:1311–1321

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Sierro F, Biben C, Martinez-Munoz L, Mellado M, Ransohoff RM, Li M, Woehl B, Leung H, Groom J, Batten M, Harvey RP, Martinez AC, Mackay CR, Mackay F (2007) Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7. Proc Natl Acad Sci USA 104:14759–14764 Snider P, Olaopa M, Firulli AB, Conway SJ (2007) Cardiovascular development and the colonizing cardiac neural crest lineage. ScientificWorldJournal 7:1090–1113 Sohy D, Parmentier M, Springael JY (2007) Allosteric transinhibition by specific antagonists in CCR2/CXCR4 heterodimers. J Biol Chem 282:30062–30069 Steiner B, Wolf S, Kempermann G (2006) Adult neurogenesis and neurodegenerative disease. Regen Med 1:15–28 Stumm RK, Rummel J, Junker V, Culmsee C, Pfeiffer M, Krieglstein J, Hollt V, Schulz S (2002) A dual role for the SDF-1/CXCR4 chemokine receptor system in adult brain: isoform-selective regulation of SDF-1 expression modulates CXCR4-dependent neuronal plasticity and cerebral leukocyte recruitment after focal ischemia. J Neurosci 22:5865–5878 Stumm RK, Zhou C, Ara T, Lazarini F, Dubois-Dalcq M, Nagasawa T, Hollt V, Schulz S (2003) CXCR4 regulates interneuron migration in the developing neocortex. J Neurosci 23:5123–5130 Stumm R, Kolodziej A, Schulz S, Kohtz JD, Hollt V (2007) Patterns of SDF-1alpha and SDF1gamma mRNAs, migration pathways, and phenotypes of CXCR4-expressing neurons in the developing rat telencephalon. J Comp Neurol 502:382–399 Sun JH, Yang B, Donnelly DF, Ma C, LaMotte RH (2006) MCP-1 enhances excitability of nociceptive neurons in chronically compressed dorsal root ganglia. J Neurophysiol 96:2189–2199 Tachibana K, Hirota S, Iizasa H, Yoshida H, Kawabata K, Kataoka Y, Kitamura Y, Matsushima K, Yoshida N, Nishikawa S, Kishimoto T, Nagasawa T (1998) The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature 393:591–594 Tanaka T, Minami M, Nakagawa T, Satoh M (2004) Enhanced production of monocyte chemoattractant protein-1 in the dorsal root ganglia in a rat model of neuropathic pain: possible involvement in the development of neuropathic pain. Neurosci Res 48:463–469 Thacker MA, Clark AK, Bishop T, Grist J, Yip PK, Moon LD, Thompson SW, Marchand F, McMahon SB (2008) CCL2 is a key mediator of microglia activation in neuropathic pain states. Eur J Pain 13(3):263–272 Thored P, Arvidsson A, Cacci E, Ahlenius H, Kallur T, Darsalia V, Ekdahl CT, Kokaia Z, Lindvall O (2006) Persistent production of neurons from adult brain stem cells during recovery after stroke. Stem Cells 24:739–747 Tissir F, Wang CE, Goffinet AM (2004) Expression of the chemokine receptor Cxcr4 mRNA during mouse brain development. Brain Res Dev Brain Res 149:63–71 Tiveron MC, Rossel M, Moepps B, Zhang YL, Seidenfaden R, Favor J, Konig N, Cremer H (2006) Molecular interaction between projection neuron precursors and invading interneurons via stromal-derived factor 1 (CXCL12)/CXCR4 signaling in the cortical subventricular zone/ intermediate zone. J Neurosci 26:13273–13278 Toews AD, Barrett C, Morell P (1998) Monocyte chemoattractant protein 1 is responsible for macrophage recruitment following injury to sciatic nerve. J Neurosci Res 53:260–267 Toggas SM, Masliah E, Rockenstein EM, Rall GF, Abraham CR, Mucke L (1994) Central nervous system damage produced by expression of the HIV-1 coat protein gp120 in transgenic mice. Nature 367:188–189 Tran PB, Miller RJ (2003) Chemokine receptors: signposts to brain development and disease. Nat Rev Neurosci 4:444–455 Tran PB, Ren D, Veldhouse TJ, Miller RJ (2004) Chemokine receptors are expressed widely by embryonic and adult neural progenitor cells. J Neurosci Res 76:20–34 Tran PB, Banisadr G, Ren D, Chenn A, Miller RJ (2007) Chemokine receptor expression by neural progenitor cells in neurogenic regions of mouse brain. J Comp Neurol 500:1007–1033 Uçeyler N, Sommer C (2008) Cytokine regulation in animal models of neuropathic pain and in human diseases. Neurosci Lett 437:194–198

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Valentin G, Haas P, Gilmour D (2007) The chemokine SDF1a coordinates tissue migration through the spatially restricted activation of Cxcr7 and Cxcr4b. Curr Biol 17:1026–1031 Wallace VC, Blackbeard J, Segerdahl AR, Hasnie F, Pheby T, McMahon SB, Rice AS (2007) Characterization of rodent models of HIV-gp120 and anti-retroviral-associated neuropathic pain. Brain 130(Pt 10):2688–2702 Wang JG, Strong JA, Xie W, Yang RH, Coyle DE, Wick DM, Dorsey ED, Zhang JM (2008) The chemokine CXCL1/growth related oncogene increases sodium currents and neuronal excitability in small diameter sensory neurons. Mol Pain 4:38 Watkins LR, Hutchinson MR, Ledeboer A, Wieseler-Frank J, Milligan ED, Maier SF (2007a) Norman Cousins Lecture. Glia as the “bad guys”: implications for improving clinical pain control and the clinical utility of opioids. Brain Behav Immun 21:131–146 Watkins LR, Hutchinson MR, Milligan ED, Maier SF (2007b) “Listening” and “talking” to neurons: implications of immune activation for pain control and increasing the efficacy of opioids. Brain Res Rev 56:148–169 Welner RS, Kincade PW (2007) Stem cells on patrol. Cell 131:842–844 Westmoreland SV, Rottman JB, Williams KC, Lackner AA, Sasseville VG (1998) Chemokine receptor expression on resident and inflammatory cells in the brain of macaques with simian immunodeficiency virus encephalitis. Am J Pathol 152:659–665 White FA, Sun J, Waters SM, Ma C, Ren D, Ripsch M, Steflik J, Cortright DN, Lamotte RH, Miller RJ (2005) Excitatory monocyte chemoattractant protein-1 signaling is up-regulated in sensory neurons after chronic compression of the dorsal root ganglion. Proc Natl Acad Sci USA 102:14092–14097 White FA, Jung H, Miller RJ (2007) Chemokines and the pathophysiology of neuropathic pain. Proc Natl Acad Sci USA 104:20151–20158 Williams KC, Hickey WF (2002) Central nervous system damage, monocytes and macrophages, and neurological disorders in AIDS. Annu Rev Neurosci 25:537–562 Xia M, Qin S, McNamara M, Mackay C, Hyman BT (1997) Interleukin-8 receptor B immunoreactivity in brain and neuritic plaques of Alzheimer’s disease. Am J Pathol 150:1267–1274 Xu J, Mora A, Shim H, Stecenko A, Brigham KL, Rojas M (2007) Role of the SDF-1/CXCR4 axis in the pathogenesis of lung injury and fibrosis. Am J Respir Cell Mol Biol 37:291–299 Yusuf F, Rehimi R, Dai F, Brand-Saberi B (2005) Expression of chemokine receptor CXCR4 during chick embryo development. Anat Embryol (Berl) 210:35–41 Zhang J, De Koninck Y (2006) Spatial and temporal relationship between monocyte chemoattractant protein-1 expression and spinal glial activation following peripheral nerve injury. J Neurochem 97:772–783 Zhang J, Shi XQ, Echeverry S, Mogil JS, De Koninck Y, Rivest S (2007) Expression of CCR2 in both resident and bone marrow-derived microglia plays a critical role in neuropathic pain. J Neurosci 27:12396–12406 Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR (1998) Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393:595–599

Chapter 10

Modulation of Neuronal Cell Cycle Proteins by Chemokine Receptors and Its Role in the Survival of Postmitotic Neurons Muhammad Z. Khan

10.1 Introduction Chemokines or chemotactic cytokines are the only subgroup of cytokines that bind and signal through 7-transmembrane G-protein coupled receptors. Chemokines are 8–13  Kd proteins and have well-conserved roles in the development and normal functioning of immune as well as nervous system through evolution. Structurally, chemokines have been divided into four groups (CXC, CC, C, and CX3C) on the basis of the pattern of cysteins in their N-terminal region. Here, I will review the current knowledge of chemokine receptor signaling focusing on CXCR4, CCR5, CXCR2, and CCR2 and their effect in the brain in the context of neuroAIDS.

10.2 General Structure of Chemokines and Their Receptors Despite low homology across the family, chemokines exhibit a remarkably similar tertiary structure as determined by NMR and X-ray crystallography studies. Their N-terminal consists of 6–10 amino acids and serves as the key signaling domain in all chemokines without any exception (Allen et al. 2007). Deletion or modification of N terminal significantly alters the signaling induction by chemokines, in many cases converting the truncated molecules into antagonists (e.g. CCL2, CCL7, CCL8) and in some instances (i.e. CXCL12) leading to a truncated chemokine that is unable to bind to its endogenous receptor (Gong and Clark-Lewis 1995; Crump et al. 1997; Loetscher et al. 1998; Zhang et al. 2003; Van Damme et al. 2004; Allen et al. 2007). Removal of N termini of chemokines through the action of specific proteases, like matrix metalloproteases (MMPs), is known to be an important, naturally existing modulatory mechanism of chemokine signaling (McQuibban et al. 2001; Guan et al. 2002; Overall

M.Z. Khan () Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102, USA e-mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_10, © Springer Science+Business Media, LLC 2010

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et  al. 2002; Guan et  al. 2004; Parks et  al. 2004; Van Damme et  al. 2004). The amino terminal is followed by an N-loop which contains important binding elements like 310 helix (from Arg20 to Val23 in CXCL12) and is considered an important docking site in receptor activation. N-loop is followed by a three stranded b-helix, and a C-terminal helix. Disulfide bonds among cysteins stabilize the overall tertiary structure (Allen et al. 2007). Many chemokines can polymerize to form dimers or higher order polymers in solution in millimolar to micromolar concentration range or upon binding glycosaminoglycans present on various cell surfaces and such interactions might be involved in chemokine gradient formation in those tissues (Handel et al. 2005; Johnson et al. 2005). Despite this, chemokines have been shown to bind their receptors as monomers to evoke cell migration (Allen et al. 2007). This result was demonstrated with either specific mutations or synthetic variations of MCP-1 (CCL2), IL-8 (CXCL8), CCL4, and CCL5 that are obligate monomers (Rajarathnam et al. 1994; Paavola et al. 1998; Laurence et al. 2000; Proudfoot et al. 2003). Though CXCL12 is unlikely to exist as a dimer in vivo because of a dimerization constant which is significantly higher than its physiological concentration, high ionic strength and binding to glycosaminoglycans at the cell surface can increase its dimerization (Baryshnikova and Sykes 2006). All chemokine receptors exhibit significant homology in their molecular structure. These receptors contain 340–370 amino acids and consist of 7-membrane spanning, helical regions, connected by extra membranous loops. N terminal and three extracellular loops are exposed outside of the cell while C terminal and three intracellular loops lie inside the cell. As with most other 7-transmembrane receptors, no chemokine receptor structure has been solved as yet. All the available models in the literature are based on bovine rhodopsin, the only G-protein coupled receptor (GPCR) whose three-dimensional structure has been resolved, which shows about 20% homology with the rest of the GPCR family members (Allen et al. 2007). Similar to other GPCRs, intracellular loops of chemokine receptors are bound to heterotrimeric G proteins (abg) through Ga and signal through these proteins. In the following sections of this chapter, a detailed discussion on the signaling pathways of specific chemokine receptors, i.e. CXCR4, CCR5, CCR2, and CXCR3 is conducted with a focus on their role in neuronal physiology and pathology, particularly in neuroAIDS.

10.3 Receptor Dynamics 10.3.1 Receptor Oligomerization GPCRs are known to exist as monomers or oligomers. Likewise chemokine receptors such as CXCR4, CCR5, and CCR2 exist as homo or heterodimers and these interactions have been studied in nonneuronal cells (Wang and Norcross 2008). CXCR4 is known to exist constitutively as a homodimer, an event that might occur

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probably soon after protein translation and receptor units moving to cell membrane as dimers (Babcock et  al. 2003; Wang et  al. 2006). Some reports suggest that CXCL12 is able to induce CXCR4 dimerization in a time dependent manner (VilaCoro et al. 1999; Babcock et al. 2003; Toth et al. 2004). There are other reports which are unable to support this hypothesis but do support the possibility of ligandinduced conformational changes in the dimerized receptor units (Percherancier et  al. 2005). CXCR4 has also been observed to heterodimerize with the other chemokine receptors like CCR5 and CCR2 and such oligomerization is shown to have important roles in cell migration (Sohy et al. 2007; Wang and Norcross 2008). Particularly, a recent study shows that in T lymphocytes, CXCR4 and CCR5 heterodimerize constitutively (Contento et  al. 2008). Such an interaction between these two receptors is important for their function at immunological synapse and is involved in T cell activation (Contento et al. 2008). Another study provides additional evidence for CCR5-CXCR4 heteromerization and shows that CCR5 agonists enhance this coupling (Isik et al. 2008). Furthermore, the interaction between these two principal HIV coreceptors could be involved in the “coreceptor switch”. In 50 % of HIV infected patients, after initial infection with CCR5-using (nonsyncytium inducing) strains, CXCR4-using (syncytium inducing) strains become predominant at the later stages of disease (Schuitemaker et  al. 1992; Richman and Bozzette 1994; Maas et al. 2000). For CCR5, specific residues in TM1 and TM4, Ile52 and Val150 in particular, have been implicated in dimerization (Hernanz-Falcon et  al. 2004). A double mutant of the receptor was unable to dimerize or signal upon stimulation. CCR5 has also been shown to make heterodimers with CCR2 and ligands of each receptor can block the signaling of the other receptor (Rodriguez-Frade et al. 2004). Earlier studies indicated that heterodimerization could be involved in modulating the chemokine receptor signaling (Mellado et  al. 2001). These data have been confirmed by more recent studies (Pello et al. 2008). Monocyte chemoattractant protein-1 (MCP-1), also known as CCL2, is able to induce the homodimerization of its receptor, CCR2 and is able to induce specific functional responses (Rodriguez-Frade et al. 1999). It has been shown that with a specific monoclonal antibody against CCR2, CCR2-01, that does not interfere with chemokine signaling or induce receptor downregulation, HIV-1 infection via CCR5 and CXCR4 could be blocked (Rodriguez-Frade et al. 2004). This was shown to be due to enhancement of oligomerization of CCR2 with CCR5 and CXCR4 using immunoprecipitation and FRET. Moreover, in humans, a specific CCR2 mutation (CCR2V64I) has been found that helps delay the AIDS development (Smith et al. 1997). Mutant CCR2 is able to heterodimerize with either CXCR4 or CCR5, which might result in delaying the AIDS progression in individuals carrying that mutation (Mellado et al. 1999), thus indicating the role of receptor heterodimerization in HIV infection. Overall, chemokine receptor oligomerization might have important regulatory roles in receptor signaling at various levels ranging from ligand binding to signaling specificity, as well as receptor trafficking, with significant therapeutic implications.

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10.3.2 Ligand Receptor Binding According to a model initially advanced by Clark-Lewis et al. (1995) and supported later by several studies as reviewed in Allen et al. (2007), chemokines possess two important sets of sites to interact with their receptors and this interaction takes place in two steps accordingly. First, N-loop region of the chemokine binds the receptor with high affinity (called the “docking step”). For certain chemokines, specific residues distributed on the entire core domain are also involved in this reaction. This event leads to binding and positioning of flexible N-terminal region to specific contact points on receptor surface (called the “triggering step”); a low affinity event that results in receptor activation.

10.3.2.1 CXCR4 Like all chemokine-receptor binding events, CXCL12 and CXCR4 interaction also takes place in two steps. The first step consists of an initial interaction between residues 12–17 of CXCL12 and 2–36 of CXCR4 (Crump et al. 1997; Huang et al. 2003). The receptor affinity for this step is enhanced by the sulfation of specific tyrosine residues in the N-terminal region of the receptor (Farzan et al. 2002; Veldkamp et al. 2006). This interaction brings about a conformational change in the receptor that facilitates the interaction between the first eight amino acid residues on the N terminal of CXCL12 and an exposed binding area of CXCR4 that includes specific residues from second and third extracellular loops (Brelot et al. 2000; Zhou et al. 2001). This step can be interrupted through the cleavage of specific N terminal residues on CXCL12 by certain proteases like cathepsin G and elastase released by neutrophils or dipeptidase 26 (CD26) during an inflammatory response as reviewed in Van Damme et al. (2004). A neutrophil elastase has also been shown to cleave N terminal domain of CXCR4 (Valenzuela-Fernandez et al. 2002). In the context of neuroAIDS, HIV-infected macrophages secrete matrix metalloproteinase-2 which is activated by a neuronal membrane type 1 matrix metalloproteinase (MT1-MMP). Activated MMP2 cleaves the first four residues from the CXCL12 N terminal (Zhang et al. 2003). This truncated CXCL12 is unable to bind CXCR4 and is also unable to induce chemotaxis and inhibition of HIV entry into the cells (Zhang et  al. 2003). Cleaved CXCL12 is upregulated in HIV/dementia patients and causes neurodegeneration by signaling through CXCR3 (Zhang et al. 2003; Vergote et al. 2006).

10.3.2.2 CCR5 Several chemokines bind and signal through CCR5, including CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), and CCL8 (MCP-2) which bind to this receptor with high affinity. The chemokines that bind CCR5 with low affinity are CCL7 (MCP-3), CCL11 (Eotaxin), and CCL13 (MCP-4). The globular core domain of

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chemokines interacts with specific extracellular regions of CCR5, including the N-terminal and second extracellular loop (Samson et  al. 1997; Blanpain et  al. 2003). The interaction of chemokines with specific aromatic residues in the second extracellular loop plays a main role in ligand specificity as suggested by mutagenesis studies including binding of chimeric chemokine MIP/RANTES to specific CCR5 mutants (Blanpain et al. 2003). Binding of chemokines to specific aromatic residues at the extracellular border between the second and third extracellular loops might be involved in receptor activation (Govaerts et al. 2003).The N-terminal of CCR5 exhibits negatively charged and aromatic residues, in particular sulfated tyrosines that provide a flexible and negatively charged area for ligand binding (Blanpain et  al. 1999; Farzan et  al. 1999). It is the N-terminal of CCR5-binding chemokines that interacts with the second transmembrane helix of the receptor. Depending upon the ligand, specific motifs play important role in receptor activation as suggested by the mutagenesis studies (Govaerts et al. 2001; Blanpain et al. 2003). Studies on several CC chemokines including MIP-1b, RANTES, MCP-1, and eotaxin indicate a common receptor binding area on the chemokine molecules involving patches of basic residues separated by a hydrophobic groove that includes N-loop and 310 helix (Blanpain et al. 2003). 10.3.2.3 CCR2 CCR2 is a receptor for b-chemokines like CCL2 (MCP-1), CCL7 (MCP-3), CCL8 (MCP-2), CCL13 (MCP-4), and CCL16, though it preferentially binds CCL2 (MCP-1). CCR2 is expressed as two alternatively spliced isoforms, CCR2a and CCR2b. These isoforms are identical except for their C-termini. CCR2 shares a 71% sequence homology with CCR5 as reviewed in Oppermann (2004). The lack of homology between CCR5 and CCR2 receptors in ligand binding regions is reflected in their mutually exclusive sets of chemokine ligands (Combadiere et al. 1996; Raport et al. 1996). In a comparative study between CCR5 and CCR2b, the second extracellular loop of CCR5 was found to be critical for high affinity binding and for functional responses, as well as ligand specificity, and in this region, 16 out of 26 residues are different between these receptors (Samson et al. 1997). For CCR2b on the other hand, in the amino terminal domain, specific acidic and hydrophobic residues play a fundamental role in binding. In this region, there is a high level of divergence in sequences between CCR2b and CCR5, which might account for their respective ligand specificities (Samson et al. 1997; Han et al. 1999). On MCP-1, along with its N-terminal region, two patches of basic residues (consisting of first disulfide bridge and residues 13–30) separated by a 35 A groove, are responsible for the 50% binding affinity, while R24 residue has the next largest impact on receptor binding (Hemmerich et  al. 1999; Jarnagin et  al. 1999). In CCR2b structure, low affinity interaction of N-terminal region of MCP-1 with the second extracellular loop as well as the first and third loops is required for receptor activation and signaling (Hemmerich et  al. 1999). Truncation of chemokine N-terminal completely abrogates chemotaxis (Zhang et al. 1994; Gong et al. 1997).

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10.3.2.4 CXCR3 CXCR3 is the receptor for chemokines i.e. CXCL4, CXCL9 (MIG), CXCL10 (IP10), and CXCL11 (I-TAC). For the main CXCR3 ligand CXCL11 (I-TAC), the data indicate that both N-terminal and N-loop regions contribute to high receptor binding affinity. N terminal region of I-TAC is critical for inducing functional responses from CXCR3 such as chemotaxis and transient calcium influx, as well as receptor internalization, since the truncation of the first three residues makes I-TAC (3-73) a CXCR3 antagonist (Clark-Lewis et  al. 2003). The second extracellular loop of CXCR3 has been shown to be essential for receptor activation by all ligands, CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). N terminus and the first extracellular loop are known to be important for CXCL10- and CXCL11-mediated receptor activation (Booth et  al. 2002; Xanthou et  al. 2003). Moreover, the third extracellular loop is required only for CXCL9 and CXCL10 signaling (Xanthou et al. 2003). Hence, these chemoking ligands mainly have two principal regions for receptor binding; the N-loop region and the residues surrounding it are involved in high affinity interaction with the receptor, while the N-terminal region is involved in receptor activation. On the receptor surface, though, N-terminal residues and those in 1–3 extracellular loops might be involved in ligand binding and activation.

10.3.3 Regulation of Signaling GPCR mediated signaling is regulated by several processes: desensitization, receptor phosphorylation, internalization, and degradation, as well as loss or inactivation of important second messengers involved in the signaling. The process of desensitization results in receptor becoming nonresponsive to the continued stimulation by its ligand and it involves phosphorylation of specific serine / threonine residues on the third intracellular loop or the cytoplasmic tail of the receptor following receptor activation as reviewed in Premont and Gainetdinov (2007). The kinases involved are G protein-coupled receptor kinases (GRKs). This specific phosphorylation leads to the binding of arrestins (arrestin 2–3) to the receptor, thereby uncoupling it from heterotrimeric G proteins and targeting the receptor for internalization (Premont and Gainetdinov 2007). 10.3.3.1 CXCR4 Upon activation by CXCL12, CXCR4 is quickly phosphorylated and internalized. Several residues in its C-terminus tail have been identified as potential phosphorylation sites by truncation and mutagenesis studies as reviewed in Busillo and Benovic (2007). Removal of 45 amino acid residues from the C-terminal of CXCR4 led to elimination of CXCL12-induced phoshorylation, enhanced receptor activity

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and attenuation of its internalization (Haribabu et  al. 1997). In another study, mutation of Ser338 and Ser339 or truncation of serines 346–348 and 351–352 led to reduction in CXCL12-mediated phosphorylation (Orsini et al. 1999). GRK2 and GRK6 have been implicated in the agonist-induced CXCR4 C-tail phosphorylation in overexpression (GRK2) and gene knock-out studies (GRK6) (Orsini et al. 1999; Cheng et al. 2000; Fong et al. 2002; Vroon et al. 2004). Overexpression and gene disruption studies indicate the role of b-arrestin-2 and -3 in CXCR4 desensitization and internalization (Orsini et al. 1999; Cheng et al. 2000; Sun et al. 2002). CXCR4 also undergoes heterologous desensitization following protein kinase C (PKC) mediated phosphorylation on specific serine residues on its C-tail (Haribabu et al. 1997; Signoret et al. 1997; Orsini et al. 1999). Phosphorylation at Ser 324 and 325, as well as Ser 338 and 339, is involved in phorbol ester-mediated CXCR4 desensitization (Signoret et al. 1998; Woerner et al. 2005). Several physiological stimuli including activation of other chemokine receptors such as CXCR1, CXCR2, and CCR5 have been shown to induce PKC mediated CXCR4 internalization (Hecht et al. 2003; Richardson et al. 2003; Suratt et al. 2004). Recent studies conducted in our lab indicate that stimulation of µ opioid receptors leads to CXCR4 desensitization by novel mechanisms involving the upregulation of the protein ferritin heavy chain (FHC) (Sengupta et al. 2009) which has earlier been reported as a negative regulator of CXCR4 signaling (Li et al. 2006). RNAi-mediated depletion of FHC in neurons is able to abrogate the negative regulation of CXCR4 by the opioid receptor (Sengupta et al. 2009). Tyrosine residues in CXCR4 have also been shown to be phosphorylated by direct activation of CXCR4 through CXCL12 and, indirectly, by cytokines like granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 (Vila-Coro et al. 1999; Wang et al. 2001). While CXCL12-mediated tyrosine phosphorylation might result in activation of the JAK / STAT pathway (Vila-Coro et al. 1999; Zhang et al. 2001), cytokine-induced phosphorylation might lead to agonist-independent internalization (Wang et al. 2001). Upon CXCL12 stimulation, CXCR4 is ubiquitinated on one of the three lysine residues in its C terminal tail, sorted to lysosomes (Marchese and Benovic 2001), and degraded, a process mediated by E3 ubiquitin ligase AIP4 and involving arrestin 2 (Marchese et al. 2003; Bhandari et al. 2007). CXCR4 is shown to be monoubiquitinated on specific lysine residues in its C-tail and mutation of these residues abrogates receptor ubiquitination and degradation (Marchese and Benovic 2001). 10.3.3.2 CCR5 Serine/threonine protein kinases like GRKs regulate phosphorylation of CCR5 at its C terminal that causes the receptor to uncouple from G proteins and thus switch the signal off. Overexpression studies as well as those involving GRK inhibition with specific antibodies show that GRK2 and GRK3 are mainly responsible for CCR5 phosphorylation, desensitization, and internalization (Aramori et al. 1997; Olbrich et al. 1999; Oppermann et al. 1999). Moreover, CCR5 ligands like CCL3, CCL4,

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and CCL5 are able to induce differential effects on receptor phosphorylation and internalization (Oppermann et  al. 1999). PKC, on the other hand, is known to be involved in heterologous desensitization of CCR5. Both GRKs and PKC phosphorylate the receptor at serines 336, 337, 342, and 349 (Oppermann et al. 1999). Serine phosphorylation on CCR5 C-tail is required for its binding to regulatory proteins b-arrestins-1 and -2 (Kraft et al. 2001). Another arrestin binding site was localized on the second intracellular loop of CCR5, which consists of Asp-Arg-Tyr motif and it is not associated with phosphorylation (Huttenrauch et al. 2002). This tri-peptide motif is well-conserved among GPCRs of the rhodopsin family. As mentioned before, b-arrestins are the principal regulators of GPCR signaling and are involved in their desensitization and clathrin-mediated endocytosis. After internalization, CCR5 accumulates in perinuclear recycling endosomes and is recycled back to the cell membrane (Signoret et  al. 2000; Pollok-Kopp et  al. 2003). Dissociation of ligand from CCR5 is not required for recycling because agonist-bound CCR5 is known to undergo several recycling steps and it passes through endosomes until the dissociation or degradation of ligand (Signoret et  al. 2000). This is in contrast to CXCR4, which is ubiquitinated upon agonist binding and undergoes lysosomal degradation (Tarasova et al. 1998; Marchese and Benovic 2001). 10.3.3.3 CCR2 MCP-1 binding to CCR2 leads to rapid receptor desensitization as seen in calcium flux response and it is associated with Ser/Thr phosphorylation of the receptor (Myers et  al. 1995; Franci et  al. 1996; Aragay et  al. 1998). In addition, CCR2 stimulation also induces the complex formation between b-arrestin and GRK-2 (Aragay et al. 1998). Overexpression of GRK-2 in HEK cells led to desensitization of CCR2B receptor (Franci et al. 1996). A change of eight Ser/Thr residues to alanine in the C-tail of CCR2b led to significant slow-down of desensitization and internalization after stimulation by the agonist (Franci et  al. 1996). These data underline the importance of C-tail phosphorylation in the regulation of CCR2. 10.3.3.4 CXCR3 The C-terminal of CXCR3 is made up of two domains, the distal Ser/Thr domain and the proximal tri-leucine domains, which are separated by 14 amino acids (Dagan-Berger et  al. 2006). This arrangement differs from that existing in many other CXC receptors including CXCR4. Similar to CXCR4, however, the third intracellular loop of CXCR3 contains an additional serine, a potential phosphorylation site, raising the possibility that it might be involved in interaction with b-arrestins (Cheng et al. 2000; Dagan-Berger et al. 2006). Like CCR2, only a few studies try to address the control of CXCR3 desensitization and internalization. In a study involving mutagenesis of C-terminus of CXCR3, it was shown that CXCL11 (I-TAC)-mediated internalization and cell migration required proximal trileucine domain, while, cell adhesion was regulated by Ser245 of the third intracellular loop

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(Dagan-Berger et al. 2006). On the other hand, internalization of the receptor by PKC activation required an intact, distal Ser/Thr domain (Dagan-Berger et  al. 2006). An earlier study involving CXC receptor chimeras indicated the role of the third intracellular loop in receptor internalization (Colvin et  al. 2004). Different results in these two studies could stem from different cell types as well as the fact that chimeric receptors might differ in conformation changes and performance in signaling as compared to the endogenous receptors.

10.4 Physiological Roles of Chemokine Receptor Signaling in Nervous System Among the known chemokine receptors, CXCR4 has been established to play a crucial role in the nervous system physiology as evidenced by either CXCL12 or CXCR4 homozygous mutants’ phenotype (Ma et al. 1998; Zou et al. 1998). The other receptors discussed in this review are primarily involved in inflammatory and/ or neurodegenerative disorders which is consistent with their roles as inflammatory chemokines. Recently CXCR7 has also been identified as a CXCL12 as well as CXCL11 (I-TAC) binding receptor that has been shown to be expressed at mRNA level in the various regions of CNS (Schonemeier et al. 2008). However, its physiological and pathological functions are still under investigation (Burns et al. 2006; Thelen and Thelen 2008). Recent evidence shows that CXCR7 does not serve as a signaling receptor for CXCR4 (Burns et al. 2006; Hartmann et al. 2008). The chemokine CXCL12 and its receptor CXCR4 are expressed constitutively and in a complementary fashion in the nervous system as reviewed by Li and Ransohoff (2008). During development, CXCR4 expression is mainly observed in ventricular and subventricular zones and also in the marginal zone. All these areas serve as specialized niches for neuronal precursors. In mature CNS, CXCL12/CXCR4 expression is found in several different areas of brain in cholinergic, dopaminergic, and vasopressinergic neurons (as reviewed in Li and Ransohoff (2008)). They play key roles in CNS development as indicated by studies with the CXCL12 or CXCR4-deficient mice that show considerable overlapping deficits in the nervous system resulting in embryonic lethality (Ma et al. 1998; Zou et al. 1998). In adult CNS, CXCR4 has been suggested to modulate neurotransmission, regulate cellular interaction, and play a critical role in neuronal survival (Rostene et al. 2007; Li and Ransohoff 2008).

10.4.1 Roles of CXCR4 During CNS Development CXCL12/CXCR4 signaling is involved in the laminar organization of brain cortex by directly regulating the migration of neuronal precursors (Tiveron and Cremer 2008). New born neuronal precursors arise in the ventricular zone and migrate radially and tangentially to form cortical layers. In the developing brain cortex

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Cajal–Retzius cells, a short-lived population of neurons, migrate first radially and then tangentially and help maintain glial scaffold to help facilitate neuronal migration (Frotscher 1998; Marin-Padilla 1998). CXCL12/CXCR4 signaling is shown to be crucial in the migration of Cajal–Retzius cells (Stumm et al. 2003; Borrell and Marin 2006; Paredes et al. 2006). CXCR4 signaling has also been implicated in the proliferation and migration of cerebellar granule cell precursors during cerebellar development (Zou et  al. 1998; Klein et  al. 2001; Reiss et  al. 2002; Yacubova and Komuro 2003). Specifically, CXCL12 is able to enhance their proliferation by Sonic hedgehog (SHH) (Klein et al. 2001). In vivo studies show that CXCR4 signaling is involved in the development and migration of dentate gyrus granule cells as well (Bagri et al. 2002; Lu et al. 2002). For both cerebellar granule and dentate gyrus precursors, CXCL12 expression is localized in the overlaying meninges, while neuronal precursors express CXCR4 receptor and the chemotactic response to the CXCL12 gradient leads to their migration and specific laminar distribution (Borrell and Marin 2006; Stumm and Hollt 2007). CXCR4 is also involved in the migration of sensory neuronal precursors in dorsal root ganglia and migration of gonadotropin-releasing hormone (GnRH) neurons from the vomeronasal organ to forebrain (Li and Ransohoff 2008; Miller et al. 2008). Migration and laminar distribution of GABAergic interneurons are regulated by CXCL12/CXCR4 signaling in the cortex (Lopez-Bendito et al. 2008). Earlier studies indicated that CXCL12 is expressed along the migratory pathways of those neurons and is involved in their tangential migration inside the cortex (Stumm et al. 2003, 2007). A recent study shows that CXCR4 regulates the expression of an isoform of glutamic acid decarboxylase 67 (GAD67), one of the key rate limiting enzymes for the synthesis of GABA via Erk and early growth response gene (Egr1) signaling in neurons (Luo et al. 2008). Thus, CXCR4 plays a key role in the development as well as maturation and location of specific sub-populations of cortical neurons. CXC12 is a well-established axon guidance cue that serves to regulate and direct axonal projections to their specific synaptic targets in order to establish complete neural circuitry during brain development (Stumm and Hollt 2007). CXCL12 does not serve as a chemoattractant for neurons; rather, it reduces axonal response to several repellents. For instance CXCL12 is able to reduce the effects of the following repellents in different locations of the nervous system: slit/robo signaling on cultured retinal ganglion cell axons (Chalasani et  al. 2007), semaphoring 3A on dorsal root ganglion sensory axons, and semaphoring 3C on sympathetic neurons (Chalasani et  al., 2003b). In slit-2 mediated growth cone collapse in  vitro, both PTX and CXCR4 antagonist AMD3100 are able to block CXCL12 effects. In such experiments, cAMP as well as protein kinase A antagonists can also abrogate CXCL12 effects (Chalasani et  al. 2003b). Therefore, CXCR4 acivity results in Gi-mediated cAMP elevation which affects axonal outgrowth via protein kinase A regulation (Chalasani et al. 2003b). In murine cerebellar granule neurons, CXCL12 is able to affect axonal length by stimulation of Rho-GTPase-dependent pathway (Arakawa et  al. 2003). Furthermore, CXCL12 stimulation reduces growth cone number and axonal outgrowth, but stimulates axonal branching in rat hippocampal neurons in  vitro (Pujol et  al. 2005). Hence CXCL12/CXCR4 signaling is an

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important regulator of axon guidance for specific types of neurons in distinct areas of the nervous system.

10.4.2 Roles of CXCR4 in Mature CNS CXCR4 is expressed constitutively in adult neurons and glia (see Chap. 12 and Chap. 7 in this volume). Several studies implicated CXCL12/CXCR4 signaling as a modulator of neuronal activity in vasopressinergic neurons, MCH neurons, and GABAergic neurons as reviewed by (Guyon and Nahon 2007). Neural stem cells or neural precursor cells (NPC) are self-renewing, multipotent cells that can differentiate into neurons, astrocytes, and oligodendrocytes during the development and in adult CNS (Gage et al. 1995; Brustle et al. 1997, 1999). These cells are associated with neurogenesis and tissue repair. CXCR4 signaling has been implicated in NPC migration in adult brain (Miller and Tran 2005; Tran and Miller 2005). Human NPC have been observed to migrate towards an infarcted region where CXCL12 is upregulated by astrocytes and endothelial cells (Imitola et  al. 2004). Several signaling pathways have been implicated in CXCR4-mediated NPC chemotaxis during migration, which include MAP kinases like Erk 1 and 2, JNK, AKT, intracellular Ca2+, and cAMP (Peng et al. 2004; Tran et  al. 2004). PI-3K/Akt and ERK1/2 signaling is also shown to be involved in CXCR4-mediated proliferation and survival of NPC (Tran and Miller 2005; Gong et al. 2006). CXCR4/CXCL12 signaling is also involved in neuronal survival in health and disease, which is discussed below particularly in the context of neuroAIDS.

10.5 Role of CCR5 and CXCR4 in AIDS Neuropathogenesis CXCR4 is well-known as a coreceptor (with CD4 as the primary receptor) for X4-using HIV isolates (Berger et  al. 1999). Cells of monocytic lineage, macrophages, and microglia in the brain, on the other hand, are infected by CCR5-using (R5-using) strains (Berger et al. 1999; Lederman et al. 2006). The HIV envelope glycoprotein 120 (gp120) exists as a trimeric structure on the surface of the virions and its initial interaction with the cellular CD4 receptor brings about specific conformational changes that facilitate its binding to a chemokine coreceptor. Once bound to both CD4 and chemokine receptor, a specific sequence of conformational events leads to the fusion of viral and cellular membranes and subsequent HIV entry into the cell (as reviewed by Pohlmann and Reeves 2006). R5-using HIV strains are involved in early HIV infection and remain as the predominant strains found in the brains and CSF of neuroAIDS patients (Gonzalez-Scarano and MartinGarcia 2005; Peters et al. 2007). In the brain, macrophages, microglia, and other mononuclear phagocytes serve as HIV reservoirs which mainly support R5-using HIV strains. With the disease progression, R5-using strains that require low expression

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of CD4 and CCR5 also appear and have been shown to be more resistant to fusion inhibitors (Gorry et al. 2005; Martin-Garcia et al. 2006). Nevertheless, importance of CXCR4 in neuroAIDS is underscored by several lines of evidence. In about 50% infected individuals, there is a switch in coreceptor usage of HIV strains from CCR5 to CXCR4, which is associated with rapid depletion of CD4+ T lymphocytes and it is shown to correlate with the onset of neuropathogenesis of AIDS (Schuitemaker et  al. 1992; Richman and Bozzette 1994; Maas et al. 2000). Moreover, in vitro studies indicate that both X4- and R5-gp120s affect the integrity, cellular permeability as well as viability of blood brain barrier adversely, leading to the infiltration of infected macrophages as well as lymphocytes in the brain parenchyma (Dallasta et al. 1999; Gonzalez-Scarano and MartinGarcia 2005). These effects involve specific signaling events such as intracellular calcium increase, and subsequent involvement of myosin light chain kinase and rho kinase, leading to the disruption of tight junctions across the blood brain barrier, thereby affecting its integrity (Kanmogne et al. 2005, 2007; Nakamuta et al. 2008). When compared with each other, X4 gp120 proteins were found to be more potent in their adverse effects on brain microvasculature endothelial cells that constitute blood brain barrier than R5 envelope proteins (Kanmogne et al. 2002, 2007). In the context of these studies, the correlation between coreceptors switch to CXCR4 during AIDS progression and the excessive permeability of the HIV across the blood brain barrier and, consequently, the onset of neurological deficits become clear. Macrophages infected with HIV are considered to be the main cell type implicated in the HIV infection of brain. Next, the microglia get infected and both these cell types support the active HIV replication and serve as HIV reservoir in the brain (Gonzalez-Scarano and Martin-Garcia 2005). Studies have indicated that though these cell types are principally infected by R5-using HIV strains, they can also support efficient replication of a subset of X4-using strains also known as dual-tropic strains (Berger et al. 1999). Furthermore, besides R5-using HIV strains, some X4 and R5X4-using strains have been isolated from HIV-infected brains that could also infect monocytes (Gorry et al. 2001; Ohagen et al. 2003; Yi et al. 2005). In light of such studies, suggestions have been made to modify the traditional concept of M and T tropism of HIV and keep it separate from the coreceptor usage of HIV, as it is known that macrophages and T lymphocytes express both CCR5 and CXCR4 on their cell surface in vivo and can be cross-infected by either X4-using or R5-using strains (Gorry et al. 2001; Goodenow and Collman 2006). Hence according to these studies, M-tropism rather than coreceptor usage predicts and is related closely with neurotropism.

10.5.1 Chemokine Receptor Activation by HIV Envelope Protein gp120 Neuropathology of AIDS is thought be a result of complex interactions of both viral and host factors that directly or indirectly cause neuronal loss as well as other manifestations associated with neuroAIDS (Jones and Power 2006). The HIV envelope

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protein gp120 can be found in the blood, cerebrospinal fluid, and extracellular matrix of brain parenchyma because of defective viral replication and release by the infected cells (Nath 2002). The envelope protein can directly interact with neuronal and glia chemokine receptors (independently of CD4) in picomolar range and mediate neurotoxicity via specific signaling cascades (Meucci and Miller 1996; Hesselgesser et al. 1997, 1998; Meucci et al. 1998; Kaul and Lipton 1999; Khan et  al. 2004; Xu et  al. 2004). The R5- and X4-using proteins induce several proapoptotic signaling pathways that compromise neuronal survival and connectivity in the brain as reviewed in Mattson et al. (2005). The most important determinant of coreceptor specificity within gp120 is its third hypervariable loop region (V3), a disulfide linked loop of about 35 amino acids that makes direct contact with the chemokine coreceptor during the entry process (Huang et al. 2005; Roux and Taylor 2007). Another gp120 region called “bridging sheet” furnishes the largest binding area to the coreceptor (Poignard et al. 2001). Highly conserved bridging sheet contains the common determinants of coreceptor recognition, while V3 region has the specific structure and charge to dictate the specificity for X4 or R5 tropism (Poignard et al. 2001; Huang et al. 2005). On the surface of chemokine receptors, the gp120 binding surface primarily consists of the N terminal, which has specific tyrosine residues that get sulfated via posttranslational modification (Berger et al. 1999). These modifications probably facilitate electrostatic interactions with the positively charged bridging sheet and the base of V3 loop (Huang et al. 2005). Another receptor domain involved in the viral entry is the second extracellular loop which interacts with the tip of V3 region (Berger et al. 1999). Earlier work showed that the signaling function of coreceptor is not required for the entry but a recently published study implicates specific Gaq protein signaling in this process downstream of CCR5 (Harmon and Ratner 2008). Another study supports the role of CXCR4-mediated actin depolymerization via Gai-independent pathway in viral nuclear localization during the latent infection of resting T cells (Yoder et al. 2008).

10.5.2 HIV Envelope gp120 – Mediated Signaling Pathways in CNS Infected monocytic macrophages and microglia shed viral proteins that can directly cause neurotoxicity (Hesselgesser et  al. 1998; Xu et  al. 2004). Studies done on neuronal cultures show that X4-using gp120 causes neurotoxicity (Brenneman et  al. 1988; Meucci and Miller 1996). This effect involves intracellular calcium dysregulation which can be inhibited by NMDA receptor antagonists as well as chemokines (Meucci and Miller 1996; Meucci et al. 1998). Collectively, these studies argue for a direct involvement of HIV envelope proteins in neuronal death associated with AIDS neuropathology. Studies on mixed glial/neuronal cerebrocortical cultures from wild type mice and CXCR4-deficient mice show that HIV envelope proteins are able to cause neurotoxicity through CXCR4 or CCR5 by the activation of p38 and specific downstream effector

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caspases (Kaul and Lipton 1999; Garden et al. 2002). However, presence of microglia is found to be important for the gp120 neurotoxicity in these studies, thus implicating indirect mechanisms that involve glial factor in HIV envelope protein induced neuronal death (Kaul et al. 2007). A dominant negative construct for p38 or Akt expression is able to abrogate neurotoxicity (Kaul et al. 2007). This is in line with the previous reports showing that chemokines, including CXCL12, activate prosurvival signaling such as Akt while gp120 is unable to activate this pathway in neurons (Meucci et al. 2000; Khan et al. 2004). Earlier studies have indicated that HIV-infected patients, who display higher concentrations of CCL3, CCL4, or CCL5 in their cerebrospinal fluid, perform better on neuropsychological treatments as compared to those with low or undetectable levels of these chemokines (Letendre et al. 1999). Hence, these chemokines might have protective roles against neurological deficits of AIDS. We and others have found CXCL12 to be neuroprotective not only against gp120 neurotoxicity but also against different types of neurotoxic insults including NMDA-mediated excitotoxicity (Meucci et  al. 1998; Chalasani et  al. 2003a; Khan et  al. 2003, 2008; Patel et  al. 2006). However, as previously discussed, cleavage of CXCL12 by MMPs leads to the generation of a highly neurotoxic product that interacts with CXCR3 (rather than CXCR4) (Zhang et al. 2003; Vergote et al. 2006). Treatment of neurons with dualtropic X4R5 gp120 has been shown to induce cytochrome c accumulation and caspase 9, 8, and 3 activation (Garden et al. 2002). Incidentally in the same studies, activated caspase 3 immunoreactivity was found in the brains of neuroAIDS patients. Furthermore, caspase 3 activation was found to be involved in the synaptic and dendritic injury (Garden et al. 2002). Chronic dendritic disruption has been correlated with the severity of HIV-related dementia (as reviewed in Ellis et al. 2007). Other studies have shown that CXCL12 and gp120IIIB induce glutamate secretion from astrocytes via TNF-a and that glutamate causes neurotoxicity in conjunction with microglia activation in vitro and in vivo (Bezzi et al. 2001). Both CXCL12 and gp120 can stimulate microglial CXCR4 to induce TNF-a production, which apart from inducing glutamate secretion by astrocytes, can directly cause neurotoxicity by binding TNFR1 receptor on the surface of neurons (Bezzi et al. 2001). Astrocytes have also been shown to produce TNF-a in response to CXCL12 (Han et al. 2001). Hence, stimulation of glial CXCR4 could play a significant role in the neuronal loss in HIV encephalitis. Earlier studies have shown that gp120 induced neurotoxicity can be prevented by NMDA receptor antagonists, thus implicating these ionotropic glutamate receptors in this process (Lipton et al. 1991; Meucci and Miller 1996; Kaul and Lipton 2004). Furthermore transgenic mice expressing X4-using gp120 (under a glial promoter) have been found to gradually develop many symptoms that are typical of HIV neuropathology including neuronal cell loss and astrogliosis (Toggas et al. 1994). This damage was found to be correlated with the amount of gp120 expressed and prevented by administering the NMDA receptor antagonist, memantine (Lipton 1992; Toggas et al. 1996). HIV-infected glia secrete inflammatory factors that can indirectly contribute to excitotoxicity (Giulian et al. 1996). The HIV envelope protein induces the secretion

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of specific neurotoxins like quinolinic acid from mononuclear phagocytes that can directly stimulate NMDA receptors (Giulian et  al. 1990, 1993). A recent study showed that gp120 could induce interleukin-1b production from glia causing phosphorylation of specific tyrosine residues on NR2B subunits of NMDA receptors and sustained intracellular calcium increase in hippocampal neurons (Viviani et al. 2006). The HIV envelope protein also increased the binding of NMDA receptor subunit to postsynaptic density protein 95 (PSD95) significantly (Viviani et  al. 2006). Thus gp120 may regulate NMDA receptor activity and its synaptic localization in vivo. Several studies implicate the role of oxidative stress in HIV-mediated cell death (Sacktor et al. 2004). Jana and Pahan have shown that CXCR4 activation of gp120 leads to NADPH-mediated superoxide production (Jana and Pahan 2004). This in turn results in the activation of neutral sphingomyelinase activity and production of ceramide, which causes death of primary human fetal neurons. Increased expression of sphingomyelin and ceramide has also been observed in the cerebrospinal fluid and brain tissues from neuroAIDS patients (Haughey et al. 2004). Together, these studies suggest that oxidative stress mediated by HIV envelope proteins can cause lipid imbalance, thereby compromising neuronal survival during HIV neuroinflammation. Besides these pathways, specific kinases like mixed lineage kinase and doublestranded RNA-activated protein kinase (PKR) have been implicated in gp120mediated neuronal death (Bodner et al. 2002; Alirezaei et al. 2007).

