Aging, Immunity, and Infection (Infectious Disease)

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Aging, Immunity, and Infection JOSEPH F. A LBRIGHT JULIA W. A LBRIGHT

Aging, Immunity, and Infection

I

n f e c t i o u s . D i s e a s e SERIES EDITOR: Vassil St. Georgiev National Institute of Allergy and Infectious Diseases National Institutes of Health

Aging, Immunity, and Infection, by Joseph F. Albright and Julia W. Albright, 2003 Handbook of Cytokines and Chemokines in Infectious Diseases, edited by Malak Kotb, PhD and Thierry Calandra, MD, PhD, 2003 Opportunistic Infections: Treatment and Prophylaxis, Vassil St. Georgiev, PhD, 2003 Innate Immunity, edited by R. Alan B. Ezekowitz, MBChB, DPhil, FAAP and Jules A. Hoffmann, PhD, 2003 Pathogen Genomics: Impact on Human Health, edited by Karen Joy Shaw, PhD, 2002 Immunotherapy for Infectious Diseases, edited by Jeffrey M. Jacobson, MD, 2002 Retroviral Immunology: Immune Response and Restoration, edited by Giuseppe Pantaleo, MD and Bruce D. Walker, MD, 2001 Antimalarial Chemotherapy: Mechanisms of Action, Resistance, and New Directions in Drug Discovery, edited by Philip J. Rosenthal, MD, 2001 Drug Interactions in Infectious Diseases, edited by Stephen C. Piscitelli, PharmD and Keith A. Rodvold, PharmD, 2001 Management of Antimicrobials in Infectious Diseases: Impact of Antibiotic Resistance, edited by Arch G. Mainous III, PhD and Claire Pomeroy, MD, 2001 Infectious Disease in the Aging: A Clinical Handbook, edited by Thomas T. Yoshikawa, MD and Dean C. Norman, MD, 2001 Infectious Causes of Cancer: Targets for Intervention, edited by James J. Goedert, MD, 2000

Infectious.Disease

Aging, Immunity, and Infection By

Joseph F. Albright, PhD and

Julia W. Albright, PhD George Washington University School of Medicine, Washington, DC

Humana Press

Totowa, New Jersey

© 2003 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Due diligence has been taken by the publishers, editors, and authors of this book to assure the accuracy of the information published and to describe generally accepted practices. The contributors herein have carefully checked to ensure that the drug selections and dosages set forth in this text are accurate and in accord with the standards accepted at the time of publication. Notwithstanding, as new research, changes in government regulations, and knowledge from clinical experience relating to drug therapy and drug reactions constantly occurs, the reader is advised to check the product information provided by the manufacturer of each drug for any change in dosages or for additional warnings and contraindications. This is of utmost importance when the recommended drug herein is a new or infrequently used drug. It is the responsibility of the treating physician to determine dosages and treatment strategies for individual patients. Further it is the responsibility of the health care provider to ascertain the Food and Drug Administration status of each drug or device used in their clinical practice. The publisher, editors, and authors are not responsible for errors or omissions or for any consequences from the application of the information presented in this book and make no warranty, express or implied, with respect to the contents in this publication. Production Editor: Mark J. Breaugh. Cover design by Patricia F. Cleary.

This publication is printed on acid-free paper. ∞ ANSI Z39.48-1984 (American National Standards Institute) Permanence of Paper for Printed Library Materials. For additional copies, pricing for bulk purchases and/or information about other Humana titles, contact Humana at the above address or any of the following numbers: Tel: 973-256-1699; Fax: 973-256-8341; E-mail: [email protected], or visit our website at www.humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $20.00 per copy is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-89603-644-8/03 $20.00]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging-in-Publication Data Albright, Joseph F. Aging, immunity, and infection / authored by Joseph F. Albright and Julia W. Albright. p. ; cm. -- (Infectious disease) Includes bibliographical references and index. ISBN 0-89603-644-8 (alk. paper) e-ISBN 1-59259-402-6 1. Developmental immunology. 2. Aged. 3. Immunosuppression--Age factors. 4. Natural immunity. 5. Infection--Age factors. I. Albright, Julia W. II. Title. III. Infectious disease (Totowa, N.J.) [DNLM: 1. Immunity--Aged. 2. Aging--physiology. 3. Infection--physiopathology--Aged. QW 540 A342a 2003] QR 184.5.A43 2003 616.07'9--dc21 2002191941