10.5.3 Role of Cell Cycle Proteins in Chemokine Receptor-Mediated Neuronal Survival Postmitotic neurons, as their name implies, do not undergo cell division under normal physiological conditions. On the other hand, under specific stresses or pathological conditions such as DNA damage or growth factor withdrawal, induction of the cell cycle machinery can take place, which leads to neuronal entry into S-phase and DNA replication (Herrup and Yang 2007). Neurons undergoing such stress conditions are not able to advance any further along the cell cycle beyond S-phase and die. Certain cell cycle-related transcription factors like E2F1 and p53, which regulate cell cycle entry, have been shown to be involved in controlling cell survival under different stress conditions (Becker and Bonni 2004). Among cell cycle regulators, the retinoblastoma protein (Rb) is known to regulate the transcriptional activity of E2F1. E2F1-mediated cell cycle arrest and/or cell death is controlled by both p53-dependent and -independent pathways as reviewed in Dasgupta et  al. (2006) and Verdaguer et al. (2007). Rb protein is regulated by the action of cyclindependent serine/threonine kinases (CDKs). A hypophosphorylated Rb keeps E2F1 transcriptional activity in check through its specific suppression activity; upon phosphorylation, Rb is unable to bind and inhibit E2F1 activity (Delston and Harbour 2006). Over a period of several years, the role of these cell cycles or cell

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cycle-related proteins has been implicated in many neurodegenerative disorders including Alzheimer’s diseases, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, ataxia-telangiectasia, etc. (reviewed in Becker and Bonni 2004; Verdaguer et al. 2007). Immunohistochemical studies first showed altered levels of E2F1 and Rb (in its inactive, phosphorylated form) in HIV/SIV encephalitis brains (Jordan-Sciutto et al. 2002). Our earlier studies on cerebellar granule neurons showed that CXCR4 stimulation by CXCL12 or gp120 could regulate Rb, in an opposite manner, i.e. while the chemokine promoted Rb activity, the HIV envelope protein increased its phosphorylation (Khan et al. 2003). Later, we found that X4-using gp120 was able to increase E2F1 levels in the postmitotic cortical neurons, and also stimulated its pro-apoptotic transcriptional activity (Shimizu et al. 2007). Furthermore, increased levels of “Rb-free” E2F1 were observed in neurons in the brains of HIV patients with neurological deficits as compared to control HIV patients. Notably, in E2F1 deficient neurons, gp120 neurotoxicity was observed to be significantly reduced leading us to conclude that E2F1 transcriptional activity plays a critical role in CXCR4-mediated neurotoxicity (Shimizu et al. 2007). As mentioned before, NMDA receptor-mediated neurotoxicity plays a significant role in neurodegenerative disorders including neuroAIDS (Kaul and Lipton 2004). In a recent study, we show that CXCR4/CXCL12 signaling upregulates Rb and its transcriptional activity in postmitotic cortical neurons (Khan et al. 2008). We also show that the neuroprotective effect of CXCR4/CXCL12 signaling is mediated by Rb. Indeed, Rb-depleted neurons were still sensitive to NMDA-induced neurotoxicity even in the presence of CXCL12, unlike control cells which were rescued by CXCL12 (Khan et al. 2008). These studies indicate that CXCR4/CXCL12 signaling regulates important cell cycle mediators that are involved in neuronal survival. Further studies in this direction might lead to the discovery of disease specific denominators that could be used either as therapeutic targets or diagnostic markers. The transcriptional activity of p53 has been associated with a variety of neurodegenerative disorders (as reviewed by Morrison et  al. 2003). This transcription factor seems to be upregulated in neurons and microglia as suggested by the studies in individuals with HIV dementia and also in SIV brains (Jordan-Sciutto et  al. 2000; Silva et al. 2003; Garden et al. 2004). Neurons obtained from p53-deficient mice are partially protected from gp120-induced neurotoxicity, when exposed to the dualtropic gp120SF2 (Garden et al. 2004). Apparently both neurons and microglia must express p53 for the induction of gp120 neurotoxicity (Garden et al. 2004; Jayadev et al. 2007). Other studies indicate that gp120-induced caspase 3 activation in neurons requires p53, while activation of caspase 8 and Bid cleavage can take place without p53 (Tun et  al. 2007). We have shown that in cortical neurons, X4-gp120 signaling involves increased levels of nuclear p53, as well as its phosphorylation (Khan et  al. 2005). Moreover, HIV envelope protein also stimulates p53 transcriptional activity and induces the production of p53-target like MDM2, p21, and Apaf-1 leading to neuronal death (Khan et al. 2005). Cyclin-dependent kinase 5 (cdk5) is a nonconventional cdk which plays important roles in the neuronal survival. Supernatants from HIV-infected macrophages

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led to NMDA receptor-mediated neuronal death which was caused by calpain activation (O’Donnell et al. 2006). Such treatment of neurons led to the calpaindependent cleavage of p35 subunit of cdk5, to its constitutively active isoform, i.e. p25 (Wang et  al. 2007). Inhibition of cdk5 p25 provided neuroprotection (Wang et al. 2007). Moreover, increased calpain activity and p25 levels were found in the midfrontal cortex of HIV patients with cognitive deficits (Wang et al. 2007). These studies did not identify any causative agent in supernatants for the observed neurotoxicity. Hence cdk5 activation by calpain could take part in HIV-mediated indirect mechanism of neuronal death. Recent investigations into the regulation of cell cycle of neural progenitor cells by chemokines have led to some potentially interesting findings. Both CXCL12 and the CCR3 ligand CCL11 (Eotaxin) inhibit cell proliferation and promote quiescence, while CCL3 (MIP-1a) does not alter the rate of proliferation (Krathwohl and Kaiser 2004). When stimulated with either X4 or R5 gp120, the cells show reduced attachment to the substratum and neuronal differentiation, decreased DNA replication resulting in less BrdU incorporation, and enhanced expression of cyclindependent kinase inhibitors, p21 and p27 (Krathwohl and Kaiser 2004). Furthermore, exposure of these neural progenitor cells to cerebrospinal fluid (CSF) from HIV-1 infected individuals with or without dementia shows that the CSF from patients without dementia had hardly any effect on BrDU incorporation, while CSF from patients with dementia showed substantial reduction in BrDU incorporation (Krathwohl and Kaiser 2004). In the case of HIV patients with dementia, the degree of proliferation inhibition was strongly correlated with the viral load in CSF. Furthermore, in post mortem hippocampal tissue from HIV patients with dementia, there was a 75% reduction in the neural progenitors as compared to that from the control group (tissue from HIV patients without dementia) (Krathwohl and Kaiser 2004). Another study shows that gp120 could also decrease proliferation of adult neural progenitors (Okamoto et al. 2007).Thus these studies correlate chemokine receptor-mediated cell cycle control with AIDS-related dementia. In postmitotic neurons, stimulation of transcriptional factors like p53 and E2F1 by gp120-mediated CXCR4 signaling leads to neuronal death (Khan et  al. 2005; Shimizu et  al. 2007). Whether it is a consequence of cell cycle entry or not is still an open question. In our studies, this cell death seemed to be associated with the pro-apoptotic transcriptional activity of p53 and E2F1 transcription factors (Khan et  al. 2005; Shimizu et al. 2007). Further studies are required to study the role of chemokine signaling in cell cycle control of postmitotic neurons.

10.5.4 A Comparison Between CXCL12 and gp120 Signaling Our studies on CXCR4 signaling in neurons indicate that CXCL12 and X4-using gp120 regulate neuronal survival pathways in an opposing manner (Meucci et al. 1998; Khan et  al. 2003, 2004, 2005). Using fluorescence activated cell sorting (FACS), we have shown that, unlike CXCL12, X4-gp120 does not cause CXCR4

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internalization under experimental conditions (from picomolar to nanomolar concentration range) that normally results in signaling and cellular toxicity (Bardi et al. 2006). However, some recent studies involving gp120 treatment of neurons in cultures (in nanomolar range) and its injection into cortical striatum of rodents, indicate that it is sequestered by neurons and transported in retrograde manner along the axon length (Bachis et al. 2006). The issue of gp120-mediated CXCR4 internalization in neurons requires further clarification by the use of single-cell techniques and low concentrations of gp120 i.e. picomolar range. Moreover, unlike gp120, activation of CXCR4 by its endogenous ligand, CXCL12, increases nuclear levels of Rb in neurons and stimulates its transcriptional activity under different pro-apoptotic conditions (Khan et al. 2003, 2008). CXCL12 is able to protect neurons against NMDA excitotoxicity; this protection is abrogated by Rb deficiency in neurons (Khan et al. 2008). In a similar manner, as compared to gp120 mediated signaling downstream of CXCR4, CXCL12 does not upregulate p53 or its transcriptional activity (Khan et al. 2005). Thus, we have observed a clear difference in the signaling pathways stimulated by the two CXCR4 ligands. Another major difference is the ability of CXCL12 (but not of gp120) to induce the phosphorylation/activation of serine/threonine kinase Akt. Akt-mediated nuclear transport of p65 NF-kB is also stimulated by CXCL12 (Khan et  al. 2004). At the level of MAPK induction (e.g. Erk, Junk, and p38) clear differences were detected in the efficacy of these two CXCR4 ligands in human astrocytes (Khan et  al. 2004). HIV envelope protein was significantly more potent in causing p38 and JNK activation when compared to CXCL12 (Khan et  al. 2004). Hence, our studies show that intrinsic efficacies of the two CXCR4 ligands are significantly different and register their effects on neuronal survival in a contrasting manner.

10.6 Role of CXCR3 and CCR2 in NeuroAIDS 10.6.1 CXCR3 CXCR3 is expressed in the immune system as two isoforms, CXCR3-A and CXCR3-B. It is an important chemokine receptor associated with various inflammatory neurodegenerative diseases (e.g. multiple sclerosis) and other models of neuronal injury as reviewed by (Liu et al. 2005). CXCR3 has also been recently implicated in the AIDS neuropathogenesis. As mentioned earlier (and more extensively discussed in the Chap. 6), MMP2truncated CXCL12 (5–67) causes neuroinflammation, neuronal loss, and behavioral abnormalities in vivo that can be prevented by a CXCR3 antagonist (Zhang et al. 2003; Vergote et al. 2006). Moreover, monocytoid cells from HIV dementia patients express high levels of both CXCL12 and truncated CXCL12 (4–67) (Vergote et  al. 2006). The truncated chemokine can also induce the expression of

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proinflammatory genes IL-1b, TNF-a, Indoleamine 2, 3-deoxygenase, and IL-10 in astrocytes and monocytoid cells (Vergote et al. 2006). Hence these studies indicate that inappropriate induction of CXCR3 by N-terminal truncated CXCL12 can adversely affect the neuronal survival in neuroAIDS.

10.6.2 CCR2 This receptor is expressed as two isoforms, CCR2A and CCR2B, and CCL2 (MCP-1) is able to bind and signal through both of them. There is significant evidence in favor of a pathological role for CCL2 in CNS. CCL2 is able to attract and activate mononuclear phagocytes, monocytes, and macrophages as well as microglia potently; hence this chemokine plays a central role in phagocytic trafficking and activation. CCL2 levels are upregulated in cerebral ischemia, multiple sclerosis, Alzheimer’s disease, as well as in HIV-related dementia (Xia and Hyman 1999; Mahad and Ransohoff 2003; Minami and Satoh 2003; Dhillon et al. 2008). CCL2 content in the CSF of AIDS patients correlates with HIV-1 replication and severity of HIV-related dementia (Cinque et al. 1998; Kelder et al. 1998; Mengozzi et al. 1999; Wu et  al. 2000; Bernasconi et  al. 1996). HIV infected monocyte-derived macrophages (MDM) secrete CCL2 and HIV replication is shown to be required for this effect (Mengozzi et al. 1999). Moreover, HIV-1 Tat protein induces CCL2 expression in astrocytes and MDM (Conant et al. 1998; Weiss et al. 1999). Because of a very close link between CCL2 expression and mononuclear phagocyte recruitment and activation, upregulated expression of this chemokine might play a very important role in HIV-encephalitis by bringing the HIV-infected mononuclear phagocytes across blood brain barrier as “Trojan horses” (Fischer-Smith and Rappaport 2005; Gonzalez-Scarano and Martin-Garcia 2005; Maslin et  al. 2005; Dhillon et al. 2008). Moreover, CCL2 is involved in the spread of infection in the brain as it might regulate viral replication as well as movement of productively infected cells to specific areas of brain (Maslin et al. 2005).

10.7 Conclusions Neurological complications of AIDS are found in a significant portion of HIVinfected patients and affect their quality of life substantially. With the prevalence of these deficits on the rise, the importance of addressing these issues therapeutically cannot be underestimated. There is considerable evidence for the significance of chemokine signaling in various aspects of HIV neuropathology, from neurotropism to dendritic dysfunction and neuronal death. Hence, further exploration into chemokine receptor dynamics and signaling should yield significant dividends of therapeutic value in this regard.

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Chapter 11

Chemokines and Primary Brain Tumors Shyam S. Rao, Mahil Rao, Nicole Warrington, and Joshua B. Rubin

Normal development of the central nervous system (CNS) is critically dependent upon the coordinated expression of chemokines and their receptors in a spatially and temporally regulated fashion (reviewed in Klein and Rubin 2004; Li and Ransohoff 2008). During development, chemokines control progenitor cell migration (Bagri et al. 2002; Klein et al. 2001; Lu et al. 2002; Lu et al. 2001), proliferation (Klein et al. 2001), and survival (Chalasani et al. 2003a) as well as modulate differentiated cell functions such as axon pathfinding and fasciculation (Chalasani et al. 2003b; Chalasani et al. 2007). Thus, it should not be surprising to discover that chemokines and their receptors also contribute to the biology of CNS neoplasms. In this chapter we will primarily review studies that have defined the role that one chemokine, CXCL12, and its receptor, CXCR4, play in brain tumor biology and how this pathway is being targeted for brain tumor therapy. In addition, we will touch on the role that brain tumor-derived chemokines play in the recruitment of inflammatory cells to sites of brain tumor growth.

11.1 The Prognostic Significance of CXCL12 and CXCR4 Expression in Brain Tumors In 1998, Sehgal et  al. described increased expression of CXCR4 in brain tumor tissue relative to that in normal brain (Sehgal et al. 1998). Increased expression was evident in  vitro in both medulloblastoma and astrocytoma (glioma) cell lines. Further, primary glioma and meningioma tumor specimens were also found to possess high levels of CXCR4 protein. As medulloblastomas, astrocytomas, and meningiomas are derived from neural, glial, and mesenchymal lineages, respectively, these observations prompted the hypothesis that CXCR4 plays a broad and important role in brain tumorigenesis (Sehgal et al. 1998).

S.S. Rao, M. Rao, N. Warrington, and J.B. Rubin  (*) Department of Pediatrics, St. Louis Children’s Hospital, Washington University School of Medicine, Campus Box 8208, 660 South Euclid Ave, St. Louis, MO 63110, USA e-mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_11, © Springer Science+Business Media, LLC 2010

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Subsequently, a number of studies have examined the patterns and significance of CXCL12 and CXCR4 expression in brain tumors. Medulloblastoma, the most common malignant brain tumor of childhood is derived from neuronal progenitors of the cerebellum (Giangaspero et al. 2000; Lee et al. 2005). Medulloblastoma has several histologic subtypes that carry prognostic significance. The desmoplastic subtype of medulloblastoma appears to arise from granule precursor cells of the external granule cell layer of the cerebellum (Lee et al. 2005; Pomeroy et al. 2002; Schuller et al. 2006). During early development, CXCL12 and CXCR4 are essential for normal patterning of the cerebellum, as they regulate GPC migration (Lu et al. 2001; Ma et al. 1998; Reiss et al. 2002; Zou et al. 1998) and proliferation (Klein et al. 2001). Consistent with this ontogeny, desmoplastic medulloblastoma samples were found to have higher amounts of CXCR4 transcript compared to other subtypes such as classic medulloblastoma, which derive from a distinct cerebellar progenitor population (Rubin et al. 2003; Schuller et al. 2005; Yang et al. 2007). Schuller also found that CXCR4 expression correlated with expression of other markers of GPC lineage including p75NTR, ATOH1, and GLI. Together, these findings suggest that CXCR4 may be an important marker that could distinguish between medulloblastomas derived from the EGL and other cerebellar locations (Schuller et al. 2005). CXCR4 is also highly expressed in astrocytomas of all histological grades. In contrast to medulloblastomas, which are all World Health Organization (WHO) grade IV tumors, astrocytomas can occur as any of the four WHO grades for brain tumors. Histological grade refers to the degree of malignancy that is apparent upon microscopic analysis. Grade I tumors have no features of malignancy, while grade IV tumors have multiple features of the malignant phenotype (nuclear atypia, mitoses, vascular proliferation, and necrosis) (Cavenee et al. 2000). Therefore, examination of the spectrum of astrocytomas affords an opportunity to gain insight into the relative importance of a specific pathway like the CXCL12/CXCR4 pathway to malignant biology. Rempel et al. (2000) and Rubin et al. (2003) have examined the expression of CXCL12 and CXCR4 in human glioblastoma. Rempel found that CXCL12 and CXCR4 were expressed at high levels in areas adjacent to necrosis, in regions between angiogenic vessels where the tumor appeared to be undergoing degeneration, and occasionally in endothelial cells of new blood vessels. They concluded that CXCL12 and CXCR4 might contribute to angiogenesis and/or modulation of immune response (Rempel et al. 2000). Rubin identified the endothelium of tumorassociated blood vessels as a rich source of CXCL12 in GBM and suggested that this could provide for a paracrine activation of CXCR4 expressed on the surface of GBM cells (Rubin et al. 2003). Salmaggi et al. (2004) and Calatozzolo et al. (2006) made similar observations regarding endothelial cell expression of CXCL12 upon examination of specimens from patients with either oligodendroglioma or oligoastrocytomas. To address whether the co-expression of CXCL12 and CXCR4 results in the activation of receptor signaling, Woerner et  al. (2005)examined astrocytomas of various grades, Yang et  al. (2007) examined medulloblastoma specimens, and Warrington et al. (2007) examined Neurofibromatosis Type-1 associated pilocytic

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astrocytomas with an antibody that recognizes a CXCL12-induced phosphorylated form of CXCR4 (P339-CXCR4). In the Woerner study, eight grade I, five grade II, eight grade III, and eight grade IV astrocytomas were examined and found to express CXCL12, CXCR4, and P339-CXCR4 (Woerner et  al. 2005). Similar to prior studies, Woerner et al. observed that CXCL12 expression was localized to the endothelium of tumor-associated blood vessels. They also found that CXCL12 was present within infiltrating microglia. CXCR4 expression was found in greater than 50% of all tumor cells, regardless of tumor grade, but was greatest in grade I gliomas. Importantly however, the extent of CXCR4 phosphorylation, reflecting the degree of ligand activation of this receptor, was greater in diffuse astrocytomas (grades II–IV) than in grade I tumors. These data suggested that the degree of CXCR4 activation, and accordingly its functional role, was greater in higher as compared to lower grade astrocytomas. Similar to findings in astrocytoma specimens, Yang et  al. (2007) found that histologic subtype of medulloblastoma was associated with differences in the phosphorylation of CXCR4. While all medulloblastomas are grade IV tumors, prognosis is also determined by histological subtype. Of the three most common subtypes of medulloblastoma, the desmoplastic variant is less aggressive, while the anaplastic large cell variant is more aggressive, than the classic subtype. In their limited series, none of the desmoplastic specimens exhibited phospho-CXCR4 staining, while 2/3 classic and 3/4 anaplastic large cell tumors did. Together with the observations regarding the phosphorylation of CXCR4 in astrocytomas, these data suggest that increasing malignancy or aggressive growth is associated with increased ligand activation of CXCR4. Expression of CXCL12 and CXCR4 is not limited to highly malignant brain tumors. Indeed, Rempel et al. (2000) and Warrington et al. (2007) detected expression of CXCL12 and CXCR4 in low-grade astrocytomas as well. Moreover, Warrington demonstrated that CXCL12 was highly expressed in vascular endothelium, infiltrating microglia and entrapped axons (Warrington et al. 2007). As in the more malignant tumors, this multiplicity of CXCL12 sources was associated with the presence of CXCR4 in a ligand-induced phosphorylated form. A common theme in cancer biology is the abnormal stimulation of growth regulatory pathways through anomalous autocrine, paracrine, or mutational activation of cell-surface receptors. While mutation of G-protein coupled receptors like CXCR4 is rarely reported in cancer biology, Schuller et  al. (2005)described two examples of medulloblastoma in which the gene encoding CXCR4 had a coding mutation. In one case, the mutation resulted in an isoleucine-to-leucine change in the first transmembrane domain. In the second case, the mutation resulted in an asparagine-to-aspartic acid change in the second transmembrane domain. It remains unclear whether these mutations affect the functionality of the receptor or the behavior of the tumors. The work of Rempel, Woerner, Bain, and Yang suggests that CXCL12 and CXCR4 play an important role in malignant brain tumor biology. One prediction that follows from these observations is that the level of CXCL12 and CXCR4 expression would have prognostic significance. Bian et  al. (2007) found that

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patients bearing CXCR4-positive tumors had significantly reduced survival compared to those with CXCR4-negative tumors. In a study of tumors from 50 patients with a WHO diagnosis of low grade glioma (grades I,II), Salmaggi et al. (2005) found that time to progression (TTP) was significantly shorter in patients whose tumors exhibited CXCL12 expression, compared to those whose tumors did not. Additionally, Calatozzolo et al. (2006) found that approximately 2/3 of oligodendrogliomas and oligoastrocytomas expressed CXCL12. For either tumor type, CXCL12 staining on either tumor or endothelial cells was associated with a shorter TTP. Importantly, CXCL12 expression was a more powerful predictor of TTP than histological subtype. In sum, CXCL12 and CXCR4 are present in brain tumors derived from multiple lineages. The most common mechanism for abnormal activation of CXCR4 in brain tumors appears to be through a paracrine relationship between tumor cells and multiple stromal elements, including endothelial cells, microglia, and entrapped axons. Finally, increased activation of CXCR4 is associated with a greater degree of malignancy and is a negative prognostic factor.

11.2 CXCR4 Activity Stimulates Brain Tumor Growth Through Diverse Mechanisms The studies described above suggest that CXCR4 plays an important role in brain tumor biology but do not identify any specific functions with regard to tumorigenesis and growth. Tumor growth is a complex process that parallels normal tissue growth in many regards. Tumors expand through the activities of a germinal pool of cells that have been most recently referred to as “tumor-initiating cells” or “tumor stem cells” (Sauvageot et al. 2007). These cells appear to be functionally related to or similar to the committed progenitor pools found in liver, GI tract, skin, and CNS. Tumor stem cells (TSCs) give rise to daughter cells that can undergo some degree of differentiation or be induced to initiate cell death. The balance of cell generation and cell death is critical but not an exclusive determinant of tumor growth; other powerful influences on tumor growth potential include the ability to remodel the extracellular matrix, stimulate angiogenesis, and regulate immune responses. Each of these processes is governed by chemokines and in the following sections we will describe the effects of CXCL12 and CXCR4 on each and brain tumor growth.

11.2.1 CXCR4 and Brain Tumor Stem Cells In the last several years, there has been escalating interest in the idea that cancer stem cells play a central role in tumor formation and spread. The cancer stem cell

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hypothesis proposes that a restricted subset of tumor cells with stem cell-like properties is responsible for the growth, maintenance, and metastasis of the tumor (Reya et al. 2001). These cells form a reservoir of cells capable of self-renewal and may be largely accountable for tumor growth, metastasis, and recurrence. Their partially differentiated progeny, in turn, constitute the bulk of the tumor and contribute to the tumor phenotype. Such tumor stem cells were first identified in acute myeloid leukemia and have since been identified in other solid tumors including breast cancer, prostate cancer, colon cancer, lung cancer, and primary brain tumors (Galli et al. 2004; Singh et  al. 2003; Singh et  al. 2004). While the tumor stem cells exhibit properties similar to normal stem cells, it remains unclear whether tumor stem cells arise from normal stem cells. Brain tumor stem cells were first identified by their ability to form characteristic spherical aggregates similar to neural stem cells under neural stem cell culture conditions, along with their expression of neural stem cell markers including CD133 and nestin. Like their normal neural stem cell counterparts, brain tumor stem cells are capable of self-renewal, as well as differentiation along multiple neural cell lineages. Furthermore these brain tumor stem cells exhibit a remarkable propensity to initiate tumors in mouse xenograft models. In a landmark study, Singh et  al. (2004) found that as few as 100 CD133+ cells isolated from human glioblastoma were capable of tumor formation when implanted into mice, while implantation of 105 CD133─ cells were incapable of tumorigenesis. In addition to their inherent self-sustaining properties, brain tumor stem cells may be more resistant to chemotherapy and radiation therapy than other tumor cells. Bao et al. (2006) found glioma stem cells (CD133+) were relatively radioresistant compared to CD133- tumor cells and preferentially activated the DNA damage checkpoint response. This relative resistance to standard treatment approaches of tumor stem cells compared to the majority of other cells within a tumor may underlie our current inability to cure patients with aggressive brain tumors such as glioblastoma. Much of our initial understanding of stem cells and tumor stem cells arises from the hematopoietic system. Detailed characterization of hematopoietic lineages has identified the central role of chemokines in hematopoietic stem cell function. Similarly, chemokines play a significant role in the function of normal neural stem cells and likely brain tumor stem cells as well. In particular, the chemokine receptor CXCR4 is expressed on neural stem cells and serves as a stem cell marker (Reiss et al. 2002). Ma et al. (2008) evaluated the expression of multiple stem cell markers in astrocytomas from 72 patients. Astrocytoma specimens showed higher expression of CD133, nestin, Sox-2, Musahi, Flt-4, CD105, and CXCR4 than normal brain tissue. Another study found that the majority of tumor stem cells isolated from glioblastoma tumorspheres express CXCR4 along with nestin (Salmaggi et al. 2006). CXCL12 has also been found to be elevated in post-surgical endocavitary fluid (Salmaggi et al. 2005). The functions of the CXCL12─CXCR4 axis in maintaining the stem cell pool might include chemo-localization to specialized stem cell niches as well as maintenance of mitotic quiescence characteristic of stem cell populations. Bone marrow

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stem cells are localized through a complex set of interactions that require CXCL12─CXCR4 (Nervi et al. 2006). The importance of CXCL12 and CXCR4 to marrow localization is underscored by the efficacy of the CXCR4 antagonist AMD3100 in mobilizing CD34+ bone marrow progenitors (Broxmeyer et al. 2005; Flomenberg et al. 2005). Brain tumor stem cells also reside in a specific niche. Cells identified as brain tumor stem cells by their expression of phenotypic markers like CD133, nestin, or Sox2 are primarily localized to the perivascular space in brain tumors. The mechanism for this localization is not clear, but the expression of CXCL12 by brain tumor associated vascular endothelium (Rubin et al. 2003) together with the high level of expression of CXCR4 on brain tumor stem cells suggests that the CXCL12─CXCR4 pathway may localize brain tumor stem cells to the perivascular niche in a manner similar to the localization of bone marrow stem cells to the marrow niche. The persistence of stem cell pools is dependent upon a regulated balance between proliferation and quiescence. It is clear from considerations of bone marrow recovery after chemotherapy that quiescent stem cells are relatively resistant to the cytotoxic effects of therapy. The reported resistance of brain tumor stem cells to DNA damaging agents could be a consequence of mitotic quiescence (Bao et al. 2006). In this regard, Khan et  al. (2003) demonstrated that CXCL12 induces a quiescent state in primary neurons characterized by decreased phosphorylation of Rb and decreased E2F transcriptional activity. A similar role is suggested for CXCL12 in bone marrow (Nie et al. 2008). It remains to be determined whether these mechanisms are important to brain tumor stem cell biology.

11.2.2 CXCL12 Stimulates Brain Tumor Cell Proliferation and Survival Sehgal et al. (1998) were the first to suggest that CXCR4 activation could stimulate brain tumor growth. They demonstrated that overexpression of CXCR4 increased the proliferation rate of three glioblastoma cell lines and increased the rate of colony formation in soft agar (a measure of transformation) by up to 90% in GB1690 cells. Furthermore, treatment of the cultures with antibodies directed against CXCR4 or CXCL12 reduced the proliferation rate of these cell lines without altering the proliferation of a non-GBM cell line. Interestingly, all of these experiments were done without the addition of exogenous CXCL12, suggesting that autocrine activation of CXCR4 was possible in glioblastoma, though subsequent studies of CXCL12 expression in primary GBM specimens have not identified tumor cells as a source of CXCL12. CXCR4 expression is regulated by several pathways commonly activated in cancer including, the hypoxia inducible factor-1a (HIF-1a) (Wang et  al. 2008; Zagzag et al. 2006), and vascular endothelial cell growth factor (VEGF) (Zagzag et  al. 2006) pathways. Hypoxia or ligand stimulation of these pathways increases CXCR4 expression through the actions of Ets1 or NF-kappaB activity

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(Maroni et al. 2007) and could enhance the proliferative capacity of CXCR4-expressing brain tumors (Hong et al. 2006). In contrast, von Hippel–Lindau factor suppresses CXCR4 expression (Staller et  al. 2003) and overexpression of Leucine-Rich Repeats Containing 4 (LRRC4), a putative glioma growth inhibitor, suppresses CXCR4 expression at baseline; this is correlated with a decrease in Erk 1/2 activation, Akt activation, and in SDF-induced proliferation (Wu et al. 2008). Thus, the functions of CXCR4 in brain tumors may be modulated by activity in parallel pathways. Paracrine activation of CXCR4 appears to be the predominant mechanism for eliciting CXCR4 effects on brain tumor growth. Accordingly, treatment with CXCL12 increases proliferation in glioblastoma cell lines in a dose-dependent fashion with maximum effect at 12.5  nM (100  ng/mL) over the course of 24  h (Barbero et al. 2002; Wu et al. 2008). Consistent with a direct effect on proliferation, CXCL12 concentrations between 100 and 200  ng/mL induce a 25–36% increase in DNA synthesis in primary cultures of human GBM specimens compared to that in untreated controls (Bajetto et al. 2006). Numerous studies have identified multiple events downstream of CXCR4 activation as mediators of CXCR4 effects on tumor cell proliferation. CXCR4, like the other chemokine receptors, is a Gi-coupled receptor, although there is some evidence to suggest that activation of Gq proteins also occurs. Activation of CXCR4 by CXCL12 leads to association with the pertussis toxinsensitive Gai subunit, which, in turn, inhibits the activity of adenylyl cyclase and leads to a drop in intracellular cyclic AMP levels. Through the action of either the beta-gamma subunits or through Gaq coupling, CXCR4 activation increases the activation of phospholipase C and causes the release of calcium from internal stores, although it does not result in intracellular calcium oscillations (Florio et al. 2006). CXCL12 treatment also transiently increases the potassium conductance of the cell, perhaps through the action of large-conductance calcium-dependent potassium channels (BKCa), as this effect is blocked by pre-treatment with either BAPTA-AM or TEA (Florio et al. 2006). The increase in potassium conductance also appears to require the action of Pyk2, a calcium-dependent cytosolic tyrosine kinase, as pre-treatment with salicylate, an inhibitor of Pyk2, blocks the effects of SDF on potassium conductance. CXCL12 treatment also leads to the activation of a MAP kinase cascade. In cortical astrocytes, Erk 1/2 phosphorylation increases 5 min after the onset of CXCL12 treatment and is sustained for greater than 30 min (Bajetto et al. 2006; Wu et al. 2008). CXCL12 also leads to the activation of PI-3kinase and Akt; phosphorylation of Akt occurs between 15 and 30 min after the onset of CXCL12 treatment (Wu et al. 2008). Many of these intracellular events are critical to the proliferative effects of CXCL12. For example, the CXCL12-induced effect on proliferation is dependent on calcium. Pre-treatment of pituitary adenoma cells with BAPTA-AM abolishes the CXCL12-induced increase in proliferation (Florio et al. 2006). The increase in proliferation also requires activation of Erk 1/2, as pre-treatment with PD98059, a MEK inhibitor, blocks the proliferative effect of CXCL12, and this is correlated with a decrease in Erk 1/2 phosphorylation. Similarly, the proliferative effects of

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CXCL12 are dependent on activation of cytosolic tyrosine kinases such as Pyk2. Use of either genistein, a cytosolic tyrosine kinase inhibitor, or salicylate, a Pyk2 inhibitor, blocks the CXCL12-induced increase in proliferation in pituitary adenoma cells. However, these two pathways appear to be independently involved in the proliferative effects of CXCL12; pre-treatment with BAPTA-AM reduced Pyk2 phosphorylation without altering Erk 1/2 phosphorylation, while pre-treatment with PD98059 did not alter Pyk2 phosphorylation but reduced Erk 1/2 phosphorylation. Lastly, the proliferative effects of CXCL12 also appear to be dependent on an increase in potassium conductance, as pretreatment with TEA abolished the proliferative effect of CXCL12 without altering Erk 1/2 or Pyk2 phosphorylation. In addition to its effects on proliferation, CXCL12 enhances brain tumor growth by promoting tumor cell survival. In fact, the in  vivo anti-tumor effect of the specific CXCR4 antagonist AMD3100 was most closely correlated with an increase in tumor cell apoptosis and not with a change in tumor cell proliferation (Rubin et  al. 2003). Systemic delivery of AMD 3100 induced apoptosis in intracranial xenografts of U87 glioblastoma and Daoy medulloblastoma cells within 24  h of treatment initiation and in the absence of any evident effect on the vascular endothelium. AMD 3100 treatment for up to 3 weeks induced apoptosis in intracranial xenografts of both Daoy medulloblastoma and U87 glioblastoma cells. The degree of apoptosis was greater in U87 cells compared to Daoy cells and this correlated with the magnitude of the anti-tumor effect, as measured by inhibition of intracranial xenograft growth, which was also greater in the U87 xenografts. The anti-tumor effect of CXCR4 inhibition was not correlated with an effect on tumor cell proliferation. While a significant reduction in tumor cell incorporation of BrdU was observed in AMD 3100 treated Daoy xenografts, no reduction of BrdU incorporation was observed in AMD 3100 treated U87 xenografts, despite the greater inhibition of growth in these tumors. CXCL12-induced survival may also underlie the formation of low-grade gliomas in the autosomal dominant tumor predisposition syndrome neurofibromatosis type 1 (NF1) (Friedman et al. 1999). Individuals with NF1 are predisposed to developing benign and malignant tumors of the peripheral and central nervous systems (Rubin and Gutmann 2005). The most common tumor in the CNS occurs in young patients and involves the optic nerve and chiasm, the so-called optic pathway glioma (OPG) (Listernick et al. 1997). The neoplastic cell in NF1-associated OPGs is a glial fibrillary acidic protein (GFAP) positive cell (astroglial cell) that sustains complete loss of neurofibromin function (Gutmann et al. 2000; Gutmann et al. 2003; Kluwe et al. 2001). Loss of the second (non-mutated) NF1 allele is likely to occur randomly, but it is clear that a second, non-random process is necessary for tumor formation. The temporal and spatial pattern of OPG formation and growth suggests it is dependent upon a factor(s) expressed along the optic pathway in a developmentally regulated fashion. Warrington et al. (2007) determined that CXCL12 might be the necessary microenvironmental co-factor for OPG formation. They demonstrated that CXCL12 is present in the optic nerves of human infants and young mice, but not in adolescents or adult mice. Further, they demonstrated that Nf1 loss leads to a dysregulated cellular response to CXCL12: wildtype astrocytes undergo apoptosis in response to

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CXCL12, while the survival of similarly treated Nf1−/− astrocytes was enhanced. Thus the growth-promoting effects of CXCL12 involve increased survival in low and high-grade astrocytomas as well as medulloblastoma. While multiple signaling pathways are implicated in mediating the proliferative effects of CXCL12, only Akt activation, p38 activation, and suppression of cAMP levels have been shown to influence the survival response. Vlahakis et al. (2002) demonstrated that CXCL12 regulates survival in CD4+ T lymphocytes via the balance of pro-survival Akt activation and pro-apoptotic p38 activation . Whether the cellular response to CXCL12 was survival or apoptosis depended on coincident activation of these pathways by other factors. Utilizing the stark difference in response of wildtype astrocytes (increased apoptosis) and Nf1−/− astrocytes (decreased apoptosis), Warrington et  al. (2007) identified differences in cAMP responses downstream of CXCR4 activation as the primary determinant of whether astrocytes exhibited pro- or anti-apoptotic responses to CXCL12. They determined that loss of neurofibromin function resulted in abnormally sustained suppression in intracellular cAMP levels as a result of inhibition of CXCR4 desensitization. The critical nature of cAMP suppression to cell survival was evident when CXL12 effects were phenocopied by pharmacological inhibition of adenylyl cyclase and cAMP production with dideoxyadensoine treatment, and CXCL12 pro-survival effects were blocked by adenylyl cyclase activation and elevation of cAMP with forskolin. CXCR4 desensitization is a counter-regulatory mechanism to limit heterotrimeric G protein dependent signaling (Gainetdinov et al. 2004). It is mediated through the actions of a family of kinases known as G protein receptor kinases or GRKs (Pitcher et al. 1998). Warrington et al. found that loss of neurofibromin inhibited GRK2 activity in an ERK-dependent fashion. Publicly available genomic datasets (geo) indicate that GBM is associated with decreased expression on GRK2, 3 and 5 suggesting that altered CXCR4 desensitization may be an important mechanism for enhancing CXCR4-dependent growth in brain tumors.

11.3 CXCL12 Functions as a Migratory/Invasive Factor for Brain Tumor Cells In addition to its effects on proliferation and survival, CXCL12 appears to promote the invasion of tumor cells into the surrounding parenchyma through the activation of matrix metalloproteinases (MMPs). In three glioma cell lines (LN827, LN215, and U373), CXCL12 increased the expression of transcripts encoding MT2-MMP, but not other MMPs (Zhang et al. 2005). This was correlated with increased cell surface expression of MT2-MMP. Increased MT2-MMP expression was also noted on resected glioma specimens compared to normal brain. The increased MT2MMP expression was correlated with invasiveness; treatment with CXCL12 increased the capacity for LN827 and U373 glioma cells to invade through matrigel, an effect that was abolished by antisense knockdown of MT2-MMP. The knockdown of MT2-MMP also appears to reduce the invasiveness of the tumor in a xenograft

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model, converting the pattern of tumor growth from infiltrative to a more discrete mass with a well-circumscribed border. The same study also demonstrated increased survival in mice bearing xenografts with MT2-MMP knockdown. Interestingly, treatment with VEGF, which has been shown to increase SDF expression, also increased cell invasion through matrigel in U251n glioblastoma cells (Hong et al. 2006). Together, these findings suggest a role for the CXCL12/CXCR4 axis in invasion of tumor cells.

11.4 CXCR4 and Angiogenesis While the intrinsic ability of tumor cells to proliferate, survive, invade adjacent tissues, and metastasize is of obvious importance to tumor growth, the local microenvironment is a key component to these processes as well. For example, because of the limits of diffusion through tissue of essential factors such as oxygen, additional blood vessels are required for tumor growth beyond a sub-millimeter scale. Angiogenesis, the process of new vessel growth via generation of new endothelium from existing vasculature has been actively explored as means to limiting tumor growth. Numerous regulators, including bFGF and VEGF, have been characterized in this process. More recently, the concept that new vessels can be formed through the recruitment of bone marrow derived endothelial precursors, or vasculogenesis has been investigated. Intriguingly, besides providing the essential function of increased tumor perfusion, new vessel growth may also promote tumor growth through a separate mechanism. Neural stem cells and likely brain tumor stem cells reside in a perivascular niche, or specialized microenvironment, that both supports the cells and controls proliferation and fate determination (Calabrese et al. 2007). New vessel growth may therefore, also support brain tumor stem cells by providing new niche sites. Chemokines play a role in tumor angiogenesis. Both CXCL12 and CXCR4 are expressed in tumor-associated endothelial cells (Rubin et al. 2003; Salmaggi et al. 2004; Warrington et al. 2007; Woerner et al. 2005; Yang et al. 2007; Zagzag et al. 2006). Human glioma cells produce the pro-angiogenic factors VEGF and IL-8 upon activation of CXCR4 by CXCL12 (Ping et al. 2007). Reciprocally, VEGF and hypoxia-inducible factor 1 (HIF) upregulate CXCR4 in glioblastoma (Zagzag et al. 2006). HIF is a transcription factor whose levels increase with hypoxia. Both VEGF and CXCR4 contain hypoxia response elements (HRE) and are positively regulated by HIF. Furthermore, VEGF and bFGF increased CXCR4 expression in endothelial cells (Zagzag et al. 2006). These finding suggest a complex pattern of paracrine/ autocrine signaling between endothelial cells and glioma cells. In addition to acting directly on endothelium, cytokines such as CXCL12 can act via other cell types to induce new vessel formation. Aghi et al. found that CXCL12, but not VEGF, recruits bone marrow derived vascular progenitor cells to induce new vasculature in rodent glioma models (vasculogenesis) (Aghi et  al. 2006). Interestingly in regions of tumor hypoxia, marrow derived pericytes and endothelial

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cells accumulated, whereas in normoxic tumor regions only marrow derived endothelial cells were found. Du and colleagues also found that HIF1, partly through CXCL12, induces recruitment of bone marrow derived cells to promote neovascularization in glioblastoma (Du et al. 2008). Both irradiation and hypoxia have been reported to promote homing of hematopoetic progenitors to gliomas via TGF-beta dependent, HIF1-mediated release of CXCL12 (Tabatabai et al. 2006). Other cytokines produced by gliomas including IL-8, TGFbeta1, NT-3, and VEGF can induce angiogenesis by attracting marrow stromal cells (Birnbaum et al. 2007). Together these studies define multiple mechanisms in which gliomas induce new vessel formation via cytokine signaling.

11.5 Therapeutic Targeting of Chemokine Pathways The widespread and abnormally high expression of CXCL12 and CXCR4 in brain tumors together with the pleiotrophic effects of CXCL12 on brain tumor growth suggests that targeting this pathway could have broad and powerful therapeutic benefit in the treatment of brain tumors. Accordingly, multiple groups, working in diverse cancer models, have examined the anti-tumor effects of CXCR4 pathway antagonism. The first report of an anti-tumor effect of CXCR4 blockade involved inhibiting the metastatic spread of breast cancer cell line implantations with an antibody directed against CXCR4 (Muller et  al. 2001). These were the first data to indicate that CXCR4 expression had a functional significance with regard to mediating the pattern of cancer spread. The objective of modulating CXCR4-mediated cell motility has two distinct applications in brain tumor therapy: limitation of brain tumor cell migration/invasion and spread through decreased CXCR4 activity and increasing the homing of stem cell-based therapeutics by increasing CXCR4 activation. Neural stem cells (NSCs) exhibit tropism for glioma in brain tumor models (Aboody et al. 2000; Ehtesham et al. 2004; Tang et al. 2003). When injected into brain regions that are distant from the site of glioma xenografts, NSCs migrate to the tumor mass and satellite tumor cells and are especially tropic for the tumor vasculature and its associated tumor stem cell population. The glioma-tropic behavior of NSCs provides a unique therapeutic opportunity to use NSCs as couriers to deliver cytotoxic agents. This strategy has proved effective in preclinical trials for glioma in which NSCs were engineered to deliver tumoricidal genes (Aboody et al. 2008; Ehtesham et  al. 2002a; Ehtesham et  al. 2002b; Herrlinger et  al. 2000; Yu et al. 2006). The success of NSC therapy will be dependent upon the efficacy of their cargo and the efficiency with which they track to tumor cells. With regard to the latter, Ehtesham et  al. 2006 found that NSC migration is dependent upon CXCR4 and can be inhibited by treatment with CXCR4 antagonists. The growth effects of CXCR4 can also be directly targeted. Rubin et al. (2003) were the first to demonstrate that systemic administration of a small molecule antagonist of CXCR4 could block the intracranial growth in xenograft models of glioblastoma and medulloblastoma. Building on these findings, Redjal et al. (2006)

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showed that combining the specific CXCR4 antagonist, AMD 3100 with the DNA alkylator BCNU resulted in a synergistic anti-tumor effect. While these results confirm the importance of this pathway to brain tumor biology and validate CXCR4 as a therapeutic target, the diverse homeostatic functions of CXCR4 in multiple tissues may preclude prolonged and generalized inhibition of this pathway in cancer treatment. For this reason, continued investigation into the downstream mediators and efforts to distinguish normal and cancer cell CXCR4 signaling will be necessary to support ongoing efforts to develop viable strategies for CXCR4 antagonism. In this regard, Yang et al. (2007) demonstrated that pharmacological and genetic blockade of CXCR4-mediated cAMP suppression blocks CXCL12-induced brain tumor cell growth in vitro and that cAMP elevating drugs can block the intracranial growth of U87 glioblastoma and Daoy medulloblastoma xenografts.