Dedication We are deeply grateful to our mentors, Takashi (Mak) Makinodan and the late James D. (Jim) Ebert who introduced us to the satisfactions and occasional frustrations of biological research. "The only way to cross this Malebolge—and without Vergil as your guide—is to tell yourself that what was is; that once young, always young, once beautiful, always beautiful; once bright, always bright; that what lived cannot die." Erwin Chargaff Heraclitean Fire

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Preface The preparation of Aging, Immunity, and Infection has been a "labor of labor." When we began, there existed a huge literature—but manageable, we thought, given our years of experience in the area often referred to as immunogerontology. However, in the time that we have been at work, the new relevant literature has increased at a prodigious rate. The more we read and tried to assimilate, the farther we fell behind. In order to have any hope of completing a book on this rapidly evolving topic, we have been forced to become increasingly selective in covering new and recent publications. We dare to hope that many readers will find the book useful and only a few will dwell on the inevitable inadequacies. We consider the book a work in progress, and welcome suggestions for future editions. Five chapters cover several aspects of infection and the decline of immunity with age. The first chapter "Human Aging: Present and Future," is devoted to demographics and theories of senescence. Chapter 2 outlines the gradual breakdown of resistance to infection in the aged individual. Chapters 3 and 4 cover changes in innate and acquired immunity. The final chapter, "Nutrition, Longevity, and Integrity of the Immune System," discusses such provocative ideas as life-span extension and nutritional intervention for the delay of immunosenescence. We acknowledge with gratitude the outstanding staff of the National Cancer Institute Scientific Library at Frederick, Maryland for maintaining a first-rate library where nearly everything is available and easy to locate.

Joseph F. Albright, PhD Julia W. Albright, PhD

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Contents Dedication ............................................................................................... v Preface .................................................................................................. vii 1 Human Aging: Present and Future .....................................................1 Demographics ...................................................................................... 1 Infectious Diseases of the Aging ......................................................... 5 Limits on Life Expectancy and Future Prospects .............................. 7 Theories of Senescence ....................................................................... 11 Chapter Summary .............................................................................. 14 References ........................................................................................... 15 2 Aging and Altered Resistance to Infection ..................................... 19 Relatively Common Bacterial Infections of Aging Humans ........... 20 Selected Examples of Age-Associated Susceptibility to Bacterial Infections .................................................................... 24 Bacterial Interactions with Mucosal Surfaces.................................. 28 Antibiotic Resistance and Bacterial Variation ................................. 39 Viral Infections in Aging Humans ................................................... 42 Protozoan Parasites in Aging Subjects ............................................ 47 Fungal Infections in Aging Subjects ................................................ 50 Chapter Summary .............................................................................. 51 References ........................................................................................... 53 3 Senescence of Natural/Innate Resistance to Infection ................. 61 Pattern Recognizing Receptors of Innate Immunity ....................... 62 Phagocytic Cells: Monocytes/Macrophages ..................................... 72 Microbial Evasion of Phagocytic Destruction ................................. 80 Age-Related Changes in Macrophages ............................................. 81 Phagocytic Cells: Neutrophils ........................................................... 96 Natural Killer/Lymphokine-Activated Killer Cells ........................ 105 Chapter Summary ............................................................................ 115 References ......................................................................................... 117 4 Aging of Adaptive/Acquired Immunity .......................................135 Aging of the Thymus and Thymus-Derived (T) Cells ................... 136 The Functions and Diversity of Peripheral T Cells ....................... 145 Summary: Known and Cognizable Effects of Aging T Cells ......... 172 Differentiation, Functions, and Aging of B Cells .......................... 183 ix

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Contents Chapter Summary ............................................................................ 195 References ......................................................................................... 197 5 Nutrition, Longevity, and Integrity of the Immune System .................................................................213 RCI-Mediated Delay of Immunosenescence ................................... 214 How Does RCI Promote Life-Span Extension? ............................. 217 Dietary Restriction vs Malnutrition .............................................. 218 References ......................................................................................... 221 Epilogue ...............................................................................................225 Index .................................................................................................... 233

Human Aging: Present and Future

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1 Human Aging Present and Future Then the Lord said, “My spirit will not contend with man forever, for he is mortal; his days will be a hundred and twenty years.” —Genesis 6:3

DEMOGRAPHICS Of all the potential disasters and scourges that threaten mankind—famine, nuclear war, collision of Earth with meteoroids, and many others—none seems as impending as the aging human population. According to data and projections released recently by the Population Division of the United Nations’ Department of Economic and Social Affairs (1), the world population in 1998 was 5.9 billion. By the time this book is published, it will be well over 6 billion. The projected, “most likely” estimate for the year 2050 is 8.9 billion. It took 12 years, from 1987 to 1999, to add 1 billion people to the world’s population. In another 50 years, 3 billion more persons will be alive. What should be considered startling, indeed alarming, are the expected increases in the aging and aged segments of the population. In 1998, 66 million persons in the world were over 80 years of age (Table 1-1). That number is projected to rise almost sixfold, to 370 million persons by the year 2050. The number of centenarians will reach 2.2 million by 2050, a 16-fold increase over the number in 1998. The life expectancy of human beings has been increasing dramatically all around the world and will continue to increase in the years ahead. Table 1-2 provides data for a few “developed” countries that show the increase in life expectancy of men and women over the 25-year period from 1965 to 1990. For reasons that remain obscure, females have lived longer than males in every era From: Aging, Immunity, and Infection By J. F. Albright and J. W. Albright © Humana Press Inc., Totowa, NJ

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Aging, Immunity, and Infection Table 1-1 Present and Projected Oldest Humans Worldwide Population in millions Group

Ages

1998

2050

Octogenarian Nonagenarian Centenarian

80–89 90–99 100+

59 7 0.1

311 57 2

Data from ref. 1.

Table 1-2 Increase in Life Expectancy over the Period 1965 to 1990 Life expectancy (years) Men Country England/Wales France Japan Sweden United States

Women

1965

1990

1965

1990

68 67 67 72 67

73 72 76 75 72

74 75 73 76 73

78 80 82 81 78

Data from ref. 1.

of history. The increase that occurred in Japan during that 25 years was profound and Japan now leads the world in life expectancy at birth (2). A similar demographic shift has been occurring in the less-developed regions around the world. In those regions, the life expectancy rose from around age 40 in the early 1950s to an average of about 62 in 1990 (3). The worldwide aging of the population is the consequence of two contributing phenomena: a) the increase in life expectancy at birth, and b) the declining rate of new births. That is illustrated by the data presented in Figure 1-1. It is projected, as the figure shows, that 50 years hence the population in regions now classed as “less-developed” will comprise equal numbers of persons aged 60 and over, and aged 14 or younger. In the regions now considered developed, more than 30% of the population will be age 60 and over and approx 15% will be 14 and under. The relatively rapid shift in the demographics of the world’s population that will occur in the next half century presents formidable challenges in many respects. One major challenge, of course, is to provide the aged and aging with

Human Aging: Present and Future

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Fig. 1-1. Projected percentage of world’s population, aged 14 and younger or aged 60 and over, who will reside in regions considered “developed” or “underdeveloped.” Developed: ≥60 (䊉), ≤14 (䊊). Underdeveloped: ≥60 (䉱), ≤14 (䉭). Redrawn from data in ref. (1).