11.6 Other Chemokines Contribute to Brain Tumor Biology CXCL12 and CXCR4 are not the only chemokine and cognate receptor pair present in brain tumors and the role of chemokines in brain tumor biology is not limited to those functions described above. CCL3L1 and its receptors CCR3 and CCR5 are expressed in the majority of glioblastomas but not lower grade astrocytomas (Kouno et  al. 2004). Overexpression of CCL3L1 in the U251 GBM cell line enhanced in vitro growth. Similarly, Zhou et al. showed that the glial growth factor GRO-a (CXCL1) was highly expressed in two primary anaplastic astrocytoma specimens (Zhou et al. 2005). Overexpression of CXCL1 in U251 cells increased the expression of migration/invasion-associated proteins like MMP2 and b1-integrin and this correlated with enhanced migratory and invasive behavior of these cells in vitro as well as increased intracranial growth capacity. These data identify these pathways as potentially important therapeutic targets. In all tissues, chemokines function to regulate leukocyte trafficking and inflammatory responses. This is true in the central nervous system as well and may also be true in brain tumors. For instance, expression of monocyte chemoattractant protein 1 (MCP-1, CCL2) is greater in high-grade astrocytomas than in low-grade astrocytomas (Desbaillets et al. 1994; Leung et al. 1997). CCL2 functions to recruit multiple types of inflammatory cells, including those of the monocyte-macrophage lineage, such as microglia (Platten et al. 2003). Microglia are bone marrow-derived resident macrophages of the CNS. They are often present in brain tumors in great numbers, along with macrophages, but whether they stimulate or inhibit growth is not clear. Also unclear is the relative importance of CCL2 in the recruitment of microglia in  vivo. Kielian et  al. were able to knock-down CCL2 expression in tumor cells without affecting the microglial infiltrate (Kielian et al. 2002). These data suggested that either CCL2 was unimportant, or that redundant signals were involved, in the recruitment of microglia. Moreover, Liang et  al. (2008) have recently suggested that CCL2 may function in an autocrine manner by binding its receptor CCR2A and stimulating glioma cell migration. Finally, recruitment of

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CCR4-expresssing regulatory T cells is also stimulated via glioma cell secretion of CCL2. This may be an important mechanism whereby gliomas suppress anti-tumor immune responses and evade immune detection (Jordan et al. 2008).

11.7 Conclusions and Future Directions Chemokines serve as the messengers in a reciprocal relationship that exists between brain tumors and their environment (Fig. 11.1). Brain tumors are dependent upon stromal sources of chemokines for their growth and spread. They also use chemokines

Fig. 11.1  Chemokines mediate both stromal regulation of brain tumor growth and brain tumor recruitment of stromal constituents. The endothelium of tumor-associated blood vessels provides a niche for brain tumor stem cells (TSC). (a) CXCL12 is produced by vascular endothelium and may serve to localize TSC and maintain their mitotic quiescence, in a manner analogous to CXCL12 functions in the bone marrow. (b) CXCL12 produced by multiple stromal elements in brain tumors including vascular endothelium, infiltrating microglia, and entrapped axons promotes brain tumor cell proliferation and survival. (c) Expression of CXCL12 by endothelium and other parenchymal elements of the brain may dictate the patterns of spread such as the perivascular spread of GBM as originally described by Scherer. (d) The secretion of chemokines such as CXCL3L1 and CCL2 may be critical to the recruitment of inflammatory cells such as macrophages (Mf) or regulatory T cells (TC). Expression of CXCL12 may be important to the recruitment of mesenchymal stem cells (MSC) including those that participate in angiogenesis

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to recruit cellular elements to their microenvironment, changing it to better support their own growth. The study of chemokines and brain tumors will be critically important in our efforts to understand how brain tumors form and grow as well as to identify key biological targets for novel brain tumor therapies. The challenge will continue to lie in the need to distinguish between brain tumor and normal brain biology before chemokine action in brain tumors may be targeted without disrupting normal chemokine functions. In this regard, a better understanding of how the various effects of chemokines in brain tumor biology are mediated will be essential. Especially important will be determining how the primary oncogenic events in brain tumorigenesis such as loss of p53, Rb or PTEN function, and gain of Sonic hedgehog, EGF, or PDGF receptor signaling regulate chemokine and chemokine receptor expression and function. Just as the brain tumor can only be understood in the context of its environment, chemokine actions in brain tumor will only be understood when they are studied in the context of the abnormal biology of brain tumors.

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Chapter 12

Chemokines as Neuromodulators: Regulation of Glutamatergic Transmission by CXCR4Mediated Glutamate Release From Astrocytes Corrado Calì, Julie Marchaland, Osvaldo Mirante, and Paola Bezzi

12.1 The CXCL12/CXCR4 Signaling Pathway In the Central Nervous System (CNS) 12.1.1 The Expression of CXCL12/CXCR4 System In the CNS Chemokines are a class of small secreted proteins that were first identified as inflammatory mediators of leukocyte chemotaxis (Luster 1998; Luther and Cyster 2001) and have been subsequently shown to possess a much larger repertoire of physiological and pathological functions (Rossi and Zlotnik 2000). Since the discovery of the first protein showing a chemotactive activity (Yoshimura et  al. 1987), the chemokine family has been expanded to approximately 50 chemokines (Laing and Secombes 2004). They are classified according to the number and spacing of the conserved cysteine residues at the N-terminal position (Murphy et al. 2000). The two largest groups are (1) the CXC, where the first two cysteines are separated by one amino acid residue and (2) the CC, where the first two cysteines are adjacent to each other (Rossi and Zlotnik 2000; Fernandez and Lolis 2002). The two small groups are (1) the C chemokines with only one cysteine in the N-terminal region and (2) the CX3C chemokine: the two cysteines are separated by three amino acid residues (Rossi and Zlotnik 2000). Phylogenic analyses showed that the CXC chemokine family is a very recent phenomenon, limited to higher vertebrates. Interestingly, homologues of CXC chemokines and their receptors are present in animals that do not possess an immune system (Huising et  al. 2003), suggesting that its ancestral role might be within the central nervous system and not within the immune system. Chemokines exert their biological effects through cell surface receptors belonging to the family of seven-membrane domain G-protein-coupled receptors (GPCRs, Chen et al. 2004). At least 22 chemokine receptors have been characterized to date, which

C. Calì, J. Marchaland, O. Mirante, and P. Bezzi () Department of Cellular Biology and Morphology (DBCM), Faculty of Medicine, University of Lausanne, Rue du Bugnon 9, Lausanne, Switzerland e mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_12, © Springer Science+Business Media, LLC 2010

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are classified according to the chemokine nomenclature. In general, chemokine GPCRs can be activated by several chemokines and can bind different G-proteins, triggering a great variety of intracellular signaling pathways (Neves et al. 2002). Although chemokines have been identified and extensively characterized in the immune system, the idea that chemokines and their receptors are important in areas beyond immunology came first from studies on inflammation and inflammatory diseases. In particular in the central nervous system (CNS) it was early recognized that glial cells such as astrocytes and microglial cells constitutively express a large variety of chemokines and their associate receptors. Interestingly, their expression can be modulated in the presence of inflammatory mediators or during acute and chronic inflammatory conditions as for instance in different neurological disorders, such as multiple sclerosis, trauma, stroke, Alzheimer’s disease, and acquired immunodeficiency syndrome-associated dementia (also called AIDS dementia complex, Streit et  al. 2001; Vila et  al. 2001; Lee et  al. 2002; McGeer and McGeer 2004; Cartier et  al. 2005). There is therefore a general consensus about the role of chemokines as mediators of neuroinflammation processes and leukocyte infiltration in the brain (Mennicken et al. 1999; Gerard and Rollins 2001; Biber et al. 2002; McGeer and McGeer 2002; Minghetti 2005; Ubogu et al. 2006; Ransohoff et al. 2007; Li and Ransohoff 2008; Rojo et al. 2008). Constitutive expression of chemokines and their receptors has later been documented in the developing brain where these proteins play a role in the migration, differentiation, and proliferation of glial and neuronal cells. One of the first important indications of the fundamental role of chemokines in the brain came from the analysis of phenotypes associated with deletion of some genes expressing chemokines or chemokines receptors. For instance it has been reported that among the CXC chemokines, the CXCL12 (also called Stromal cell Derived Factor-1 or SDF-1) and its related receptor CXCR4 are essential for life during development. Deletion of genes encoding both CXCR4 and CXCL12 genes (Nagasawa et al. 1996; Ma et al. 1998; Tachibana et al. 1998; Zou et al. 1998) is lethal for mice soon after birth, with several abnormalities affecting many organs, including the CNS. In particular, the integrity of the CXCL12/CXCR4 system is indispensable for normal neuronal cell migration and pattering in the developing cerebellum and hippocampus (Ma et al. 1998; Zou et al. 1998; Lu et al. 2002). Basically all of the deficits observed in the knockout mice can be explained by the role of CXCR4 signaling in regulating the migration of different types of progenitor cells. In addition to its role in the progenitor migration, development and organogenesis, the CXCR4 signaling seems to have tissue specific roles; for instance CXCR4 signalling can work as an axon guidance cue (Chalasani et al. 2003, 2007; Lieberam et al. 2005). Recently, another receptor for the CXCL12 has been described in T lymphocytes (CXCR7, Balabanian et al. 2005), but to date, there is no evidence of its presence in the normal brain. An emerging area of interest for the physiological action of the brain of chemokine is the intercellular communication system between neuronal and glial cells. Indeed, recent in vitro and in situ studies indicated that the chemokines together with their receptors are constitutively expressed by glial cells and neurons in mature brain (Asensio and Campbell 1999; Cho and Miller 2002; Tran and Miller 2003; Rostene

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et al. 2007). Although it is widely recognized that astrocytes and microglia are the primary source of chemokines, there are evidences that neurons express and secrete chemokines as well, confirming a neuronal contribution to chemokine signaling. Among chemokines with a possible role in fast communication system in the brain, the best evidence concerns the CXCL12 chemokine and its receptor CXCR4. The CXCL12 chemokine has been first described as a secreted product of the bone marrow stromal cell line (Tashiro et al. 1993). Recent evidences show that it is differentially expressed in the developing and mature CNS (Rostene et al. 2007) and exists in three isoforms (a, b, g) arising from alternative splicing (Gleichmann et al. 2000; Pillarisetti and Gupta 2001; Stumm et al. 2002). The CXCR4 receptor is a G-protein coupled receptor (GPCR) that is highly expressed both during development and in the mature brain (Jazin et al. 1997; Lavi et al. 1997; McGrath et al. 1999), as well as its natural ligand CXCL12. In vitro, neuronal and astrocytic expression of CXCL12 and CXCR4 receptor was revealed in cultured cortical, hippocampal and cerebellar neurons (Ohtani et  al. 1998; Bajetto et  al. 1999; Klein et al. 1999; Bezzi et al. 2001; Tham et al. 2001). In situ, studies performed by in situ hybridization and dual immunohistochemistry demonstrated that CXCL12/ CXCR4 system is constitutively expressed in astrocytes, microglia and neurons, in discrete neuroanatomical regions such as the cerebral cortex, substantia innominata (where CXCR4 is colocalized with choline acetyltransferase; Banisadr et al. 2002), supraoptic and paraventricular hypothalamic nuclei (where CXCR4 expressed in arginine–vasopressin (AVP) neurons; Banisadr et  al. 2003), lateral hypothalamus (where CXCR4 is colocalized with neurons expressing the melanin-concentrating hormone (MCH); Guyon et  al. 2005b), ventromedial thalamic nucleus (Banisadr et al. 2002) and hippocampus (Banisadr et al. 2003). Further studies have shown that the chemokine CXCL12, like CXCR4, is expressed on GABAergic neurons of the pars reticulate (Guyon et al. 2006) and in the Purkinje neurons and granule cells in the cerebellum (Ragozzino et al. 2002). The overall reported results together clearly showed that the chemokine CXCL12 and its receptor CXCR4 are differentially expressed; CXCL12 is highly expressed in glial cells (mostly on astrocytes) and CXCR4 in both glial (astrocytes and microglia) and specific subsets of neuronal cells (Asensio and Campbell 1999). The pattern of distribution, therefore, supports the idea that CXCL12/CXCR4 system is involved in the bidirectional communication between glia and neurons.

12.1.2 The Effect of CXCR4-Mediated Signaling Pathway on Neuronal Activity and Neurotransmitter Release Number of papers to date have shown that the CXCR4 receptors expressed in both neuronal and glial cells are functional and coupled to multiple intracellular pathways (Lazarini et al. 2003). The CXCR4 through pertussis toxin (PTX)- sensitive G proteins is coupled to at least two distinct signaling pathways: (1) the first pathway, involving the activation of phosphatidylinositol- 3 (PI-3) kinase and extracellular signal

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regulated kinase (ERK)1⁄2, has been described in astrocytes, neuronal progenitors, and cortical neurons (Bacon and Harrison 2000; Lazarini et al. 2000; Bajetto et al. 2001; Bezzi et al. 2001; Bonavia et al. 2003); (2) the second pathway, coupling the activation of the phospholipase C to the intracellular increases of calcium, has been described in astrocytes, cortical neurons, and cerebellar granule cell, as well as in primate fetal neuron and microglia in vitro (Bajetto et al. 1999; Klein et al. 1999; Zheng et al. 1999b). The signaling arising from the CXCL12-CXCR4 interaction can exert diverse important biological effects in different brain areas (Lazarini et al. 2003). Indeed, it has been reported that the activation of CXCR4 receptor induces migration and proliferation of cerebellar granule cells (Lazarini et al. 2003), chemo-attracts microglial cells (Bajetto et al. 2001; Rezaie et al. 2002), acts as neuromodulator (Guyon and Nahon 2007) and stimulates cytokine production and glutamate release from astrocytes (Bezzi et al. 2001). Evidences supporting the participation of CXCL12/ CXCR4 system to the intercellular communication between glia and neurons are, however, limited; it includes activation by CXCL12 of intracellular events involving calcium elevations in both astrocytes and neurons (Zheng et al. 1999a; Zheng et al. 1999b) and some modulatory effects of such chemokine on neuronal electrical properties and synaptic function (Limatola et al. 2000; Guyon and Nahon 2007). In contrast, one of the best characterized biological effect of CXCL12/CXCR4 signalling concerns the in  vitro studies performed on the development of nervous system. These results has been recently supported by in vivo studies performed on knockout mice (Ma et al. 1998; Zou et al. 1998) where a clear abnormality in the development of the cerebellum (Klein et al. 2001; Klein and Rubin 2004) and the hippocampal dentate gyrus (DG; Bagri et al. 2002; Lu et al. 2002) was reported. In neuronal populations of different brain areas, the CXCL12 chemokine interacts with neuronal activity by directly binding CXCR4 through different multiple pathways including: (1) modulation of the plasma membrane ionic channels such as the sodium, the potassium and particularly the high threshold calcium channels (Zheng et  al. 1999b) resulting in the intracellular calcium increase and PYK2 activation (Lazarini et  al. 2000); (2) activation of the G-protein-activated inward rectifier potassium current (Guyon et al. 2005a, 2005b) and (3) increase in neurotransmitter release (GABA, glutamate and dopamine) often through calcium-dependent mechanisms (Zheng et  al. 1999b; Limatola et  al. 2000; Guyon et  al. 2006). It is interesting to note that the CXCL12/CXCR4 system shows a convergent presynaptic activity in different brain areas; for instance the application of CXCL12 chemokine causes increase on glutamate and/or GABA synaptic activities in lateral hypothalamus (Guyon et al. 2005a), hippocampus (Zheng et al. 1999b), cerebellum (Limatola et  al. 2000) and substantia nigra (Guyon et  al. 2006). The CXCL12mediated pre-synaptic effect, however, can be exerted by two different mechanisms: (1) by a direct interaction with pre-synaptic CXCR4 receptor (for instance in the substantia nigra) or (2) by an indirect mechanism involving glutamate release from peri-synaptic glial cells. In particular, Purkinje neurons from cerebellar acute slices responded to CXCL12 applications by an increase in spontaneous GABAergic activity and by slow inward currents (SICs) that appeared only when synaptic transmission was

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inhibited. Moreover, the SICs were almost abolished by antagonists of ionotropic glutamate receptors (Ragozzino et al. 2002), suggesting that they were mediated by extrasynaptic glutamate, most likely released by surrounding glial cells (Limatola et al. 2000). Despite the extensive studies performed to elucidate the biological effects of the receptor/chemokine interaction, the intracellular biochemical pathways required to achieve such a variety of biological effects remain to be further elucidated.

12.1.3 Activation of CXCR4 Induces Release of Chemical Transmitters (Gliotransmitters) from Glial Cells The first demonstration that astrocytes signal to neurons in response to activation of plasma membrane GPCRs came in 1994 from the group of Philip Haydon (Parpura et al. 1994). In this pioneering paper, authors described calcium rises and glutamate release from astrocytes mediated by the activation of astrocytic bradykinin receptors in mixed neuron-glia cell culture. Further evidences supporting this first in  vitro study came few years later, from the group of Giorgio Carmignoto; by stimulating neuronal afferent fibers in acute brain slices, they found that a bidirectional communication between neuron and glia based on glutamate release from astrocytes exists in intact brain tissue (Pasti et al. 1997). A parallel study by Andrea Volterra’s group (Bezzi et al. 1998) provided the direct demonstration that activation of astrocytic metabotropic glutamate receptors (mGluR) both in cell cultures and in situ triggered glutamate release via calcium-dependent mechanisms involving prostaglandins (PGs) production. More recently a wide range of studies (from our and other groups) have confirmed these initial observations, strongly supporting the theory that the calciumdependent glutamate pathway is triggered by activation of diverse GPCRs (such as chemokine receptor CXCR4, Bezzi et  al. 2001; or purinergic receptor P2Y1, Domercq et al. 2006) and required release of calcium from internal stores (Sanzgiri et al. 1999; Jeremic et al. 2001; Kang et al. 2005; Takano et al. 2005). In 2001 we discovered that, in addition to classical transmitters, such as glutamate or ATP, the CXCL12 chemokine induces calcium-dependent glutamate release from astrocytes through a direct activation of the GPCR CXCR4 (Bezzi et al. 2001). Interestingly, the chemokine-mediated glutamate release process seemed to involve a long chain of intracellular and extracellular events related to the release of two chemical mediators, the tumor necrosis factor-alpha (TNFa) and the PGs (Fig 12.1). During the first events of such intracellular pathway, the CXCR4 activation induced by its natural ligand CXCL12 caused intracellular calcium elevations and triggered a sequence of molecular events that resulted in the extracellular release of soluble TNFa. Such events included calcium-dependent stimulation of the ERK-MAPK pathway (ERK1/2, possibly via the calcium-dependent kinase PyK2) followed by activation of a surface metalloproteinase such as the TNF-converting enzyme (TACE), ultimately responsible for shedding of TNFa from its plasma membrane precursor.

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Fig.  12.1  CXCR4-mediated signalling pathway leading to glutamate release from astrocytes. Cartoon in this figure shows the involvement of TNFa and PGs on the CXCR4-mediated glutamate release. (1) CXCL12 chemokine binds its receptor, CXCR4, on the astrocytic membrane. (2) CXCR4 activation induces somatic Ca2+ increases. (3) Ca2+-dependent activation of TACE (TNFa converting enzyme) is responsible for the shedding of TNFa from its plasma-membrane precursor. (4) Shedded TNFa can bind TNFR1 both on the same membrane it is coming from (autocrine pathway) or in the surrounding astrocytes (paracrine pathway), leading the production of PGE2. (5) Released PGs bind its receptor EPRs on the same astrocyte they were released (autocrine pathway) or in the surrounding astrocytes (paracrine pathway). (6) EPRs activation results in somatic Ca2+ leading to glutamate release from astrocyte

The interaction of TNFa with its cell-surface receptors, in particular TNFR1, initiated a second sequence of intracellular signaling events leading to the production of PGE2 via a calcium-independent mechanism. PGE2 then activates a third series of events leading to calcium elevation followed by glutamate release. It is important to note that the mechanism by which PGE2 controls the latter process is largely unknown and may involve extracellular release of the PG followed by interaction with one of its cell-surface receptors (EP receptors). The role of the extracellular mediators TNFa and PGs in the CXCR4-mediated glutamate release process has been recently suggested in a different study by our group where the glutamate release process

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was induced by the activation of purinergic P2Y1 receptor (Domercq et al. 2006). The shedding of the cytokine TNFa as well as the formation of PGE2 apparently represents primordial signalling events accompanying glutamate release. Indeed, the amount of glutamate was strongly diminished in the presence of pharmacological inhibitors of TNFa and PGs and in astrocytes cultures from TNFa and TNFR1 knockout mice (Domercq et al. 2006). The overall results indicated that TNFa and PGs act as extracellular amplifiers of the intracellular calcium response first and of glutamate release process thereafter; whether their role as external amplifier could be mediated by a fast release upon activation of CXCR4 or by a tonic basal release, remains to be elucidated. Indeed, the fast glutamate release kinetics indicates that the production and the release of the two mediators should occur very soon after the GPCR activation. Alternatively, a basal release of both mediators might tune the secretory apparatus for a more efficient glutamate release. The TNFa and PGs can act on the same cell they are released from (autocrine amplification) or it is equally likely that they act on neighboring astrocytes, therefore propagating the signal in a paracrine fashion (Fig. 12.1). Importantly, we found that CXCR4 is also expressed in microglia and stimulation of CXCR4 in these cells results in strong TNFa release, several-fold higher than in astrocytes, but occurring only when microglia are in a reactive state. Stimulation of microglial CXCR4 does not induce glutamate release (Bezzi et al. 2001; Vesce et al. 2007). From the overall data, it is becoming increasingly apparent that astrocytes, by releasing chemical transmitters (the so called “gliotrasmitters”, Bezzi and Volterra 2001), now appears to be critically implicated in rapid communication systems in the brain.

12.2 Regulated Exocytosis of Glutamate from Astrocytes can Modulate Synaptic Transmission 12.2.1 Mechanisms of Gliotransmitters Release: Evidence on CXCR4-Mediated Glutamate Exocytosis from Astrocytes Because of their strategic localization, astrocytes play a crucial role in maintaining the extracellular ionic homeostasis, provide energetic metabolites to neurons and remove excess of neurotransmitter in schedule with synaptic activity. In addition, the strategic location of astrocytes allows them to carefully monitor and control the level of synaptic activity. Indeed, number of papers during the last 15 years have shown that cultured astrocytes can respond to a variety of neurotransmitters with a variety of different patterns of intracellular calcium increases (Verkhratsky et  al. 1998). Later on, studies performed in intact tissue preparations (acute brain slices) further established that the plasma membrane receptors can sense external inputs (such as the spillover of neurotransmitters during intense synaptic activity) and transduce them as intracellular calcium elevations, mostly via release of calcium from internal stores (Dani et al. 1992; Murphy et al. 1993; Porter and McCarthy

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1995a, b; Pasti et  al. 1997; Latour et  al. 2001; Grosche et  al. 2002; Perea and Araque 2005; Serrano et  al. 2006; Jourdain et  al. 2007). Recently, two studies performed in the living brain have revealed that astrocytes of the somatosensory cortex undergo calcium elevations in response to the neuronal activity triggered by sensory stimulation (Wang et  al. 2006; Winship et  al. 2007), providing the first direct evidence that astrocytes are activated during physiological functioning of the brain circuitry. The intracellular calcium elevations in astrocytes are a complex phenomenon involving multiple and varied spatio-temporal patterns of calcium peaks most likely representing a cellular “code” for generation of diverse output. The real significance of the calcium “code” generated in astrocytes by neuronal activity remains mysterious and waits to be deciphered. One of the better characterized output generated by the complex calcium signalling in astrocytes is the release of gliotransmitters (Bezzi and Volterra 2001; Volterra and Meldolesi 2005; Santello and Volterra 2008). Over the last few years, the number of recognized gliotransmitters has increased; for instance glutamate, ATP and its metabolites, D-serine, TNFa and prostaglandins (PGs) are now considered as chemical mediators participating to the intercellular communication system, including astrocyte-to-neuron signalling (Araque et al. 1999). The molecular mechanisms leading to release of gliotransmitters from astrocytes are still widely debated and remain to be fully elucidated (Nedergaard et al. 2003). Among gliotransmitters the amino acid glutamate is the better characterized one; number of papers to date have shown that it can be released from astrocytes (1) by both calcium-dependent and calcium-independent mechanisms, (2) through multiple pathways activated under different conditions, (3) at different loci and (4) with different modalities (Vesce et  al. 2007). The calcium-independent mechanisms include (1) reversed operation of reuptake carriers, notably under ischemic conditions (Rossi et  al. 2000), (2) exchange with cystine, the essential substrate for astrocytic production of glutathione, mediated by specific cystine-glutamate antiporters (Warr et  al. 1999), and (3) molecular permeation of large pore channels, including P2X7 receptors (Duan et al. 2003), gap-junction hemichannels (Ye et al. 2003) and volume-sensitive organic anion channels (Kimelberg et  al. 1990), although for the latter a calcium-dependent mechanism has also been described (Takano et al. 2005). The evidence for the existence of a calcium-dependent exocytosis pathway in astrocytes has been provided for the first time by our group, some years ago (Bezzi et al. 2004). By means of ultrastructural studies, we provided the first proof that astrocytes in adult brain tissue (in the hippocampus) contain a vesicular compartment competent for storing and releasing glutamate. The astrocytic vesicles, found in perisynaptic astrocytic processes, strictly resemble synaptic vesicles (SV) in glutamatergic nerve terminals with a diameter of 30–50 nm, express vesicular glutamate transporters (VGLUT1/2) and SV SNARE proteins such as cellubrevin (Bezzi et  al. 2004; Crippa et  al. 2006; Jourdain et  al. 2007; Ni et  al. 2007). By complementing the ultrastructural studies in situ with dynamic total internal reflection fluorescence (TIRF) real-time imaging studies in cultured astrocytes, we could directly document individual vesicle fusion events accompanied by glutamate

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release in response to mGluR activation (Bezzi et al. 2004). Such exocytic fusions occurred within hundreds of milliseconds from GPCR stimulation and were abolished by a classical blocker of exocytosis, tetanus neurotoxin (TeNT). The presence of regulated exocytosis in astrocytes has now been documented with a wide range of experimental approaches, including optical detection techniques different from TIRF (Kang et al. 2005; Crippa et al. 2006), membrane capacitance measurements (Kreft et al. 2004; Zhang et al. 2004b), electrochemical amperometry (Chen et al. 2005), as well as by selective interference with proteins of the exocytotic machinery (Montana et al. 2004; Zhang et al. 2004a). In spite of these findings, it has never been investigated whether the activation of the CXCR4 receptor triggers the release of glutamate by regulated exocytosis. This is an important issue that can clarify the physiological role of chemokines in the brain especially after the recent works suggesting that glutamate exocytosis from astrocytes can modulate synaptic transmission (Fellin et  al. 2004; Fiacco and McCarthy 2006; Jourdain et al. 2007; Perea and Araque 2007; Navarrete and Araque 2008). Investigating the spatio-temporal features of the CXCR4-mediated glutamate release in astrocytes may therefore elucidate whether chemokines can participate in fast brain signaling. In our recent work, we studied CXCR4-mediated exocytosis in astrocytes (Calì et al. 2008) by taking advantage of a state-of-art imaging methodology recently developed to investigate SV recycling processes in nerve terminals (Zenisek et al. 2002; Aravanis et al. 2003; Richards et al. 2005; Harata et al. 2006). We adapted the imaging approach in order to study the properties of CXCR4mediated glutamate exocytosis in astrocytes at the single-vesicle level. Thanks to the limited penetration of the evanescent wave (EW, about 100  nm; Domercq et  al. 2006) illumination in TIRF microscopy and the properties of the red fluorescent styryl dye FM 4-64, we selectively loaded recycling SLMVs and directly visualized and monitored the CXCR4-dependent exocytosis. The loading protocol and the single vesicle analysis provided detailed information about the recycling processes. We found that glutamatergic SLMVs in astrocytes, monitored with TIRF and confocal microscopies, have an average diameter of about three pixels (about 378 nm), compatible with those of fluorescent beads of 40 nm of diameter and with those of SVs visualized with TIRF microscopy (Zenisek et al. 2000) and thus coherent with the diameters of astrocytic SLMVs identified in hippocampal tissue by electron microscopy (Bezzi et al. 2004; Jourdain et al. 2007). Interestingly, we found that the glutamatergic SLMVs underwent recycling in different cellular compartments (Calì et al. 2008). Indeed, about 65% of SLMVs, most likely representing those undergoing a local recycling (such as kiss-and-run or kiss-and-stay, Harata et  al. 2006; Marchaland et al. 2008), remain in proximity of the plasma membrane; the remaining 35%, most likely representing those undergoing an intra-cytoplasmic recycling (such as clathrin-coat-dependent process or bulk retrieval, Rizzoli and Jahn 2007), were instead found deep in the cytoplasm. These results suggest that astrocytes, similarly to neurons and neurosecretory cells, show at least two modes of exocytosis and recycling processes (Chen et al. 2005; Marchaland et al. 2008). Single fusion events of glutamatergic vesicles have been then monitored by following FM 4-64 destaining (Fig. 12.2), a sequence of the stereotyped fluorescence

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Fig 12.2  CXCR4-mediated glutamate exocytosis from astrocytes. (a) Sequence of images showing a typical exocytic event of a VGLUT-positive SLMV revealed by FM4-64 de-staining. On the left, a SLMV expressing VGLUT2-EGFP. Images in gray scale represent the same vesicle loaded with FM4-64 during a single fusion event. Times represent milliseconds before (−50 ms) and during (50, 250, 400 ms) the fusion event. Bars: 500 nm. Under gray scale images, are represented the “radial sweeps” calculated by plotting the FM fluorescence intensity against the distance from the center of the fluorescent spot. Red lines represent the best fits of Gaussian functions. (b) Temporal distribution of fusion events during CXCL12 application, obtained by plotting the number of fusions (counted as FM4-64 de-staining) against time. (c) CXCR4 and Ca2+ dependency of SDF-1a-evoked exocytosis. Fusion events are reduced to the basal level (CTRL) if astrocytes were stimulated in the presence of AMD3100 (100  ng/ml, n = 5 cells), were pre-incubated with BAPTA/AM (50 mM, n = 5 cells), with thapsigargin (Thapsi 50  nM, 20  min; n = 6 cells) or with cyclopiazonic acid (CPA 10 mM, 15 min; n = 6 cells). In contrast, the absence of external Ca2+ (0 Ca2+ with 5 mM of EGTA; n = 5 cells) failed to produce any significant effect. Image modified from Calì et al. 2008

intensity changes that is well characterized for SVs (Zenisek et  al. 2000; Harata et al. 2001). The activation of CXCR4 receptor with nM concentrations of its natural ligand CXCL12, induced a burst of exocytosis with the rate of fusion events increasing up to 174-fold over the basal level in the first 500 ms (Fig. 12.2). During the exocytic burst, about 60% of SLMVs located within 100 nm from the plasma membrane (in the EW) before the stimulus, underwent exocytosis in about 4 s. The exocytosis triggered by CXCL12 was mediated by the activation of CXCR4 receptors and involved the release of calcium from internal stores (Fig. 12.2), consistent

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with the reported coupling of CXCR4 receptor with 1,4,5-triphosphate-(IP3)dependent intracellular calcium elevations (Zheng et al. 1999b). One important peculiarity of the CXCR4-dependent stimulus-secretion coupling that leads to SLMVs exocytosis is its rapidity. It occurs with a relatively fast time scale in the order of few hundred milliseconds, longer than what is recorded in SVs but analogous to events reported for clear vesicles of neurosecretory cells (Kasai 1999). Interestingly, the exocytic burst unfolds in a time scale much shorter than that of dense-core granules in neurosecretory cells, which are governed by voltagegated calcium channels (VGCCs, Kasai et al. 1999). Taking into account that astrocytes are electrically non-excitable and thus exocytosis in these cells cannot be triggered by the opening of strategically distributed VGCCs (Carmignoto et  al. 1998), our results are remarkable. Indeed, the kinetics of the stimulus-secretion coupling differs enormously among secretory cell types, but in non-electrically excitable cells the process is usually much slower than in excitable cells (Chow et al. 1994; 1996; Kasai 1999). One of the main reasons is that non-excitable cells similarly to astrocytes rely only on the activation of GPCRs, a signaling pathway that involves signaling steps necessary for the release of calcium from the internal stores. Our results indicates that the exocytosis time scale in response to GPCR stimulation in astrocytes is remarkably faster than in any other non-excitable cell (Bezzi et al. 2004; Calì et al. 2008; Marchaland et al. 2008). The stimulus-secretion coupling in astrocytes appears to reflect an adaptation of astrocytes (the most abundant non electrically-excitable cells in the brain) for rapid information transmission at the frequencies characteristic of synapses. As there is a direct correlation between the spatial organization of the sites of exocytosis and sites of calcium increase with kinetics of secretion (Penner and Neher 1988; Kasai 1999; Augustine 2001; Gundelfinger et  al. 2003; Schneggenburger and Neher 2005), the rapid exoendocytosis pathway in astrocytes is of difficult interpretation without considering the existence of a local morphological and functional interaction between SLMVs and sites of Ca2+ release. The GPCR-induced signalling pathway underlying the fast exocytosis have been investigated in a different work from our group and seems dependent to localized morphological and functional microdomains of calcium (Marchaland et al. 2008). In view of our results and the strategic localization of CXCL12 and its receptor (discussed in Sect. 12.1.1), it is intriguing to speculate that the CXCL12/CXCR4 system might be involved in a fast bidirectional communication system between astrocytes and neurons.

12.2.2 Localized Calcium Microdomains Control Exocytosis of Glutamate in Astrocytes In the central nervous system (CNS) the concept that localized microdomains of calcium are responsible for triggering vesicle fusion generally refers to neurons (Rizzuto and Pozzan 2006). Concerning glial cells although the existence of structural

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calcium microdomains has been described in the fine profiles of the cerebellar Bergmann glia (Grosche et al. 1999, 2002), very little is known about their physiological implications. Previous works monitoring calcium responses to GPCR activation in these cells focused their observations to the somatic cell region (Porter and McCarthy 1996; Pasti et al. 1997, 2001; Bezzi et al. 1998; Parri et al. 2001; Fiacco and McCarthy 2006). The observed somatic calcium elevations in astrocytes upon the GPCR-activation showed too slow kinetics (1.5 s in the case of mGluR activation) to underlie the rapid burst of SLMVs exocytosis recently reported (Bezzi et al. 2004; Calì et al. 2008; Marchaland et al. 2008). In order to explain the rapidity of the burst of exocytosis in astrocytes (Bezzi et al. 2004; Jourdain et al. 2007; Calì et al. 2008), we hypothesize the existence of localized domains of calcium in the sites of exocytosis. Indeed, recently obtained results by our group confirmed our hypothesis (Marchaland et  al. 2008); we found that endoplasmic reticulum (ER) tubules are located within~400 nm from the nearest SLMVs in the tiny structural domains of fl volume space. By looking at ER in astrocytes with a TIRF illumination (Fig. 12.3) we can appreciate the complex spatial organization of the tubules and cisterns approaching the plasma membrane; they form, together with SLMVs

Fig. 12.3  Sub-membrane calcium microdomains control exocytosis of glutamate from astrocytes. (a) Confocal images showing an astrocyte double transfected with ER-GFP and VGLUT1mCherry. ER forms a complex network of tubules and cisterns within the whole cell. On the right, cartoon showing that spatial localization of mGluR, ER tubules, sub-membrane calcium events and glutamatergic SLMVs could define a functional Ca2+ microdomain. (b). TIRF images of an astrocyte transfected with ER-GFP and VGLUT1-mCherry. On the right, high magnification image confirms that SLMVs within the EW field are close to the portion of ER approaching plasma membrane

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and plasma membrane, complex and peculiar structures of sub-micrometer space that might limit diffusion exchange of signaling molecules, including calcium (Fig. 12.3). This organization might provide the structural basis for local and eventually global calcium signaling patterns necessary to control diverse intracellular processes simultaneously. Moreover, ER structures and SLMVs lie in the submembrane compartment in tight spatial proximity, with an average distance of 300–500 nm. These results are in line with recent observations where ER tubules have been considered as spatially distinct compartments, structurally and functionally coupled to the plasma membrane via cytoskeletal scaffold proteins (Tse et al. 1997; Blaustein and Golovina 2001; Sala et al. 2005; Wu et al. 2006). The organization of cytoskeleton in astrocytes could be similar to that of glutamatergic synapses, where the scaffold protein Homer provides a molecular link between IP3 receptors at the tip of the ER cisternae and mGluRs on the plasma membrane (Tu et al. 1998; Xiao et al. 1998; Sala et al. 2005). What is the functional implication of the subplasma membrane ER domain? By using fast acquisition rate TIRF imaging we found that, upon stimulation of group I of mGluR, submicrometer localized calcium elevations (hot spots) are generated in the sub-plasma membrane domains of ER. Interestingly, in most cases the sub-membrane calcium events occurred at/near sites where SLMVs underwent exocytosis (interdistance: £280 nm), and were in strict temporal correlation with the fusion events. Indeed, the two types of sub-plasma membrane event displayed similar, biphasic distribution, with the two peaks of calcium events preceding the corresponding peaks of fusion events (Marchaland et al. 2008). The localized sub-membrane calcium events in astrocytes is reminiscent of the elementary signals of calcium due to the opening of a spatially restricted group of IP3Rs channels, the so called “calcium puffs” (Bootman et al. 2001), that have been described in different cell lines (Rizzuto and Pozzan 2006). The calcium puffs similar to the sub-membrane calcium events in astrocytes typically consist of a fast elevation of the intracellular calcium (~50 ms) with limited spatial spread (~2–6 mm, Bootman et al. 1997; Thomas et al. 2000). As for the astrocytic signals, whether the localized hot spots of calcium in astrocytes represents a structural organization of IP3R clusters, or a convergence of modulatory inputs (for instance the mGluR clusters), remains to be investigated. Interestingly, in several cell types, the calcium puffs act as “pacemaker” and control the frequency of repetitive calcium spiking (the phenomenon called calcium oscillations). Sometimes, the cumulative recruitment of calcium puffs can lead to the initiation and propagation of a global cytosolic calcium signal (Tovey et al. 2001). In astrocytes we cannot exclude the existence of such a phenomenon; indeed, we found that some local sub-plasma membrane calcium hot spots propagate from the site where they originate long distances in the EW (up to 14 mm) causing a sub-membrane calcium wave. It is likely that the sub-membrane calcium events merge into propagating waves even in the z direction (out of the EW) causing the mGluR-mediated global cytosolic calcium signal with the typical slower rise (time to peak about 1.5  s). It is therefore possible that local (sub-membrane) and global calcium signaling in astrocytes control different calcium-dependent cellular processes.

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The findings recently reported can be relevant to understand the functional role of transmitter release from astrocytes in brain function. Interestingly, a recent report shows for the first time the occurrence in vivo of fast astrocytic calcium events in response to neuronal activity, peaking with millisecond time-scale from sensory stimulation (Winship et al. 2007). This finding suggests that the fast calcium events we have recently identified are not a peculiarity of astrocytes in cell culture but may correspond to events taking place in astrocytes of the living brain. Establishing whether fast calcium events in vivo are associated to transmitter release via SLMV exocytosis, becomes, therefore, of the outmost importance in order to define the type of modulatory influence exerted by astrocytes on neighboring neuronal circuits.

12.2.3 Glutamate Exocytosis from Astrocytes Induces Synaptic Modulation It is now clear that peri-synaptic astrocytes play an active role in maintaining and modifying synaptic activity (reviewed in Bezzi and Volterra 2001; Volterra and Meldolesi 2005; Haydon and Carmignoto 2006; Santello and Volterra 2008). Indeed, the astrocyte-released gliotransmitters are able to activate neuronal receptors and thereby modify the neuronal electrical excitability and synaptic transmission (Bezzi and Volterra 2001; Schipke and Kettenmann 2004; Volterra and Meldolesi 2005; Santello and Volterra 2008). Perisynaptic astrocytes receive chemical signals, “translate” them into their own language as intracellular calcium changes, and “talk back” to synapses by releasing neuroactive substances (Bezzi and Volterra 2001; Volterra and Meldolesi 2005). Thus, release of gliotransmitters (especially glutamate) from astrocytes now appears to be critically implicated in rapid communication between astrocytes and other adjacent cells in the brain. Such a conceptual breakthrough makes this field of investigation one of the hottest in neuroscience, as it calls for a revision of past theories of brain function as well as for new strategies of experimental exploration (Bezzi and Volterra 2001). Recent works have shown that glutamate released from astrocytes during physiological synaptic activity targets neuronal receptors in either axonal terminals or dendrites, exerting in the two cases a different type of neuromodulatory action (Santello and Volterra 2008). A recent paper from Andrea Volterra’s group have shown that at perforant path–granule cell (PP–GC) synapses in the hippocampal dentate gyrus, astrocytes of the outer molecular layer sense synaptic activity, elevate their intracellular calcium and release glutamate via exocytosis of SLMVs (Jourdain et al. 2007). Astrocytic glutamate is released at presynaptic level in close proximity of NR2B-containing NMDA receptors; the activation of such receptors results in an increased synaptic transmitter release and in the strengthening of synaptic transmission (Jourdain et  al. 2007). Interestingly, this is the only neuromodulatory action of astrocytes for which a precise ultrastructural correlate has been established. The authors found that the distribution of such NR2B subunits is particularly

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abundant in the extrasynaptic terminal membrane opposed to astrocytic processes containing SLMVs. This morphological evidence together with the electrophysiological results suggest a focal mode of astrocyte-to-synapse communication where the interaction between astrocytic transmitter and terminal NMDARs may occur with a spatial precision similar to that observed at neuronal synapses (Santello and Volterra 2008). In another region of the hippocampus, the CA1 region, astrocytes of the stratum radiatum similarly sense the activity of Schaffer collateral afferents and respond to it with intracellular calcium elevations and release of glutamate. In this case, however, the astrocytic glutamate acts on extrasynaptic NR2B-containing NMDA receptors located on the dendrites of CA1 pyramidal cells inducing large SICs in the pyramidal cells (Angulo et al. 2004; Fellin et al. 2004; Perea and Araque 2005). The generation of synchronous SICs has been proposed to depend on a single episode of glutamate release in one astrocyte that is sensed simultaneously by different pyramidal cells sitting in the territory of that astrocyte (Halassa et al. 2007). The astrocyte-evoked SICs has been reported also in other brain regions, including the thalamus and the nucleus accumbens (Parri et al. 2001; D’Ascenzo et al. 2007) but not in response to the activity of PP–GC synapses (Santello and Volterra 2008).

12.3 The Calcium-Dependent Glutamate Release From Astrocytes is Deregulated in Pathological Conditions With an Inflammatory Component The appreciation that brain activity involves interactive signaling between neurons and glia opens new perspectives for understanding the pathogenesis of brain diseases (Bezzi and Volterra 2001). Affecting neuron-astrocyte interactions might turn out to have a more profound impact on the functionality of the associated neuronal circuits than previously thought and, in some cases, even to be the primary cause of the neuropathology. The brain inflammation is known to cause major morphological and functional changes in glial cells, particularly astrocytes and microglia, broadly defined as “reactive gliosis” (Bezzi and Volterra 2001; Vesce et al. 2007). The signals exchanged between the two glial cell types during these events are largely unknown, yet their transition from the resting to the activated state appears to be associated with a marked up regulation of several genes and the secretion of factors like cytokines, eicosanoids, reactive oxygen species, nitric oxide, and excitatory amino acids (Perry et al. 1995). The inflammation is not only found to produce profound alterations in the structural relations between neurons and astrocytes (Fitch et al. 1999), but also between glial networks. In particular, in the presence of inflammatory cytokines such as interleukin-1b (IL-1b) and TNFa, changes in the expression of junctional proteins (John et al. 1999; Duffy et al. 2000), in propagation of intracellular calcium waves (Liu et al. 2000) and glutamate release (Bezzi et al. 2001) have been observed. Thus, when local inflammatory reaction is triggered in the brain (as it may occur, for example, in Alzheimer’s disease, in the

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AIDS neuropathology or after brain ischemia), microglial cells rapidly migrate to the site of injury (Davalos et al. 2005; Nimmerjahn et al. 2005), become activated and start releasing a number of mediators such as IL-1b, IL-6, PGs and TNFa, deeply altering the properties of glial networks (Bezzi and Volterra 2001). Moreover, in view of the control exerted by PGs and TNFa on calcium-dependent glutamate release from astrocytes (Bezzi et  al. 1998, 2001; Domercq et  al. 2006), overproduction of these mediators during neuroinflammation might favor an increased and deleterious glutamatergic input from astrocytes to neurons (Vesce et  al. 2007). Consequently, the neuron-glial signaling might be perturbed. This hypothesis is substantiated by direct experimental evidence.