proper health care; and the attendant challenge, which is outside the scope of this book (fortunately, for us!), of how to pay for that health care. To more firmly grasp the magnitude of the challenges, consider the statement, “each month, the world sees a net gain of 800,000 people over 65, 70% of whom are in the developing world” (3). From the perspective of health care, it is the population aged 65 and over who have, far and away, the greatest needs. The greater the age, the greater the needs. To further emphasize the challenge, look again at Table 1-1, which provides a summary of the anticipated growth in the “oldestold” segment of the world’s population in the next 50 years. Future Health and Research The social and economic problems associated with the aging population in the developed nations are clearly illustrated by the current situation in Japan (reviewed by Oshima, ref. 2). The problems facing the, as yet, underdeveloped regions of the world are clearly and succinctly presented by Holden (3). In reviewing the situation in the United States, Schneider (4) has presented a compelling case for vigorous programs in aging research, leading to significant advances in preventing and treating the diseases of the elderly, as the

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Aging, Immunity, and Infection Table 1-3 Projections of the Older Population of the United States: Years 2000–2050 Number of persons (in thousands) aged Year

60–64

65–74

75–84

85–100

>100

2000 2050

45,363 99,459

34,709 78,859

16,574 44,127

4,259 18,223

72 834

Data from refs. 5 and 6.

key to coping with the impending crisis in health care for the older population. The dimensions of the problem for the United States are summarized in Table 1-3 using projections by the US Bureau of the Census in 1996 (5,6). The data presented in Table 1-3 leave little doubt that the entitlement program, Medicare, “will be stressed by the large numbers of eligible older Americans” (4). Schneider argues that through strong, adequately supported programs of research aimed at better understanding aging and the prevention and treatment of diseases of the elderly, the likely result would be that “the average health of a future 85-year-old in the year 2040 resembles that of a current 70-year-old with relatively modest needs for acute and long-term care.” Indeed, research leading to effective means to retard and prevent the debilitating effects of aging, which are neither too complex nor costly, may be the only hope for enabling much of the world’s population to age with dignity and relative independence. Research leading to good sanitation, good nutrition, and the control of communicable diseases has led to the phenomenal increase in life expectancy and is largely responsible for getting us into the present dilemna. It seems, therefore, paradoxical to assert that more research represents the best hope for the way out. The fact is, however, that the research that led to extension of life expectancy was not concerned with understanding aging. Research to elucidate the causes and possible moderation of aging is relatively new on the biomedical scene but already has made considerable progress. In this book, we consider an important outcome of aging, viz., the heightened susceptibility to infections, and explore the underlying causes, the consequences and the prevention of infectious diseases in the elderly. Infectious diseases remain an important cause of the morbidity and mortality of aging humans especially in the developing nations. Advances in the ability to cure and prevent those diseases will greatly improve the health and independence of the aging population and decrease the expenditures for health care.

Human Aging: Present and Future

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Table 1-4 Rates of Death From the 10 Leading Causes, United States, 1996 Rank

Cause of death

Deaths/100,000 population

Ages 45–64, both sexes, all races

1 2 3 4 5 6 7 8 9 10

All causes

703.6

Malignant neoplasms Heart diseases Accidents (motor vehicle & other) Cerebrovascular diseases Chronic obstructive pulmonary disease Diabetes mellitus Chronic liver disease & cirrhosis HIV infection Suicide Pneumonia and influenza

244.7 190.5 31.1 28.8 23.9 23.6 20.0 15.0 14.4 10.6

Ages >65 years, both sexes, all races 1 2 3 4 5 6 7 8 9 10

All causes Heart diseases Malignant neoplasms Cerebrovascular diseases Chronic obstructive pulmonary disease Pneumonia and influenza Diabetes mellitus Accidents (motor vehicle & other) Alzheimer’s disease Kidney diseases Septicemia

5061.1 1808.0 1131.1 414.9 270.1 221.4 137.0 91.0 62.2 61.6 51.2

From Communicable Diseases Center. Vital Statistics Report 1998;47(9):26–36.

INFECTIOUS DISEASES OF THE AGING Table 1-4 presents the 10 leading causes of death in the United States among humans aged 45–64 and over 65 years of age. Pneumonia and influenza are listed as 10th in the 45–64 age group and septicemia does not appear on the list. On the list of the over-65 age group, pneumonia and influenza appear as the 5th leading cause and septicemia is 10th. Among persons over 65, pneumonia and influenza and septicemia are responsible for the deaths of approximately three persons per thousand in the United States.

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Aging, Immunity, and Infection

Fig. 1-2. Increase in numbers of patients over age 65 diagnosed as having an infectious disorder; based on US hospital discharges over the decade 1980 (䊐) to 1990 (䊐).

Morbidity resulting from infections among US residents over age 65 is illustrated in Fig. 1-2 where the index is the rate of discharge from hospitals of patients who had been treated for one of the three infections shown. The rates are shown for the years 1980 and 1990. Notice that the rate was almost 4 times greater in 1990 than 1980 for septicemia, 2 times greater for urinary tract infection, and approx 1.5 times greater in the case of pneumonia. Those increases reflected more frequent contacts between elderly patients and physicians, more aggressive treatment regimens, and an increasing proportion of the older-old among the over-65 population. As Fig. 1-2 shows, in 1980 the rate of hospitalization of persons over 65 for three leading infections was about 745 per 10,000 elderly persons or 7.5/100. In 1990, the rate was about 1365 per 10,000 elderly persons or about 13.7/100. Thus, it is abundantly clear that if there are no new or improved programs for preventing and managing those infections among the US population over 65 years of age, the toll of suffering and the drain on medical resources will be enormous in the years ahead. To illustrate: if nothing changes in the next 50

Human Aging: Present and Future

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years except the increase in number of person over 65, in the year 2050 there will be 20 million elderly persons hospitalized for pneumonia, septicemia, and/ or urinary tract infections. That, of course, is a serious underestimate because as Table 1-4 shows, the greatest increase will occur in the oldest-old age groups in which the rate of hospitalization increases dramatically. The preceeding discussion of infections among the elderly in the United States is approximately true of other developed regions of the world. It is far from representative of the less-well-developed regions. In many of the underdeveloped regions, infections such as influenza and pneumonia are secondary to much more formidable scourges. The leading killers in most of the world are malaria, tuberculosis, leishmaniasis, and a host of diseases caused by enteric pathogens. The impact of those pathogens, which are so prevalent and to which the elderly are inordinately susceptible (discussed more fully later), on the health of emerging elderly populations is impossible to foresee. This monograph is intended to be a review of current knowledge about the susceptibility of the elderly to infections in relation to the immune and allied systems that decline in competence associated with aging. In order to deal effectively with that broad subject, it is necessary to include information drawn from the fields of nutrition, biochemistry and molecular biology, cellular and systems physiology, and others. LIMITS ON LIFE EXPECTANCY AND FUTURE PROSPECTS Because we do not understand the mechanisms of biological aging or the reasons for aging, estimating the limits of human life expectancy is highly empirical. There is a strong evolutionary and genetic influence on life expectancy (7–9). The forces of natural selection decrease with advancing age because, in natural populations, few individuals survive past the reproductive ages. Therefore, among the survivors, random mutations (alleles) will accumulate and their detrimental effects will be expressed after reproductive activity has ceased. In recognition of those ideas, Williams (10) proposed an “antagonistic pleiotropy” hypothesis, which suggests that disadvantageous genes in a population will not be selected against if they arise after the reproductive phase that is required to maintain the population. A related theory is that of the “disposable soma” theory (11), the concepts of which were summarized by Holliday (12) as follows: The environment is hostile, and individuals are competing for natural resources. This competition results in the natural selection of the fittest. In these circumstances the probability of an organism surviving and reproducing for a long period becomes very small, so potential immortality confers very little, if any, adaptive advantage. In other words, such organisms are not necessarily the fittest because resources are used to maintain the soma for a long period of time. It

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Aging, Immunity, and Infection is a better strategy for the survival of an organism’s lineage to invest resources into growth to adulthood and reproduction, rather than in long-term maintenance of the soma. Thus, the organism that evolves a soma with a limited survival time is at an advantage over one that attempts to maintain the soma indefinitely. This disposable soma theory neatly explains the early origins of aging in animals.