12.3.1 The Case of HIV-Associated Dementia Infection with human immunodeficiency virus-1 (HIV-1) can destroy the immune system and lead to acquired immunodeficiency syndrome (AIDS); the virus can induce severe and debilitating neurological problems as well (Ellis et al. 1997; Kaul et al. 2001, 2005; Power et al. 2002). The syndrome of cognitive and motor dysfunction observed in the presence of HIV-1 has been designated HIV-associated dementia (HAD) or AIDS-dementia complex (McArthur et  al. 1993; Kaul et  al. 2001; Gendelman and Persidsky 2005). Although highly active antiretroviral therapy (HAART) has decreased the incidence of HAD, it does not seem to provide complete protection from or reversal of HAD (Dore et al. 1999; Major et al. 2000). Currently there is no specific treatment for HAD, mainly because of an incomplete understanding of the mechanisms by which HIV infection causes neuronal injury and apoptosis (Kaul et al. 2001). It widely accepted that HIV entry into CNS occurs via infected monocytes cells (macrophages and microglial cells, Koenig et al. 1986; Gartner 2000; Persidsky et al. 2001) however, the injury and apoptotic cell death occur only in neurons (Eilbott et  al., 1989). Once in the brain, infected macrophages or microglia release viral envelope glicoproteins (gp120), cytokines (for example TNFa) and chemokines, which in turn activate uninfected glial cells that start to release neurotoxic substances (Giulian et al. 1996; Wesselingh et al. 1997) such as quinolinic acid and other excitatory amino acids (EAAs, such as glutamate), L-cysteine, arachidonic acid, PAF, free radicals and TNFa (Kaul et al. 2001; Kaul et al. 2005). These substances, including the gp120 released from infected cells induce neuronal injury (Miller and Meucci 1999), dendritic and synaptic damage and apoptosis (Masliah et al. 1997; Everall et al. 1999) through direct (Toggas et al. 1994) or indirect routes (for instance via release of glutamate from astrocytes, Bezzi et al. 2001). Neuronal death is therefore thought to occur via interactions with infected microglial cells as well as with the astrocytes (Meucci and Miller 1996; Toggas et al. 1996; Miller and Meucci 1999; Kaul et al. 2001). In support to these findings, the topographic distribution of neuronal apoptosis is correlated with evidence of structural atrophy and closely associated with markers for microglia activation. Infection of macrophages and lymphocytes by HIV-1 can occur after binding of the viral envelope protein gp120 to one of the several possible chemokine receptors

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in conjunction with CD4 (Miller and Meucci 1999; Kaul et al. 2005). Generally, T cells are infected via the a-chemokine receptor CXCR4 and/or the b-chemokine receptor CCR5. In contrast, macrophages and microglia are primarily infected via the b-chemokine receptor CCR5 or CCR3, but the a-chemokine receptor CXCR4 may also be involved (He et  al. 1997; Michael and Moore 1999). The HIV coreceptors CCR5 and CXCR4, among other chemokines receptors, are also present on neurons and astrocytes (Rottman et al. 1997; Zhang et al. 1998). As CXCR4 receptor serves as a HIV co-receptor (Feng et  al. 1996) and is expressed on glial and neuronal cells, several in vitro studies have suggested that CXCR4 is directly involved in HIV-associated neuronal damage while CCR5 may additionally serve a protective role (Hesselgesser et al. 1998; Meucci et al. 1998; Kaul and Lipton 1999). In cerebrocortical neurons and neuronal cell lines from humans and rodents, picomolar concentrations of HIV-1 gp120 can induce neuronal death via CXCR4 receptors (Hesselgesser et  al. 1998; Ohagen et  al. 1999; Chen et al. 2002). In mixed neuronal/glial cerebrocortical cultures that mimic the cellular composition of the intact brain, this apoptotic death appears to be mediated predominantly via the release of microglial toxins rather than by direct neuronal damage (Bezzi et al. 2001; Garden et al. 2004). By using mixed neuronal/glial cerebrocortical cultures, acute brain slices, and an in vivo model of HAD, we have further investigated the role of CXCR4 chemokine receptor in the neurotoxicity of gp120. The rapid CXCR4-triggered signaling cascade (Fig. 12.2) displays the involvement of TNFa. Indeed, data obtained not only in cell cultures but also in the TNFa knockout mice demonstrate the essential involvement of the cytokine in the coupling of GPCRs to exocytotic glutamate release from astrocytes (Bezzi et al. 2001; Domercq et al. 2006). In the pathological brain, such as during HAD, astrocytes and microglia often form local foci of reactive cells around the sites of lesion or infection. In a recent paper, we mimicked this condition by adding lypopolissaccaride (LPS) activated microglia to astrocyte-pure cultures in about the same ratio (1:10) existing in the brain. Simultaneous stimulation of CXCR4 with its natural ligand CXCL12 or with the glycoprotein gp120IIB (a T-tropic form of the HIV envelope protein) in reactive microglia and astrocytes resulted in a dramatic amplification of TNFa release from both cells; the TNFa amplification, as a consequence, resulted in strong potentiation of calcium-dependent glutamate release from the astrocytes (Bezzi et  al. 2001, Fig.  12.4). Since shedding of viral proteins in the HIV-1 infected brain is an uncontrolled process, it is likely that an over stimulation of glial CXCR4 receptor could lead to both an excessive glutamate release from astrocytes and the excitotoxic neuronal cell death. Indeed, in separate set of experiments performed in hippocampal cultures containing neurons, astrocytes and activated microglia, we could demonstrate that the microglia- and TNFa-dependent potentiation of astrocyte glutamate release had neurotoxic consequences, inducing slow apoptotic death of neuronal subpopulations. Finally, experiments done at the whole animal level confirmed that the identified CXCR4-acitvated cascade is part of the mechanism responsible for the toxic action of gp120 in the brain. In agreement with previous findings obtained by Gigi Bagetta’s group (Bagetta et  al. 1999), subchronic intracerebroventricular microinfusions of gp120IIIB (100 ng/daily for 7 days) consistently induced early

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Fig  12.4  TNFa-dependent alteration of CXCR4-dependent glutamate release from astrocytes during inflammatory conditions. During HAD, a local inflammatory reaction is triggered in the brain; this phenomenon induces microglial cells to converge to the site of injury, to become activate and to start releasing a number of mediators, notably inflammatory cytokines such as TNFa, that alter profoundly the properties of astrocytes. HIV-infected microglia become reactive and shed viral particles, including the envelope glycoprotein gp120 which can act as a ligand of CXCR4 receptors that are present in astrocytes and also in the reactive microglia itself, thus inducing release of higher concentrations of TNFa compared to the physiological conditions. The increase of TNFR1 activation in astrocytes results in massive and ultimately excitotoxic release of glutamate

microglial activation and delayed apoptotic cell death in the rat brain neocortex. When the CXCR4 antagonist or anti-TNFa antibodies were co-administered with the viral glycoprotein, the number of apoptotic cells observed was significantly reduced (Bezzi et  al. 2001). We therefore discovered that gp120 acts as CXCR4 agonist in both astrocytes and microglia and, similar to the endogenous CXCL12, triggers potent TNFa-dependent glutamate release from astrocytes (Fig. 12.4). The dramatic amplification of the cytokine-induced response in the presence of activated microglia is consistent with a switch from physiology to excitotoxicity. In view of their broad significance, the TNFa-dependent synergism between reactive microglia and astrocytes leading to excitotoxicity might be operative not only in HAD but also in other neurodegenerative diseases.

12.3.2 The Case of Alzheimer’s Disease In the past few years an increasing set of evidence has converged on the major role of glial cells and alterations in their function in the pathway toward

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neuronal degeneration (Saez et  al. 2006; Vesce et  al. 2007; Rojo et  al. 2008). Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the progressive loss of cognitive function. One of the best-known biochemical hallmarks in the AD is the accumulation of the amyloid-b (Ab) protein into amyloid plaques that can be found in the extracellular space of forebrain regions (Glenner and Wong 1984). Chronic inflammatory glial cell reactions, involving both microglial and astrocytic cells, are well documented around Ab plaques (WyssCoray 2006). While it is not completely understood whether inflammation plays a causative role in AD, recent data from animal models suggest that some inflammatory processes might at least accelerate the development of the disease. For instance, a-secretase, which is the Ab -generating enzyme, was found in reactive astrocytes from aged Tg2576 mice (Hartlage-Rubsamen et al. 2003), a transgenic AD mouse model expressing the double mutation K670N-M671L of the amyloid precursor protein APP. The proximity of these astrocytes to the plaques raises the possibility that they promote Ab accumulation. Inflammation is a process that has been actively related with the onset of several neurodegenerative disorders, including AD. However, the precise implications of the inflammatory response for neurodegeneration have not been elucidated. A current hypothesis considers that an extracellular insult to neurons could trigger the production of inflammatory cytokines by astrocytes and microglia such as IL-1b, TNFa, and IL-6, that in turn might affect the normal behavior of neuronal cells (Arai et al. 1990). In spite of these indications, the precise role of cytokines in AD is not fully understood (Rojo et  al. 2008). It has been described that cytokines secreted by microglial cells, astrocytes and/or neuronal cells may induce synthesis of certain acute-phase proteins including the amyloid precursor protein or APP (Del Bo et al. 1995; Blasko et al. 2000). On the other hand, the Ab itself can induce the expression of cytokines in astrocytes and microglial cells in culture (Gitter et al. 1995; Chong 1997). Interestingly, high levels of proinflammatory cytokines have been detected in the brain of AD patients as well (Zhao et al. 2003). In particular for IL-6, it was observed that (1) its secretion by peripheral blood mononuclear cells was increased specifically in patients with AD as opposed to those suffering from other brain disorders such as vascular dementia (Ravaglia et al. 2006) and that (2) overexpression of IL-6 in the brain of transgenic mice (Campbell 1998) is associated with a variety of neuropathological findings, including gliosis and disruption of cholinergic neurotransmission in the hippocampus (Heyser et al. 1997). The observation that the TNFa signaling is implicated in the glutamate release process from astrocytes (Bezzi et al. 2001; Domercq et al. 2006) together with the fact that the expression of both TNFa and TNFR1 receptors is enhanced in the brain of AD patients (Zhao et  al. 2003; Del Villar and Miller 2004) suggested to the Volterra’s group to investigate possible alterations of the TNFa-dependent pathway of glutamate release in PDAPP mice, a transgenic model of AD (Rossi et al. 2005). The authors used transgenic mice ubiquitously overexpressing the familial AD-linked human V717F mutation of the amyloid precursor protein (APP) gene (PDAPP mice (Games et al. 1995)). These animals show age-related deficits in spatial learning and memory retention (Dodart et al. 1999; Chen et al. 2000) and reproduce several neuropathologi-

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cal features of human AD, notably deposition of Ab plaques and associated reactive gliosis (Suzuki et al. 1994; Masliah et al. 1996; Reilly et al. 2003). Changes in the calcium-dependent glutamate release have been investigated in acute brain slices from hippocampus of PDAPP mice by direct application of TNFa. Surprisingly, the authors found a considerable reduction of TNFa-dependent glutamate release compared to adult control animals (Rossi et al. 2005). The defect was region-selective as the glutamate release response from cerebellar slices of aged PDAPP mice was identical to that of controls. Interestingly, the secretory process appeared intact, since stimulation with PGE2, which acts as downstream of TNFa, evoked normal glutamate release. Therefore, the alteration must take place at the level of the stimulus-secretion coupling mechanism. An important intracellular mediator of TNFa signaling is the protein DENN/MADD, which binds TNFR1 and triggers multiple downstream signaling pathways, including cytosolic phospholipase A2 that is coupled to arachidonic acid release and prostaglandin production. Interestingly a reduced expression of DENN/ MADD was reported in the AD patients (Del Villar and Miller 2004). Volterra’s group found that DENN/MADD is defective in the hippocampus of PDAPP mice as well (Rossi et al. 2005), suggesting that the defect of this protein expression may account for reduced glutamate release in PDAPP mice. Downregulation of DENN/MADD may result from the chronic inflammation that characterizes the slow progression of AD and might be a consequence of long-term overstimulation of TNFR1 by excessive TNFa. This condition is very different from the in vitro acute inflammation model that we used in the studies concerning CXCR4-evoked glutamate release (Bezzi et  al. 2001). In that case, we tested glutamate release within 24  h from adding reactive microglia to astrocyte cultures. It is therefore likely that acute and chronic TNFa overproduction cause opposite alterations of glutamate release from astrocytes.

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Chapter 13

Role of CX3CL1 in Synaptic Activity and Neuroprotection Davide Ragozzino, Clotilde Lauro, and Cristina Limatola

13.1 Introduction 13.1.1 CX3CL1/CX3CR1 Pair: Structure and Signalling Fractalkine, named CX3CL1 in accordance with the official chemokine nomenclature (Murphy et  al. 2000), has a distinctive transmembrane structure only shared by another chemokine, CXCL16 (Matloubian et  al. 2000). The presence of a long mucin stalk headed by a chemokine domain at the N-terminus has been implicated in the adhesive function of CX3CL1 on the luminal surface of the vascular endothelium to recruit circulating leukocytes: a systematic analysis of the properties of the mucin-like stalk revealed that it may have only bestowal function (Fong et  al. 2000). The long membrane-adhering trail can be cleaved by metalloproteinases (Chapman et  al. 2000), whose enzymatic activity can be either inducible, like ADAM17 (Garton et  al 2001; Tsou et  al. 2001), or constitutive, like ADAM10 (Hundhausen et al. 2003) and, as recently described in the spinal cord, by cathepsis S (Clark et al. 2007), generating a soluble molecule with chemotactic activity. The CX3CL1 receptor, CX3CR1, first cloned in rat as orphan RBS11 (Harrison et al. 1994) and subsequently in human as V28 (Raport et al. 1995) or CMKBRL1 (Combadiere et al. 1995), was recognized as the specific one for CX3CL1 by Imai et  al. (1997). CX3CR1 is reported to be coupled to the Gai protein (Imai et  al. 1997; Maciejewski-Lenoir et  al. 1999), with the inhibition of the adenyl cyclase (Ragozzino et  al. 2006), and the activation of both the mitogen-activated protein kinase ERK and the phosphatidylinositol-3 kinase (PI-3K)/Akt pathways (Meucci et al. 1998, 2000). One exception is reported for murine CX3CR1 where a polymorphism, introducing a proline residue at the C-terminal region (aa326), abolishes PI-3K/Akt coupling (Davis and Harrison 2006). More debated is CX3CR1 coupling to the stress-activated protein kinase (SAPK) 2, p38, whose phosphorylation is not D. Ragozzino, C. Lauro, and C. Limatola () Department of Physiology and Pharmacology, University Sapienza, Piazzale A. Moro, 5, 00185, Rome, Italy e-mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_13, © Springer Science+Business Media, LLC 2010

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observed in neurons (Meucci et  al. 1998; Deiva et  al. 2004), is controversial on microglial cells (Chen et al. 2002; Zhuang et al. 2007) and is reported to be either stimulated or inhibited in Mono Mac 6 cells (Cambien et  al. 2001; Vitale et  al. 2004). Like all the signalling chemokine receptors cloned to date, CX3CR1 activation is coupled to intracellular Ca2+ increase induced by phosphatidylinositol,4,5 bisphosphate-specific PLC activation (Imai et al. 1997; Combadiere et al. 1998a, 1998b). In case of CX3CR1, an additional Ca2+ source is the entry of Ca2+ from the extracellular space (Kansra et  al. 2001; Deiva et  al. 2004) which, at least when CX3CR1 is expressed in the CHO cell line, may require the receptor-induced PI3-K activity (Kansra et al. 2001).

13.1.2 Distribution in the Nervous System Since the original cloning in 1997 (Bazan et  al. 1997) CX3CL1 appeared to be highly expressed in the brain. A detailed analysis of CX3CL1 localization in the brain disclosed a widespread expression, with maximal levels observed in the cortex, striatum and hippocampus (Tarozzo et al. 2003). A microarray analysis performed on developing hippocampus revealed that the expression of CX3CL1 gene is upregulated in the hippocampus between p16 and p30, thus suggestive of a possible modulatory role of the chemokine on mature synapses (Mody et al. 2001). Specifically, CX3CL1 expression is very well recognized in neurons both in the brain and in the spinal cord (Harrison et al. 1998; Nishiyori et al. 1998; Schwaeble et al. 1988; Boddeke et al. 1999; Verge et al. 2004) but it is also reported in astrocytes and microglial cells, where CX3CL1 level can be modulated by combinations of locally produced cytokines under pathophysiological conditions (MaciejewskiLenoir et  al. 1999; Yoshida et  al. 2001; Pereira et  al. 2002; Hughes et  al. 2002; Hulshof et al. 2003; Sunnemark et al. 2005; Lindia et al. 2005; Lee et al. 2007). CX3CR1 is also expressed throughout the nervous system: microglial cells are the cells that most abundantly express CX3CR1 in the brain and in the spinal cord (Harrison et al. 1998; Nishiyori et al. 1998; Verge et al. 2004). Astrocytes have been reported to express CX3CR1 either constitutively or upon cytokine induction (Jiang et  al. 1998; Maciejewski-Lenoir et  al. 1999; Dorf et  al. 2000; Chen et  al. 2002; Hulshof et al. 2003). Nevertheless, even if astrocyte treatment with CX3CL1 does not activate a classical migratory response, it does induce Ca2+ signalling and the release of factor(s) that stimulate microglial cells proliferation (MaciejewskiLenoir et  al. 1999). The expression of CX3CR1 on neurons is instead a debated issue: though its presence in neuronal preparations is rarely described, (Meucci et al. 2000; Hatori et al. 2002; Hughes et al. 2002) different functional assays testify for clear neuronal responses to CX3CL1 (Meucci et al. 1998, 2000; Gillard et al. 2002; Oh et al. 2002; Deiva et al. 2004; Limatola et al. 2005; Ragozzino et al 2006; Lauro et al. 2006). However, the generation of knock-in mice where CX3CR1 is deleted and the EGFP fluorescent protein is expressed under the CX3CR1 promoter (CX3CR1GFP/GFP, Jung et al. 2000) revealed only green microglial cells in the brain,

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thus supporting the view that microglia represent the main cell population expressing CX3CR1 in the CNS.

13.2 CX3CL1 Functions in Physiological and Pathological Conditions 13.2.1 CX3CL1 and Synaptic Activity There is growing evidence of neuromodulatory actions of chemokines in the CNS where they may modulate several types of ion channels, thus contributing to the control of neural transmission (de Haas et al. 2007). Specifically, CX3CL1 has also been shown to play a role in neuromodulation since both pre- and post-synaptic effects have been reported in different neuronal preparations (Meucci et al. 1998; Deiva et al. 2004; Limatola et al. 2005; Ragozzino et al. 2006). Ca2+ transients upon CX3CL1 application have been detected in cultured hippocampal neurons (Meucci et al. 1998; 2000), dorsal root ganglion cells (Oh et al. 2001), cerebellar granules and Purkinje cells (Gillard et  al. 2002), mixed neuronal-glial cultures and in SK-N-SH neuroblastoma cells (Deiva et al. 2004), although most of the times only a minority of the tested cells had a clear response to CX3CL1, even within putative homogeneous neuronal populations (Oh et al. 2001; Gillard et al. 2002). CX3CL1 has been reported to act pre-synaptically, reducing synaptic glutamate release in hippocampal cultured neurons (Meucci et  al. 1998). In these cells, CX3CL1 reduces the frequency of spontaneous Ca2+ transients and of excitatory post-synaptic currents, both likely due to the activity of cell network in the culture. Since CX3CL1 causes a decrease in the frequency of synaptic events without a corresponding shift in the amplitude distribution, this pattern is indicative of a reduced rate of synaptic release. Consistently with the pre-synaptic action of the chemokine, CX3CL1-induced inhibition of voltage-activated Ca2+ currents are reported (Meucci et  al. 1998). Similarly, CX3CL1 inhibits Ca2+ currents in a reconstituted cellular system and, also to a minor extent, in dorsal root ganglion neurons but not in dissociated septal neurons (Oh et al. 2002). The observation that CX3CL1 effects on Ca2+ current modulation are sensitive to pertussis toxin (PTX) indicates that this chemokine controls neurotransmitter release through one of the most common mechanisms of pre-synaptic neuromodulation. Moreover, as reported for other chemokines that directly affect neuronal excitability both modulating the properties of action potential and changing membrane potential (Guyon and Nahon 2007), CX3CL1 favours neuron excitability on dorsal root ganglion cells (Oh et al. 2001). In addition to the pre-synaptic effects, CX3CL1 modulates the functional properties of ligand-gated channels at post-synaptic sites. In SK-N-SH cells, a human neuroblastoma cell line, CX3CL1 reduces the amplitude of NMDA-induced calcium transients (Deiva et  al. 2004). In these cells, CX3CL1 application causes a PTX insensitive transient increase in the intracellular Ca2+ concentration dependent on

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the influx from the extracellular space (see also Kansra et al. 2001). Yet, CX3CL1 strongly reduces the amplitude of Ca2+ transients caused by subsequent NMDA application and this effect is specific for NMDA-induced Ca2+ influx, since acetylcholine-induced Ca2+ transient is unaltered. However, it is not clear whether this phenomenon involves a direct modulation of NMDA receptors, affecting current amplitude. Other post-synaptic neuromodulatory effects of exogenous CX3CL1 have been reported in hippocampal neurons, where CX3CL1 reduces the amplitude of AMPAevoked glutamatergic currents through post-synaptic mechanisms (Limatola et al. 2005; Ragozzino et al. 2006). This down-regulation of AMPA-currents is mediated through CX3CR1, being absent in CX3CR1-deficient mice (Bertollini et al. 2006; Ragozzino et al. 2006), and being blocked by neurons treatment with anti-CX3CR1 antibody (Ab) (Limatola et  al. 2005; Bertollini et  al. 2006). Furthermore, it has been reported that (1) the CX3CR1 activation causes a Ca2+- and PTX-dependent reduction of GluR1 receptor phosphorylation at Ser845, a protein kinase A target, an event attributed to both a decrease in cAMP accumulation and a shift in favour of phosphatase activity on this site; and (2) CX3CL1-induced current depression occurs only when AMPA receptors are active and intracellular Ca2+ is not tightly buffered by intracellular BAPTA, requiring a simultaneous synaptic stimulation, and suggesting that this phenomenon may depend on the recent story of synaptic activity (Ragozzino et al. 2006). This down-regulation of AMPA receptors is associated with a reduction in the amplitude of evoked and spontaneous excitatory postsynaptic currents, as well as the amplitude of field potential in hippocampal slices (Limatola et al. 2005; Ragozzino et al. 2006; Bertollini et al. 2006). In addition, the synaptic depression induced by CX3CL1 apparently shares common mechanisms with long-term depression (LTD) of synaptic activity, which is also associated with AMPA receptor dephosphorylation. In fact, when LTD is saturated by repetitive trains of electrical stimulation, the effect of CX3CL1 on the field potentials is absent, showing an occlusion between CX3CL1-induced synaptic depression and LTD (Bertollini et  al. 2006). Altogether, these data strongly suggest that soluble CX3CL1 production, in the brain, could modulate both short- and long-term synaptic plasticity events, with functional implications in synaptic transmission, and these results are summarized in Fig. 13.1a. Lacking clear evidence of CX3CR1 expression in neurons, experiments have been addressed to see whether any CX3CL1-induced effect could be mediated by non-neuronal cells, namely microglia. CX3CL1-induced depression of AMPAcurrents is abolished by treatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a specific antagonist of adenosine receptor type 1 (A1), indicating that the activation of A1 is involved in the neuromodulatory effects of CX3CL1. Interestingly, these effects are mimicked by exogenous adenosine (Lauro et  al. 2008). Moreover, the medium conditioned by microglial cells stimulated with CX3CL1 also reduces the amplitude of AMPA-currents in CX3CR1-deficient neurons (CX3CR1GFP/GFP), and this current depression is abolished by DPCPX treatment (Lauro et al. 2008). These results imply that the stimulation of microglial cells by CX3CL1 causes the release of diffusible factor(s), which modulate glutamate

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neuroprotection Fig. 13.1  Neuroprotection and neuromodulation by CX3CL1. (a) CX3CL1 reduces glutamatergic synaptic transmission acting both pre- and post-synaptically. (1). In cultured hippocampal neurons, CX3CL1 reduces voltage activated calcium currents and glutamate release, activating Gi/ G0 protein coupled receptors. (2). In hippocampal neurons, CX3CL1 reduces the amplitude of currents through active AMPA type glutamate receptors. Current depression is Ca2+- and phosphatase- dependent and mediated through Gi/G0-mediated reduction in cAMP accumulation and GluR1 dephosphorylation at Ser845. (3). In SK-N-SH neuroblastoma, CX3CL1 reduces Ca2+ transients induced by NMDAR activation. This effect is independent of pertussis toxin. (b) CX3CL1 protects neurons against excitotoxic insults. CX3CL1 is expressed on neurons in membraneanchored form and is released (1) by metalloproteinases cleavage, upon excitotoxic insult. The soluble form acts on microglial cells (2), reducing the release of inflammatory cytokines and on neurons (3), activating ERK and PI3k/Akt pathways and modulating AMPA and NMDA receptors. Both microglial and neuronal pathways activated by CX3CL1 are involved in chemokine-induced neuroprotection

receptors activity on neurons. To be effective on neurons, such putative factor(s) require the presence of tonically active A1 (as depicted in Fig. 13.2), acting as permissive elements, as already demonstrated for the vasointestinal inhibitory peptide (VIP)-induced GABA release (Cunha-Reis et al. 2008).

13.2.2 CX3CL1 and Pain Modulation Pain sensation is modulated by glial cells communication with neuronal cells (reviewed by Scholz and Woolf 2007). The involvement of the CX3CL1/CX3CR1 pair in pain modulation has been recently demonstrated in different examples of experimental neuropathic pain induced by peripheral nerve injury or inflammation

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Fig. 13.2  Adenosine receptor 1 (A1) involvement in CX3CL1 actions on neurons. Many of the effects of CX3CL1 require the activation of A1 and are mediated by microglia-released factors, suggesting the involvement of microglial cells and adenosine receptors in CX3CL1-induced neuromodulation and neuroprotection. (1) CX3CL1 reduces microglial release of inflammatory cytokines; (2), causes the release of adenosine and probably of other diffusible trophic factors. Adenosine may act on A1 on presynaptic terminals (3), reducing glutamate release; postsynaptic membranes (4), modulating the properties of glutamate receptors; microglial cells (5) or astrocytes (6), inducing the release of neuroprotective factors

(Milligan et al. 2004, 2005; Verge et al. 2004; Lindia et al. 2005; Sun et al. 2007). Intrathecal injection of CX3CL1 is reported to induce mechanical allodynia and thermal hyperalgesia (Milligan et  al. 2004, 2005), through CX3CR1 activation, since CX3CL1 effects are blocked by anti CX3CR1 Ab (Milligan et al. 2004; Clark et  al. 2007). Furthermore, anti-CX3CR1 Ab strengthens the effects of chronic intratechal morphine when co-injected (Johnston et al. 2004). CX3CL1 expression levels are up-regulated in injured nerves (Lindia et al. 2005; Clark et  al. 2007; Luongo et  al. 2008), and this effect is often paralleled by an increased CX3CR1 expression on locally recruited microglial cells, like shown in facial motor neurons after axotomy (Harrison et al. 1998), and in the spinal cord upon inflammatory or traumatic nerve injury (Verge et  al. 2004; Ji et  al. 2005; Lindia et  al. 2005; Sun et  al. 2007). In the spinal cord, an increase of soluble CX3CL1 has been explained by cleavage of the membrane bound form by cathepsin S released from the microglia (Clark et al. 2007). In its turn, soluble CX3CL1 enhances pain sensation through the activation of p38 phosphorylation on microglial cells (Zhuang et al. 2007), and also through an increased release of inflammatory cytokines, since CX3CL1 effects are blocked by IL-1 and IL-6 antagonists and by NO synthase inhibitors (Milligan et  al. 2005), and spinal cord tissues stimulated with CX3CL1 show an increased release of interleukin-1 (Johnston et al. 2004).

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CX3CL1

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pain

allodynia hyperalgesia

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allodynia

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2 lesion

3 CX3CL1

Fig. 13.3  CX3CL1 and pain modulation. CX3CL1 and CX3CR1 are involved in pain transmission, although their exact role still need to be clarified. (1) CX3CL1 is up-regulated in injured nerves after axotomy, inflammatory or traumatic injury; in the spinal cord, the increase of soluble CX3CL1, after injury, is due to the cleavage of the membrane bound form by cathepsin S, released from microglia. Soluble CX3CL1 enhances pain sensation through the activation p38 and the increased release of inflammatory cytokines (IL-1, IL-6) and NO by microglial cells. In parallel, locally recruited microglial cells show increased CX3CR1 expression. Intrathecal injection of CX3CL1 (2) induces CX3CR1-dependent mechanical allodynia and thermal hyperalgesia. Conversely (3), intra-neural CX3CL1 administration reduces allodynia in the spinal nerve injury (SNI) mice model of neuropathic pain

In further support of a pro-nociceptive role of CX3CL1 are data showing that the direct injection of CX3CL1 in the periaqueductal grey, a brain region mostly involved with analgesic responses, albeit un-effective by itself, results in inhibition of the antinociceptive effects induced by m, d, and k opioid agonists (Chen et al. 2007). In contrast with the above reported results, a recent study demonstrates an opposite role for CX3CL1 on nociception. Actually Holmes and co-authors (Holmes et al. 2008) reported that the intra-neural CX3CL1 administration has an antinociceptive role in mice, reducing allodynia in the spinal nerve injury (SNI) model of neuropathic pain. Furthermore, CX3CR1 knockout mice have increased allodynia in the same model of neuropathic pain (Holmes et al. 2008). These results would imply either that peripheral versus central CX3CR1 activation may have opposite roles on nociception (Fig. 13.3) or, that the effect of CX3CL1 on nociception may vary between rats and mice. However, overall, they indicate that the exact role of the pair of CX3CL1/CX3CR1 in pain sensation still needs to be clarified.

13.2.3 CX3CL1 and Neuroprotection In recent years increasing evidence have accumulated that CX3CL1 may have important neuroprotective functions in the nervous system. The emerging concept is that the pair of CX3CL1/CX3CR1 plays several modulatory roles regulating cytokine production upon different pathophysiological conditions. The roles of CX3CL1 in neuroprotection have been documented by a series of experiments

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performed both in  vitro and in  vivo in different models of neuronal injury and neurotoxicity. 13.2.3.1 HIV Infection Several observations are suggestive of a potential intriguing defensive role of CX3CL1 against the detrimental effects of HIV infection in the nervous system. Even before its identification as a specific receptor for CX3CL1, V28/CX3CR1 was established as one of the chemokine receptors with functions as HIV coreceptor (Reeves et al. 1997; Rucker et al. 1997; Combadiere et al. 1998a, 1998b; Garin et  al. 2003). Furthermore, soluble CX3CL1 was shown to inhibit the HIV co-receptor activity of CX3CR1 (Combadiere et al. 1998b) and an up-regulation of CX3CL1 protein is observed, in neurons, in the brain of HIV-infected pediatric patients, with a specific localization in vesicular structures (Tong et al. 2000) and, in astrocytes, in the brain of adult HIV-infected patients (Pereira 2001). This high levels of soluble CX3CL1, also reported in other studies (Cotter et  al. 2002; Erichsen et al. 2003), provide a potential putative role for CX3CL1 as a functional competitor of virus entry and cell infection. Moreover, a possible correlation between a CX3CR1 structural variant, the I249/M280 haplotype (which has been associated with a lower risk of cardiovascular diseases, Moatti et al. 2001) and the progression to AIDS in HIV-infected patients, is questioned (Faure et  al. 2000; McDermott et al. 2000; Vidal et al. 2005). The first evidence of a direct neuroprotective function of CX3CL1 appeared in a paper published by Meucci et  al. (1998) where this and other chemokines are shown to possess protective activities towards HIV-gp120 induced neurotoxicity on cultured hippocampal neurons. Afterwards, the neurotoxic effects of two others HIV-related proteins, tat and PAF, on cerebellar neurons, are shown to be antagonized by CX3CL1 (Tong et  al. 2000). The protective activity against gp120IIIb protein neurotoxicity is shown to be mediated by PI3K/Akt signalling pathway, being completely abolished when the activation of PI3-K by CX3CL1 is prejudiced by the use of specific pathway inhibitors (Meucci et al. 2000). 13.2.3.2 Glutamate Excitotoxicity and Models of Brain Ischemia A protective effect of CX3CL1 against glutamate receptor over-activation has been described in human, rat and mouse neurons and seems to rely, at least in part, on the ability of CX3CL1 to down-modulate glutamate-mediated responses, either reducing the NMDA-mediated Ca2+ influx, as shown on human embryonic neurons and SK-N-SH neuroblastoma cells (Deiva et al. 2004), or depressing the AMPAtype mediated currents, as shown on rat hippocampal neurons (Meucci et al. 1998; Limatola et al. 2005; Ragozzino et al. 2006). The direct modulation of glutamate receptors activation by CX3CL1 cannot account for all the neuroprotective activities described for CX3CL1, since it maintains its neuroprotective capabilities even

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when given 8 hours after the induction of the excitotoxic insult, when the glutamate-mediated receptor(s) activation is largely exhausted (Limatola et  al. 2005) (Fig.  13.1b). In addition, other evidence indicate that both the early and the late protective effects of CX3CL1 against glutamate-induced excitotoxicity require the activation of different intracellular mechanisms, since the inhibitors of signal transduction pathways (PI3-K and ERK), which are active in blocking the protection induced by early treatment (Deiva et al. 2004; Limatola et al. 2005), are completely ineffective on protection induced by late CX3CL1 treatment (Limatola et al. 2005). Moreover, CX3CL1 is also capable of reducing Purkinje neurons death induced by glial feeder layer removal (Gillard et  al. 2002) and excitotoxicity of cerebellar granule neurons (Limatola et al. 2005). The role of microglial cells in mediating the neuroprotective effect of CX3CL1 was recently evaluated in CX3CR1GFP/GFP mice both in  vivo (Cardona et  al. 2006; Huang et al. 2006, see below) and in vitro (Lauro et al. 2008). Hippocampal neurons obtained from CX3CR1GFP/GFP mice can be rescued from glutamate-induced neurotoxicity if treated with a medium conditioned by wt microglial cells stimulated with CX3CL1 (Lauro et  al. 2008). This effect is dependent on the activation of A1, being completely abolished by neuron treatment with DPCPX. Furthermore CX3CL1-treated microglial cells and hippocampal mixed cultures release adenosine (Lauro et  al. 2008), which has very well known neuroprotective activities through the activation of A1 (Ribeiro 2005). Thus, it is possible to hypothesize that microglial cells release adenosine that, together with not yet identified factors, are responsible for CX3CL1-mediated neuroprotection acting either directly on neurons or indirectly through astrocytes (Fig.  13.2). In vitro experiments demonstrate that neuron over-stimulation with glutamate, a situation that may mimic glutamateinduced excitotocixity which occurs upon brain ischemia, induces a rapid CX3CL1 cleavage from the plasma membrane of rat and human neurons increasing soluble CX3CL1 (Chapman et al. 2000; Erichsen et al. 2003; Limatola et al. 2005), with a mechanism that requires matrix metalloproteinase activity (Chapman et al. 2000), again suggestive of a cellular attempt to protect damaged tissue. Intriguingly, urokinase-type plasminogen activator (uPA), which is commonly used in the treatment of ischemic stroke, has been demonstrated to increase the expression of several cytokines, among which is CX3CL1 (Lee et al. 2007). Considering the protective effect of CX3CL1, this led to the hypothesis that the activity of this thrombolytic substance goes far behind its very well known role as plasminogen activator in stroke therapy. In brain ischemia, glutamate-induced excitotocixity is involved in the modulation of neuronal cell death. Experiments of transient focal brain ischemia, in rats, describe an increased CX3CL1 immunoreactivity in the perifocal area 1 or 2 days after ischemia, likely reflecting a cellular attempt to survive in the ‘penumbra’, an area proximal to the primary ischemic region which became successively damaged due to the release of toxins from dead cells (Tarozzo et al. 2002). No early changes (6  h) in CX3CL1 mRNA are observed upon permanent MCA occlusion in rats (Chapman et  al. 2000). However, using CX3CL1-deficient mice (CX3CL1−/−) Soriano et  al. (2002) demonstrated that permanent cerebral ischemia caused by

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experimentally induced middle cerebral artery occlusion (MCAO) produces a significant reduction in the brain ischemia volume in comparison with wt mice, suggesting a detrimental role for CX3CL1 in the brain, during cerebral ischemia, in contrast with the data reported in vitro. 13.2.3.3 Modulatory Effects on Neuronal Precursors The neuroprotective potential of CX3CL1 is further testified by the observation that it reduces neuronal precursor cells death induced by deprivation of growth factors which form the culture/cultural medium (Krathwohl and Kaiser 2004). Furthermore, CX3CL1 may drive mesenchymal stem cells (MSC) trafficking towards impaired brain regions, like the hypoglossal nucleus, where CX3CL1 expression is increased upon injury (Ji et al. 2004) thus serving as a guide for the directional migration of stem cells potentially involved in the repair process in the adult brain. MSC injected into the brain also efficiently engraft in the hippocampal region likely in a CX3CR1-dependent way, since in  vitro experiments show that MSC migration towards neurospheres is blocked by specific anti-CX3CR1 Ab (Lee et al. 2006). Under hypoxic conditions, astrocytes down-regulate CX3CL1 expression while up-regulate other cytokines and chemokines potentially involved in the migration of neuronal precursors towards hypoxic regions to replace the damaged cells (Xu et al. 2007). Together with the observation that CX3CL1 has an inhibitory role on basal migration and on SDF-1/CXCL12-induced chemotaxis of immature hippocampal and cerebellar neurons (Lauro et al. 2006) these results could implicate a facilitative role of CX3CL1 down-regulation, upon ischemia, in favour of the chemotactic activity induced by other locally up-regulated chemokines. 13.2.3.4 Neuroinflammation and Neurodegeneration Models An additional input to the description of the neuroprotective actions of CX3CL1 came from the observations that it has notable roles in fading microglial response to the pro-inflammatory endotoxin LPS, reducing the production of cytokines like TNF-a, NO, IL-6 and IL-1 (Zujovic et al 2000, 2001; Mizuno et al. 2003; Cardona et al. 2006). Consistently, neuronal death induced by cytokines released by microglial cells stimulated with LPS and IFN-g is greatly abolished by CX3CL1 treatment (Mizuno et  al. 2003). CX3CL1 also protects microglial cells from Fas ligandinduced cell death, with a PI3-K-dependent mechanism (Boheme et al. 2000). Several reports describe that CX3CL1 and CX3CR1 expression is up-regulated in the CNS by inflammatory stimuli or during pathological conditions (Pan et  al. 1997; Hughes et al. 2002; Sunnemark et al. 2005). The cerebrospinal fluid (CSF) of patients affected with different central and peripheral neuroinflammatory diseases contains an increased amount of CX3CL1 compared with patients with non-inflammatory neurological diseases (Kastenbauer et al. 2003). Furthermore, at the peak of experimental autoimmune encephalopathy (EAE), a strong increase of soluble CX3CL1 is detected, with no differences in the level of mRNA (Huang et al. 2006),

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suggesting increased shedding of the soluble form. Soluble CX3CL1 levels are reported to increase even in the serum of Alzheimer’s disease (AD) patients with mild cognitive impairment (Kim et al. 2008), and in multiple sclerosis (MS) patients (Kastenbauer et al. 2003). Noteworthy, the ratio between CSF and serum CX3CL1 levels reported for MS patients versus control subjects does not change, thus suggesting that the increased CSF CX3CL1 levels might be due to blood–brain barrier disruption in addition to the local intrathecal production (Kastenbauer et al. 2003). Recently, two studies reported intriguingly roles for the CX3CL1/CX3CR1 pair in different models of neurodegenerative diseases in  vivo: Huang et  al. (2006) reported that CX3CR1 knockout mice, injected with MOG to induce EAE, had a more severe phenotype in terms of the onset of the disease and severity and mice mortality in comparison with wt animals, and that this effect may be, at least in part, due to a reduced immunitary response in term of selective deficiency of NK cells recruitment in the CNS of CX3CR1-deficient mice. Similarly, in different animal models of inflammatory and neurodegenerative insults (intraperitoneal LPS injection, an inflammatory insult; intrastriatal MPTP administration, a rodent model of Parkinson’s disease; and a genetically modified mouse as a model of amyotrophic lateral sclerosis), CX3CR1-deficient mice had and increased neuronal death rate in comparison with control mice (Cardona et al. 2006). The increased neuronal death reported for CX3CR1 knockout mice might be explained by a loss of an inhibitory signal for IL-1b production by CX3CR1-deficient microglia cells since they produce more IL-1b upon LPS treatment while IL-1Ra co-injection reverts the phenotype of CX3CR1−/− microglial cells. These data gave a solid input to the hypothesis of a role for the pair CX3CL1/CX3CR1 in reducing microglial neurotoxicity during inflammatory neuropathologies.

13.3 CX3CL1 as Mediators of Intercellular Communication in the Nervous System One of the first evidence of a modulatory role of CX3CL1 in mediating neurons microglial communication was obtained by Harrison et al. (1998) on the experimental model of facial motor neuron axotomy. Results obtained in these experiments led to the suggestion that neuron injury, causing CX3CL1 release, could recruit microglial cells in the damaged area taking part to the reparative or destructive processes. When, 2 years later (Jung et al. 2000), this same model was applied to CX3CR1GFP/GFP mice, it became evident that the CX3CL1/CX3CR1 pair is not essential for microglial chemotactic response towards injured regions since CX3CR1GFP/GFP mice have an unaltered microglial response to facial motor neuron axotomy (Jung et  al. 2000), and also to laser-induced brain parenchyma injury (Davalos et  al. 2005). Nevertheless, other reports highlighted that the migratory behaviour of CX3CR1 GFP/GFP microglial cells depends on their activation state (Cardona et al. 2006), thus indicating a modulation of CX3CR1 activity in microglial cells by different local stimuli. This evidence is further supported by the contrasting data that recently emerged on the role of CX3CL1 in pain modulation,

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where different kinds of administration, namely central or peripheral, may generate either nociceptive or analgesic effects (Milligan et al. 2004; Holmes et al. 2008). In line with this evidence, CX3CL1 in the brain has in general an inhibitory activity on microglia function, in terms of inflammatory cytokine release while, in the spinal cord, CX3CL1 stimulates the release of cytokines from microglia. Overall, considering the data described in this chapter, at this stage of knowledge CX3CL1 could be considered as a neuronal messenger, whose soluble form increases upon several kinds of toxic stimuli, primarily acting on microglial cells in a way that could differ as a consequence of the local cytokine milieu, possibly regulating the activation and the potential toxicity of microglia. Acknowledgments  The authors thank Drs. Fabrizio Eusebi and Flavia Trettel for helpful discussions during the writing of this chapter.

Abbreviations Ab AD AMPA AMPA-current CSF DPCPX EAE ERK IL-1Ra IL-1b LPS LTD MCAO MPTP MS PI-3K PTX SAPK uPA wt

Antibody Alzheimer’s disease a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid AMPA-evoked currents Cerebrospinal fluid 8-Cyclopentyl-1,3-dipropylxanthine Experimental autoimmune encephalopathy Extracellular signal-regulated kinase IL-1 receptor antagonist Interleukin 1 b Lipopolysaccharide Long-term depression Middle cerebral artery occlusion 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Multiple sclerosis Phosphatidylinositol-3 kinase Pertussis toxin Stress-activated protein kinase Urokinase-type plasminogen activator Wild type

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Scholz J, Woolf CJ (2007) The neuropathic pain triad: neurons, immune cells and glia. Nat Neurosci 10:1361–1368 Soriano SG, Amaravadi LS, Wang YF et al (2002) Mice deficient in fractalkine are less susceptible to cerebral ischemia-reperfusion injury. J Neuroimmunol 125:59–65 Sun S, Cao H, Han M et al (2007) New evidence for the involvement of spinal fractalkine receptor in pain facilitation and spinal glial activation in rat model of monoarthritis. Pain 129:64–75 Sunnemark D, Eltayeb S, Nilsson M et al (2005) CX3CL1 (fractalkine) and CX3CR1 expression in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis: kinetics and cellular origin. Neuroinflammation 2:17 Tarozzo G, Bortolazzi C, Crochemore S et  al (2003) Fractalkine protein localization and gene expression in mouse brain. J Neurosci Res 73:81–88 Tarozzo G, Campanella M, Ghiani M et al (2002) Expression of fractalkine and its receptor, CX3CR1, in response to ischaemia-reperfusion brain injury in the rat. Eur J Neurosci 15:1663–1668 Tong N, Perry SW W, Zhang Q et al (2000) Neuronal fractalkine expression in HIV-1 encephalitis: roles for macrophage recruitment and neuroprotection in the central nervous system. J Immunol 164:1333–1339 Tsou CL, Haskell CA, Charo IF (2001) Tumor necrosis factor-converting enzyme mediates the inducible cleavage of fractalkine. J Biol Chem 276:44622–44626 Verge GM, Milligan ED, Maier SF et al (2004) Fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) distribution in spinal cord and dorsal root ganglia under basal and neuropathic pain conditions. Eur J Neurosci 20:1150–1160 Vidal F, Viladés C, Domingo P et al (2005) Spanish HIV-1-infected long-term nonprogressors of more than 15 years have an increased frequency of the CX3CR1 249I variant allele. J Acquir Immune Defic Syndr 40:527–531 Vitale S, Schmid-Alliana A, Breuil V et al (2004) Soluble fractalkine prevents monocyte chemoattractant protein-1-induced monocyte migration via inhibition of stress-activated protein kinase 2/p38 and matrix metalloproteinase activities. J Immunol 172:585–592 Zhuang ZY, Kawasaki Y, Tan PH et al (2007) Role of the CX3CR1/p38 MAPK pathway in spinal microglia for the development of neuropathic pain following nerve injury-induced cleavage of fractalkine. Brain Behav Immun 21:642–651 Zujovic V, Benavides J, Vigé X et  al (2000) Fractalkine modulates TNF-alpha secretion and neurotoxicity induced by microglial activation. Glia 29:305–315 Zujovic V, Schussler N, Jourdain D et al (2001) In vivo neutralization of endogenous brain fractalkine increases hippocampal TNFalpha and 8-isoprostane production induced by intracerebroventricular injection of LPS. J Neuroimmunol 115:135–143 Yoshida H, Imaizumia T, Fujimotoa K et al (2001) Synergistic stimulation, by tumor necrosis factora and interferon-g, of fractalkine expression in human astrocytes. Neurosci Lett 303:132–136 Xu Q, Wang S, Ishida X et al (2007) Hypoxia-induced astrocytes promote the migration of neural progenitor cells via vascular endothelial factor, stem cell factor, stromal-derived factor-1alpha and monocyte chemoattractant protein-1 upregulation in  vitro. Clin Exp Pharmacol Physiol 34:624–631

Section III

Chapter 14

Interaction Between Opioid and Chemokine Receptors in Immune Cells: Implications for HIV Infection Christine Happel, Changcheng Song, Mathew J. Finley, and Thomas J. Rogers

14.1 Introduction Both opioid and chemokine receptors belong to the G-protein-coupled receptor (GPCR) superfamily of over 1,000 members. Generally, GPCRs are synthesized in a single chain of about 400–500 amino acids in length and become integrated into a lipid bilayer, most commonly the cell membrane. Seven transmembrane alpha helixes span through the lipid bilayer to create three extracellular and three intracellular protein loops. The extracellular N-terminal domain is generally posttranslationally modified by glycosylation, an event that can substantially change the molecular weight of the protein. Several signaling events can be initiated through heterotrimeric G proteins with a, b, and g subunits to mediate the extracellular to intracellular signal transduction. Ga subunits are categorized into four families, Gas, Gai, Gaq/11, and Ga12/13, based on their downstream effector proteins. G protein functions as molecular switches to alternate Ga subunit between an inactive guanosine diphosphate (GDP) and active guanosine triphosphate (GTP) bound state. The Ga-GTP and disassociated bg subunits can interact with effector proteins in initiating diverse signaling pathways. The signaling is terminated with the hydrolysis of GTP to GDP, and the reassociation of the abg complexes. Ultimately, the GPCRs regulate a broad spectrum of cell processes and biochemical events including adenyl cyclase and phospholipase C (PLC), modulation of calcium and potassium channels, activation of adhesion molecules, and the induction of chemotaxis. There are known to be three opioid receptors, designated m, k, and d, and these receptors share a high degree of amino acid sequence homology. The m-opioid receptor (MOR) is the primary receptor, which is activated by the nonselective

C. Happel, C. Song, M.J. Finley, and T.J. Rogers () Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelhia, PA, 19140, USA e-mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_14, © Springer Science+Business Media, LLC 2010

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opioid agonist morphine, and by the highly selective agonists, endomorphin-1 and endomorphin-2. The enkephalins are relatively nonselective opioid receptor agonists, and activate MOR, DOR, and KOR. Finally, the endogenous peptide agonist dynorphin is a highly-selective KOR agonist. The functions of the opioid receptors for the immune system have been the subject of intense research over the last 20 years. Many aspects of the immune response are modulated following the activation of the opioid receptors, including both B and T lymphocyte function, phagocytic cell activity, and the production of a variety of cytokines by numerous cell populations (reviewed in McCarthy et al. 2001, Bidlack, 2000, Eisenstein and Hilburger 1998, Rogers and Peterson 2003). We have previously reviewed literature that suggests that the activation of MOR and KOR may have opposing effects on the immune response, and that the overall effect of activation of KOR may be strongly anti-inflammatory (Rogers and Peterson 2003). Chemokine receptors are classified into four primary families based on the terminal cysteine amino acid sequence of their respective ligand (C, CC, CXC, and CX3C). The angiotensin receptors are most likely the closest family member to the chemokine receptors as are the opioid, somatostatin, and melanin concentrating hormone (MCH) receptors (Lio and Vannucci 2003). Chemokine receptors have also been found to be involved in disease processes such as inflammatory conditions, cancer, and viral infections. This creates an exciting avenue for the exploration of novel therapeutic targets and strategies for the treatment of various diseases. As an example, the chemokine receptors CCR5 and CXCR4 are the major coreceptors for the human immunodeficiency virus-1 (HIV-1) infection. CCR5 is responsible for T-lymphocyte tropic HIV-1 infection (Doranz et  al. 1996), while CXCR4 is involved with macrophage-tropic HIV-1 infection (Dragic et  al. 1996). Increased numbers of coreceptors on the cell surface can lead to an advanced disease state. The chemokine receptors were originally identified for their role in mediating the chemotaxis of leukocytes, and the chemokines are now known to play a vital role in the function of the immune system. Chemokines are a group of small proteins that regulate the cellular trafficking of leukocytes and are involved in mounting an immune response, initiation of wound healing, hematopoiesis, apoptosis and cellular growth. Chemokines are classified by the locations of two conserved cysteine resdues located on the amino terminus on the proteins, and are divided into four main families: CC, CXC, C, and CX3C. Chemokines are known to play a key role in HIV infection and progression through their function as a chemoattractant for NK cells, T cells, monocytes, neutrophils, fibroblasts, and endothelial cells. There is considerable evidence that the opioid and chemokine receptors interact at both the level of gene expression and protein function. As a part of the crossregulation between these receptor sub-families, the opioid receptors control the expression of many members of the chemokine sub-family as well, adding to the degree of cross-regulation between the opioid and chemokine sub-families. Finally, it should be appreciated that the cross-regulation between these receptors and their ligands occurs both in the immune system as well as in the brain, where numerous opioid and chemokine ligands are produced, and where a great number of their receptors are expressed.

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14.2 Regulation of Chemokine Expression by Opioids Opioid receptors can be activated endogenously as well as exogenously by alkaloid opiates, the prototype of which is morphine. Heroin is the most commonly abused opiate and is a metabolite of morphine. The activation of MOR by opioid agonists, including morphine, has been shown to alter the release of cytokines and chemokines important for the inflammatory response (McCarthy et  al. 2001). The m-selective synthetic agonist, [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) has the capability to increase the expression of several proinflammatory chemokines, including CCL2, CCL5, and CXCL10, in nonactivated and PHA-stimulated human peripheral blood mononuclear cells (PBMCs). This increase in chemokine expression was seen at both the RNA and protein levels and pretreatment with a MOR-selective antagonist, CTAP, abolished the DAMGO-induced increase of CCL2 and CCL5 (Wetzel et al. 2000). Additionally, Happel et al. (2008) demonstrated that the DAMGO-induced expression of CCL5, but not CCL2 or CXCL10, is mediated through the cytokine, TGF-b. This suggests that the mechanism of DAMGO induction of CCL2, CCL5, and CXCL10 is distinct. It is very likely that the effects of MOR activation are dependent on many different factors including the nature and duration of the stimulus, responding cell type, and target cell population(s). Rock et al. (2006) showed that morphine stimulates CCL2 production at both the mRNA and protein level in neurons. In addition, the MOR antagonist, b-funaltrexamine (b-FNA), was found to block the morphine-mediated increase in CCL2 expression, demonstrating that this effect is mediated through the µ-opioid receptor (Rock et  al. 2006). Caco-2 cells, an intestinal epithelial cell line, were found to constitutively express MOR and KOR (Neudeck and Loeb 2002, Neudeck et  al. 2003). Activation with the endogenous µ-tetrapeptide, endomorphin-1, resulted in a significant increase in CXCL8 secretion, and this effect could be reversed by pretreatment with the antagonist, b-FNA (Neudeck and Loeb 2002). Opioid receptors are also highly expressed in the brain by cells such as astocytes and microglial cells (Ruzicka et al. 1995, Bodnar 2007). These cell types are also a major source of CCL2 in the brain, and a natural site for the production of endogenous µ-opioids (Conant et  al. 1998). Therefore, studies of opioid-mediated regulation of chemokines in astrocytes are particularly significant in understanding the potential mechanism of monocyte recruitment into the CNS and HIV infection. El-Hage et al. (2005) demonstrated that murine astrocytes treated with morphine and the HIV-1 nuclear protein, Tat1–72, resulted in a synergistic increase in CCL2 and CCL5 production compared with treatment of morphine or Tat alone. Pretreatment with b-FNA blocked this effect while pretreatment with the k-opioid antagonist, nor-binaltorphimine (nor-BNI) had no effect, showing that the affects of morphine are mediated by MOR (El-Hage et  al. 2005). Chemotaxis assays were also used to demonstrate that the combined treatment of morphine and Tat1–72 resulted in enhanced cell migration of microglial cells (El-Hage et  al. 2006). Furthermore, this increase in cell motility was attenuated through the use of a neutralizing CCL2 antibody (El-Hage et al. 2006). Taken together, we suggest that one

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consequence of the opioid induction of chemokine expression would be to increase the susceptibility of the CNS to HIV infection by recruiting monocytes and macrophages to the site of infection through the synergistic increase of CCL2 and CCL5 (Rogers and Peterson 2003). Although the µ-opioid receptor has been shown to increase proinflammatory chemokine expression, the activation of the KOR has been shown to have an opposing effect despite a high degree of sequence homology between these receptors, and the similarity in the signaling capacities of these GPCRs. Activation of KOR by the k-opioid selective synthetic agonist U50,488H, (trans-3,4-dichloro-N-methyl-N[2(1-pyrolidinyl)cyclohexyl]benzeneacetamide methanesulfonate) in Caco-2 cells has been shown to decrease CXCL8 secretion in the presence of IL-1b (Neudeck et al. 2003). Studies in primary human astrocytes demonstrate that U50,488H has the ability to down-regulate CCL2 production when stimulated by Tat (Sheng et al. 2003), and the KOR-selective antagonist nor-BNI completely blocked the inhibitory effect of U50,488. HIV-Tat has previously been shown to activate the transcription factor, nuclear factor-kB (NF-kB) (Demarchi et al. 1996, Conant et al. 1996), and Sheng et al. (2003) demonstrated that U50,488 inhibited Tat-induced activation of the transcription factor nuclear factor-kB (NF-kB) and that this effect could be reversed by pretreatment with PDTC (an inhibitor of NF-kB) (Sheng et al. 2003). Not only do these studies highlight the disparate effects of MOR and KOR on chemokine expression but they also implicate a role for NF-kB in mediating the regulation of chemokine expression. Interestingly, evidence seems to suggest that NF-kB signaling may be involved in opioid-mediated immune responses regardless of opioid receptor subtype, even though the mechanisms functioning in each case may be different. NF-kB is one of the most widely studied transcription factors due to its complex signal transduction pathways and its role in inflammation, innate and adaptive immunity and the cellular-stress response. NF-kB also participates in leukocyte trafficking through the regulation of chemokine expression. NF-kB target genes include the chemokines CCL2, CCL3, CCL4, CCL5, CCL11, CCL17, CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9, CXCL10 and CXCL12 (Hoffmann et  al. 2003, Ahn et  al. 2004, Grove and Plumb 1993, Widmer et  al. 1993). The NF-kB family of transcription factors is also an integral player in the regulation of chemokine expression, although the effects are diverse and cell-type dependent. Many studies have helped to elucidate the role of NF-kB in the regulation of chemokine expression. The 5¢ regulatory regions, and the NF-kB element, present in many NF-kB target genes (including the chemokine genes) vary greatly, and the role for NF-kB may be quite diverse (Saccani et al. 2001). For purpose of our review, we will focus on the function of NF-kB activation during induction of CCL2 gene expression, since this chemokine is known to be a particularly important participant during HIV infection and progression. The promoter of the CCL2 gene contains multiple NF-kB-binding sites in both its proximal and distal promoter region, and regulation of CCL2 has been shown to be mediated through the activation and binding of NF-kB (Ueda et  al. 1997, Ueda et  al. 1994).

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Furthermore, studies have shown that the NF-kB subunit p65 is necessary for the recruitment of proteins to the enhancer region of the CCL2 promoter and induction of CCL2 expression following TNF-a induction (Saccani et al. 2001, Ping et al. 1999). In addition to TNF-a-inducted activity, NF-kB is also required for the IL-1b- and TPA-induced enhancer activity of CCL2 (Ueda et  al. 1997). Finally, NF-kB is not only responsible for increased transcriptional activity but also for changes in local chromatin structure and in the recruitment of additional binding partners (Boekhoudt et al. 2003). Activation of GPCRs often results in the activation or repression of NF-kB signaling through a number of different pathways including the cAMP/PKA/CREB, PI3K/Akt and PLCb/PKC signaling pathways (Ye 2001). Many members of the Gi sub-family of receptors have been reported to mediate activation of NF-kB, including the receptors for fMLF, CXCL12, CXCL1 and CXCL8 (Ye 2001). Recently, NF-kB has also been implicated in the transcriptional regulation of MOR (Kraus et al. 2003), DOR (Chen et al. 2006b) and KOR (Law et al. 2004). Opioid receptor regulation of NF-kB signaling was first revealed to play an important role in the regulation of cytokine receptor expression in morphine-treated rhesus monkeys (Carr et al. 1995). Recent studies have shown that DAMGO treatment is able to increase NF-kB DNA-binding activity in primary rat cortical neurons (Hou et al. 1996). The endogenous µ-selective ligands, endomorphins 1 and 2, have been shown to induce substantial potentiation of NF-kB binding activity in THP-1 cells which were differentiated to a macrophage-like cellular phenotype (Azuma and Ohura 2002). Additionally, both endomorphin-1 and -2 significantly potentiated LPS-stimulated NF-kB binding (Azuma and Ohura 2002). Roy et  al. (1998) demonstrated that morphine treatment modulates LPS-induced expression IL-6 and TNF-a through the regulation of NF-kB in macrophages. Interestingly, nanomolar concentrations of morphine resulted in an increase in NF-kB DNA binding activity while morphine treatments at micromolar concentrations led to a significant decrease in NF-kB activation (Roy et al. 1998). Our laboratory is currently investigating the role of NF-kB in the DAMGO-induced activation of MOR and subsequent regulation of CCL2 expression. Our preliminary results indicate that the MOR-induced activation of CCL2 is dependent on the activation of NF-kB. Taken together; these data indicate that NF-kB is relatively a common participant in the MOR-induced regulation of immune responses. Although the effects can be diverse and often cell-type dependent, NF-kB appears to be a critical component in opioid function and receptor gene expression (Chen et al. 2006a). It is well established that NF-kB signaling plays a critical role in inflammation and immunity. Understanding the mechanism of NF-kB involvement in opioid receptor activation and chemokine expression may provide a vital key to understanding this complex signaling network. However, the elucidation of molecular mechanisms following activation of the opioid receptor family could aid in the development of future therapeutics for immune system-related and inflammatory diseases, drug addiction and HIV infection.

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14.3 Regulation of Chemokine Receptor Expression by Opioids The molecular mechanism of opioid-mediated up-regulation and down-regulation of gene expression levels of chemokine receptors has been studied by several groups. As an example, it has been clearly documented that morphine treatment of immune cells of the blood as well as cells of the nervous system results in an increased level of CCR5 mRNA and protein levels. An added complication in understanding this is the fact that activation of opioid receptors often results in increased expression of the very same opioid receptor (Suzuki et al. 2000, 2001). This has been demonstrated at both the mRNA and protein levels, and not unexpectedly, an increased number of opioid receptors translate into an increased number of opioid-binding sites and thus an increased signal transduction potential of this pathway. An important set of observations were reported by Nair et al. (2000) demonstrating increased mRNA levels of the chemokine receptor CCR5 in peripheral blood mononuclear cells (PBMCs) following treatment with cocaine. This suggested the possibility that the regulation of chemokine receptor expression may represent a target for the effects of abuse of drugs. Coincidental studies evaluating the effects of morphine on Simian Acquired Immunodeficiency Syndrome virus (SIV) infection revealed an increased viral infection rate in primary primate PBMCs following morphine administration. Further investigation demonstrated that an explanation for these findings may be attributed to the increased expression of the CCR5 coreceptor (Suzuki et al. 2002a, b). Further studies showed that both CCR5 mRNA and protein expression was increased following morphine treatment. These findings provided an explanation for the increased severity of HIV infection, which can be observed in individuals who are also intravenous drug abusers. Up to this point, it was unclear which opioid receptor was involved in the up-regulation of CCR5 due to the fact that morphine does not exclusively bind a single opioid receptor class. Studies by Steele et  al. (2003) showed that either the MOR-selective agonist DAMGO, or morphine, up-regulated CCR5 and CXCR4 expression in primary human PBMCs. More recent studies have demonstrated that KOR has a very different effect on the expression of the major HIV-1 coreceptors. Treatment of primary human PBMCs with the KOR-selective agonist U50,488H results in a profound decrease of CCR5 and CXCR4 mRNA and protein levels (Finley et al. 2008a). In contrast to these results, studies using murine developing T-cells, or thymocytes, show that U50,488H treatment up-regulates CCR2 mRNA expression (Zhang and Rogers 2000). These results show that KOR activation leads to the up-regulation of some chemokine receptors, decreases expression of other receptors, and has no effect on still others. However, these results do point out the opposing effects of MOR and KOR in the regulation of the immune system. Exploitation of these intricate mechanisms, through the further development of opioid-based therapeutics, may prove to be beneficial for the treatment of inflammatory and viral diseases.

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The mechanistic basis for the effects of the opioid receptors on chemokine receptor expression has been the subject of research in cells of both neuronal and immune systems. Using primary normal human astrocytes, morphine treatment results in increased mRNA for both CCR5 and CCR3, which is consistent with observations reviewed above for leukocytes (Mahajan et  al. 2002). Further work demonstrated that the morphine treatment of the U87 atrocytoma cell line results in the up-regulation of CXCR2 mRNA. Furthermore, the human brain-derived astrocytoma/glioblastoma cell line, U373, also exhibited increased levels of CCR5 and CCR3 following morphine treatment. These results indicate that the opioid-induced regulation of chemokine receptor expression is likely to be similar in cells of both the immune and nervous systems. Possible intracellular mechanisms that are responsible for these findings are still under study. It has been established using differential display real-time polymerase chain reaction (ddRT-PCR), a method of comparing cDNA sequences of a control population with a treated population, that the transcription factor Kruppel-Like Factor 7 (KLF7) is increased with morphine treatment (Suzuki et  al. 2003). The up-regulation of KLF7 by morphine treatment could be blocked with pretreatment for 30 minutes with the opioid antagonist, naloxone. KLF7 is an essential transcription factor of the zinc finger family. It is normally expressed during development of the nervous system and is a close relative of the specificity protein 1 (SP-1) transcription factor. Both KLF7 and SP-1 bind to an element termed the GT-Box that can be found in tandem in the promoter regions of many genes. The consensus binding sequence that both KLF7 and SP-1 share is CACCC, an element that is found in abundance within the proximal promoter of CXCR4. Most recently, Happel et al. (2008) further described the mechanism responsible for the up-regulation of CCR5 and CXCR4 in primary human PBMCs following DAMGO treatment. These studies demonstrated that the expression of TGF-b is required for the DAMGO-mediated up-regulation of CXCR4. When cells are treated with an anti-TGF-b antibody, the up-regulation of CXCR4 following DAMGO treatment can be effectively blocked. Furthermore, TGF-b is not required for the DAMGO-induced up-regulation of CCR5, although it may still play a partial regulatory role. In fact, our preliminary studies suggest that the regulation of CCR5 by following MOR activation is likely dependent on the function of a number of transcription factors, including AP-1 and NF-kB, and there are several consensus sequences for these transcriptional elements in the CCR5 promoter. In contrast to the function of the MOR, we have found that KOR activation results in a significant down-regulation of the expression of both CCR5 and CXCR4. Preliminary studies from our laboratory have demonstrated the interferon signaling pathway might be involved in the KOR-mediated down-regulation of these chemokine receptors. Using a transcription factor binding array, we have determined that primary human PBMCs that were treated with U50,488H exhibit increased expression levels of interferon regulatory factors (IRFs) and gamma activated sequence interferon regulatory element (GAS/ISRE) activity compared to control untreated cells. These effects occur as early as 45 min post KOR activation indicating that a rapid transcriptional mechanism is most likely evoked. We propose

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a model in which following KOR activation, a positively acting interferon regulatory factor (IRF1), which is ubiquitously expressed, undergoes competitive binding with a negatively acting interferon regulatory factor (IRF2). IRF2 is produced following an increase in the signal derived from interferon gamma binding to the cell surface interferon gamma receptor. How interferon gamma is produced following KOR activation remains unknown and is currently under study. We have hypothesized that IRF2 then abolishes expression of target genes, including IRF1, CCR5 and CXCR4, due to the role of interferon gamma. A diagram summarizing our current model is shown here (Fig. 14.1).

KOR-Induced Down-Regulation of CXCR4 IFN

βγ

Gα

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Receptor a chain

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Fig. 14.1  Propposed model to explain the regulation of CCR5 and CXCR4 gene expression by KOR. The kappa opioid receptor agonist U50,488H selectively binds to the kappa opioid receptor (KOR) triggering an intricate signaling cascade. Part of this mechanism includes transcriptional activation of the interferon gamma (IFN-g) gene as illustrated. Secretion of the interferon gamma protein results in binding to IFN-g receptors (potentially in an autocrine manner). Activated interferon gamma receptor allows for active JAK formation and phosphorylation of the IFN-g receptor itself. This provides a docking site for STAT proteins to bind and be phosphorylated by JAK. Once STAT1 is phosphorylated, STAT1 can homodimerize to itself (or heterodimerize to other STAT family members) and with dimerization, the protein complex translocates into the nucleus and binds to STAT elements, thus regulating transcription of target genes. One gene that is activated by the interferon gamma depended STAT1 activation is IRF1. IRF1 and IRF2 bind an identical IRF element (IRFE). IRF1 is able to trigger production of IRF2, which functions as a transcriptional repressor for IRF1 as well as several other genes. Since IRFE’s have been found in the promoter of CXCR4 and CCR5, it is possible that IRF1 is bound constitutively and with U50,488H mediated up-regulation of IRF2, IRF2 replaces IRF1 on the IRFE within the promoters of CXCR4 and CCR5. The end result of this transcriptional mechanism is dramatically decreased levels of CXCR4 and CCR5 mRNA transcription

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Oligomerization between chemokine and opioid receptors has been studied and found to be a form of additional functional control (Suzuki et al. 2002b). Alterations in the expression of chemokine and opioid receptors can play a vital role in this function since the close proximity of the two types of receptors can be changed by decreased or increased total cell surface numbers of the receptors. The latter study evaluated the oligomerization of CCR5 with KOR, MOR and DOR, and the results show that the activation of one receptor (e.g., KOR), triggers the activity of a second receptor when present in an oligomeric complex. Since MOR activation results in increased levels of CCR5, one would predict that increased chemokine signaling will occur, while KOR activation would result in the reverse outcome.

14.4 Cross-talk Between Opioid and Chemokine Receptors The activation of a GPCR typically results in the rapid down-regulation of that receptor through the process of homologous desensitization. The activation of many GPCRs also induces the cross-inactivation of unrelated GPCRs through heterologous desensitization. The selective nature of the heterologous desensitization process provides a level of receptor regulation that allows GPCR ligands to limit responsiveness to certain “less-desirable” ligands. The cross-desensitization phenomenon has significant implications in the regulation of both neuronal and immune responses, particularly for the generation of inflammatory responses and developing immunity to infectious diseases. In this chapter, we review the interactions at the level of protein function, between opioid receptors and chemokine receptors, using both in vitro and in vivo model systems. As a part of this discussion, we also describe our current understanding of the biochemical pathway(s) that are involved in the heterologous desensitization process between these groups of receptors.

14.4.1 Certain Chemokine Receptors Cross-Desensitize Opioid Receptors It has been observed that the accumulation of chemokines at inflammatory response sites is typically associated with an increase in the perception of pain, suggesting the possibility that opioid receptors may be down-regulated. One potential mechanism which would explain the inhibition of opioid receptor function is the crossdesensitization of opioid receptors following chemokine receptor activation at the site of inflammation. Indeed, we have found that the activation of CCR2, CCR5, CCR7, or CXCR4 rapidly induces desensitization of the activities of both MOR and DOR (Szabo et al. 2002a, Rogers et al. 2000). Specifically, this work demonstrates that these chemokine ligands cross-desensitize the ability of MOR or DOR to mediate a chemotactic response when expressed by either, primary human monocytes, primary

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human T cells, or a number of leukocyte, fibroblast or keratinocyte cell lines (Szabo et al. 2002). More recent studies have shown that treatment with CCL3 of HEK-293 cells expressing CCR1 and MOR, induces inactivation of MOR functional activity (as measured by impairment of MOR-mediated inhibition of cAMP accumulation, and MOR-induced calcium mobilization) (Zhang et al. 2004). These studies showed inhibition of MOR functional activity of primary neurons pretreated with CCL2, CCL3, CCL5, or CXCL8 (Zhang et  al. 2004). These studies also showed that pretreatment with CCL3 induced partial internalization of MOR from the cell surface. However, this internalization process does not fully account for the cross-desensitization of MOR activity following activation of these chemokine receptors. In an effort to examine the physiological consequences of the desensitization of opioid receptors, in collaboration with the M. W. Adler laboratory, we attempted to determine the effect of chemokines on the function of opioid receptors in the brain. We carried out experiments in which we administered various chemokines into the periaqueductal grey matter (PAG) of the brain, and then measured the ability of the opioid receptors to elicit an analgesic response. Stimulation of the opioid receptos in the PAG is known to elicit a signal which leads to a depressed sensation of pain. In the initial set of experiments, we determined that the pretreatment of the PAG with agonists for either CCR5 or CXCR4 blocked the ability of MOR to mediate a normal analgesic response to a MOR agonist (Szabo et al. 2002). Additional studies demonstrated that the desensitization (or inactivation) of the MOR analgesic response was apparent for a period of up to 120 min (with pretreatment of CCR5 agonists) or 240 min (when pretreated with a CXCR4 agonist) (Szabo et al., 2002). More recent work has demonstrated that administration of either morphine (a nonselective opioid receptor agonist), CCL5 (a CCR1/5 agonist), or CXCL12 (a CXCR4 agonist) induces desensitization of both MOR and DOR (Chen et  al. 2007a). Moreover, the administration of a CX3CR1 agonist (CX3CL1/fractalkine) induces cross-desensitization of MOR, DOR, and KOR (Chen et  al. 2007b). Finally, a very recent set of studies shows that activation of CXCR4 results in crossdesensitization of KOR (Finley et al. 2008b). These findings suggest that cross-talk between a number of chemokine receptors and each of the opioid receptors, suggesting an extensive degree of cross-regulation of the function of the opioid receptors in the brain. These results have several potential implications, which bear on our understanding of the regulation of the function of opioid receptors. Through the process of heterologous desensitization, chemokine ligands for CXCR4 and CCR5 can inactivate the normal neuronal signaling pathway involved in reducing the sensation of pain. Moreover, there is growing evidence that chemokines participate in a substantial way in normal physiological processes, particularly in the brain. First, a number of chemokine receptors are selectively expressed constitutively in numerous regions of the brain (Horuk et al. 1997, Banisadr et al. 2002), and are expressed by neurons as well as by glial cells. Moreover, both CXCL12 and its receptor CXCR4 are required for normal brain development, based on the abnormal neuronal organization in the cerebellum of CXCR4 or CXCL12-deficient mice (Ma et al. 1998). Several chemokines, including CXCL12 and CCL5 are normally produced in the

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brain, and the expression of these and other chemokines are greatly up-regulated as a part of several neuroinflammatory or neuropathogenic processes (Ambrosini and Aloisi 2004, Mizuno et al. 2003). The up-regulation of these chemokines, and the potential cross-inactivation of the opioid receptors, may account for the increased sensitivity to pain seen in some of these pathological conditions. Finally, studies carried out in several model systems, have established that the chemokine receptors alter or regulate responses that are mediated by several neurotransmitters and neuropeptides, and this has led Adler and Rogers (2005) to propose the hypothesis that the chemokine/chemokine receptor system represents the third major transmitter in the brain.

14.4.2 Opioid Receptors Cross-Desensitize Chemokine Receptors Early evidence showed that in vivo and in vitro morphine administration inhibited the chemotactic response of neutrophils and microglial cells to complement components (Chao et  al. 1997, Liu et  al. 1992). More recently, Grimm et  al. (1998) reported that both met-enkephalin and morphine inhibited the chemotactic response of human neutrophils to CXCL8, or the response of human monocytes to CCL5, CCL3, or CCL2. This cross-desensitization was shown to be selective since opioid treatment had no effect on responses to fMLF in either neutrophils or monocytes. The use of more highly selective opioid agonists, and selective opioid receptor antagonists, established that both MOR and DOR were capable of inducing desensitization of the chemokine receptor-mediated responses (Grimm et  al. 1998). Importantly, treatment with met-enkephalin was found to induce cross-phosphorylation of both CXCR1 and CXCR2, suggesting the involvement of a protein kinase in the cross-desensitization pathway. However, results based on both radiolabeled ligand binding analysis and laser-scanning confocal microscopy showed that opioid treatment did not alter the surface expression of either CXCR1 or CCR5. Additional studies have shown that treatment with met-enkephalin, MOR-, and DOR-selective agonists results in the inhibition of the chemotactic response of monocytes or opioid receptor-expressing Jurkat T cells to CCL5 (Rogers et al. 2000, Szabo et al. 2003). As CCL5 is an agonist for both CCR1 and CCR5 (and to a lesser degree CCR3), experiments were carried out to determine whether CCR5 or CCR1, specifically, is a target for opioid-induced cross-desensitization using cell lines transfected to express both MOR and either CCR5 or CCR1. These studies demonstrated that both CCR5 and CCR1 are targeted by MOR and DOR for cross-desensitization using a variety of cell lines for these studies (Szabo et al. 2003, Zhang et al. 2003). In contrast, neither MOR nor DOR activation appears to be capable of cross-desensitization of CXCR4 in either monocytes or T cells (Szabo et al. 2003). Finally, using flow cytometry, radiolabeled binding analysis, and confocal microscopy we found that the heterologous desensitization of CCR5 was not associated with a significant degree of receptor internalization in either primary human monocytes or in stably transfected cell lines (Szabo et al. 2003).

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As noted above, we have observed that neither MOR nor DOR is able to cross-desensitize CXCR4, and we interpreted this as an indication of the relative resistance of this chemokine receptor to cross-desensitiztion. In an effort to extend these studies, we carried out studies to examine the potential interaction between KOR and CXCR4. Somewhat surprisingly, we found that KOR is able to substantially cross-desensitize CXCR4, and we believe this is an indication of the relative strength of KOR to carry out heterologous desensitization (Finley et  al. 2008b). Coupled with the capacity of KOR to induce down-regulation of the expression of CXCR4 (reviewed above), our results suggest that KOR has considerable inhibitory activity for this chemokine receptor.

14.4.3 Molecular Mechanism of Heterologous Desensitization between Opioid and Chemokine Receptors A number of studies have shown that the mechanism underlining the heterologous desensitization between opioid receptors and chemokine receptors involves target receptor phosphorylation. This phosphorylation event appears to typically be different from the G-protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation that is a common aspect of homologous desensitization. Following homologous desensitization, the phosphorylated receptors may, or may not, be internalized. In contrast, heterologous desensitization is almost always dependent on target receptor phosphorylation, and this process is believed to be typically carried out by a second messenger-dependent kinase (reviewed in Steele et al. 2002). For Gi coupled-receptors, which include both the chemokine and opioid receptors, this kinase is typically one or more of the family of PKC enzymes. Recent studies by Zhang et  al. (2003) showed that opioid-mediated downregulation of the CCR1 function could be blocked by the general PKC inhibitor calphostin C, but not by the calcium-dependent classic PKC inhibitor Go6976. Further work showed that opioid receptor activation preferentially resulted in the activation of calcium-independent PKCs, suggesting opioids achieve desensitization of CCR1 via a unique pathway, involving only calcium-independent PKC isotypes (Zhang et  al. 2003). In more recent studies, we have found that the biochemical mechanism of MOR-induced cross-desensitization of CCR5 is dependent specifically on the activation of PKCz, a member of the atypical PKC sub-family (Song et al., submitted for publication). It is currently uncertain whether the cross-desensitization of other chemokine receptors is dependent on the same PKC family members. It is also not clear whether other opioid receptors may mediate cross-desensitization by a signaling pathway that is dependent on the same or other kinases.

14.4.4 Implications for HIV Infection We carried out experiments which show that the activation of either MOR or DOR leads to the cross-desensitization of CCR5 but not CXCR4 (Steele et  al. 2003).

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Studies carried out with both primary monocytes and CHO cells transfected to express both MOR and CCR5 show that the inactivation of CCR5 function is not due to detectable receptor endocytosis, based on both radiolabeled binding analysis and laser-scanning confocal microscopy. We examined the ability of CCR5 to function as a coreceptor for HIV following MOR-induced heterologous desensitization, and our results show that the susceptibility of monocytes, or monocyte-derived macrophages, to infection by R5 strains of HIV is significantly inhibited following cross-desensitization. In contrast, the susceptibility of cells to infection with X4 strains is not altered, consistent with the failure of MOR to cross-desensitize CXCR4 (Steele et al. 2003). We do not have an explanation for the inability of cross-desensitized CCR5 to function as a coreceptor for HIV, since the receptor in this case remains on the cell surface. However, the inability of CCR5 to signal may impair the coreceptor activity. More importantly, it is known that the cross-desensitized CCR5 is phosphorylated, and the phosphorylated CCR5 may be unable to participate in the ability of the virus to productively attach and internalize to the target cell. It is possible that the cross-phosphorylated CCR5 is unable to carry out the normal cell surface-viral envelope membrane fusion which is required for viral infection. The biochemical basis for the inability of MOR-cross-desensitized CCR5 to function as an HIV coreceptor is the subject of additional work in our laboratory. The inhibition of CCR5 coreceptor activity following MOR-induced heterologousdesensitization is consistent with recently reported studies showing that the B-oligomer of pertussis toxin induces cross-desensitization of CCR5 in human cells, and this coincides with a dramatic reduction in the susceptibility of cells to HIV-1 infection (Alfano et al. 1999). In these studies, the activation of the pertussis toxin B-oligomer receptor results in cross-desensitization of CCR5 signaling, without altering the level of binding of CCR5 ligands. Moreover, the B-oligomer induces heterologous desensitization of CCR5 without altering CXCR4 function, and reduces susceptibility to R5, but not X4, strains of HIV. It is now clear that CCR5 coreceptor function is susceptible to heterologous desensitization induced by several unrelated GPCR ligands. Wang and his colleagues (Shen et al. 2000, Li et al. 2001, Le et al. 2001), in a series of studies with a number of formyl peptide receptor agonists, have also shown that cross-desensitization of CCR5 results in reduced susceptibility to HIV.

14.5 Conclusions Opioid regulation of chemokine and chemokine receptor expression has several disease- related implications. Viral infection by HIV-1 can be enhanced with opioids that activate MOR while the opposite can be true for opioids that activate KOR. Increased levels of HIV-1 coreceptors, such as CCR5 and CXCR4, can promote viral binding and trafficking of HIV-1 virally infected cells. It is likely that this allows the viral disease to progress in the immune cells of the blood in addition to neurological reservoirs. Alterations of chemokine receptor expression will not only affect viral infection, but also alter immune system function. In certain diseases

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these mechanisms could be exploited using opioid agonist therapeutics as to fine tune the immune response, whether it is increased levels of chemokine receptors as demonstrated with MOR activation, or decreased by activation of KOR. The ability of the opioid and chemokine receptors to cross-talk by heterologous desensitization has additional implications for the function of the nervous and immune systems. The elevated level of chemokines associated with episodes of inflammation and tissue injury in the brain would be expected to result in altered neuronal function, and specifically, in reduced MOR-mediated analgesia. It is well established that exaggerated pain (hyperalgesia) occurs as a part of inflammatory stress reactions (Watkins et al. 1995, Junger and Sorkin 2000). This pain response is a condition that often occurs with systemic inflammatory “flu-like” reactions, with symptoms of joint and muscle aches, fever, malaise, somnolence, and decreased locomotion. The possibility that a reduction in analgesia may contribute to the pain in the periphery associated with a variety of inflammatory disease states including rheumatoid arthritis, dental caries, and certain infectious diseases, should be investigated further. Our studies suggest the hypothesis that the cross-desensitization of MOR induced by chemokines may provide a basis for the hyperalgesia associated with inflammatory reactions in general. Acknowledgments  This work was supported by the National Institutes of Health grants DA06650, DA16544, DA13429, DA14230, and T32DA07237.

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Junger H, Sorkin LS (2000) Nociceptive and inflammatory effects of subcutaneous TNFalpha. Pain 85:145–151 Kraus J, Borner C, Giannini E, Hollt V (2003) The role of nuclear factor kappaB in tumor necrosis factor-regulated transcription of the human mu-opioid receptor gene. Mol Pharmacol 64:876–884 Law PY, Loh HH, Wei LN (2004) Insights into the receptor transcription and signaling: implications in opioid tolerance and dependence. [Review] [123 refs]. Neuropharmacology 47(Suppl 1): 300–311 Le Y, Wetzel MA, Shen W, Gong W, Rogers TJ, Henderson EE, Wang JM (2001) Desensitization of chemokine receptor CCR5 in dendritic cells at the early stage of differentiation by activation of formyl peptide receptors. Clin Immunol 99:365–372 Li BQ, Wetzel MA, Mikovits JA, Henderson EE, Rogers TJ, Gong W, Le Y, Ruscetti FW, Wang JM (2001) The synthetic peptide WKYMVm attenuates the function of the chemokine receptors CCR5 and CXCR4 through activation of formyl peptide receptor-like 1. Blood 97:2941–2947 Lio P, Vannucci M (2003) Investigating the evolution and structure of chemokine receptors. Gene 317:29–37 Liu Y, Blackbourn DJ, Chuang LF, Killam KF Jr, Chuang RY (1992) Effects of in vivo and in vitro administration of morphine sulfate upon rhesus macaque polymorphonuclear cell phagocytosis and chemotaxis. J Pharmacol Exp Ther 263:533–539 Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, Kishimoto T, Bronson RT, Springer TA (1998) Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc Natl Acad Sci USA 95:9448–9453 Mahajan SD, Schwartz SA, Shanahan TC, Chawda RP, Nair MP (2002) Morphine regulates gene expression of alpha- and beta-chemokines and their receptors on astroglial cells via the opioid mu receptor. J Immunol 169:3589–3599 McCarthy L, Wetzel M, Sliker JK, Eisenstein TK, Rogers TJ (2001) Opioids, opioid receptors, and the immune response. [Review] [128 refs]. Drug Alcohol Depend 62:111–123 Mizuno T, Kawanokuchi J, Numata K, Suzumura A (2003) Production and neuroprotective functions of fractalkine in the central nervous system. Brain Res 979:65–70 Nair MP, Chadha KC, Hewitt RG, Mahajan S, Sweet A, Schwartz SA (2000) Cocaine differentially modulates chemokine production by mononuclear cells from normal donors and human immunodeficiency virus type 1-infected patients. Clin Diagn Lab Immunol 7:96–100 Neudeck BL, Loeb J, Buck J (2003) Activation of the kappa-opioid receptor in Caco-2 cells decreases interleukin-8 secretion. Eur J Pharmacol 467:81–84 Neudeck BL, Loeb JM (2002) Endomorphin-1 alters interleukin-8 secretion in Caco-2 cells via a receptor mediated process. Immunol Lett 84:217–221 Ping D, Boekhoudt GH, Rogers EM, Boss JM (1999) Nuclear factor-kappa B p65 mediates the assembly and activation of the TNF-responsive element of the murine monocyte chemoattractant-1 gene. J Immunol 162:727–734 Rock RB, Hu S, Sheng WS, Peterson PK (2006) Morphine stimulates CCL2 production by human neurons. J Neuroinflammation 3:32 Rogers TJ, Peterson PK (2003) Opioid G protein-coupled receptors: signals at the crossroads of inflammation. [Review] [43 refs]. Trends Immunol 24:116–121 Rogers TJ, Steele AD, Howard OM, Oppenheim JJ (2000) Bidirectional heterologous desensitization of opioid and chemokine receptors. [Review] [51 refs]. Ann NY Acad Sci 917:19–28 Roy S, Cain KJ, Chapin RB, Charboneau RG, Barke RA (1998) Morphine modulates NF kappa B activation in macrophages. Biochem Biophys Res Commun 245:392–396 Ruzicka BB, Fox CA, Thompson RC, Meng F, Watson SJ, Akil H (1995) Primary astroglial cultures derived from several rat brain regions differentially express mu, delta and kappa opioid receptor mRNA. Brain Res Mol Brain Res 34:209–220 Saccani S, Pantano S, Natoli G (2001) Two waves of nuclear factor kappaB recruitment to target promoters. J Exp Med 193:1351–1359 Shen W, Li B, Wetzel MA, Rogers TJ, Henderson EE, Su SB, Gong W, Le Y, Sargeant R, Dimitrov DS, Oppenheim JJ, Wang JM (2000) Down-regulation of the chemokine receptor CCR5 by activation of chemotactic formyl peptide receptor in human monocytes. Blood 96:2887–2894

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Sheng WS, Hu S, Lokensgard JR, Peterson PK (2003) U50, 488 inhibits HIV-1 Tat-induced monocyte chemoattractant protein-1 (CCL2) production by human astrocytes. Biochem Pharmacol 65:9–14 Steele AD, Henderson EE, Rogers TJ (2003) Mu-opioid modulation of HIV-1 coreceptor expression and HIV-1 replication. Virology 309:99–107 Steele AD, Szabo I, Bednar F, Rogers TJ, Steele AD, Szabo I, Bednar F, Rogers TJ (2002) Interactions between opioid and chemokine receptors: heterologous desensitization. [Review] [142 refs]. Cytokine Growth Factor Rev 13:209–222 Suzuki S, Chuang AJ, Chuang LF, Doi RH, Chuang RY (2002a) Morphine promotes simian acquired immunodeficiency syndrome virus replication in monkey peripheral mononuclear cells: induction of CC chemokine receptor 5 expression for virus entry. J Infect Dis 185:1826–1829 Suzuki S, Chuang LF, Doi RH, Bidlack JM, Chuang RY (2001) Kappa-opioid receptors on lymphocytes of a human lymphocytic cell line: morphine-induced up-regulation as evidenced by competitive RT-PCR and indirect immunofluorescence. Int Immunopharmacol 1:1733–1742 Suzuki S, Chuang LF, Doi RH, Chuang RY (2003) Identification of opioid-regulated genes in human lymphocytic cells by differential display: upregulation of Kruppel-like factor 7 by morphine. Exp Cell Res 291:340–351 Suzuki S, Chuang LF, Yau P, Doi RH, Chuang RY (2002b) Interactions of opioid and chemokine receptors: oligomerization of mu, kappa, and delta with CCR5 on immune cells. Exp Cell Res 280:192–200 Suzuki S, Miyagi T, Chuang TK, Chuang LF, Doi RH, Chuang RY (2000) Morphine upregulates mu opioid receptors of human and monkey lymphocytes. Biochem Biophys Res Commun 279:621–628 Szabo I, Chen XH, Xin L, Adler MW, Howard OM, Oppenheim JJ, Rogers TJ (2002) Heterologous desensitization of opioid receptors by chemokines inhibits chemotaxis and enhances the perception of pain. Proc Natl Acad Sci USA 99:10276–10281 Szabo I et al (2003) Selective inactivation of CCR5 and decreased infectivity of R5 HIV-1 strains mediated by opioid-induced heterologous desensitization. J Leukoc Biol 74:1074–1082 Ueda A, Ishigatsubo Y, Okubo T, Yoshimura T (1997) Transcriptional regulation of the human monocyte chemoattractant protein-1 gene. Cooperation of two NF-kappaB sites and NF-kappaB/Rel subunit specificity. J Biol Chem 272:31092–31099 Ueda A, Okuda K, Ohno S, Shirai A, Igarashi T, Matsunaga K, Fukushima J, Kawamoto S, Ishigatsubo Y, Okubo T (1994) NF-kappa B and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene. J Immunol 153:2052–2063 Watkins LR, Maier SF, Goehler LE (1995) Immune activation: the role of pro-inflammatory cytokines in inflammation, illness responses and pathological pain states. [Review] [135 refs]. Pain 63:289–302 Wetzel MA, Steele AD, Eisenstein TK, Adler MW, Henderson EE, Rogers TJ (2000) Mu-opioid induction of monocyte chemoattractant protein-1, RANTES, and IFN-gamma-inducible protein-10 expression in human peripheral blood mononuclear cells. J Immunol 165:6519–6524 Widmer U, Manogue KR, Cerami A, Sherry B (1993) Genomic cloning and promoter analysis of macrophage inflammatory protein (MIP)-2, MIP-1 alpha, and MIP-1 beta, members of the chemokine superfamily of proinflammatory cytokines. J Immunol 150:4996–5012 Ye RD (2001) Regulation of nuclear factor kappaB activation by G-protein-coupled receptors. [Review] [136 refs]. J Leukoc Biol 70:839–848 Zhang L, Rogers TJ (2000) Kappa-opioid regulation of thymocyte IL-7 receptor and C-C chemokine receptor 2 expression. J Immunol 164:5088–5093 Zhang N, Hodge D, Rogers TJ, Oppenheim JJ, Zhang N, Hodge D, Rogers TJ, Oppenheim JJ (2003) Ca2+-independent protein kinase Cs mediate heterologous desensitization of leukocyte chemokine receptors by opioid receptors. J Biol Chem 278:12729–12736 Zhang N, Rogers TJ, Caterina M, Oppenheim JJ (2004) Proinflammatory chemokines, such as C-C chemokine ligand 3, desensitize mu-opioid receptors on dorsal root ganglia neurons. J Immunol 173:594–599

Chapter 15

Chronic Morphine’s Role on Innate Immunity, Bacterial Susceptibility and Implications in Wound Healing Josephine Martin and Sabita Roy

15.1 Introduction Drug abuse is a significant social problem in the United States (US) and abroad. An estimated 2 million people in the US alone have or will abuse a drug in their lifetime. Over a billion dollars are spent annually to treat direct and indirect effects associated with opioid abuse. Immunosuppression has been observed in individuals diagnosed as chronic users. Clinical correlations between wound complications stemming from improper healing and infection and opioid abuse have been shown. Further exaggeration of improper healing and infection is prevalent when the abuser is HIV-1 positive. The model concerning wound healing occurs in two phases; (1) pro-inflammatory responses (Glaser and Kiecolt-Glaser 2005; Moore 1999; Tidball 2005; Whelan et al. 2005) which are needed to ensure adequate clearance of pathogen at the site of tissue injury, as well as, (2) re-epithelialization and neovascularization events (Frantz et  al. 2005; Moore 1999; Naldini and Carraro 2005; Olah and Caldwell 2003; Whelan et al. 2005) to ensure proper wound closure. It is important to note that the resolution of pathogen clearance is essential in order for the wound closure processes to take place (Robson 1997).

15.2 Wound Healing and Innate Immunity 15.2.1 Pro-inflammatory Response Following tissue injury, the innate immune response is the first line of defense against possible pathogen infections. Through evolutionary pressures, the body automatically assumes infection which will be present post injury. Innate immunity J. Martin and S. Roy () Department of Surgery and Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA e-mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_15, © Springer Science+Business Media, LLC 2010

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Table 15.1  Cellular functions of innate immunity Cell type Function Neutrophil Derived from bone marrow progenitor cells Macrophage Phagocytoses and kills invading pathogens, especially bacteria Mast cell/Basophil Tissue resident cells derived from monocytes Eosinophil Phagocytoses cellular and foreign debris Involved in chronic inflammation Antigen presenting cell Induces fever (IL-1, -6, TNF-a) Facilitates neutrophil migration (MIP-2, IL-1b, -8) Stimulates wound contraction (PDGF) Releases histamine and leukotrienes Defends against parasites

components are presented to launch an attack against a broad range of possible infectious agents. Innate immune cells are equipped to recognize several extracellular and intracellular components that are foreign to the host (van Bruggen et al. 2007). These cell types work in concert for the eradication of pathogens (see Table 15.1). Resident macrophages, for example, are able to recognize the natural polysaccharide cell wall component of gram-negative bacteria, lipopolysaccaride (LPS) (Cross et al. 1995). LPS initially binds to LPS binding proteins (LBP) forming a complex, recognized by toll like receptor four (TLR4) located on the surface of macrophages. Bound TLR4 to the endotoxin complex initiates TLR4 interactions with CD14:MD-2 at the membrane (Vasl et al. 2008). Activation of this complex in macrophages elicits downstream effects of increased cytokine production and secretion, instrumental for sustaining a pro-inflammatory response, recruitment of systemic neutrophils to the site of injury/inflammation and up-regulation of intracellular signaling cascades which further increases the expression of pro-inflammatory mediators. Neutrophils, released from the bone marrow stores in response to injury/ inflammatory cues, migrate and elicit killing via oxygen-dependent and oxygenindependent mechanisms (Miyasaki et  al. 1986). Neutrophils also produce and secrete potent chemokines instrumental in the activation and migration of bone marrow-derived macrophages (Daley et al. 2005). These systemic macrophages not only aid in the killing of bacteria, but are also necessary for the debridement of apoptotic neutrophil and bacteria debris (Leibovich and Ross 1975). The net effect of these processes lead to an eradication of infectious agents. Resolution therefore is dependent on pro-inflammatory cues necessary for promoting activation, migration and pathogen recognition by leukocytes. As observed by Celus, a Roman physician during the first century A.D., the four cardinal signs characterize inflammation: swelling, redness, heat, and pain. Swelling/edema occurs in response to the accumulation of fluids from damaged capillaries following injury. Increased fluid accumulation leads to increase capillary permeability. Histamine, produced and released by resident mast cells, also

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facilitates the increased permeability and vasodilation of capillaries near the damaged tissue site. Interestingly, studies have shown histamine to induce neutrophil adhesion to endothelial cells (Schaefer et al. 1998; 1999). The culmination of these effects contributes to the redness and heat observations present during inflammation. Simultaneously, Hageman factor (Factor XIIa) is activated and facilitates the production of bradykinin and the degradation of fibrin matrixes near the site of injury (Maeda et  al. 1996; Perkins et  al. 2008). Bradykinin B1 (constitutively expressed) and B2 (induced expression in response to LPS) receptors have been shown to induce pain perception. Degradation of fibrin facilitates leukocyte chemotaxis across blood vessels into injured tissues. During the first phase of wound healing, varying populations of cells migrate to the site of the wound in a sequential fashion (Boyce et al. 2000; Lee et al. 2001; Park and Barbul 2004). Platelets are the initial responders post injury followed by neutrophil, macrophage, lymphocyte and fibroblast populations (Park and Barbul 2004). Platelets, peaking 12 h post wounding, are required for coagulatory events, whereas neutrophils and macrophages (peaking on day 1 and 3, respectively) are pro-inflammatory populations key in migratory and proliferatory events. In addition to their individual contributions to wound healing, each cell type has been found to produce and secrete potent chemotactic factors that enable the migration and activation of subsequent cell populations (Man et al. 2007). Chemokines and growth factors, such as platelet-derived growth factor (PDGF) (Lutgens et al. 2005), macrophage inflammatory protein 2 (MIP-2) (Ao et al. 2006) and monocyte chemotactic protein 1 (MCP-1) (Ishikawa and Miyazak 2005; Li et al. 2005) are known chemoattractants that produce a chemical gradient at the site of injury to not only promote migration and activation of neutrophils and macrophages, but to also increase the expression of adhesion molecules necessary for the sticking and translocation of rolling systemic leukocytes at the site of injury (Li et al. 2005). Other pro-inflammatory factors produced and secreted by these cell populations such as interleukins (IL) -1, -6, -12, and tumor necrosis factor alpha (TNF-a) are instrumental in the resolution of bacterial clearance (Daley et  al. 2005; Ishikawa and Miyazak 2005; Kudo et al. 2005; Man et al. 2007; Wang et al. 2005; Warner and Srinivasan 2004).