Finch (9) has reviewed the relationships between life-span and reproductive ability, and between life-span and hostility of the environment for a number and animal and plant species. He cites examples of organisms such as benthic fishes and bristlecone pine trees that live for hundreds or even several thousand years. In such cases, it appears that the environments to which such organisms have adapted are not threatening and that reproductive activity of those organisms is quite prolonged. In recognition of this important relationship between environment and life-span, Finch has written (9): “one may consider that the recent expansion of human life-spans parallels that of bristlecones at high altitude, and may be due, in our case, to improvements of hygiene and nutrition that adventitiously favored greater life spans.” That brings us to the questions: What is the current practical limit of human longevity? Can it be extended? For many years, the Gompertz function, or “Gompertz hazard function” (14), first formulated by Benjamin Gompertz in 1825, has seemed to describe best the relationship between human age and mortality. This relationship shows the increasing probability of death with the increasing age of a population (e.g., ref. 15). Over the range of age from approx 45 to 85, the rate of death increases steadily and can be represented graphically by a straight line. As is discussed later, data gathered in the last three decades indicate that the death rate in the oldest segment of the population (beyond age 80, approximately) has slowed considerably. As a consequence the Gompertz relationship needs to be revised and new models developed; this has become apparent as a result of the increasing human survivorship beyond age 85 (14). The development of new models is a complex undertaking owing in part to the dearth of data extending over a protracted time from which to extract factors for relative risk of mortality at given ages and how those risks may vary among different elements (cohorts) of the population. The complexity that a satisfactory model must assume is illustrated by the following (14): The model must describe the effects on mortality of internal mechanisms of physiological change with age operating under genetic constraints. It must also show how genetically constrained processes evolve with age as a result of the stochastic impact of environmental shocks, and how the operation of physiological mechanisms evolve to respond to and modify the organism’s internal environment because of those shocks.

The Gompertz formulation cannot accommodate the leveling out of the relationship between mortality and aging that has become evident in the human

Human Aging: Present and Future

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population in recent years. Data obtained from the US population over the last 25 years have shown that the major change contributing to extended life expectancy has been in the segment of the population over 50 years of age. It has been stated (16) that: “Most of the declines in mortality and gains in life expectancy during this recent mortality transition were achieved in the elderly population—a phenomenon so unexpected and unexplained that it has been referred to as a new stage in the epidemiologic history of developed nations.” Is there, at present, a realistic estimate of the mean human life expectancy? If so, how is it obtained? Several methods (reviewed in ref. 16) have provided estimates in the range of 85 to 99 years. The US Social Security Administration has forecast life expectancy in the year 2050: for women, the figure is 82.9 years, for men, 77.5 years. These figures are based on extrapolation of the data accumulated over the last quarter century (approximately). The advantages and pitfalls of projections based on extrapolation have been reviewed (17). The most reliable estimates place life expectancy at birth at around age 85. Only recently has the steady trend toward increasing life-expectancy beyond age 50 shown any indication that life-span is approaching an upper limit (16,17). As noted in ref. 16 very recent data suggest that “a biological limit to life is operating.” That suggestion comes from evidence of “mortality compression.” The latter refers to a change in shape of survival curves toward rectangularization. Figure 1-3 is a hypothetical example of a survival curve beyond age 50 as it might look today for a population with a life expectancy at birth of 80 years compared with a curve for a population in the year 2050 having a life expectancy at birth of 100 years. The second curve shows evidence of mortality compression. The rectangularization results from prolonging the phase of the curve in which few deaths occur followed by a relatively short phase in which the rate of mortality is high and the number of survivors declines precipitately. The rectangularization of the survival curve suggests that the life expectancy of the population may be approaching an upper limit. Retangularization also means that a proportion of the population will live somewhat longer than is the case when it does not occur. The combined trends of longer life expectancy at birth and longer life expectancy beyond age 50 argue strongly that the risk of death in later years has been declining over the last 20–30 years. This might be explained by a version of the antagonistic pleiotropy theorem based on the supportable conclusion that the random accumulation of mutations is not necessarily deleterious but relatively neutral. Mutations that are potentially deleterious may remain latent until some extrinsic (environmental) or intrinsic (pathogenic) event triggers their expression. Changes in any number of factors including better nutrition, reduced incidence and severity of infections, reduced exposure to certain xenobiotics (e.g., carcinogens) and others, singly or in combination, could result in improved environments for the aging population.