15.2.2 Pro-angoigenic Response Upon resolution of the pathogen, the second phase of wound healing occurs where re-epithelialization and neovascularization are essential for wound closure. Formation of new blood vessels (angiogenesis) (Barcelos et al. 2005; Roy et al. 2008), fibrin matrices (Midwood et al. 2006), and collagen deposits (Seppinen et al. 2008) are all events that promote the wound closure process. New blood vessels, formed from pre-existing vessels, occurs 7 days post wounding (Park and Barbul 2004). Potent angiogenic factors such as vascular endothelial

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growth factor (VEGF) (Roy et al. 2006a; Roy and Loh 1996), matrix metalloproteinases (MMP) (Harper and Moses 2006; Moses 1997; Roy et al. 2006b), and the transcription factor hypoxia inducible factor 1 alpha (HIF-1a) (Lee et al. 2004) have been found to be involved in promoting neovascularization (Ushio-Fukai and Nakamura 2008). VEGF, produced and secreted by neutrophils and macrophages, is not only an inducer of angiogenesis, but has also been shown to stimulate proliferation, migration, and permeability of endothelial cells. Although several isoforms of VEGF have been identified with evidence of functional redundancy, a predominate isoform involved during wound healing is VEGF-A. VEGF-A mediated angiogenesis acts through VEGF type-1 (VEGFR1/Flt1) and type-2 receptors (VEGFR2/Flk-1). In the presence of a VEGF gradient, newly formed vessels sprout in patterns which follow along the extracellular matrix (ECM). In order to accommodate the outgrowth of these vessels, degradation of the ECM’s basement membrane must occur. MMPs are major players during this degradative process and are also secreted by the innate immune responders, neutrophils and macrophages. Transcription of growth factors and matrix metalloproteinases are mediated through the transcription factor HIF-1a (Ahn et  al. 2008; Misra et  al. 2008; Surazynski et al. 2008). In response to injury and a drop in oxygen tension (hypoxia), HIF-1a protein becomes stabilized by avoiding rapid ubiquitin-mediated degradation. As stable HIF-1a proteins migrate from the cytosol into the nuclear compartment, it dimerizes with HIF-1b/ARNT to recognize and bind its response element on DNA. Once bound, the HIF complex promotes the transcription of growth factors and MMPs. In concert, all of these factors have been shown to promote the angiogenic arm of wound repair (Eming et al. 2007).

15.3 Opioids Tissue injury and inflammation increases the excitability of sensory neurons called nociceptors. Excitation of these receptors leads to an increased perception of pain. In response to chemical stimuli, as seen by kinins (previously discussed), peripheral terminals of nociceptors become excited via a GqPCR mechanism. Excitation of these receptors leads to increased intracellular calcium levels through inositol triphosphate and prostaglandin production yielding to an increase in synaptic action potentials/firings. It is the net effect of increased firings that lead to increased perceptions of pain. Interestingly, nociceptors and chemokine receptors are members of the GPCR family (Marinissen and Gutkind 2001). It is through this similarity that many in the scientific community believe opioids regulate immune function directly and indirectly. m opioid agonist (endogenous and exogenous) induce analgesic effects by regulating both the pre-synapse and post-synapse of sensory neurons (Griffin 2008). At the pre-synapse, opioids bound to m opioid receptors block voltage-gated calcium

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(Ca2+) channels and hence, block Ca2+ influx. Lower intracellular Ca2+ leads to an inhibition of excitatory neurotransmitter release from pre-synaptic vesicles. Opioids bound to MOR on postsynaptic terminals promote the efflux of potassium (K+) via K+ channels. The net effect of active MOR receptor results in hyperpolarization of the post-synapse causing inhibition of neuronal firing. Studies have shown MOR effects at the pre- and post-synapse synergistically decreases the perception of pain (Glaum et  al. 1994; Kohno et  al. 1999; Williams et  al. 2001; Yoshimura and North 1983).

15.3.1 Endogenous Opioids Three endogenous opioids have been identified; enkephalins, dynorphins and beta-endorphins. These opioid peptides selectively bind to the seven transmembrane GPCRs delta (d), kappa (k), and mu (m). Although dynorphin binds predominately to the k receptor, b-endorphines and enkephalins bind to m and d opioid receptors. It is important to note that the analgesia induced by opioids is mediated predominately through the m opioid receptor. In vitro studies have shown a decrease in the immune function and proliferation following b-endorphin administration in rodents (Ray and Cohn 1999) and that the immunosuppressive effects by b-endorphins are steroid-independent (Berkenbosch et al. 1984; Nelson et al. 2000).

15.3.2 Exogenous Opioids Morphine and its derivatives continue to be considered the gold standard for alleviating pain. Morphine is metabolized in the liver via N-dealkylation and glucoronidation at the third (M3G) or sixth position (M6G). Although M3G are the most common metabolites (accounts for 50% of the metabolites produced), they elicit no biological activity when bound to MOR. It is the M6G metabolite (accounts for 10% of the metabolites produced) that elicits the nociceptive/analgesic effect upon binding to the m opioid receptor (Dahan et al. 2008). M6G is predominately eliminated via renal excretion. The synthetic morphine derivatives fentanyl and heroin have similar efficacy and addictive properties as morphine, yet these two drugs differ in their onset and duration of action properties. Rapid onset of fentanyl and heroin are attributed to their highly lipophilic profiles making these drugs readily available to cross the blood-brain barrier. It is important to note that while fentanyl is an approved analgesic therapeutic, heroin is not acceptable for treating pain in the United States (US). However, heroin use amongst the drug abusing population is on the rise at an alarming rate in the US.

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15.3.3 Opioid Antagonists The opioid antagonists’ naloxone and naltrexone bind to all three opioid receptors, m, k, and d. These compounds are antagonists due to their inability to elicit downstream effects of these receptors once bound (Sarton et al. 2008; Yaksh and Rudy 1977). Interestingly, both antagonists have a high binding affinity for MORs. Naloxone is used to reverse the effects of an acute opioid overdose because of its rapid onset of action. Naltrexone elicits similar actions, but has a longer onset and duration of action and hence, is used for the maintenance of treatment for opioid addicts.

15.4 Opioids and Innate Immunity 15.4.1 Morphine and Immunosuppression Morphine’s modulatory role as an immunosuppressive agent has been well established over the past 50 years. The consensus of morphine’s immunosuppressive actions being multifaceted adds to the complexity of identifying its pro-inflammatory targets when establishing a working model. Most of the literature to date has attributed the immunosuppressive effects of chronic morphine treatment to an indirect, down-regulation of potent pro-inflammatory chemotactins and cytokines (Johnston et al. 2004; Volk et al. 2004; Wang et al. 2005). However, morphine may directly initiate its suppressive effects on innate immune cells by binding to m opioid receptors expressed on macrophages and neutrophils. Sedqi et al. and Chuang et al. were the first laboratories to eloquently demonstrate immune cell expression of m opioid receptors (Chuang et  al. 1995; Sedqi et al. 1995). Later studies also provided evidence of the presence of opioid receptors on cells involved in host defense and immunity and that these MOR on immune cells have a higher binding affinity for morphine, but not for the endogenous opioid peptides. Taken together, these findings shed light on the possible roles both endogenous and exogenous opioids may have on immune function.

15.4.2 Morphine and Neutrophils Although controversial, findings as to how chronically administered morphine modulates neutrophil chemotaxis and function, a growing consensus believes that morphine is suppressive in the recruitment and functional aspects of these cells during an innate immune response. When peripheral human blood neutrophils were pretreated with exogenous opioids, IL-8-induced chemotaxis was inhibited (Grimm et al. 1998). Conversely, Simpkins et al. reported an increase in neutrophil chemotaxis

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following b-endorphin treatment (Simpkins et  al. 1984). The discrepancy of the later finding may in part be explained by the fact previously stated that morphine has a greater MOR binding affinity on immune cells than does b-endorphins. Cytokines, produced and secreted by neutrophils, believed to regulate wound healing by controlling host resistance to infection has also been shown to be inhibited following morphine treatment (Clark et al. 2007). Skin samples taken following a murine hind paw incision, showed decreased levels of interleukins1a, -6, tumor necrosis factor alpha (TNF-a), granulocyte colony stimulating factor (G-CSF) and keratinocyte-derived cytokine (KC) when morphine was administered. This study also showed a significant reduction of the neutrophil bactericidal agent, myeoperoxidase (MPO). It is important to note that the morphine suppression discussed in the Clark et  al. study is representative of an acute effect.

15.4.3 Morphine and Macrophages Several studies to date have reported macrophage modulation following morphine treatment. Morphine is shown to act as a suppressor of macrophage recruitment and function during an innate immune response. A study carried out by Grimm et  al. showed a significant decrease in macrophage chemotaxis when morphine, heroin or the potent MOR agonists DAMGO were administered in rats (Grimm et  al. 1998). They concluded that morphine’s inhibition of subsequent macrophage chemotaxis occurs upon direct binding to the macrophage m opioid receptors and that this activation of MOR leads to the phosphorylation and desensitization of chemokine receptors CCR1, CCR2, CXCR1 and CXCR2. Desensitized chemokine receptors are therefore unable to elicit a response when their ligands are present. Concerning macrophage function, both an ability to phagocytose pathogens and produce and secrete potent pro-inflammatory factors was attenuated in the presence of morphine. Engulfment and killing of yeast (Candida albicans) by peritoneal macrophages was attenuated following morphine treatment (Pacifici et  al. 1993; Rojavin et  al. 1993). This inhibition of phagocytosis by morphine however was reversed following naltrexone administration. Interestingly, Tomei et al. makes the argument that the inhibition of macrophage-mediated phagocytosis is dependent on the dose and exposure duration of morphine. They report an inhibition of phagocytotic activity by peritoneal macrophages in vitro during early time points. However, following 16  h exposure to morphine, no effect on phagocytosis was seen. They attribute this removal of inhibition following chronic exposure to a desensitization of the m opioid receptors on macrophages (Tomei and Renaud 1997). Clearly, further studies are needed to determine what roles chronic morphine plays on phagocytotic activity. When looking at macrophage function in mediating inflammatory responses, several groups have shown either a marked induction or suppression of cytokine and

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transcription factor expression. An induction of nitric oxide (NO) and interleukin-10 (IL-10) production by macrophages was seen in response to chronic morphine or heroin treatments (Singhal et  al. 1998; Weber et  al. 2004). While up-regulations may seem beneficial in regards to bacterial killing, these groups report a detrimental outcome. Increased NO production was found to enhance macrophage apoptosis, as well as, increased killing of non-infected cells. In the presence of the endotoxin LPS, suppression of cytokines IL-6 and TNF-a were seen following morphine treatment (Roy et al. 1998). The transcription factor NFkB, responsible for up-regulation of several cytokines including IL-6, TNF-a, NO and IL-10, was also suppressed following morphine treatment. Taken together, the complexity by which morphine acts as an immunosuppressor on the migration and functional activity of innate immune responders, particularly neutrophils and macrophages, poses a compromising environment that proves detrimental for the hosts’ ability to eradicate pathogens.

15.4.4 Morphine and Wound Healing The Center for Disease Control and Prevention conducted a randomized epidemiological study on patients who had received morphine nerve paste post-operatively for pain management purposes. Ninety-four percent of the patients used in the cohort presented themselves with surgical-site complications such as edema and inflammation 24 days (median) post-operation. Upon culturing of the wounds, 64% tested positive for bacterial infection. It is important to note that all of the patients were found to have residual morphine paste on board indicative of a chronic morphine state (Sacerdote et al. 2000). More recently we have, through the standardization of an in  vivo wound murine model, characterized the suppressive effects of morphine on wound healing events in the presence of an infection. Our model is the first to mimic the clinical manifestations seen in opioid user and abuser populations. We hypothesized that in the presence of an inflammatory inducer lipopolysaccharide (LPS), chronic morphine treatment would suppress leukocyte migration by suppressing the chemical cues necessary for migration. Our findings show a marked decrease in wound closure, wound integrity, and bacterial sepsis following chronic morphine administration. A 48 h delay and suppression of neutrophil migration was observed and was attributed to altered chemokine expression independent of MIP-2. Our studies also revealed a suppression of macrophage infiltration is attributed to suppressed levels of the potent macrophage chemoattractant MCP-1. Taken together, our findings indicate that although the mechanism by which morphine exerts its immunosuppressive effects on pro-inflammatory responses varies depending on the cell type, the net result is a significant suppression of wound healing events (Martin J and Roy S. unpublished observations)

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15.5 HIV and Innate Immunity Epidemiological studies of opioid abuse within injection drug user (IDU) populations have shown overwhelming evidence that correlates opioid abuse with increased HIV-1 viral replication and rapid disease progression (Nath et al. 2002). To date, the majority of animal in vivo and in vitro studies on opioids, HIV and immunity have been carried out in the brain. These studies support the growing consensus that opioids pose detrimental effects on the neurological immune cell function of microglia and astrocytes (Peterson et al. 1990; 1994; 2004). However, little is known concerning the ramifications opioids may have on peripheral functions. Human immunodeficiency virus 1 (HIV-1) infection begins with attachment and entry into the target cells (Bagnis 2007). Cells expressing CD4 antigen and cytokine receptors CXCR4 and/or CCR5 are ideal targets for HIV infection. The target cells that meet the above antigen and receptor expression motifs are T cells, dendritic cells, and macrophages. Further preference for a particular target cell depends on the HIV strain; for example, T-tropic and M-tropic strains prefer T cells and macrophages, respectively. Following gp120-mediated viral entry, genomic RNA is reversed transcribed initiating a cDNA viral copy which is transcribed into the host genome. This provirus copy is then spliced and translated into viral proteins group antigens (Gag), RNA polymerase (Pol) and envelope (Env). Following further proteolytic cleaving, the Gag protein gives rise to capsid, matrix, and nucleocapsid proteins. The Pol protein, upon cleavage, gives rise to protease, reverse transcriptase and integrase. Interestingly, protease is involved in further cleavage of Gag and Pol precursor proteins while integrase is involved in viral integration. Env protein codes for gp160 proteins (gp120 and gp41) that, upon further cleavage, give rise to the proteins constituting the viral envelope. Other HIV proteins include: regulator of viral expression (Rev), negative effectors (Nef), viral protein R (Vpr), viral protein U (Vpu), viral infectivity factor (Vif) and transactivator protein (Tat). These proteins are instrumental in viral mRNA expression, viral replication and transactivation, viral release and maturation, viral infection, and maintenance of viral transcript activation and expression, respectively (Tripathi and Agrawal 2007). Tat is an 86 amino acids protein that increases the expression of all genes turned on by human immunodeficiency virus type 1 (HIV-1) long terminal repeats (LTR). TAT interacts with the LTR’s cis-activation targeting sequence. Tat genes are divided into two coding exon regions which result in the translation of the Tat protein. The first coding exon of Tat can sufficiently transcribe a fully active protein (Kim and Panganiban 1993).

15.5.1 HIV and Macrophages: The Trojan Horse During the early stages of HIV infection, macrophages play an important role in viral survival. Infected macrophages enable the virus to evade killing for a long duration of time (Gartner et al. 1986), as well as, induce activated T cell apoptosis

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by promoting chemotaxis of T cells to areas where infected macrophages are present (Banda et al. 1992). Recently, HIV viral entry into macrophages has also been shown to occur through the binding of gp120 to the macrophage’s mannose receptor (MR) (Trujillo et al. 2007). Upon binding, macrophages are able to eradicate viral replication via degradation and antigen presentation. Nevertheless, this occurrence of gp120:MR interactions are lesser than the occurrence of gp120 interacting with CD4, CXCR4, and CCR5 on macrophages. Overall, in the presence of HIV, infected macrophages act as viral reservoirs, aid in viral evasion of killing, induce T cell apoptosis, and mediate T cell depletion. Once macrophages become virally infected, they begin a vicious cycle of immune activation and HIV dissemination.

15.5.2 HIV and the Host Innate Immunity Response The natural killer cells (NK) are the host’s primary innate immune responders against viral infections. Studies have shown morphine to suppress the cytolytic activity of NK cells (Shavit et al. 2004). In vivo studies carried out in the Indian rhesus macaques looked at chronic morphine administration and SIV; the equivalent of HIV in apes. This group concluded that morphine contributed to the pathogenesis of Simian Immunodeficiency Virus (SIV) infection and that this contribution occurred in conjunction with the replication of viral proteins including Tat (Noel and Kumar 2006; Noel et al. 2006). Innate immune response to viral infections is predominately through interferonalpha, -beta (IFN-a and –b) induction and activation of natural killer (NK) cells. Although viral replication can induce IFN-a and –b expression, macrophages are capable of producing and secreting cytokines which also induce the production of these type I interferons (Falk 2001). Bound IFNa and b to its receptors on NK cells increases its ability to lyse virally-infected cells. NK cells elicit viral killing directly by lysing the virus, or indirectly by NK-mediated antibody-dependent cytotoxic killing and activation of the immune system. HIV has escaped viral killing from host NK cells mainly by suppressing the NK cell’s ability to kill the virus based on antibody-dependent cytotoxicity. Although the majority of NK cells do not possess the CD receptor necessary for HIV entry, a subset of NK cells (CD4+) have been found to function as a reservoir for HIV particles (Valentin et al. 2002). This poses a greater disadvantage for the host immune system in eradicating viral infection.

15.5.3 Opioids, HIV and Wound Healing Heroin, a synthetic derivative of the m opioid receptor agonist morphine, is a welldocumented narcotic that alters mood and rewarding behaviors. To date, heroin is the most abused opioid with an estimated 750,000–1,000,000 hardcore users in the

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United States alone. In addition to poor nutrition and poor hygienic practices, heroin addicts present themselves with compounding non-healing wounds stemming from injection site-related complications. Although chronic morphine treatment is well established as an immunosuppressant and mechanisms by which wound healing occurs is well documented, the underlying mechanisms concerning these phenomena seen in heroin-addicted patients with delayed wound healing in the presence of an infection has not been explored. Several studies have correlated the presence of human immunodeficiency virus (HIV) infection with an increased risk of wound complications leading to improper wound healing. A retrospective, blind cohort examining wound infection incidences following implant surgery of patients testing either negative or positive for HIV found comparable incidences of wound infection (5.0% and 3.5%, respectively) when preoperative contamination was not a factor. Conversely, in the presence of preoperative contamination, HIV-positive patients were four times as likely to develop infection at the site of wound compared to the HIV-negative population (42% and 11%, respectively). Although correlations between HIV and poor wound healing have been implicated, the possible compounding affects of heroin abuse or chronic morphine use, that it may have on wound healing in HIV-positive patients has yet to be elucidated. We show (unpublished observation) that in the presence of both inflammatory inducers LPS and Tat, chronic morphine sustains a persistent elevation of neutrophil influx resulting in a continued production/secretion of MCP-1. Although MCP-1 is a potent chemoattractant for macrophage recruitment, morphine alters macrophage’s ability to respond to these cues. This results in an (1) increased number of neutrophils causing tissue necrosis and pus formation, (2) sustained elevation of MCP-1 signal, and (3) inadequate resolution of neutrophil debridement. These results are consistent with clinical epidemiological findings, that report substantial susceptibility to bacterial infections and improper wound healing in chronic opioid user and abuser populations that are HIV positive.

15.6 Proposed Model Overall, tissue injury and infection will trigger a cascade of events leading to an up-regulation of pro-inflammatory factors required for the first arm of wound healing; pathogen clearance. Resident macrophages produce and secrete several chemokines (including MIP-2, IL-8, Sdf-1, and KC) involved in establishing a chemical gradient necessary for the attraction and activation of neutrophils. These neutrophils in turn, elicit bacterial killing of pathogens while producing and secreting chemokines (including MCP-1, MIP-1a, MIP-1b, and MIP-3) necessary for the recruitment of subsequent pro-inflammatory cell types including macrophages. Macrophages migrating in response to these secreted cues aid in the killing of pathogens, engulf apoptotic neutrophils, and secrete factors instrumental in promoting the second arm to wound healing: wound repair. Although pro-angiogenic

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factors are produced during the pro-inflammatory phase, it is only until pathogens are resolved that the pro-angiogenic events will occur. Researchers speculate that IL-12 acts as a switch in coordinating when phases are turned on and off. When IL-12 is expressed, the pro-inflammatory response is on leaving pro-angiogenic events off. Our data show that various stimuli (LPS, hypoxia, or Tat), chronic morphine treatment will lead to decreased wound healing as a result of a suppression in neutrophil and macrophage migration and function and hence maintain a brake on the formation of new blood vessels.

15.7 Summary Overwhelming evidence indicates that the mechanism by which morphine exerts its immunosuppressive effects on pro-inflammatory responses varies depending on the immune cell type and depending on the type of infectious agent present. Morphine’s suppression of early stage innate immunity, critical in determining the outcome of pathogen resolution in wounds, shifts the outcome to a persistence of pathogen onboard, prolonged inflammation, pathogen dissemination and inadequate wound healing.

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Chapter 16

Opioids, Astroglial Chemokines, Microglial Reactivity, and Neuronal Injury in HIV-1 Encephalitis Kurt F. Hauser, Nazira El-Hage, Annadora J. Bruce-Keller, and Pamela E. Knapp

16.1 Introduction 16.1.1 The Endogenous Opioid System The endogenous opioid system consists of multiple receptor [µ (MOP), d (DOP), k (KOP), and NOP (also known as the “opioid receptor-like (ORL1) clone-1”)] and peptide (proenkephalin, prodynorphin, proopiomelanocortin, and nociceptin/orphanin FQ) gene families with overlapping functions (Akil et  al. 1984; Evans et  al. 1988; Sibinga and Goldstein 1988; Mogil and Pasternak 2001). “Opiates” [a term that refers to opium alkaloids derived from the opium poppy, Papaver somniferum (e.g., opium and heroin)] act by mimicking endogenous opioid ligands and by activating opioid receptors. Most opiate drugs with abuse liability preferentially activate MOP, and MOP are the principal receptor type involved in addiction. Heroin is rapidly deacetylated to morphine in the serum and in the nervous system. A majority of heroin actions in the central nervous system (CNS) are due to its conversion to morphine (Wright 1941; Jaffe and Martin 1985), making morphine a prototypic drug of choice to study opiate drug action and addictive processes. Since initial studies identified opioid receptors on T-lymphocytes (Wybran et al. 1979), the effects of opioids on immune function have been extensively studied. Details of these studies have been exhaustively reviewed (Madden et al. 1991; Adler et al. 1993; Peterson et al. 1998; Donahoe and Vlahov 1998; Roy et al. 2006), and will only be briefly mentioned here. In general, opioids suppress immune function. Peripheral leukocytes, including lymphocytes and peripheral blood mononuclear cells (PBMCs) can express the four major opioid receptor types, MOP, DOP, KOP,

K.F. Hauser  (*) N. El-Hage, A.J. Bruce-Keller, and P.E. Knapp Department of Pharmacology and Toxicology, Virginia Commonwealth University School of Medicine, 1217 East Marshall Street, Richmond, VA 23298-0613, USA e-mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_16, © Springer Science+Business Media, LLC 2010

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and recently NOP, and are important cellular targets of opiate drug abuse (Carr et al. 1990; Bryant et al. 1990; Madden et al. 1991, 1998; Eisenstein and Hilburger 1998; Sharp 2003, 2006; Tomassini et  al. 2004; Roy et  al. 2004; Finley et  al. 2008a).

16.2 Opioid–Chemokine Interactions 16.2.1 Chemokine Ligands and Receptors Details regarding structural/functional differences among chemokines and their receptors are discussed elsewhere in this volume. Briefly, chemokines (and their cognate receptors) consist of four main classes (CC, CXC, CX3C and C) based on the number and spacing of at least four conserved cysteine residues (Murphy 2002). Opioid–chemokine interactions occur at the molecular level. For example, opioid and chemokine receptors can form heterodimers (Rogers and Peterson 2003; Bidlack et al. 2006; Finley et al. 2008b). Examples of such heterodimers include: CCR5–MOP (Chen et al. 2004); CXCR4–MOP (Burbassi et al. 2008); CXCR4–KOP (Finley et al. 2008a); CXCR4–DOP (Pello et al. 2008); and CXCR2–DOP (Parenty et al. 2008). This list is incomplete and will likely continue to grow. The resultant dimer pairs likely result in signaling events that are different than those that occur through stimulation of either receptor alone, and may directly contribute to heterologous cross-sensitization/desensitization. Besides direct GPCR interactions, opioids and chemokines interact at other levels, including intracellular signaling downstream from GPCRs and at the level of transcription. For example, MOPnuclear factor-kB (NF-kB) transcriptional interactions have been documented in several cell types (Kraus et al. 2003; Borner et al. 2004; Roy et al. 2006). Pu.1, a leukocyte specific promoter, targets MOP expression in hematopoietic cells including monocyte-derived macrophages and microglia (Hwang et al. 2004). This suggests a molecular basis for inherent opioid interactions with innate (macrophages and granulocytes) and adaptive (B and T lymphocytes) immune cells (Singh et al. 1999).

16.2.2 Pain/Nociception Considerable evidence for opioid–chemokine interactions comes from studies of pain and inflammation, where the inherent relationships between pain, inflammation, and the counteracting antinociceptive influences of opioids have considerable biomedical implications. The adaptive changes in immune and nervous system function with chronic inflammation and pain further reveal the inherent interrelatedness between opioids (Ossipov et al. 2003; Evans 2004; Roy et al. 2006; Christie 2008)

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and chemokines (DeLeo et al. 2004; Stefano et al. 2005; Machelska and Stein 2006; Rittner et al. 2008; Stefano and Kream 2008). In the above situations, chemokines originating from the nervous system are largely glial in origin. The relationship of opioids, chemokines, and more recently glia, as related to peripheral inflammation and pain has been extensively reviewed elsewhere (Stein et al. 2003; Machelska and Stein 2006; Watkins et al. 2007; Hutchinson et al. 2008).

16.3 NeuroAIDS The CNS is highly vulnerable to infection by human immunodeficiency virus type 1 (HIV-1 or HIV). HIV encephalitis (HIVE) and neurological complications are frequent manifestations of HIV infection and occur in one-third to one-half of the individuals with AIDS. Prior to the advent of highly active antiretroviral therapy (HAART), an estimated 10–30% of patients experienced full-blown HIV-associated dementia (HAD) (Kandanearatchi et al. 2003; Ghafouri et al. 2006; Anthony and Bell 2008). The use of antiretroviral agents and protease inhibitors caused an initial decline in the incidence of HIVE and HAD, and changed the pattern of neurological and psychological impairment (Cysique et al. 2004; Gonzalez-Scarano and Martin-Garcia 2005). Increasing patient survival combined with the selection of more virulent and resistant forms of HIV, however, contributed to recently reported increases in the severity and prevalence of HIVE (Neuenburg et al. 2002; Ghafouri et al. 2006; Ances and Ellis 2007; Anthony and Bell 2008).

16.3.1 Glia are the Principal Targets of HIV in the CNS Since HIV-1 does not infect neurons directly, neuropathological changes are secondary to “cellular toxins” (e.g., cytokines, glutamate, reactive oxygen and nitrogen species) and “viral toxins” (e.g., toxic viral proteins, such as gp120 and Tat) originating from infected or virally exposed glia. This principally includes microglia, monocyte-derived macrophages that are the major site of viral infection in the CNS, but also includes astroglia, which can be infected latently or persistently, and can release cellular toxins in response to intact virions or viral proteins. The extent that immature neurons and glia (Ensoli et al. 1994; McCarthy et al. 1995; Krathwohl and Kaiser 2004), or glial progenitors, which express CXCR4 (Ni et al. 2004), are present in the adult, and can be infected with HIV-1 (Lawrence et al. 2004), contribute to the pathogenesis is uncertain. Other glial types such as oligodendroglia may also respond directly to virotoxins or cellular toxins, or secondarily to neuronal or perhaps astroglial injury (Stys and Lipton 2007; Hauser et al. 2008). HIV-1 protein “virotoxins” (Nath 2002), released from infected cells, have direct bystander effects on neighboring glia and neurons and cause many of the deleterious consequences of HIV-1. Residual viral proteins, including Tat, gp120 and Vpr,

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are formed because the stoichiometry of viral assembly in infected neural cells is inexact and excess proteins can be actively released (e.g., Tat) or are released when infected cells die (Brack-Werner 1999; Nath 1999; Avison et al. 2003). For example, the small numbers of astroglia that become latently or persistently infected, may produce disproportionate amounts of individual proteins (Brack-Werner 1999; Kramer-Hammerle et al. 2005b). Despite the inability of the virus to infect neurons, HIV type-1 (HIV-1) proteins such as gp120 (Dreyer et al. 1990), Tat (Sabatier et al. 1991; Haughey et al. 1999), Nef, Rev (Avison et al. 2003), and Vpr (Piller et al. 1998) that are released from infected cells are directly neurotoxic (Kaul et  al. 2001; Bonavia et  al. 2001; Trillo-Pazos et  al. 2004; Mattson et  al. 2005). Importantly, increased levels of detection of Nef and Rev in astrocytes coincides with increased HIV dementia (Ranki et al. 1995) and the overexpression of the nef gene in the CNS was proposed some time ago as a marker for HIV-1 infection in astrocytes in pediatric neuroAIDS (Saito et al. 1994). Neurotoxicity can therefore be thought of as resulting in large part from the response of neurons to viral and cellular toxins originating from glia. Tat is proposed to interact with a variety of molecular targets on neurons and glia including a subset of integrins [avb3 and a5B1 (Barillari et  al. 1993; Vogel et al. 1993)], vesicular endothelial growth factor (VEGF) receptor (Morini et al. 2000), lipoprotein-related protein receptor (Liu et al. 2000), as well as chemokines receptors such as CCR2 (Albini et  al. 1998), and perhaps CXCR4 (Watson and Edwards 1999; King et al. 2006). Because of its highly basic nature and its ability to translocate biological membranes, Tat is likely to interact in a semi-selective manner with a variety of molecular targets (Prochiantz and Joliot 2003; Fittipaldi and Giacca 2005). As noted below, soluble gp120 interacts with CXCR4 and CCR5 chemokine receptors, as well as other potential targets, to induce CNS toxicity and inflammation (Moore 1997; Kaul and Lipton 1999; Khan et  al. 2004; Misse et al. 2005).

16.4 Opiate–Immune Interactions in the Brain are Unique Evidence for biomedically relevant opioid–chemokine interactions come from the studies of examining the comorbidity of opiate drug abuse in HIV-infected individuals. As will be discussed in this review, chemokines are important mediators of HIV-1 neuropathogenesis (Miller and Meucci 1999). Moreover, concurrent substance abuse exacerbates HIV encephalitis largely through exacerbating chemokine release. Although the discussion here is limited to opiate drug abuse, methamphetamine, cocaine, and alcohol also worsen disease progression by exaggerating the production of cytokines and chemokines from microglia and astroglia (Nath et  al. 1999a, 2000, 2002; Hauser et  al. 2007). The opioid– chemokine connection is exemplified by studies of HIVE, where acute and chronic opioid exposure can cause synergistic increases in the production of chemokines that are key players in driving CNS inflammatory changes, reactive

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gliosis, and neuronal injury. The CNS consequences of HIV-1 infection, opiate exposure, and their comorbid effects as related to glia are considered in the discussion below. Unlike peripheral immune responses, opiates appear to exacerbate many aspects of the pathophysiological effects of HIV infection in the CNS (Donahoe 2004; Hauser et  al. 2007). Although experimental studies addressing opioid– HIV-1 interactions using murine and human cells in vitro, and nonprimate models of HIV encephalitis in  vivo, typically support direct comorbid interactions (discussed below), studies in nonhuman primates have been more controversial. Recent findings afford some explanation for certain inconsistencies reported between the CNS effects of opiates on simian immunodeficiency virus (SIV) or chimeric simianhuman immunodeficiency virus (SHIV) disease progression (Burdo et al. 2006). Some inconsistencies have been attributed to the differences in the pharmacodynamics of opioid administration (Donahoe et al. 1993), and differences in the relative neurovirulence of SIV/SHIV strains used (Suzuki et  al. 2002; Kumar et  al. 2004; Marcario et  al. 2004; Chuang et  al. 2005). Recent studies in nonhuman primates increasingly show preferential CNS infection and/or injury with morphine coadministration (Kumar et  al. 2006; Noel. et  al. 2006; Perez-Casanova et al. 2007), although there are considerable differences among outcome measures between individual studies (Noel and Kumar 2007; Marcario et al. 2008). In general, chronic morphine worsens the neuropathology and neurocognitive defects resulting from neurovirulent strains of SIV/SHIV. How opiate abuse aggravates HIVE is not completely understood, but likely reflects the unique response of astroglia and microglia to opiates. Importantly, opioid receptors have been localized at the cellular level on every glial type in the CNS, including astrocytes (Gurwell et al. 1996; Hauser et al. 1996; Stiene-Martin et al. 1998, 2001), oligodendrocytes (Knapp and Hauser 1996; Knapp et al. 1998, 2001; Hauser et al. 2008), macrophages/microglia (Turchan-Cholewo et al. 2008), ependymal cells (Hauser, unpublished), and/or radial glia (Sargeant et al. 2007). In addition to unambiguously colocalizing opioid receptors on specific populations of various glial types, there are now numerous studies providing functional evidence of MOP, DOP, and KOP expression in enriched-primary populations of astrocytes (Stiene-Martin and Hauser 1990, 1991, 1998; Eriksson et al. 1990, 1991, 1992; Stiene-Martin et al. 1991; Gurwell et al. 1996; Hauser et al. 1996; Belcheva et al. 1998, 2003, 2005), microglia (Peterson et al. 1993, 1995; Chao et al. 1993, 1996a, b), and most recently oligodendrocytes (Knapp and Hauser 1996; Knapp et al. 1998, 2001). Glial precursors in the ventricular and subventricular zones can also express opioid receptors (Zhu et al. 1998; Reznikov et al. 1999; Stiene-Martin et al. 2001; Kim et al. 2006; Tripathi et al. 2008). Immature glia and adult progenitors express opioid receptors, which affect cellular maturation (Stiene-Martin and Hauser 1990, 1991; Stiene-Martin et al. 1991; Persson et al. 2003, 2006) and cell fate decisions (Kim et  al. 2006). Importantly, glial precursors (Khurdayan et  al. 2004; Lawrence et  al. 2004; Buch et al. 2007), and especially immature oligodendroglia (Khurdayan et al. 2004; Hauser et al. 2008), also appear to be vulnerable to opioids and HIV-1 proteins.

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Unlike their mature counterparts, glial progenitors can be readily infected with HIV (Lawrence et al. 2004).

16.5 Microglia As noted earlier, because HIV-1 does not directly infect neurons, most of the neurotoxic effects are the inevitable consequences of virally infected microglia and to a lesser extent latently/persistently infected astroglia. Although astrocytes can become latently infected and harbor HIV-1, the principal cell type infected with HIV-1 in the brain is microglia. Overactivated microglia are principal contributors to chronic inflammation and neurodegenerative effects of HIV infection (Block et al. 2007; Gao and Hong 2008). Much of the pathogenesis in the CNS originates from infected monocyte-derived macrophages and HIV-1-infected resident microglia (Kaul et al. 2001; Hauser et al. 2005; Deshpande et al. 2005; Kaul and Lipton 2005; Kadiu et  al. 2005). Toxic production of ROS and nitric oxide by HIV-1exposed macrophages likely exacerbate metabolic compromises in neurons and astroglia caused by viral infection and the resultant inflammatory response (Koka et al. 1995; Nath 1999; Kaul et al. 2001; Persidsky and Gendelman 2003). Thus, microglia are the principal immune effector and principal cell type infected by HIV-1 in the CNS. The innate response of microglia to external insults relies on recognizing “pathogen-associated molecular patterns” through a growing list of “patterned response receptors” (Akira et  al. 2006), including Toll-like receptors (TLRs) (Aravalli et al. 2007; Carpentier et al. 2008; Falsig et al. 2008), scavenger receptors (Murphy et  al. 2005; Dunn et  al. 2008), and the macrophage antigen complex-1 (MAC1) receptor (Block et al. 2007; Gao and Hong 2008), which may act alone or in concert.

16.5.1 Opioids and Microglia Monocytes, macrophages, and microglia have long been known to be direct targets for opioids (Peterson et al. 1991, 1998; Chao et al. 1996b; Sheng et al. 1997; Roy et al. 1998; Rogers and Peterson 2003). Isolated macrophages (Singhal et al. 1992; Hagi et al. 1994; Makman 1994; Dobrenis et al. 1995; Roy et al. 1998; Eisenstein and Hilburger 1998) and brain-derived microglia (Peterson et al. 1993, 1995; Chao et al. 1993, 1996a, b) express opioid receptors Perhaps most importantly, MOP agonists can potentiate HIV propagation in lymphocytes, monocytes, and monocyte-derived macrophages/microglia (Peterson et al. 1990; Carr and Serou 1995). This includes morphine (Peterson et al. 1990, 1999, 2004; Hu et  al. 2005), methadone (Douglas 2001), and the MOP peptide agonist endomorphin (Peterson et  al. 1999). The effects of morphine on HIV

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replication depend on the duration of exposure and implicate adaptive processes including drug tolerance in mediating the response (Peterson et al. 1990, 2004; Sheng et  al. 1997). Moreover, in addition to increasing viral replication, macrophage/ microglial numbers are increased in some brain regions with drug abuse and this may be exaggerated by HIV (Bell et al. 2002). More recently, this observation has been extended to suggest that opiate abuse may hasten the turnover of perivascular macrophages in individuals with HIVE (Anthony et al. 2005). To add to the complexity of opioid–microglial interactions, unlike the effects of MOP activation, KOP stimulation with U50,488, or opioid receptor blockade with naltrexone can significantly reduce HIV-1 replication (Chao et al. 1996a, 2001; Gekker et al. 2001) and also attenuate oxyradical production by HIV-1-infected macrophages/microglia (Hu et al. 1998; Liu et al. 2002). By contrast to viral replication, opioids are typically described as inhibiting leukocyte function including the migration of human polymorphonuclear leukocytes (Makman et  al. 1995; Miyagi et  al. 2000), human blood monocytes (Stefano et  al. 1993; Miyagi et  al. 2000) and macrophages/microglia (Singhal et al. 1996; Chao et al. 1997, 1999; Grimm et al. 1998b; Hu et al. 2000; Clark et al. 2007). In fact, opium was described as suppressing chemotaxis of guineapig macrophages about a century ago (reviewed by Vallejo et  al. 2004). By contrast, increased motility has also been reported (Takayama and Ueda 2005), including some early studies that showed opioids at very low concentrations increase chemoattraction depending on the particular opioid receptor–effector signaling pathway that is activated and culture conditions (van Epps and Saland 1984; Grimm et al. 1998b). Singhal and coworkers have shown that opioids can affect macrophage-programmed cell death (Singhal et  al. 1998, 2002; Malik et al. 2002; Bhat et al. 2004), which likely alters a variety of functions including differences in chemotaxis resulting from differences in chemokine production (Malik et al. 2002). We find that the response of microglia to opioids is highly contextual and is likely reprogrammed by cytokines/chemokines. For example, MOP activation generally inhibits the motility of isolated macrophages/microglia; however, if macrophages/ microglia are cultured with astrocytes, or with conditioned medium from astrocytes, they show the opposite response to morphine and their motility is markedly increased (El-Hage et al. 2006b). We propose that high levels of proinflammatory cytokines and especially chemokines produced by astrocytes, including RANTES/ CCL5 and MCP-1/CCL2, can reprogram macrophages/microglia to respond differently to opiates. Elimination of CCL2/MCP-1 either by immunoneutralization or by using conditioned medium from astrocytes from CCL2/MCP-1 knockout mice significantly attenuates the chemotactic effects of morphine on the macrophages/ microglia (El-Hage et al. 2006b). Moreover, CCR2 gene deletion markedly attenuates the recruitment/activation of macrophages/microglia toward the epicenter of stereotaxically-injected Tat into the brain (El-Hage et al. 2006a). Others report similar contextual changes in macrophage responsiveness. For example, IL-6 exposure causes an opposite response of macrophages to MOP and DOP agonists (Grimm et al. 1998a).

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Preliminary data from our group show that morphine synergistically increases Tat-induced ROS production in both cultured N9 microglial cells and primary microglia (Turchan-Cholewo et al. 2009; Bruce-Keller et al. unpublished). In isolated microglia, opiates potentiate HIV-1 Tat-induced reactive oxygen species (ROS)/reactive nitrogen species (RNS) production, while attenuating HIV-1 Tat-induced cytokine production. Opiates also increase 3-nitrotyrosine immunoreactive products in inducible Tat transgenic mice, further supporting the notion that opiates selectively increase free radical production in macrophages/microglia (Bruce-Keller et al. 2008). The paradoxical reductions in cytokine release appear to result from coordinated inhibition of proteasome proteolytic activity in microglia (Turchen-Cholewo et al. 2009). While elevated ROS typically triggers proinflammatory events in microglia and/or monocytic cells (Bruce-Keller et  al. 2001), extremely high ROS concentrations can inhibit cytokine production (Li and Engelhardt 2006). Interestingly, divergent ROS signaling and cytokine production in microglia has been reported in response to several classes of environmental toxins (Block et al. 2007). Importantly, temporary, nontoxic proteasome inhibition is sufficient to increase Tat-induced protein oxidation, while reducing Tat-induced cytokine/chemokine release (Bruce-Keller, unpublished). Furthermore, morphine potentiates both responses to Tat. The findings support a role for the proteasome in regulating microglial inflammatory signaling, and suggest that morphineinduced increases in oxidative stress may ultimately underlie the typically suppressive effects of morphine on macrophage immune responsiveness.

16.5.2 Neuron-Microglia Chemokine Signaling In addition to responding to viral proteins and signals originating from bystander microglia and astroglia (discussed below), microglia may respond to chemokine signals originating from neurons. Compromised neurons may also be important sources of proinflammatory chemokines such as interferon-g, fractalkine (Streit et  al. 2005), and CXCL10 (IP-10) (Sui et  al. 2004, 2006). Recently, it has been shown that human neurons in culture can release CCL2 following exposure to morphine (Rock et al. 2006), suggesting a means by which morphine-exposed neurons activate macrophages/microglia directly. By contrast, CX3CL1 (fractalkine) can act as a neuroprotective signal preventing microglia from targeting neurons (Meucci et  al. 2000; Cardona et  al. 2006; Ransohoff et  al. 2007), indicating that neuron– microglial interactions can either oppose or promote neural degeneration.

16.6 Astroglia Astrocytes are the most abundant cell type in the CNS and provide essential structural, metabolic, and trophic support for neurons (Parpura et al. 1994; Araque et al. 1999; Fields and Stevens-Graham 2002; Perea and Araque 2005). Astrocytes are

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proposed as critical for normal neurotransmitter function through “gliotransmission” at tripartite synapses (Araque et  al. 1999). Astrocytes directly respond to neuronal injury and dysfunction (Fields and Stevens-Graham 2002), through spatial buffering of excess extracellular glutamate and potassium, and via the production of inflammatory cytokines/chemokines. Disrupting astroglial function is likely to disrupt ion homeostasis (Araque et al. 1999; Fields and Stevens-Graham 2002; Sykova 2005; Sykova and Vargova 2008) and “gliotransmission” (Araque et al. 1999). Astroglia respond to a variety of innate microbial and environmental insults, and can express many of the same patterned response receptors as macrophages/microglia (Falsig et al. 2006, 2008; Farina et al. 2007). Their response includes the production of proinflammatory cytokines and chemokines, which alert and enhance an immune surveillance. Heightened immune surveillance can have deleterious or beneficial consequences. The initial proinflammatory response to pathogens such as LPS or HIV-1 Tat is tightly controlled by cascades involving autocrine and paracrine cytokine signals among astrocytes themselves, which appear to precede chemokine release (Chung and Benveniste 1990; Norris et  al. 1994; Benveniste et al. 1995; Benveniste and Benos 1995; Barnes et al. 1996; Luo et al. 2000, 2002, 2003; Zhang et al. 2002; Kim et al. 2004; Falsig et al. 2006, 2008). This involves the activation of NF-kB and the rapid production of key proinflammatory cytokines including IL-1b, TNF-a, and IFN-g or IFN-a (depending on the species). The initial response likely affects nearby astrocytes, as well as neighboring neurons and microglia. Although not fully understood, the purpose of the initial response may serve to amplify the proinflammatory signal and act as a validation step for subsequent chemokine release.

16.6.1 Astroglia and HIV-1 HIV-1-infected, reactive astroglia can contribute directly to the neurotoxicity (Deshpande et  al. 2005; Fischer-Smith and Rappaport 2005; Kramer-Hammerle et al. 2005a). Astroglia are a significant target for HIV and their function is markedly impacted by the disease (Brack-Werner 1999; Nath 1999, 1999b; Gorry et al. 2003; Wang et al. 2004). Besides overt toxicity and loss of astrocytes through apoptosis (Shi et  al. 1996; Adamson et  al. 1996; He et  al. 1997; Fischer-Smith and Rappaport 2005), astroglia can be latently or persistently infected by HIV-1 (Kramer-Hammerle et al. 2005b), and there is evidence that astroglia can significantly modify the response of microglia and neurons to HIV (Vitkovic and da Cunha 1995b; Rappaport et  al. 1999; McManus et  al. 2000; Kramer-Hammerle et al. 2005b). Exposure to intact HIV-1, or gp120 and Tat proteins causes release of inflammatory cytokines [e.g., including TNF-a, IL-1b, interferons (IFN), IL-6] and chemokines (e.g., including CCL2, CCL5, MIP-1a, and MIP-1b) (Wahl et al. 1991; Vitkovic and da Cunha 1995a; Conant et al. 1996; Pulliam et al. 1998; Conant et al. 1998;

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Nath et  al. 1999b; Speth et  al. 2002; Mahajan et  al. 2005) and disrupts ion homeostasis in astroglia (Pulliam et al. 1993; Benos et al. 1994a, b; Holden et al. 1999). Although gp120–CD4-coreceptor interactions are essential and well characterized for viral entry in CD4+ leukocytes, mechanisms underlying gp120 actions unrelated to viral entry are perhaps less well understood and can signal both apoptosis and the release of proinflammatory chemokines (Meucci et al. 1998; Miller and Meucci 1999; Khan et al. 2004). Interestingly, gp120 can interact with CXCR4 independently of CD4, and the resultant agonist-selective signal is fundamentally different from that seen with the endogenous CXCR4 ligand SDF-1 (Khan et al. 2004). Moreover, gp120 or SDF-1 signals downstream of CXCR4 are cell typespecific (Bodner et al. 2003). This may partially rely on agonist-selective receptor-Ga, Gbg, and/or b-arrestin interactions and novel downstream signaling, which have been described for a variety of G-protein-coupled receptors (Kelly et al. 2008), including MOP (Gainetdinov et al. 2004; Bohn et al. 2004; Zheng et al. 2008). HIV proteins can also disrupt ion homeostasis in astrocytes, which compromises neuronal function (Pulliam et al. 1993; Benos et al. 1994a, b; Holden et al. 1999). Intact HIV-1 virions or gp120 also markedly inhibit glutamate uptake by astrocytes and cause reductions in excitatory amino acid transporter-2 (EAAT2) mRNA and protein levels (Wang et al. 2003). The inability of astrocytes to buffer extracellular glutamate is likely to decrease the excitotoxic threshold of bystander neurons. The prototypic increases in TNF-a, IL-1b, and IFN-g alluded to earlier likely trigger the release of specific chemokines, and collectively the response has been characterized as a generalized “inflammatory transcriptome” and is thought to underlie reactive astrogliosis (Falsig et  al. 2006). Importantly, should read “transcriptome” TNF-a, IFN-g, and IL-1b, are greatly increased in HIVE and are thought to contribute to the pathogenesis of the disease (Mayne et al. 1998; Kadiu et al. 2005). Astrocytes alone may be sufficient for the production of chemokines in the brain in response to pathogens; it is argued that other cell types such microglia need not be present (Chung and Benveniste 1990; Norris et al. 1994; Benveniste et al. 1995; Benveniste and Benos 1995; Barnes et al. 1996; Luo et al. 2000, 2002, 2003; Zhang et al. 2002; Kim et al. 2004).

16.7 Opioids, HIV, and Astroglial-derived Chemokines As mentioned, HIV-1 gp120 and/or Tat exposure increases chemokine expression and/or release by astrocytes (Conant et al. 1998; Nath et al. 1999b; El-Hage et al. 2005; Mahajan et al. 2005). HIV-1 Tat is reported to interact with CCAAT/enhancerbinding protein-beta (C/EBPb) to cause MCP-1 expression in astrocytes (Abraham et al. 2005). We have found that morphine, acting through MOP, synergistically increases the release of CCL2 and CCL5 by HIV-1 Tat-exposed astrocytes (El-Hage et al. 2005), thereby increasing macrophage recruitment and microglial activation (El-Hage et al. 2006b). The MOP-selective antagonist, b-funaltrexamine blocks

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cytokine production by murine (El-Hage et al. 2005) and human (Davis et al. 2007) astrocytes, further suggesting a role for MOP in signaling chemokine release by astrocytes. Similar increases in chemokine expression were seen in a morphine and gp120-coexposed human astroglial cell line (Mahajan et al. 2005), suggesting that the ability of morphine to cause exaggerated release of chemokines may be a generalized effect of MOP on inflammatory signals originating from astrocytes. Moreover, increases in macrophage/microglial chemotaxis in response to intrastriatal Tat injection ± morphine are significantly reduced in CCR2-null mice compared to wild-type controls (El-Hage et al. 2006a). Thus, the MCP-1 released by HIV-1 Tat and opiate-exposed astrocytes causes microglial motility in  vitro and appears to have a functional effect on chemotaxis in vivo as well. Unlike the effects of MOP activation, KOP stimulation typically has limited or opposing effects on astrocyte function. For example, KOP activation markedly reduces Tat-induced MCP-1 expression in human astrocytes (Sheng et al. 2003) or fails to affect CCL2 release by murine astrocytes (El-Hage et al. 2005). Morphine and Tat differently affect patterns of MOP and KOP expression in astrocytes, suggesting fundamental differences in the regulation of each receptor type (TurchanCholewo et al. 2008). The differences likely reflect the unique actions of MOP and KOP in astrocytes (Bohn et  al. 2000; Belcheva et  al. 2005), and are likely to be important for HIV-1 neuropathogenesis. We propose that the activated “inflammatory transcriptome” acts as a switch, triggering the release of chemokines from astrocytes (Barnes et al. 1996; Conant et al. 1998; Guo et al. 1998; Oh et al. 1999; Luo et al. 2003). On the basis of the above discussion and findings from our laboratories, we hypothesized that an inflammatory cascade exists in astrocytes to trigger the release of chemokines, which recruit/activate macrophages/microglia (Fig.  16.1) (El-Hage et  al. 2005, 2006a, b, 2008; Hauser et al. 2007). We have been testing whether key regulatory steps in this cascade mediate HIV-1 Tat ± morphine-induced reactive astrogliosis and microgliosis (El-Hage et al. 2006a, 2008). CCL5, for example, reportedly regulates CCL2 production and release (Luo et al. 2000, 2002, 2003). Murine CXCL1 (KC/GRO-a) also appears to stimulate RANTES, MCP-1, and IL-6 production (Luo et  al. 2000). KC itself is activated by TNF-a, IL-1b, or MIP-1a, but not RANTES or MCP-1, while deletion of the cognate receptor for KC, CXCR2, prevents increases in RANTES and MCP-1. Ultimately, this suggests that KC may control the production of both chemokines (Luo et al. 2000; El-Hage et al. 2008). Moreover, findings that combined Tat and opiates no longer increase the number of CCL2-immunoreactive astrocytes in CCL5(−/−) mice implicate increases in RANTES production by astrocytes as a key site of opiate–HIV-1 Tat protein interactions (El-Hage et al. 2008). Other investigators similarly describe the coordinated actions of IL-1b and IFN-b as necessary for CCL5 expression by astrocytes including human astrocytes (Oh et al. 1999; Kim et al. 2004). Although it is suggested that CCL5 is an important intermediary in astroglial inflammation, CCL5 by itself is reportedly insufficient to cause leukocyte chemotaxis across the blood–brain barrier (Eugenin et  al. 2006). Importantly, CCL2 by itself can recruit HIV-1-infected leukocytes into the brain (Eugenin et  al. 2006).

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Fig.  16.1  Summary of proinflammatory events involved in HIV-1 Tat and opiate-induced chemokine release from astrocytes. Our past and ongoing studies suggest that opiates potentiate the inflammatory effects of HIV-1 Tat causing synergistic increases in CCL5 (RANTES) and CCL2 (MCP-1) chemokine production (El-Hage et al. 2005, 2006a, b). By contrast, opiates interact less with Tat-induced increases in TNF-a, IL-1b, or interferon-g (IFN-g), although opiates may interact with alternative viral proteins to affect these cytokines. CCL5 gene deletion significantly reduced the reactive astroglial and macrophage/microglial changes in response to intrastriatal Tat ± systemic morphine exposure, indicating that CCL5 mediates reactive gliosis and inflammation (El-Hage et  al. 2008). Moreover, CCL5 knockout mice lacked Tat ± morphine-induced increases in astroglial CCL2 (El-Hage et al. 2008), suggesting that CCL5 regulates CCL2 production in astrocytes as has been suggested from cell culture studies (Luo et al. 2002; also see text). We propose that HIV-1 and chronic opiate abuse contribute to CNS pathology through a cascade that involves HIV-1-induced activation of an “inflammatory transcriptome” (Falsig et  al. 2006; also see text) involving the coordinated production of cytokines TNF-a, IL-1b, or interferon-g (IFN-g), which triggers chemokine release. Chronic opiate abuse potentiates the ensuing chemokine production, especially CCL5 and CCL2 release from astrocytes, which recruits and activates macrophages/microglia, Additional factors that are less well substantiated (?) are also likely to contribute to the proinflammatory cascade. Note, autocrine/paracrine feedback amplification (dashed arrow) likely occurs at multiple points within the cascade that are not shown (reproduced with permission from El-Hage et al. 2008)

Considering previous findings that CCR2 gene deletion largely eliminated Tat ± morphine-induced astrogliosis and macrophage recruitment/microglial activation (El-Hage et al. 2006a), we explored whether CCL5 could modulate Tat ± opiateinduced increases in CCL2 in astrocytes that were essential for glial activation. Findings in CCL5 knockout mice support this assertion by showing that astroglial and microglial activation was significantly attenuated, and that the proportion of CCL2 immunoreactive astroglia at 7 days was reduced following intrastrial injection of Tat ± continuous exposure to morphine during the last 5  days (El-Hage et  al. 2008). The results indicate that CCL5 regulates the astrogliosis and reactive microglial changes caused by Tat ± morphine. Furthermore, a significant reduction in the proportion of CCL2 immunoreactive astrocytes near the site of Tat injection suggested that CCL5 was contributing to increases in CCL2 expression in astrocytes (El-Hage et al. 2008). Collectively, our findings suggest that CCL5 regulates Tat

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and morphine-induced increases in CCL2 in astrocytes and subsequent increases in macrophage recruitment and microglial reactivity. By further destabilizing the function of astrocytes already compromised by HIV, we put forward that opiates hijack the otherwise neuroprotective actions of astrocytes (Hauser et  al. 2007). Chronic, opiate-abused astrocytes appear unable to provide adequate metabolic and trophic support for neurons. This includes the disruption of multiple transporters (e.g., the cysteine-glutamate Xc antiporter and EAAT2), whose dysfunction is likely to result in the inability to adequately buffer extracellular glutamate (Suzuki et  al. 2006; Tai et  al. 2007). Astrocyte-derived trophic factors such as brain-derived neurotrophic factor (BDNF) and glial cell linederived neurotrophic factor (GDNF) are disrupted by HIV-1 proteins (Nosheny et  al. 2005, 2006, 2007). Moreover, opiates further disrupt BDNF and neurotrophin-3 signaling (Numan et al. 1998), which may be an additional target for opiate– HIV interactive neurotoxicity. The exaggerated, comorbid interactions between opiates and HIV are not limited to astroglia. As noted earlier, since opiates potentiate HIV-1 Tat-induced ROS and RNS production in macrophages/microglia, increased recruitment by astrocytes is likely to contribute to increased oxidative stress. The paradoxical reductions in cytokine release by microglia may be of limited consequence if astroglial-derived cytokines predominate. In preliminary studies, we find that tissue levels of CCL5 and CCL2 mRNA and/or proteins are chronically elevated in Tat transgenic mice only if morphine is coadministered (El-Hage, unpublished). Thus, opiate–HIV interactive cytotoxicity targets each cell type differently, with the net result that the collective effects are qualitatively different and more severe than the effects at any cell type alone. Thus, we propose that opiate drugs exacerbate HIV-induced increases in key inflammatory events causing reactive gliosis and neuronal injury. In large part, this appears to result from MOP-dependent increases in astrocyte-derived chemokines, although considerable oxidative and nitrosative stress likely originates from newly recruited macrophages and reactive microglia. Studies that identify the cell targets and molecular sites, where opiates and HIV converge, should be instrumental in designing therapeutic strategies to limit/prevent the deleterious effects of neuroAIDS and opiate substance abuse. Acknowledgment  We gratefully acknowledge the support of the National Institute on Drug Abuse grant DA P01 19398.

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Stefano GB, Kream R (2008) Endogenous opiates, opioids, and immune function: evolutionary brokerage of defensive behaviors. Semin Cancer Biol 18:190–198 Stefano GB, Digenis A, Spector S, Leung MK, Bilfinger TV, Makman MH, Scharrer B, Abumrad NN (1993) Opiate-like substances in an invertebrate, an opiate receptor on invertebrate and human immunocytes, and a role in immunosuppression. Proc Natl Acad Sci USA 90:11099–11103 Stefano GB, Fricchione G, Goumon Y, Esch T (2005) Pain, immunity, opiate and opioid compounds and health. Med Sci Monit 11:MS47–MS53 Stein C, Schafer M, Machelska H (2003) Attacking pain at its source: new perspectives on opioids. Nat Med 9:1003–1008 Stiene-Martin A, Hauser KF (1990) Opioid-dependent growth of glial cultures: suppression of astrocyte DNA synthesis by Met-enkephalin. Life Sci 46:91–98 Stiene-Martin A, Hauser KF (1991) Glial growth is regulated by agonists selective for multiple opioid receptor types in vitro. J Neurosci Res 29:538–548 Stiene-Martin A, Gurwell JA, Hauser KF (1991) Morphine alters astrocyte growth in primary cultures of mouse glial cells: evidence for a direct effect of opiates on neural maturation. Dev Brain Res 60:1–7 Stiene-Martin A, Zhou R, Hauser KF (1998) Regional, developmental, and cell cycle-dependent differences in m, d, and k-opioid receptor expression among cultured mouse astrocytes. Glia 22:249–259 Stiene-Martin A, Knapp PE, Martin KM, Gurwell JA, Ryan S, Thornton SR, Smith FL, Hauser KF (2001) Opioid system diversity in developing neurons, astroglia, and oligodendroglia in the subventricular zone and striatum: impact on gliogenesis in vivo. Glia 36:78–88 Streit WJ, Davis CN, Harrison JK (2005) Role of fractalkine (CX3CL1) in regulating neuron– microglia interactions: development of viral-based CX3CR1 antagonists. Curr Alzheimer Res 2:187–189 Stys PK, Lipton SA (2007) White matter NMDA receptors: an unexpected new therapeutic target? Trends Pharmacol Sci 28:561–566 Sui Y, Potula R, Dhillon N, Pinson D, Li S, Nath A, Anderson C, Turchan J, Kolson D, Narayan O, Buch S (2004) Neuronal apoptosis is mediated by CXCL10 overexpression in simian human immunodeficiency virus encephalitis. Am J Pathol 164:1557–1566 Sui Y, Stehno-Bittel L, Li S, Loganathan R, Dhillon NK, Pinson D, Nath A, Kolson D, Narayan O, Buch S (2006) CXCL10-induced cell death in neurons: role of calcium dysregulation. Eur J NeuroSci 23:957–964 Suzuki S, Chuang AJ, Chuang LF, Doi RH, Chuang RY (2002) Morphine promotes simian acquired immunodeficiency syndrome virus replication in monkey peripheral mononuclear cells: induction of CC chemokine receptor 5 expression for virus entry. J Infect Dis 185:1826–1829 Suzuki M, Narita M, Narita M, Suzuki T (2006) Chronic morphine treatment increases the expression of vesicular glutamate transporter 1 in the mouse spinal cord. Eur J Pharmacol 535:166–168 Sykova E (2005) Glia and volume transmission during physiological and pathological states. J Neural Transm 112:137–147 Sykova E, Vargova L (2008) Extrasynaptic transmission and the diffusion parameters of the extracellular space. Neurochem Int 52:5–13 Tai YH, Wang YH, Tsai RY, Wang JJ, Tao PL, Liu TM, Wang YC, Wong CS (2007) Amitriptyline preserves morphine’s antinociceptive effect by regulating the glutamate transporter GLAST and GLT-1 trafficking and excitatory amino acids concentration in morphine-tolerant rats. Pain 129:343–354 Takayama N, Ueda H (2005) Morphine-induced chemotaxis and brain-derived neurotrophic factor expression in microglia. J Neurosci 25:430–435 Tomassini N, Renaud F, Roy S, Loh HH (2004) Morphine inhibits Fc-mediated phagocytosis through mu and delta opioid receptors. J Neuroimmunol 147:131–133 Trillo-Pazos G, Kandanearatchi A, Eyeson J, King D, Vyakarnam A, Everall IP (2004) Infection of stationary human brain aggregates with HIV-1 SF162 and IIIB results in transient neuronal damage and neurotoxicity. Neuropathol Appl Neurobiol 30:136–147

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Tripathi A, Khurshid N, Kumar P, Iyengar S (2008) Expression of delta- and mu-opioid receptors in the ventricular and subventricular zones of the developing human neocortex. Neurosci Res 61:257–270 Turchan-Cholewo J, Dimayuga FO, Ding Q, Keller JN, Hauser KF, Knapp PE, Bruce-Keller AJ (2008) Cell-specific actions of HIV-Tat and morphine on opioid receptor expression in glia. J Neurosci Res 86:2100–2110 Turchan-Cholewo J, Dimayuga FO, Gupta S, Keller JN, Knapp PE, Hauser KF, Bruce-Keller AJ (2009) Morphine and HIV-Tat increase microglial-free radical production and oxidative stress: possible role in cytokine regulation. J Neurochem 108:202–215 Vallejo R, de Leon-Casasola O, Benyamin R (2004) Opioid therapy and immunosuppression: a review. Am J Ther 11:354–365 van Epps DE, Saland L (1984) Beta-endorphin and met-enkephalin stimulate human peripheral blood mononuclear cell chemotaxis. J Immunol 132:3046–3053 Vitkovic L, da Cunha A (1995) Role for astrocytosis in HIV-1-associated dementia. Curr Top Microbiol Immunol 202(105–16):105–116 Vogel BE, Lee S, Hildebrand A, Craig W, Pierschbacher MD, Wong-Stall F, Ruoslahti E (1993) A novel integrin specificity exemplified by binding of the a∨b 5 integrin to the basic domain of the HIV Tat protein and vitronectin. J Cell Biol 121:461–468 Wahl SM, Allen JB, McCartney-Francis N, Morganti-Kossmann MC, Kossmann T, Ellingsworth L, Mai UE, Mergenhagen SE, Orenstein JM (1991) Macrophage- and astrocyte-derived transforming growth factor beta as a mediator of central nervous system dysfunction in acquired immune deficiency syndrome. J Exp Med 173:981–991 Wang Z, Pekarskaya O, Bencheikh M, Chao W, Gelbard HA, Ghorpade A, Rothstein JD, Volsky DJ (2003) Reduced expression of glutamate transporter EAAT2 and impaired glutamate transport in human primary astrocytes exposed to HIV-1 or gp120. Virology 312:60–73 Wang Z, Trillo-Pazos G, Kim SY, Canki M, Morgello S, Sharer LR, Gelbard HA, Su ZZ, Kang DC, Brooks AI, Fisher PB, Volsky DJ (2004) Effects of human immunodeficiency virus type 1 on astrocyte gene expression and function: potential role in neuropathogenesis. J Neurovirol 10(Suppl. 1):25–32 Watkins LR, Hutchinson MR, Ledeboer A, Wieseler-Frank J, Milligan ED, Maier SF (2007) Norman Cousins Lecture. Glia as the “bad guys”: implications for improving clinical pain control and the clinical utility of opioids. Brain Behav Immun 21:131–146 Watson K, Edwards RJ (1999) HIV-1-trans-activating (Tat) protein: both a target and a tool in therapeutic approaches. Biochem Pharmacol 58:1521–1528 Wright CI (1941) The enzymatic deacetylation of heroin and related morphine derivatives by blood serum. J Pharmacol Exp Ther 71:164–177 Wybran J, Appelboom T, Famaey JP, Govaerts A (1979) Suggestive evidence for receptors for morphine and methionine-enkephalin on normal human blood T lymphocytes. J Immunol 123:1068–1070 Zhang Y, Zhai Q, Luo Y, Dorf ME (2002) RANTES-mediated chemokine transcription in astrocytes involves activation and translocation of p90 ribosomal S6 protein kinase (RSK). J Biol Chem 277:19042–19048 Zheng H, Chu J, Qiu Y, Loh HH, Law PY (2008) Agonist-selective signaling is determined by the receptor location within the membrane domains. Proc Natl Acad Sci USA 105:9421–9426 Zhu Y, Hsu MS, Pintar JE (1998) Developmental expression of the mu, kappa, and delta opioid receptor mRNAs in mouse. J Neurosci 18:2538–2549

Chapter 17

Regulation of Neuronal Chemokine Receptor CXCR4 by m-Opioid Agonists and Its Involvement in NeuroAIDS Rajarshi Sengupta and Olimpia Meucci

17.1 CXCR4 and Opioid Receptors in the CNS: Distribution, Signaling, and Involvement in Neuropathology The opioid and the chemokine systems constitute two vital signaling networks in the brain and periphery and a brief background on these individual pathways in the CNS would help appreciate the relevance of their cross-talk in normal as well as diseased brain. As reviewed in previous chapters, CXCR4 belongs to the large superfamily of G-protein-coupled receptors (GPCR) that binds to the CXC chemokine CXCL12. Both CXCL12 and CXCR4 are constitutively expressed in the brain and act as key regulators of CNS development and homeostasis. However, their expression can also be induced in certain neuropathological conditions such as HIV-associated dementia, ischemia, and brain tumor (Rempel et al. 2000; Miller et al. 2005; Peng et  al. 2006). The importance of this chemokine/receptor pair is evident from the studies of knockout mice (both CXCL12 and CXCR4) that show severe impairment in cerebellar and hippocampal development (Ma et al. 1998; Zou et al. 1998; Bagri et al. 2002; Lu et al. 2002; Stumm et al. 2003; Paredes et al. 2006). Their role in mediating cellular migration, proliferation and survival is supported by a complementary expression pattern frequently observed in the brain (Ma et al. 1998; Zou et al. 1998; Bagri et al. 2002; Lu et al. 2002; Reiss et al. 2002). Early during development, CXCR4 expression is detected in ventricular, subventricular, and marginal zones, areas that nurture the survival and proliferation of precursor cells (McGrath et al. 1999; Tissir et al. 2004; reviewed by Klein and Rubin 2004; Li and Ransohoff 2008). During CNS development, CXCL12/CXCR4 controls the complex laminar organization of the brain by directly regulating the migration of neuronal precursors

R. Sengupta and O. Meucci () Departments of Pharmacology and Physiology & Microbiology and Immunology, Drexel University College of Medicine, 245 N 15th St. New College Building, 8804, Philadelphia, PA, 19102, USA e-mail: [email protected] O. Meucci (ed.), Chemokine Receptors and NeuroAIDS: Beyond Co-Receptor Function and Links to Other Neuropathologies, DOI 10.1007/978-1-4419-0793-6_17, © Springer Science+Business Media, LLC 2010

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including cortical Cajal–Retzius cells (CR), cerebellar granule precursor cells, and dentate gyrus granule precursor cells (reviewed by Li and Ransohoff 2008). In addition, they also play a pivotal role in axonal guidance and pathfinding of motor neurons, retinal ganglion cells, primary afferents, and sympathetic neurons (Chalasani et al. 2003; Lieberam et  al. 2005; Li and Ransohoff 2008). CXCL12/CXCR4 is also involved in adult neurogenesis by regulating the migration and proliferation of neuronal precursor cells (Li and Ransohoff 2008). Furthermore, both chemokine and receptor expression was demonstrated in diverse areas of the mature brain where they can regulate neuronal survival, transmission, and neuro-glial communication (Ragozzino et al. 2002; Bezzi et al. 2001; Bachis and Mocchetti 2004). The chemokine CXCL12 is often co-expressed with classical neurotransmitters such as acetylcholine or dopamine and neuropeptides like vasopressin (AVP) or melaninconcentrating hormone (MCH) indicating a role in the regulation of neuronal activity (Banisadr et al. 2002; Banisadr et al. 2003; Callewaere et al 2006; Guyon and Nahon, 2007). Recent studies have demonstrated a protective effect of CXCL12 after different neurotoxic insults. For example, CXCL12 was shown to rescue cortical and hippocampal neurons from NMDA-induced neurotoxicity as well as from apoptosis induced by trophic factor withdrawal (Meucci et  al. 1998; Patel et  al. 2006; Khan et al 2008). CXCL12 was also found to rescue primary cortical cells from H2O2-induced cell death (Shyu et  al. 2008). These prosurvival actions of CXCL12 are mediated by CXCR4-induced intracellular pathways, such as activation of PI3K/PKB (Akt) and MAPKs (ERK1/2) (Meucci et al. 1998; Khan et al. 2004; Patel et al. 2006). However, as mentioned earlier, CXCR4 also acts as a coreceptor for the T-tropic HIV-1, interacting specifically with the viral envelope glycoprotein gp120. Gp120 binding to CXCR4 triggers several apoptotic pathways that finally lead to caspase activation and cell death (Meucci et al. 1998; Yi et al. 2004; Singh et al. 2005; Khan et al. 2004; Shimizu et al. 2007). Notably, gp120induced neuronal apoptosis can be significantly reduced by CXCL12 while drugs of abuse such as opioids can exacerbate HIV-induced cell death (Corasaniti et al. 2001; Hu et al 2005). Thus, depending on the nature of the ligand as well as the environmental conditions, signaling through CXCR4 can lead to either neuroprotection or neuronal death. Indeed, both CXCL12 and CXCR4 have been associated with several neurodegenerative disorders such as HIV-associated dementia (HAD), Alzheimer’s, Multiple sclerosis as well as brain tumors (Vergote et al. 2006; McCandless et al., 2008a, b; also reviewed by Li and Ransohoff 2008). In summary, CXCL12/ CXCR4 represents a crucial chemokine receptor pair in brain and cellular and environmental factors regulating CXCR4 expression, function and/or interactions with its natural ligand would significantly impact CNS physiology under normal and pathological conditions. The endogenous opioids are another family of peptides involved in different physiological processes including pain regulation, respiratory control, stress responses, appetite, thermoregulation, and humoral and cellular immune function (Bodnar RJ., 2008). Opioids act through their receptors, which are also members of the GPCR family, and are expressed in the central and peripheral nervous system as well as on cells of the immune system (Henriksen and Willoch 2008; Hauser

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et  al. 2005; Sharp 2006). Three different classes of opioid receptors have been cloned and classified as mu, delta, and kappa (MOR, DOR, and KOR). They are highly homologous to one another in the trans-membrane and the intracellular regions (>60%) but differ significantly at their N-terminal domains (Knapp et al. 1995). The endogenous ligands for these receptors include the endorphins (MOR), enkephalins (DOR/MOR), the dynorphins (KOR). Opioid receptors modulate pain by inhibiting presynaptic voltage-gated Ca2+ channels and opening K+ (GIRK) channels, which result in reduced Ca2+ influx and diminished release of neurotransmitter (Piros et al. 1995; Blanchet and Luscher 2002). This inhibits the formation and propagation of an action potential providing the molecular basis for the analgesic action of opioids. Endogenous opioids produced by central and peripheral neurons act in concert to exert analgesic effects on incoming pain stimuli in the spinal cord dorsal horn (Mason 2005; Chen and Pan, 2006). As discussed in the previous chapters, opioids have been shown to modulate immune system function by at least two different pathways: Immune cell dysregulation by opioid receptors on the same cell and indirect modulation of the neuroendocrine axes by opioid receptors in the CNS that subsequently causes changes in immune system function (Grimm et al. 1998; Peterson et al. 1998). Recent evidence has suggested a third pathway involving interactions with the chemokine receptor system. These interactions may play a role in the increased rate of HIV-1 infection and disease progression to HAD seen in opiate users (Miyagi et al. 2000; Mahajan et al. 2002; Steele et al. 2002; Suzuki et al., 2002a, b, c). Cross-talk between opioid and chemokines has been studied extensively in the immune system. Current research also indicates a direct interaction between opioid receptors and the HIV co-receptors, CXCR4 and CCR5, in brain with alterations in receptor expression/ function or even chemokine production. Thus, intravenous opioid abuse such as consumption of heroin is not only a major cofactor in the spread of HIV, but may also significantly contribute to the neurological complications associated with HIV-1 infection (Heimer et al. 2008; Nath et al. 2002). The following sections of this chapter will review our current knowledge on heterologous desensitization of GPCRs while focusing on the interaction between opioid and chemokine receptors in the central nervous system.

17.2 Heterologous Desensitization of G-protein-Coupled Chemokine Receptors GPCR signaling is strictly regulated through a highly controlled cycle of activation and deactivation. Such regulation may occur at different stages from: (1) modulation of agonist levels through alterations of its synthesis or degradation, to (2) alteration in receptor mRNA or protein levels, and (3) receptor desensitization. Receptor inactivation or desensitization prevents overstimulation of intracellular signaling, thus restoring basal conditions following stimulus responses. Desensitization can be achieved through multiple mechanisms several of which have been revealed

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in recent studies (reviewed by Steele et al. 2002): Ligand binding may induce receptor endocytosis leading to internalization and eventually degradation (or recycling to the membrane) of the receptor. In other cases, desensitization occurs through a direct inactivation of receptor function, for example, through phosphorylation of the receptor (Grimm et  al. 1998; Chen et  al., 2004). Desensitization may also involve inhibition of downstream molecules that are directly involved in the signaling pathway (Ali et al., 1997). Notably, such desensitization could be either (a) homologous: ligand binding to GPCR1 leading to its own inactivation; or (b) heterologous: activation of GPCR1 leading to the inactivation of an unrelated GPCR2. Homologous desensitization usually involves G-protein receptor kinase (GRK) induced receptor phosphorylation followed by b-arrestin and dynamin-mediated internalization (Haribabu et al., 1997). Several molecular mechanisms have been proposed for heterologous desensitization of GPCRs such as receptor phosphorylation through protein kinases, receptor oligomerization, G-protein phosphorylation by ser/thr or tyrosine kinases, as well as modification of downstream effector molecules such as phosphorylation of phopholipase-Cb. Studies performed in immune cells have clearly demonstrated a bi-directional cross-talk between opioid and chemokine receptors indicating a hierarchy among opioid and chemokine receptors in producing cross-desensitization (reviewed by Steele et  al. 2002). Currently, there are three well-accepted molecular mechanisms associated with heterologous desensitization of chemokine receptors by opioids: (1)  Receptor phosphorylation – Studies in immune cells have shown that chemokine receptors such as CCR5, CXCR1, and CXCR2 can be phosphorylated by opioid agonists such as DAMGO or met-enkephalin resulting in a loss of receptor function (reviewed by Steele et al. 2002; Chen et al. 2004). The involvement of classical PKA, PKC, or GRKs as well as tyrosine kinases like src, lyn has been suggested. Furthermore, we recently reported that in cortical neurons, prolonged MOR stimulation can block ligand-induced CXCR4 phosphorylation on serine 339 residue (Sengupta et al., 2009). Receptor phosphorylation on this residue is critical for internalization. We proposed that this inhibition of receptor phosphorylation can alter receptor internalization and recycling to the membrane thus affecting CXCR4 function. (2)  Receptor oligomerization – Heterodimerization between opioid and chemokine receptors has been described in several studies. Suzuki et  al. (2002a, b, c)suggested that the oligomerization of CCR5 and opioid receptors on lymphocyte membranes can regulate receptor function. Recently, Pello et al. (2008) reported that simultaneous activation of DOR and CXCR4 by their agonists blocked signaling through either receptor because of the formation of nonfunctional heterodimers between DOR and CXCR4. Dimerization or higher order oligomerization is often required for proper functioning of GPCRs including opioid and chemokine receptors. Different combinations of ligands can promote formation of either homo- or heterodimers that lead to enhancement or impediment of downstream signaling.

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(3)  Receptor downregulation – Internalization of chemokine receptors can be a potential mechanism to accomplish heterologous desensitization. As shown in earlier studies by Deng et al. (1999), and more recently by Finley et al. (2008), CXCR4 can get internalized in response to a separate GPCR activation (FPRL1, KOR) leading to a loss of function. Furthermore, a recent study in brain cells suggested that growth factor receptors (BDNF receptor, TrkB) can downregulate CXCR4 levels as well (Bachis et al. 2003).

17.3 Cross-talk Between Opioid and Chemokine Receptors As noted previously, the opioid and chemokine receptors are both part of the GPCR superfamily. In addition, both receptors signal via similar Gi/o (also Gq for CXCR4, Rochdi and Parent 2003) type heterotrimeric G-proteins and are both widely distributed in the brain and periphery. These receptors appear to activate similar signaling pathways. Earlier studies demonstrated morphine’s ability to enhance HIV-1 expression in chronically infected promonocytes (co-cultured with human fetal glia and neurons). However, morphine can inhibit the chemotactic and phagocytic functions of microglia (Peterson et al. 1994; Chao et al. 1997; Sowa et al. 1997). Later studies reported that the endogenous MOR agonist endomorphin-1 also potentiated HIV-1 expression in a mixed neuronal/glial and microglial fetal human brain cell cultures (Peterson et al. 1999). These reports indicated a relationship between increased HIV replication and MOR activation in cultured human brain cells, which provided the basis for subsequent investigation of a cross-talk between the opioid and chemokine receptor systems. While numerous studies focusing on the immunosuppressive role of opioids have demonstrated interactions between opioid and chemokine receptors that lead to compromised immune cell migration, our current knowledge of opioid and chemokine receptor cross-talk in the nervous system is still limited. The first report that opioids (m opioids in particular) can desensitize chemokine receptors on central neurons came from our lab in a study performed by Patel et al in 2006 showing that prolonged stimulation of MOR significantly inhibited CXCR4induced downstream signaling in neurons such as activation of ERK1/2 and Akt. More recent studies demonstrated that this was a direct effect on neuronal CXCR4 (independent of glia) observed both in vitro and in vivo (Sengupta et al., 2009). The desensitization of CXCR4 by opioids might have significant physiological relevance considering the inhibitory effect of the endogenous m-opioid endomorphin-1 on CXCR4 pathways (Patel et al. 2006). Neuronal Akt activation is instrumental for the pro-survival action of chemokines (Meucci et  al. 1998). By inhibiting CXCL12induced Akt or ERK1/2 activation m opioids may compromise the neuroprotective function of this chemokine. Indeed, CXCL12 failed to rescue neurons pre-exposed to DAMGO from NMDA toxicity (Patel et  al. 2006). MOR-induced changes in CXCL12 responses were not due to a reduction in CXCR4 expression or receptor internalization. Therefore, the ability of CXCR4 to interfere with HIV proteins (such as gp120) would not be impaired. Interestingly, CXCL12-induced Ca flux was not

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significantly affected by opioid treatment, suggesting a “selective” regulation of CXCR4 signaling by MOR. This is in agreement with other reports concerning the effect of morphine on chemokine receptor, CCR5. Studies by Hu et  al. (2000) showed that morphine inhibited CCR5 function on human microglial cells as determined by their migration toward RANTES (ligand for CCR5). However, morphine had no effect on RANTES-induced intracellular Ca++ changes suggesting that morphine’s inhibitory effect on chemotaxis occurs independently of Calcium flux. Our data suggest that endogenous levels of m opioids may contribute to the fine-tuning of CXCL12 activity in the brain and control CXCL12-mediated responses during inflammatory states. Inhibition of the trophic function of CXCR4 by opioids can also explain the HIV-associated neurological impairment in drug abusers. An investigation of the molecular mechanism underlying MOR-induced CXCR4 inhibition revealed that the desensitization required prolonged opioid treatment and an upregulation of the intracellular iron-binding protein ferritin heavy chain (FHC). Opioids also caused a reduction in the CXCL12-induced phosphorylation of CXCR4 C-terminus (serine 339). Phosphorylation of CXCR4 on serine 339 occurs as a consequence of ligand activation and is critical for the arrestin-mediated internalization and hence recycling of the receptor (Orsini et  al., 1998; Woerner et  al. 2005). CXCR4 internalization terminates Gai-induced signaling but receptors can still signal through dynamin-Ras-Raf-mediated pathways. Internalized receptor/arrestin complexes have been shown to act as scaffolds for different molecules such as MAPKs and Akt and signal through these molecules (DeFea et  al. 2000; Luttrell et al. 1999: reviewed by DeWire et al 2007). As a consequence, inhibition of CXCR4 phosphorylation by opioids may lead to a deficit in this G-protein independent signaling and compromise receptor function. The upregulation of FHC by opioids is a novel finding. FHC has been recently identified as a negative regulator of CXCR4 signaling in cell lines (Li et  al. 2006) because of its ability to interact with the C-terminus (CT) of CXCR4. In line with this result, we found increased association of FHC with CXCR4 after opioid treatment. Although purely speculative at this point, it is possible that opioids enhance the binding of FHC to the CT of CXCR4 whereby inhibiting its ability to activate the G-protein, or associate with kinases (GRKs or PKC) that phosphorylates the receptor. This would inhibit the binding of the internalization machinery such as arrestin and dynamins to CXCR4 and hence block the overall functioning through the receptor. Interestingly, serum ferritin levels are significantly higher in male opiate addicts than in control groups (Verde Mendez et al. 2003), and increased levels of ferritin were also recently found in the CSF of HIV patients presenting with acute neurological episodes (Phuapradit et  al. 1996; Deisenhammer et al. 1997). Moreover, altered FHC levels have been associated with other neurological disorders such as RLS, Parkinson’s, and Alzheimer’s disease (Sultana et al. 2007; Kondo et al. 1996; Clardy et al. 2006). In summary, these studies demonstrate that heterologous desensitization of chemokine receptors (or GPCRs in general) is much more complicated than we originally contemplated. It might be interesting, in future, to examine whether other families of receptors (RTKs, nuclear receptors) can undergo similar regulation. Moreover, there are several additional aspects that require further investigation. For example, the possibility of opioid-induced

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altered G-protein coupling is an intriguing concept which may theoretically explain some of the characteristics of CXCR4 desensitization. Opioids may cause a switch from CXCR4-Gi to CXCR4-Gq coupling which would block the survival signaling through CXCR4 (activation of ERK/PI3K) but still induce Ca influx through PLCdependant pathways. Also, worth investigating are the effects of prolonged MOR activation on other opioid receptor levels. Previous studies have shown that DOR or KOR may desensitize CXCR4 function through heterodimerization. Prolonged MOR stimulation may downregulate MOR levels but reciprocally upregulate KOR or DOR levels which then might associate with CXCR4 and prevent signaling. Finally, it should be kept in mind that there might be discrepancies in the nature of opioid–chemokine cross-talk in different tissue (for instance CNS vs the immune system) or cell types because of differences in effectors and second messenger systems that couple to these receptors in different tissue. In line with this theory, Szabo et al. reported no changes in CXCR4 function in monocytes exposed to DAMGO. Although opioid treatment in these studies were relatively short compared to ours (1 versus 24 h), these data may support the cell-specific nature of such interactions. Our studies show that prolonged opioid treatment leading to long-term modifications and de  novo protein synthesis is crucial to morphine’s action on neuronal CXCR4 (Sengupta et al., 2009). However, within the brain there seem to be differences in the effects of opioids on chemokine signaling between neuronal and nonneuronal cells as suggested by our studies (Sengupta et al., 2009). Finally, irrespective of cell types, transfected/overexpressed receptors may behave differently than endogenous receptors that are physiologically expressed by cells. The stoichiometry of receptorG-protein–effector complex may vary between endogenous and exogenous systems. It is possible that exogenous receptors can couple to some, but not all of the intracellular pathways that are normally activated by endogenous CXCR4. Finally, there may be differences among the different chemokine receptors in their ability to be desensitized. Recent research has revealed a significant role of the interaction between chemokine and opioid systems in the pathophysiology of pain, associated with both peripheral and central nervous system. In vitro studies have shown that cultured DRG neurons express several chemokine receptors and can release pain-related neurotransmitters such as substance P or CGRP in response to chemokine excitation (Qin et al. 2005; Jung et al., 2008; reviewed by White et al. 2007). In addition, in vivo models of neuropathic pain have demonstrated upregulation of several chemokine receptors as well as the respective chemokines in sensory neurons in close vicinity to the injury (Sun et al. 2006; Jung et al., 2008; Bhangoo et al. 2007; rev. White et al. 2007). On a cellular level, chemokines are known to excite DRG neurons through transactivation of TRP channels and inhibition of K+ conductances (Jung et al., 2008; Ruparel et al., 2007; Bandell et al., 2004; Zhang et al., 2005). Thus, chemokines and their receptors can contribute to chronic pain conditions through the hyperexcitation of nociceptors by directly stimulating these neurons. In addition, chemokine interactions with the endogenous opioid system may indirectly contribute to pain behavior. One possibility, as discussed next, is through the heterologous desensitization of opioid receptor function resulting from elevated levels of chemokines that is produced during inflam-

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matory states. Interaction between the chemokine receptor CCR1 and MOR was demonstrated in dorsal root ganglia (DRG) neurons (Zhang et al. 2004). Long-term exposure to pro-inflammatory chemokines such as CCL2, CCL3, CCL5, and CXCL8 compromised the function of MOR on sensory neurons in DRG potentially thorugh the internalization of cell surface receptors. Thus, in addition to their role in the recruitment/activation of macrophages and microglia to the nerve tissue, chemokines could also contribute to neuropathic pain states through the inhibition of opioidinduced analgesia. Interestingly, repeated exposure to opioids such as morphine can lead to an increasing sensitivity to noxious stimuli (loss of analgesia), a condition known as opioid-induced hyperalgesia (OIH). Chronic morphine treatment was shown to upregulate CXCL12 and CXCR4 levels in sensory neurons while administration of the CXCR4 antagonist AMD3100 completely reversed OIH (Wilson et al., 2008). These data clearly indicate the role of CXCL12 and CXCR4 in morphineinduced increased pain behavior. Recent studies have demonstrated that several spinal pro-inflammatory chemokines (such as CX3CL1, MIP-1a, and MCP-1) as well as cytokines are released after morphine exposure, and are responsible for the opposition of opioid analgesia (Hutchinson et  al 2008, 2009). The authors demonstrated that cytokines, chemokines, and glial activation are important mediators in the development of tolerance, allodynia, and hyperalgesia following chronic opioid treatment. It is possible that elevations of chemokine levels in response to opioid treatment is a part of a physiological negative feedback loop, whereby opioids upregulate the levels of inflammatory chemokines, which then subsequently downregulate opioid signaling through heterologous desensitization of opioid receptors. Such a feedback circuitry may be extremely important to maintain nociceptive homeostasis. Opioid-induced chemokine release and enhanced chemokine signaling is therefore vital to OIH and blocking chemokine receptors may help attenuate such enhanced pain states and help potentiate opioid’s analgesic effects. Paradoxically, analgesic actions of chemokines have also been reported, mediated through the release of opioid peptides by chemokines. Chemokines such as CXCL2/3 have been shown to induce release of opioid peptides in a Ca-dependent manner (reviewed by Rittner et al. 2008a, b). This chemokine-induced opioid released from leukocytes, for example, can bind to opioid receptors on peripheral sensory neurons leading to analgesia. The authors suggested that tonic release of opioids within injured or inflamed tissue activates peripheral opioid receptors and helps attenuate clinical pain. Considering the role of chemokines in trafficking of opioid-containing cells and the subsequent release of opioids to the injured tissue suggests that antichemokine therapy, in this case, may however exacerbate pain. Thus, according to current research, chemokines can potentially act as either proalgesic or analgesic mediators through their interactions with the peripheral opioid pathways. How these diverse pathways partake in chronic pain behavior is still incompletely understood and needs to be investigated further. However, it is clear that targeting specific players involved in the cross-talk between chemokine and opioid signaling networks may provide novel forms of therapeutic interventions into states of chronic pain. As one might expect, similar interactions between chemokine and opioid systems have been observed in the central nervous system as well. In 2002, Szabo et  al.

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reported that the activation of chemokine receptors such as CCR5 and CXCR4 may desensitize brain MOR function and alter pain perception in the CNS (Szabo et al. 2002). The authors hypothesized that prior exposure to chemokines may inhibit MOR signaling in brain, which would subsequently suppress the analgesic properties of m opioids. The periaqueductal gray (PAG) region of the brain is strongly associated with the analgesic properties of opioids. The effect of chemokines such as CXCL12, CCL5 and CCL2 on MOR-mediated analgesia was measured in rat PAG. Animals pre-injected with CXCL12 showed a dose-dependent reduction in DAMGO-induced analgesic responses over a 2-h period of the experiment. This inhibitory effect of the chemokine was attributed to the heterologous desensitization of MOR by CXCR4 following CXCL12 administration. Additional results suggested that the desensitization of opioid receptors can be maintained as long as there is a sustainable source of the chemokine at the site of inflammation. This is one of the first in vivo demonstrations of chemokine–opioid cross-talk in the brain indicating that chemokines can compromise the normal neuronal signaling pathway involved in reducing the sensation of pain. On a molecular level, the authors proposed that chemokine–induced phosphorylation of MOR could account for the desensitization effect. The same group recently reported that CXCL12, CCL5 or even CX3CL1 administered into the rat PAG blocked the aninociceptive effect of MOR, DOR, and KOR agonists suggesting that multiple chemokines in the brain can regulate pain pathways (Chen et al. 2007). Interestingly, the opioid antagonist naloxone was shown to act as an analgesic in a transgenic mouse model of sickle cell anemia (Lunzer et al. 2007). The authors demonstrated that this effect of naloxone was mediated through a novel receptor (not MOR) and involved a downregulation of brain CCR5 expression. This again emphasizes the role of chemokines in the CNS as endogenous regulators of opioid analgesia and tolerance. Evidently, interactions between chemokine and opioid pathways in the brain have serious implications. Neuroinflammation associated with HAD or other CNS injury causes a significant upregulation of brain chemokine levels. Such high levels of inflammatory chemokines may override the analgesic effects of opioids leading to the increased sensitivity to pain. The cross-desensitization of opioid receptors by chemokines seems to shift the balance between analgesia and hyperalgesia. Astrocytes and microglia are the major sources of chemokines in the brain (in addition to neurons which can also synthesize certain chemokines), while their receptors are expressed by multiple CNS cell types including neurons. Upregulation of chemokines during inflammation and subsequent inhibition of neuronal signaling pathways suggest that opioid and chemokines may act as chemical messengers involved in neuron-glia communication. Notably, chemokines have been recently demonstrated to act as neuromodulators directly regulating neuronal transmission in both brain and periphery (Rostene et al., 2007). The regulation of pain pathways by chemokines provide additional evidence that chemokines may participate in the function of the nervous system. Apart from their role in regulating chemokine signaling, opioids can also modulate chemokine or chemokine receptor expression levels in the brain. Studies suggest that morphine can alter the gene expression of both a and b chemokines and their

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receptors such as CCR3, CCR5, and CXCR2 on astroglial cells through the activation of MOR (Mahajan et al. 2002; Mahajan et al., 2005a, b). The authors demonstrated that in vitro treatment of human astrocytes with morphine enhances the expression of chemokine receptors/HIV coreceptors, while it downregulates the expression of the respective a and b chemokines with HIV-1 protective activity. Considering the fact that microglia and astrocytes are the only cells in the brain that are directly infected by HIV-1 these studies suggest that in brain MOR activation may lead to increase HIV-1 coreceptor expression thus promoting viral binding, trafficking of HIV-1-infected cells, and enhanced disease progression. Furthermore, astrocytes are the major cellular sources of chemokines in brain and they regulate transendothelial migration of monocytes to the CNS. Hence, alterations of chemokine or its receptor levels by morphine may modulate chemotaxis and contribute to HIV neuropathology. In addition, morphine was also shown to upregulate mRNA and protein levels of neuronal CCL2, a chemokine that plays a distinct role in the recruitment of inflammatory cells in the brain (Rock et al. 2006). This effect of morphine was neuron-specific since there was no effect on CCL2 production in astrocyte or microglial cell cultures. However, it is difficult to interpret these effects since upregulation of chemokines by opioids may be either neuroprotective, considering the neurotrophic nature of chemokines (Meucci et al. 1998; Eugenin et al. 2003; Khan et al. 2004), or deleterious through the recruitment of virus-infected leukocytes into the brain (Eugenin et  al. 2006). Notably, recent studies have demonstrated increased astrocyte activation by opioid and HIV-1 tat which is mediated by glial CCL2 production (El-hage et al 2006). Increased chemokine production by brain cells in response to opioids (alone or in concert with viral proteins) significantly increase glial activation in the brain potentially contributing to HIV neuropathogenesis. As previously reported, recent data from our laboratory showed heterologous desensitization of neuronal CXCR4 by morphine. However, we saw no changes in CXCR4 function in glia following opioid treatment (Sengupta et  al., 2009). It is possible that there are differences in the actions of opioids on chemokine pathways in neuronal vs nonneuronal cell types. While opioids can specifically interfere with chemokine signaling and desensitize chemokine-induced pathways in neurons, they may preferentially alter the chemokine and/or receptor levels in glia. Since glia are the major sources of chemokines in the brain whereas neurons (expressing the receptors) can respond to a multitude of chemokines through the activation of intracellular cascades, this may be one of the most efficient ways by which opioids interact with chemokine systems in the brain.

17.4 Opioid–Chemokine Interaction in HIV Neuropathology Opiate drug exposure has a significant impact on HIV infection as well as progression to HIV-associated dementia. On a cellular level it is comprehendible that drugs of abuse such as opioids would reduce the threshold for neurotoxicity such that a marginally toxic insult would now be exacerbated and lead to cell death or injury

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(Gurwell et  al. 2001; Hu et  al. 2005). Studies have shown that opiate exposure potentiate key apoptotic signals that are induced by gp120(or tat)–CXCR4 interaction (reviewed by Hauser et al. 2006). When human fetal neuronal/glial cells were exposed to gp120IIIB in the presence of morphine, there was a significant increase in the gp120-induced neuronal apoptosis (Hu et al 2005). Gp120IIIB binds to cellular CXCR4 while activating different pro-apoptotic pathways such as the p38 MAPKs, c-Terminal Jun kinase (JNK), p53 as well as the transcriptional factor E2F1 (Kaul et al., 2007; Singh et al. 2005; Khan et al., 2005; Shimizu et al. 2007). Combined exposure of neuron/glial cultures to morphine and gp120 significantly enhanced neuronal apoptotic cell death compared to gp120 alone, through the activation of the p38 MAPKs (Hu et  al. 2005). Microglial cells (which contributed approximately 3–7% of the neuronal/glial cultures used in this study) were shown to be involved in mediating the synergistic effects of opioids and the viral protein. In addition, opiates can enhance the cytotoxicity of HIV-1 viral protein gp120 via mechanisms that involve intracellular calcium modulation resulting in direct actions on astroglia, making them important cellular targets for HIV–opiate interactions (Mahajan et  al., 2005a, b). These results along with others show similar synergistic neurotoxicity between opioids and viral proteins and provide a molecular basis whereby opioid abuse can promote HIV neuropathology. Studies from different laboratories have reported similar observations. For example, rat cortical neurons treated with DAMGO followed by gp120 treatment showed robust potentiating of neuronal apoptosis, as measured by caspase-3 staining and nuclear morphology, when compared to neurons treated with gp120 alone (Patel and Meucci, unpublished). The effects of DAMGO on gp120-induced neurotoxicity do not appear to be related to alterations in gp120-mediated Ca++ mobilization; since both untreated and DAMGO- pretreated neurons mobilized Ca++ in response to the viral protein in a similar percentage of neurons. Interestingly, gp120-induced MAPK activation was unaffected by DAMGO pretreatment (Sengupta and Meucci, unpublished). There is evidence in literature suggesting the involvement of PTXinsensitive G-proteins (Gq family) in the action of gp120 (Del Corno et al. 2001). One speculation is that opioids block neuroprotective signaling through CXCR4-Gi whereas potentiate neurotoxic signaling through Gq. However, further research is required to understand the possibility of such interactions in the brain. Also, in most cases opioids by themselves did not cause significant cell death but remarkably exaggerated the viral protein-induced neuronal apoptosis. Several converging pathways may contribute to these synergistic effects of opioids and gp120 on neuronal degeneration. As discussed before, m opioid agonists can inhibit the prosurvival pathways such as activation of Akt and ERK1/2 induced by CXCL12. PI3Kinduced Akt phosphorylation is a major survival cue for neurons, working through the inactivation of key apoptotic substrates such as BAD, caspase-9 and GSK3-b. Interestingly, gp120 can activate GSK3-b and subsequently inhibit the activation of protective factors such as NFAT, HSF-1, etc. (Maggirwar et al. 1999; Everall et al. 2002; Dou et al 2003; also reviewed by Hauser et al. 2006). Hence, there are essentially two events that occur simultaneously as a consequence of opioid exposure: (1) inhibition of the prosurvival effects of CXCL12; and (2) viral protein–induced

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upregulation of apoptotic mediators, ultimately leading to significant neuronal damage. Several other elements can potentially contribute to the gp120-induced neurotoxicity. Some of these factors include CXCR4-induced activation of the JNK, as well as the release of excitatory glutamate from astrocytes and microglia. Studies have shown that inhibition of p38 or JNK activation can prevent gp120/ tat-induced caspase-3 activity (Singh et  al. 2005). The GPCR-binding protein b-arrestin has been shown to modulate JNK or p38 function in the context of MOR or CXCR4 activation (McDonald et al. 2000; Sun et al. 2002; Bohn et al. 1999). It is possible that MOR activation in association with gp120 or other viral proteins augments p38 or JNK-mediated apoptotic pathways through b-arrestin signaling. Studies in immune cells have shown that opioids can intrinsically modify the host response to HIV through the downregulation of chemokine production whereas upregulation of co-receptor expression. Such interactions can also occur in the brain and contribute to the enhanced disease progression to HIV dementia. Another important brain function affected by opioid-induced inhibition of CXCR4 with possible implications in HIV dementia is adult hippocampal neurogenesis (reviewed by Venkatesan et al. 2007). Evidence in literature suggests that adult neurogenesis can play a significant role in learning and memory and in the maintenance of cognitive function. CXCL12, expressed by mature neurons and glia while CXCR4 present on neuronal precursor cells may influence NPC migration in the adult hippocampus. Drugs of abuse such as cocaine and opiates can significantly downregulate NPC proliferation and survival (Yamaguchi et  al. 2004; Yamaguchi 2005; Eisch et  al 2000; Kahn et  al. 2005). It has been shown that morphine can induce premature mitosis in NPCs through alterations in cell cycle proteins potentially leading to decreased proliferation and survival (Mandyam et al. 2004). Such inhibition of cellular functions by opioids can be due to inhibition of CXCR4-induced pathways, a phenomenon that is worsened during HIV infection. It was shown that hCD4/gp120 complexes can block CXCL12-mediated chemoattraction and proliferation in neural progenitor cells. Thus, opioid-induced alteration of NPC migration in adult hippocampus play a vital role in HIV associated dementia. Impairment of CXCL12/ CXCR4 functioning may be the underlying molecular mechanism in this phenomenon. Of note, we recently showed that CXCL12 promotes (via CXCR4) Rb function in embryonic neuronal cultures thus supporting their survival (Khan et al. 2008).

17.5 Concluding Remarks There is a definite trend of bidirectional cross-talk between opioid and chemokine receptors in the central nervous system. In vitro, as well as in  vivo studies, have shown desensitization of CXCR4 by MOR and thus prevent the neuroprotective action of this chemokine. Although the precise molecular mechanism underlying this cross-talk is still under investigation, based on the evidences in literature several possible pathways can be expected to act independently or in concert and lead to the deficit of CXCR4 function. Our studies have shown that m opioids can increase the brain levels of FHC which can subsequently block CXCR4 signaling. Further studies

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on the mechanisms and implications of FHC regulation by opioids and its consequence on neuronal function during development and adult life may offer valid and novel therapeutic approaches for multiple neurodegenerative disorders. Acknowledgments  The authors thank the National Institute of Health and National Institute of Drug Abuse for their continuous support (grants DA15014 & DA19808).

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opioid receptor heterodimers reveals a mechanism for immune response regulation. Eur J Immunol 38:537–549 Peng H, Erdmann N, Whitney N, Dou H, Gorantla S, Gendelman HE, Ghorpade A, Zheng J (2006) HIV-1-infected and/or immune activated macrophages regulate astrocyte SDF-1 production through IL-1beta. Glia 54:619–629 Peterson PK, Gekker G, Hu S, Anderson WR, Kravitz F, Portoghese PS, Balfour HH Jr, Chao CC (1994) Morphine amplifies HIV-1 expression in chronically infected promonocytes cocultured with human brain cells. J Neuroimmunol 50:167–175 Peterson PK, Molitor TW, Chao CC (1998) The opioid-cytokine connection. J Neuroimmunol 83:63–69 Peterson PK, Gekker G, Hu S, Lokensgard J, Portoghese PS, Chao CC (1999) Endomorphin-1 potentiates HIV-1 expression in human brain cell cultures: implication of an atypical mu-opioid receptor. Neuropharmacology 38:273–278 Phuapradit W, Taeepanichskul S, Jetsawangsri T, Chaturachinda K, Khupulsup K, Kunakorn M (1996) Serum ferritin levels in normal and HIV-1 infected pregnant women. Aust N Z J Obstet Gynaecol 36:24–26 Piros ET, Prather PL, Loh HH, Law PY, Evans CJ, Hales TG (1995) Ca2+ channel and adenylyl cyclase modulation by cloned mu-opioid receptors in GH3 cells. Mol Pharmacol 47: 1041–1049 Qin X, Wan Y, Wang X (2005) CCL2 and CXCL1 trigger calcitonin gene-related peptide release by exciting primary nociceptive neurons. J Neurosci Res 82:51–62 Ragozzino D, Renzi M, Giovannelli A, Eusebi F (2002) Stimulation of chemokine CXC receptor 4 induces synaptic depression of evoked parallel fibers inputs onto Purkinje neurons in mouse cerebellum. J Neuroimmunol 127:30–36 Reiss K, Mentlein R, Sievers J, Hartmann D (2002) Stromal cell-derived factor 1 is secreted by meningeal cells and acts as chemotactic factor on neuronal stem cells of the cerebellar external granular layer. Neuroscience 115:295–305 Rempel SA, Dudas S, Ge S, Gutierrez JA (2000) Identification and localization of the cytokine SDF1 and its receptor, CXC chemokine receptor 4, to regions of necrosis and angiogenesis in human glioblastoma. Clin Cancer Res 6:102–111 Rittner HL, Brack A, Stein C (2008a) Pain and the immune system. Br J Anaesth 101:40–44 Rittner HL, Brack A, Stein C (2008b) The other side of the medal: how chemokines promote analgesia. Neurosci Lett 437:203–208 Rochdi MD, Parent JL (2003) Galphaq-coupled receptor internalization specifically induced by Galphaq signaling. Regulation by EBP50. J Biol Chem 278:17827–17837 Rock RB, Hu S, Sheng WS, Peterson PK (2006) Morphine stimulates CCL2 production by human neurons. J Neuroinflammation 3:32 Rostène W, Kitabgi P, Parsadaniantz SM (2007) Chemokines: a new class of neuromodulator? Nat Rev Neurosci (11):895–903 Ruparel NB, Patwardhan AM, Akopian AN, Hargreaves KM (2007) Pain, 10.1016/j.pain.2007. 08.014 Sammarco MC, Ditch S, Banerjee A, Grabczyk E (2008) Ferritin L and H subunits are differentially regulated on a post-transcriptional level. J Biol Chem 283:4578–4587 Sengupta R, Burbassi S, Shimizu S, Cappello S, Vallee RB, Rubin JB, Meucci O (2009) Morphine increases brain levels of ferritin heavy chain leading to inhibition of CXCR4-mediated survival signaling in neurons. J Neurosci 29(8):2534–2544 Sharp BM (2006) Multiple opioid receptors on immune cells modulate intracellular signaling. Brain Behav Immun 20:9–14 Shimizu S, Khan MZ, Hippensteel RL, Parkar A, Raghupathi R, Meucci O (2007) Role of the transcription factor E2F1 in CXCR4-mediated neurotoxicity and HIV neuropathology. Neurobiol Dis 25:17–26 Shyu WC, Lin SZ, Yen PS, Su CY, Chen DC, Wang HJ, Li H (2008) Stromal cell-derived factor-1 alpha promotes neuroprotection, angiogenesis, and mobilization/homing of bone marrowderived cells in stroke rats. J Pharmacol Exp Ther 324:834–849

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Singh IN, El-Hage N, Campbell ME, Lutz SE, Knapp PE, Nath A, Hauser KF (2005) Differential involvement of p38 and JNK MAP kinases in HIV-1 Tat and gp120-induced apoptosis and neurite degeneration in striatal neurons. Neuroscience 135:781–790 Sowa G, Gekker G, Lipovsky MM, Hu S, Chao CC, Molitor TW, Peterson PK (1997) Inhibition of swine microglial cell phagocytosis of Cryptococcus neoformans by femtomolar concentrations of morphine. Biochem Pharmacol 53:823–828 Steele AD, Szabo I, Bednar F, Rogers TJ (2002) Interactions between opioid and chemokine receptors: heterologous desensitization. Cytokine Growth Factor Rev 13:209–222 Stumm RK, Zhou C, Ara T, Lazarini F, Dubois-Dalcq M, Nagasawa T, Hollt V, Schulz S (2003) CXCR4 regulates interneuron migration in the developing neocortex. J Neurosci 23:5123–5130 Sultana R, Boyd-Kimball D, Cai J, Pierce WM, Klein JB, Merchant M, Butterfield DA (2007) Proteomics analysis of the Alzheimer’s disease hippocampal proteome. J Alzheimers Dis 11:153–164 Sun Y, Cheng Z, Ma L, Pei G (2002) Beta-arrestin2 is critically involved in CXCR4-mediated chemotaxis, and this is mediated by its enhancement of p38 MAPK activation. J Biol Chem 277:49212–49219 Sun JH, Yang B, Donnelly DF, Ma C, LaMotte RH (2006) MCP-1 enhances excitability of nociceptive neurons in chronically compressed dorsal root ganglia. J Neurophysiol 96:2189–2199 Suzuki S, Chuang AJ, Chuang LF, Doi RH, Chuang RY (2002a) Morphine promotes simian acquired immunodeficiency syndrome virus replication in monkey peripheral mononuclear cells: induction of CC chemokine receptor 5 expression for virus entry. J Infect Dis 185:1826–1829 Suzuki S, Chuang LF, Yau P, Doi RH, Chuang RY (2002b) Interactions of opioid and chemokine receptors: oligomerization of mu, kappa, and delta with CCR5 on immune cells. Exp Cell Res 280:192–200 Suzuki S, Carlos MP, Chuang LF, Torres JV, Doi RH, Chuang RY (2002c) Methadone induces CCR5 and promotes AIDS virus infection. FEBS Lett 519:173–177 Szabo I, Chen XH, Xin L, Adler MW, Howard OM, Oppenheim JJ, Rogers TJ (2002) Heterologous desensitization of opioid receptors by chemokines inhibits chemotaxis and enhances the perception of pain. Proc Natl Acad Sci USA 99:10276–10281 Tissir F, Wang CE, Goffinet AM (2004) Expression of the chemokine receptor Cxcr4 mRNA during mouse brain development. Brain Res Dev Brain Res 149:63–71 Venkatesan A, Nath A, Ming GL, Song H (2007) Adult hippocampal neurogenesis: regulation by HIV and drugs of abuse. Cell Mol Life Sci 64:2120–2132 Verde Mendez CM, Diaz-Flores JF, Sanudo RI, Rodriguez Rodriguez EM, Diaz Romero C (2003) Haematologic parameters in opiate addicts. Nutr Hosp 18:358–365 Vergote D, Butler GS, Ooms M, Cox JH, Silva C, Hollenberg MD, Jhamandas JH, Overall CM, Power C (2006) Proteolytic processing of SDF-1alpha reveals a change in receptor specificity mediating HIV-associated neurodegeneration. Proc Natl Acad Sci USA 103:19182–19187 White FA, Jung H, Miller RJ (2007) Chemokines and the pathophysiology of neuropathic pain. Proc Natl Acad Sci USA 104:20151–20158 Wilson NM, Jung H, Ripsch MS, Miller RJ, White FA (2008) Opioid-induced tactile hypernociception is attenuated by the CXCR4 antagonist, AMD3100. Soc Neurosci Abstr 34:270–214 Woerner BM, Warrington NM, Kung AL, Perry A, Rubin JB (2005) Widespread CXCR4 activation in astrocytomas revealed by phospho-CXCR4-specific antibodies. Cancer Res 65:11392–11399 Yamaguchi M (2005) Analysis of neurogenesis using transgenic mice expressing GFP with nestin gene regulatory regions. Chem Senses 30(Suppl 1):i117–i118 Yamaguchi M, Suzuki T, Seki T, Namba T, Juan R, Arai H, Hori T, Asada T (2004) Repetitive cocaine administration decreases neurogenesis in adult rat hippocampus. Ann N Y Acad Sci 1025:351–362 Yi Y, Lee C, Liu QH, Freedman BD, Collman RG (2004) Chemokine receptor utilization and macrophage signaling by human immunodeficiency virus type 1 gp120: implications for neuropathogenesis. J Neurovirol 10(Suppl 1):91–96 Zhang N, Rogers TJ, Caterina M, Oppenheim JJ (2004) Proinflammatory chemokines, such as C-C chemokine ligand 3, desensitize mu-opioid receptors on dorsal root ganglia neurons. J Immunol 173:594–599

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Zhang N, Inan S, Cowan A, Sun R, Wang JM, Rogers TJ, Caterina M, Oppenheim JJ (2005) Proc Natl Acad Sci USA 102:4536–4541 Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR (1998) Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393:595–599

About the Editor

Olimpia Meucci, MD, PhD is a Professor of Pharmacology and Physiology & Microbiology and Immunology at Drexel University College of Medicine in Philadelphia, PA. Since her seminal discovery about the regulation of neuronal signaling by chemokines, her research has primarily focused on the physio-pathological roles of this important class of neuroimmune modulators in the central nervous system and their involvement in neuroAIDS. These studies have significantly contributed to current understanding of the cellular and molecular mechanisms of HIV-related neuropathology including the interaction of the chemokine system with drug of abuse, namely opiates, which continues to be a major area of investigation in the Meucci lab.

399

About the Book

Chemokines and their receptors are being recognized as an integral component of the nervous system implicated in fundamental aspects of development and homeostasis, such as neurotransmission, proliferation, differentiation, and neuronal–glial communication. Thus, their involvement in HIV neuropathology goes far beyond the co-receptors role and entails complex interactions of the chemokine system with different cell types and other regulators of neuronal function. The major goal of this volume is to review these topics in order to highlight alterations of chemokine physiology that may contribute to neuroAIDS and other neuropathologies. This book should be of interest to neuroscientists in general, neurologists, virologist, immunologists, pharmacologists, as well as students in these fields.

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Index

A Acquired immunodeficiency syndrome (AIDS), 286 Acute inflammatory demyelinating polyneuropathy (AIDP), 58 AIDS neuropathogenesis CNS signaling pathways caspase 3 activation, 234 HIV envelope proteins, 233 NMDA receptors, 235 neuronal survival calpain activity, 237 CXCR4-mediated neurotoxicity, 236 E2F1 transcriptional activity, 235 neural progenitor cells, 237 p53 transcriptional activity, 236 retinoblastoma protein (Rb), 235 Allodynia, 183, 184 Alpha chemokines, 14–15 Alzheimer’s disease, 311 amyloid-β (Aβ), 289 DENN/MADD, 290 proinflammatory cytokines, 289 TNFα signalling, 290 Amyloid precursor protein (APP), 289 Angiotensin receptors, 320 Anti-apoptotic effect, 42 Antigen presenting cells (APC), 97 Antinociceptive effect, 183, 184 Antiretroviral toxic neuropathy (ATN) clinical presentation, 57 pathology AZT, 72 DNA polymerase γ hypothesis, 71–72 L-acetyl carnitine, 72 treatment toxicity, 57 Aorta-gonad-mesonephros (AGM), 198 Aplaviroc therapy, 43 Astroglia. See also Opioids



gliotransmission and ion homeostasis, 360–361 HIV-1 chemokine production, 362 gp120 interactions, 361–362 proinflammatory response, 361 Autocrine/paracrine modulatory action, 183 Autonomic neuropathy, 62 B Beta chemokines, 15 Blood-brain barrier (BBB), 96, 121 Bradykinin, 339 Brain-derived neurotrophic factor (BDNF), 75 Brain tumors CCL3L1 CCL2 glioma cell secretion, 265 microglia, 264 CXCL12 and CXCR4 astrocytomas, 254 cancer biology, 255 GBM cells, 254 medulloblastomas, 253 paracrine relationship, 256 therapeutic targets, 263–264 C Cajal-Retzius cells (CR), 380 Calbindin, 10 Calcium microdomains, sub-membrane, 282 Cannabinoid agonists, 183 CCL2 β-chemokines, 225 CNS, 239 CCR receptor, 174–175 CD26. See Dipeptidyl peptidase (DPP)

403

404 Cell cycle protein modulation AIDS neuropathogenesis CNS signaling pathways, 233–235 CXCL12 and gp120 signaling, 237–238 HIV envelope protein gp120, 232–233 neuronal survival, 235–237 CCR2 ligand receptor binding, 225 neuroAids, 239 signaling regulation, 228 CCR5 ligand receptor binding, 224–225 signaling regulation, 227–228 chemokine and receptor structure, 221–222 CXCR3 ligand receptor binding, 226 neuroAIDS, 238–239 signaling regulation, 228–229 CXCR4 CNS development, 229–231 ligand receptor binding, 224 mature CNS, 231 signaling regulation, 226–227 oligomerization, 222–223 Cellular toxins, 355–356 Cellular trafficking and immune response, 320 Central nervous system (CNS) autoimmunity. See also Leukocyte trafficking CCR5 and ligands CCR5∆32 allele, 131 demyelination, 129 immunosuppressive drug treatment, 130, 131 lymphocytes trafficking, 128 PBMC, 130 T cell infiltration, 129 CXCL12 and CXCR4 AMD3100, 126 CXCL12β, BBB expression, 127 cytokine interleukin (IL)-1β, 127 oligodendrocytes, 128 phospho-specific antibody, 127–128 Cerebrospinal fluid (CSF), 237, 311 Chemokine proteolysis aminoterminal process, 153 antagonists receptors, 150 anti-HIV properties, 156 enzymatic mechanisms, 149 evolutionary advantages, 164–165 glycosaminoglycans (GAG), 153 HIV-induced proteases

Index cathepsins, 156 CD26/DPP IV, 155 MMP, 153–154 MMP processed CXCL12 CXCL12 cleavage, 157–159 effects, in vivo model, 163–164 immunogenic properties, 162–163 neuronal membrane physiology, 161 neurotoxicity, 161–162 receptor affinity, 159–160 properties, 153 proteolytic cleavage, 150 secretases, 150 Chemokines and chemokine receptors, 320 agonists, 184–185 expression CC chemokines, 178 CX3CL1, 179 CXC chemokines, 178–179 HIV-1-associated CNS disease astrocyte function and chemokine expression, 41–42 direct mechanisms, neuronal injury and apoptosis, 38–40 monocyte/macrophage recruitment, 40–41 neuropathology, 37 HIV-1 infection and disease cellular trafficking and survival pathways, 37 HAART therapy, 36 progression, 35–36 resistance mechanism, 37 transmission, 34–35 HIV infection CD4, 33 second receptors, 33–34 neuromodulatory effect neurotransmission, 181–182 nociception, 182–184 receptor expression CCR family, 174–175 CX3CR1, 177 CXCR family, 176–177 regulatory role, 179, 180 signaling (see Signaling mechanism) survival pathways, monocyte/macrophage reservoirs, 42–43 therapeutics, trials and tribulations, 43 Cholinergic neurons, 174 Chronic inflammatory demyelinating polyneuropathy (CIDP), 58 Clade C infection, 42 Compound motor action potential (CMAP), 59

Index COUP-TF interacting protein 2, 102–103 Cryoglobulinemia, 59 C-terminus (CT), 384 CX3CL1 brain ischemia excitotocixity, 308 glutamate-mediated responses, 308 ischemic stroke, 309 microglial cells, neuroprotective effect, 309 CX3CR1 pair, structure and signalling, 301 HIV infection, 308 intercellular communication, 311–312 modulatory effects, 310 nervous system, 302–303 neuroinflammation and neurodegeneration models, 310–311 neuroprotection, 307–308 pain modulation CX3CR1, 303–304 inflammatory/traumatic nerve injury, 306–307 nociception, 307 synaptic activity, 305 adenosine receptor type 1 (A1), 304, 306 AMPA receptors, 304 long-term depression (LTD), 304 NMDA, 303-304 pre-and post-synaptic effects, 303–305 CX3CR1 receptor, 177 CXCL12 CNS development, 229 expression, 255 vs.gp120 signaling, 237–238 microglia, 273 migratory/invasive factor, 261–262 Sonic hedgehog (SHH), 230 T lymphocytes, 272 treatment, 259 vascular endothelium, 265 CXCR4 receptor angiogenesis endothelial cells, 262 marrow stromal cells, 263 paracrine/autocrine signaling, 262 bone marrow stem cells, 258 brain tumor biology, 256 CD133-tumor cells, 257 cell proliferation and survival, CXCL12 Erk 1/2 phosphorylation, 259 Gi-coupled receptor, 259 glioblastoma cell lines, 258 intracranial xenograft, 260

405 neurofibromin, 261 OPG formation, 260 paracrine activation, 259 proliferative effects, 260 tumor cell proliferation, 259 chemo-localization, 257 CNS development Cajal-Retzius cells, 230 CXCL12 signaling, 231 GABAergic interneurons, 230 desensitization, 261 expression, 258 hematopoietic system, 257 ligand activation, 255 mitotic quiescence, 258 neural stem cells, 257 phosphorylation, 255 signaling regulation C-terminus tail, 226 heterologous desensitization, 227 tumor-initiating cells, 256 CXCR7 receptor, 4 CXCR receptor, 176–177. See also specific receptor Cyclin-dependent kinase 5 (cdk5), 236 Cyclin dependent kinase 9 (CDK9), 104 Cyclophosphamide therapy, 130 D Dicer activity, 105 2’,3’-dideoxycytidine (ddC), 183 Diffuse infiltrative lymphocytosis syndrome (DILS), 61–62 Dipeptidyl peptidase (DPP), 155 Distal symmetric polyneuropathy (DSP), 56–57, 192 DNA polymerase γ hypothesis, 71–72 Dopaminergic neurons, 174 Dorsal root ganglia (DRG) neurons, 174, 386 Dysimmune inflammatory polyneuropathy, 52 E Early growth response gene (Egr1), 230 Endogenous opioid system, 353 Εnkephalins, 320 Excitatory amino acid transporter-2 (EAAT2), 362 Experimental autoimmune encephalopathy (EAE), 122, 126–128, 310

406 F Feline immunodeficiency virus (FIV), 75, 154 Ferritin heavy chain (FHC), 227, 384 Fluorescence activated cell sorting (FACS), 237 Fractalkine receptor, 177 G Genetic polymorphism, 35 Glatiramer therapy, 130 Glial fibrillary acidic protein (GFAP), 260 Gliotransmitters release amino acid glutamate, 278 gliotransmission, 361 glutamate exocytosis, astrocytes, 280 GPCR stimulation, 281 intracellular calcium, 277 recycling processes, 279 synaptic vesicles, 278 TIRF microscopy, 279 Glycosaminoglycans (GAG), 153 Gonadotropin-releasing hormone (GnRH), 230 gp120 protein chemokine interactions, 12–13 N-methyl-D-aspartate receptor (NMDAR) interactions, 13 G-protein coupled receptor (GPCR), 222, 271, 379 cell process and biochemical events, 319 ligands, heterologous desensitization, 327 NF-κB signaling activation and repression, 323 synthesis, 319 G-protein receptor kinase (GRK), 382 Grimm, M.C., 329 Guillain−Barre syndrome (GBS), 58 H Hematopoietic stem cells (HSCs), 197 Highly active antiretroviral therapy (HAART), 6, 19–20, 36, 87, 191, 286, 355 Histone acetyl transferases (HATs), 102 Histone methyl transferases (HMTs), 102 HIV-1-associated CNS disease astrocyte function and chemokine expression, 41–42 direct mechanisms, neuronal injury and apoptosis CXCL12 stimulation, 39 macrophage (M) and T-cell (T)-tropic virus, 38–40

Index macrophages/microglia infection, 38 monocyte/macrophage recruitment, 40–41 neuropathology, 37 HIV-1-associated neurocognitive disorders (HAND), 96 HIV-associated dementia (HAD), 150, 355 cognitive impairment, 5–6 HAART, 6 HIV-1-associated dementia, 96 lypopolissaccaride, 287 neuronal apoptosis, 286 neuronal/glial cerebrocortical cultures, 287 TNFα-dependent synergism, 288 HIV-associated peripheral neuropathy antiretroviral toxic neuropathy (ATN) clinical presentation, 57 pathology, 71–72 treatment toxicity, 57 autonomic neuropathy, 62 chemokine receptors HIV strains, 67–68 neuropathic pain, 73–74 diffuse infiltrative lymphocytosis syndrome (DILS), 61–62 distal symmetric polyneuropathy (DSP), 56–57 HIV seroconversion, 58 inflammatory demyelinating polyradiculoneuropathy, 11–12 model cell culture model, 74 feline immunodeficiency virus (FIV), 75 mononeuropathy multiplex (MM) clinical presentation, 60 occurrence, 59 motor neuron disease, 63 neuropathogenesis, 77 neurotoxicity dorsal root ganglion (DRG) neurotoxicity, 68–69 immunopathogenic factors, 70–71 local axonal toxicity, 69–70 pathology DRG, 66 DSP, 63–64 epidermal nerve fiber, 67 patient management principles, 51–52 prevalence, 55–56 progressive polyradiculopathy (PP) characteristics, 60–61 incidence, 60 inflammation and necrosis, 61 recognition, 52–55

Index risk factors, 55 treatment HAART, 76 L-acetyl carnitine, 76–77 narcotic analgesics, 76 HIV co-receptors antagonism, 135 chemokines and chemokine receptor, 119 CNS autoimmunity CCR5 and ligands, 128–131 CXCL12 and CXCR4, 126–128 multiple sclerosis (MS), 130 pro-inflammatory chemokines, 126 leukocyte trafficking, CNS blood-brain barrier (BBB), 121 CCR5, 122, 123 chemokine ligands, 121 CXCR4, 122, 123 homeostatic chemokine, 122 tissue inflammation, 120–123 neuroAIDS HIV-1 infection and replication, 123, 124 human consideration, 124–125 macaque model, 125 T cell-tropic viruses, 124 West Nile virus encephalitis CCR5, 133–135 CXCL12, 132–133 CXCR4 antagonists, 132–133 manifestation, 132 HIV latency and reactivation bone marrow cell populations, 98–99 cellular transcription factors, 103 chromatin determinants COUP-TF interacting protein 2 (CTIP2), 102–103 histone deacetylase-1 (HDAC1), p50, 102 histone-methylation, 102 nucleosome, 101 viral integration machinery, 100–101 CNS cells astrocytes, 97 microglial cells, 96–97 perivascular macrophages, 96–97 dendritic cells (DCs), 97–98 feature, 87–88 longevity, 88 maintenance, 99–100 monocyte-macrophage latency env gene, 93 monocyte-derived macrophages (MDMs), 95–96

407 residual viremia, 93 virological synapse, 96 neuropathogenesis and reseeding bone marrow, 106 CD14low/CD16+ monocytes, 106–107 peripheral blood, 106 resistant viral variants, 105–106 post-integration latency and resting CD4+ T cell, 90–94 potential minor reservoirs, 99 pre-integration latency APOBEC3G protein, 89 proviral DNA genome, 88, 89 uracil-DNA-glycosidases, 90 viral infectivity factor, 89 RNA interference, 105 supervised treatment interruption (STI), 107 viral proteins HAT, 104 SWI/SNF, 104–105 TAR, 103–104 viral Rev protein, 105 hNT neurons, 174 Homeostatic chemokine, 122 Human herpesvirus-6 (HHV-6), 98 Human immunodeficiency virus (HIV), 286 biology and invasion chemokine expression, endothelial cells, 8 gp120-chemokine receptor binding, 8–9 HIV tropism, 8–9 monocyte, 7–8 neuronal injury, 9 virus replication, 7 chemokines and glutamate-mediated excitotoxicity, 18–19 clinical manifestations and epidemiology HAART, 6 neurocognitive impairment, 5–6 NRT, 6 human immunodeficiency virus type 1 (HIV-1), 87 infection CD4, 33 chemokine receptors, 33–34 HAART therapy, 36 progression, 35–36 resistance mechanism, 37 transmission, 34–35 neurodegeneration chemokines and chemokine receptor, 14–15

408 Human immunodeficiency virus (HIV) (cont.) excitotoxins and N-methyl-D-aspartate receptors, 16–18 neurotoxicity, 12–14 neuropathogenesis immunohistochemistry, 10–11 MRS, 10–11 NAA/Cr ratio, 11 neuronal damage, 9 SIV/Delta B670 swarm, 9–10 neuropathology, 3–4 (see also HIVassociated peripheral neuropathy) therapeutic considerations, 19–20 Human immunodeficiency virus encephalitis (HIVE), 178 astroglia (See Astroglia) microglia (See Microglia) neuroAIDS, 355–356 opioid-chemokine interactions chemokine ligands and receptors, 354 pain/nociception, 354–355 opioid-immune interactions astroglia and microglia response, 357–358 chemokine release, 356 endogenous opioid system, 353–354 SIV/SHIV strains, 357 Hypoxia inducible factor--1α, (HIF-1α), 258, 262 Hypoxia response elements (HRE), 262 I Inflammatory demyelinating polyradiculoneuropathy (IDP), 58 Inflammatory transcriptosome, 362–364 Integrins, 120 Intercellular adhesion molecule (ICAM)-1, 121 Ion hemeostasis, 361–362 K Kaul, M., 12 Kennedy, J.M., 75 Keswani, S.C., 71, 74 Khan, M.Z., 258 Kindberg, E., 135 L Leucine-rich repeats containing 4 (LRRC4), 259 Leukocyte chemotaxis, 320

Index Leukocyte trafficking. See also Central nervous system autoimmunity blood-brain barrier (BBB), 121 CCR5, 122, 123 chemokine ligands, 121 CXCR4, 122, 123 homeostatic chemokine, 122 tissue inflammation, 120–123 Lipopolysaccharide (LPS), 310–311 Localized calcium microdomains calcium puffs, 283 endoplasmic reticulum tubules, 282 SLMV exocytosis, 284 Long term depression (LTD), 304 Long terminal repeats (LTRs), 99 Lymphocytic pleocytosis, 59, 61 M Maraviroc therapy, 43 Matrix metalloproteinases (MMPs), 221, 261 CXCL12 process CXCL12 cleavage, 157–159 effects, in vivo model, 163–164 immunogenic properties, 162–163 neuronal membrane physiology, 161 neurotoxicity, 161–162 receptor affinity, 159–160 HIV coreceptors, 154 Medulloblastoma, 254 Melanin concentrating hormone (MCH), 273, 380 Methylprednisolone treatment, 130 Microglia chemokine signals, 360 immune effector, 358 opoids CCL2/MCP-1 elimination, 359 chemotactic effects, 359 HIV replication, 358–359 Tat-induced ROS production, 360 pathogen-associated molecular pattern, 358 Middle cerebral artery occlusion (MCAO), 178, 310 Molecular switch, 319 Monocyte chemoattractant protein 1 (MCP-1), 223, 264 See also CCL2 Monocyte-derived macrophages (MDM), 239 Mononeuropathy multiplex (MM) clinical presentation, 60 occurrence, 59

Index Mononuclear phagocytes (MPs), 38 µ-opioid receptor (MOR), 340–341 agonist activation, 321 heroin, 346–347 NF-κB activation, 323 primary receptor, 319 Morphine, 320 CCL2 production stimulation, 321 drug abuse, 337 endogenous opioids, 341 exogenous opioids, 341 HIV infection, 345 innate immune response, 346 macrophages, 345–346 opioids and wound healing, 346–347 proteins, 345 immunosuppression, 342 LPS-induced expression, 323 macrophages, 343–344 µ opioid agonist, 340–341 neutrophils, 342–343 nociceptor excitation, 340 opioid antagonists, 342 pro-angoigenic response HIF-1α protein, 340 vascular endothelial growth factor (VEGF), 340 wound closure, 339–340 pro-inflammatory response injury healing, 339 innate immune response, 337–339 proposed model, 347–348 SIV infection, 324 Tat1-72 treatment, 321 wound healing, 344 Motor neuron disease, 63 Multicenter AIDS Cohort Study (MACS), 57 Murine leukemia virus (MLV), 100 N Natural killer cells (NK), 346 Neural precursor cells (NPC), 231 Neural stem cells (NSCs), 263 Neurodegeneration chemokines and chemokine receptors CCL12 expression, 15 CXCR receptor, 14–15 excitotoxins and N-methyl-D-aspartate receptors (NMDAR) antagonists, 18 CCL2 expression, 18 glutamate, 16

409 neurotoxic factors, 17 NR1 and NR2 subunits, 16–17 quinolinic acid (QUIN), 17–18 HIV protein neurotoxicity gp120, 12–13 Tat, 13–14 Neuromodulators calcium-dependent glutamate release Alzheimer’s disease, 288–290 HIV-associated dementia, 286–288 chemical transmitters, CXCR4 astrocytic bradykinin receptors, 275 glutamate release, astrocytes, 276 prostaglandins production, 275 CXCL12/CXCR4 signalling pathway, CNS arginine-vasopressin (AVP) neurons, 273 astrocytes, 273 glial cells, 272 G-protein coupled receptor, 273 inflammatory mediators, 271 intracellular signalling pathways, 272 microglia, 273 phylogenic analyses, 271 T lymphocytes, 272 neurotransmitter release, CXCR4 GABA synaptic activities, 274 pertussis toxin (PTX), 273 slow inward currents (SICs), 274 synaptic transmission gliotransmitters release, 277–281 localized calcium microdomains, 281–284 synaptic modulation, 284–285 Neuronal chemokine receptor CXCR4, µ-opioid agonists crosstalk, opioid receptors α and β chemokines, 387 chronic opioid treatment, 386 desensitized chemokine receptor, 383–384 DRG neurons, 385 endogenous and exogenous systems, 385 ferritin heavy chain (FHC), 384 MOR heterologous desensitization, 387 morphine treatment, 385–386 neuronal CCL2, 388 proalgesic/analgesic mediators, 386 RANTES, 384 G-protein coupled chemokine receptors (GPCR) down-regulation, 383 homologous desensitization, 382 intracellular signaling, 381 oligomerisation, 382 phosphorylation, 382

410 Neuronal chemokine receptor CXCR4, µ-opioid agonists (cont.) interaction, HIV neuropathology gp120, 388–389 HIV associated dementia, 390 opioid receptors brain disease, 379 cell death, 380 CXCL12/CXCR4, 379–380 immune function, 380–381 neuropathology, 379 Neuropathic pain. See also Nociception chemokines, 73–74 neuroinflammation and neuroimmune activation, 73 NRTI, 74 treatment, 76 Neurotransmission CCL22 and CX3CL1, 182 CXCL8, 181–182 CXCL12, 181 NF-κB signaling binding activity, 323 chemokine expression regulation, 322–323 opioid receptor regulation, 323 N-methyl-d-aspartate (NMDA), 39 Nociception CCL2 receptor, 183–184 CX3CR1 receptor, 184 gp 120-CXCR4 binding, 182–183 hyperalgesia, 182 Nociceptors, 340 Nucleoside reverse transcriptase inhibitors (NRTIs), 6, 55, 183 O Oligodendrocyte progenitors (OPs), 204 Opiates. See Opioids Opioid–chemokine receptor interaction chemokine expression regulation KOR activation, 322 MOR activation, 321–322 NF-κB role, 322–323 chemokine receptor expression regulation intercellular mechanism, 325 KOR activation, 325–326 model, 326 molecular mechanism, 324 oligomerization, 327 chemokine receptors classification, 319–320 function, 320

Index cross-talk cross-desensitized chemokine receptors, 329–330 cross-desensitized opioid receptors, 327–329 HIV infection, 330–331 molecular mechanism, heterologous desensitization, 330 neuronal function, 331–332 opioid receptor functions, 320 G-protein coupled receptor (GPCR) superfamily, 319 types, 319–320 Opioid-induced hyperalgesia (OIH), 386 Opioids. See also Microglia; Morphine antagonists, 342 chemokine interactions chemokine ligands and receptors, 354 pain/nociception, 354–355 endogenous opioids, 341 exogenous opioids, 341 HIV and astroglial-dervied chemokines CCL5 and CCL2 activation, 363–365 inflammatory transcriptosome, 363–364 KOP stimulation, 363 metabolic and tropic factors, 365 MOP activation, 362–363 immune interactions astroglia and microglia response, 357–358 chemokine release, 356 endogenous opioid system, 353–354 SIV/SHIV strains, 357 innate immunity immunosuppression, 342 macrophages, 343–344 neutrophils, 342–343 wound healing, 344 µ-opioid receptor, 340–341 reactive gliosis and neuronal injury, 365 Optic pathway glioma (OPG), 260 P Papaver somniferum, 353 Parkinson’s and Alzheimer’s disease, 384 Pathogen-associated molecular patterns, 358 Periaqueductal grey (PAG), 387 Peripheral blood mononuclear cells (PBMC), 35, 100, 321, 324, 353

Index Peripheral nerve injury, 305–306 Perivascular infiltrate, 121 Pertussis toxin (PTX), 303 Polymerase chain reaction (PCR), 100 Predominant plasma clone’ (PPC), 93 Primary-progressive multiple sclerosis (PPMS), 126 Primordial germ cells (PGCs), 198 Progressive multifocal leukoencephalopathy (PML), 35 Progressive polyneuropathy, 52 Progressive polyradiculopathy (PP) characteristics, 60–61 incidence, 60 inflammation and necrosis, 61 Protein-associated factor (PCAF), 94 Proteolysis. See Chemokine proteolysis P-selectin, 121 Pulmonary fibrosis (PF), 204 Pyramidal neurons, 174 Q Quantitative sensory testing (QST), 76 R Reactive nitrogen species (RNS), 360 Reactive oxygen species (ROS), 40, 360 Redjal, N., 263 Relapsing-remitting multiple sclerosis (RRMS), 126 Rempel, S.A., 254, 255 Retroviruses, 7 S SDF/CXCL12, 4 Secondary-progressive multiple sclerosis (SPMS), 126 Selectins, 120 Seroconversion, 58 Signaling mechanism adult neurogenesis CCL21 expression, 203 GABA, 201 mesenchymal stem cells, 199 pulmonary fibrosis, 204 SVZ and OB, 203 nervous system development AGM, 198 AMD3100, 198 CXCR4, 194 dentate gyrus (DG), 195

411 DRG neuron, 197 expressing progenitors, 195 HSCs, 197 phenotypes, 194 properties and functions, 192 SDF-1 regulated stem cell migration, 197 pathological pain states acute nociception, 205 CCR2, 210 chronic pain syndromes, 205 CXC3CL1 (fractalkine), 209 inflammatory response, 206 MCP-1 expression, 208 mechanical allodynia, 207 neurons, DRG, 209 opiates, 205 Wallerian degeneration (WD), 207 peripheral nervous system (PNS), 191 Simian acquired immunodeficiency syndrome virus (SIV), 7, 41, 324 Simian-human immunodeficiency virus (SHIV), 357 Sonic hedgehog (SHH), 230 Spinal nerve injury (SNI), 307 Stromal cell derived factor-1 ( SDF-1), 194. See also CXCL12 receptor Supervised treatment interruption (STI), 107 Synaptic modulation astrocytic glutamate, 284 NMDA receptors, 285 peri-synaptic astrocytes, 284 pyramidal cells, 285 Synaptophysin, 10 T TAR-RNA binding protein (TRBP), 105 Tat-associated kinase (TAK), 104 Tat protein binding sites, 13 neuromodulating effects, 13–14 U937 monocytic cell, 13 Tick-borne encephalitis flavivirus (TBEV), 135 Time to progression (TTP), 256 Total internal reflection fluorescence (TIRF), 278 Transactivation-responsive region (TAR), 103 Trojan Horse hypothesis, 96 Tumor necrosis factor-α (TNF-α), 40, 97, 275 Tumor stem cells (TSCs), 256

412 U Uracil-DNA-glycosidases, 90 Urokinase-type plasminogen activator (uPA), 309 V Vascular cell adhesion molecule (VCAM)-1, 121 Vascular endothelial cell growth factor (VEGF), 258 Vasointestinal inhibitory peptide (VIP), 305 Vicriviroc therapy, 43 Virotoxins, 355–356

Index W Wallerian degeneration (WD), 207 West Nile virus encephalitis (WNV) CCR5 CCR5∆32 allele, 135 viral infection, 133–134 CXCL12, 132–133 CXCR4 antagonists, 132–133 manifestation, 132 World Heath Organization (WHO), 254 Z Zalcitabine, 71