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Aging, Immunity, and Infection

Fig. 1-3. Rectangularization of a survival curve and illustration of mortality compression. Curve 1 reflects mortality from various causes such as infectious diseases, cardiovascular diseases, cancers, etc. Curve 2 indicates that aging has become a leading cause of mortality because deaths from other major causes have been reduced or eliminated.

What are the prospects for further prolongation of life expectancy? If there exists an upper limit, is it possible to extend that limit? If it could be extended, should it be? Consider, first, the consequences on life expectancy of eliminating some of the major diseases of the aged. Working with data gathered from the US population and made available by government agencies in 1985, Olshansky and associates (16) estimated the impact on life expectancy were it possible to eradicate only cancer, only ischemic heart disease, both cancer and ischemic heart disease, or all cancer, cardiovascular disease, and diabetes. The results were quite informative. The data provided in 1985 indicated life expectancy at birth for males of 71.2 years, and for females of 78.3 years. Elimination of all forms of cancer would have increased life expectancy by approx 3.2 years for both males and females. Elimination of both cancer and ischemic heart disease would have raised life expectancy by 8.1 years for males and 7.0 years for females. Elimination of all cardiovascular diseases, cancer, and diabetes would have increased life expectancy at birth by about 15.3 years for males and 15.8 years for females. Thus, elimination of those

Human Aging: Present and Future

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major diseases (responsible for 71.3% of all deaths in 1985) would have raised the average life expectancy to 86.5 years for males and 94.1 years for females. The cohort of the population aged 50 in 1985 was estimated to survive on average for another 25.5 years (males) or 30.9 years (females). Elimination of those major diseases would have raised the average remaining life expectancy by 15.1 years for males and 15.3 years for females. Thus, the best available analyses of the impact of eliminating the major diseases from the human population (16) provide little hope for extending life expectancies beyond 90–95 years of age. Major modification in the genetic and/or physiological regulation of longevity of the human probably will be required to extend life expectancy beyond age 100. There is now ample evidence that less complex forms of life—nematodes, fruit flies, medflies, yeast, and even rodents—are subject to considerable extension of longevity by genetic and physiological modifications. Those are discussed in later sections. Although the subject is outside the scope of this book, it is nevertheless appropriate to wonder whether or not it would be wise to prolong the human life-span beyond that which could be achieved by eliminating all major human diseases. Even that endeavor may not be so wise. As pointed out by Olshansky and associates (16): If improvements in risk factors for fatal degenerative diseases are responsible for the observed declines in old age mortality, then mortality and disability may exhibit commensurate declines. These declines would occur only if improvements in risk factors have the same effect on postponing the onset of morbidity and disability as they have on postponing mortality. However, advances in medical treatment, more than improvements in risk factors, may be allowing elderly persons who are frail and who suffer from fatal degenerative diseases to survive longer after the onset of the disease than was the case in the past. In this case, age-specific morbidity and disability rates and their duration would increase substantially.

Discussions such as the preceding are highly relevant to the formulation of medical, social, and fiscal plans in the developed regions of the world. They must, however, seem abstract and virtually irrelevant to the underdeveloped regions where the immediate problem is how to provide health and medical care to the increasing elderly population when such care is limited or not available. For much of the world, providing therapy and care to an elderly population suffering from infectious diseases looms as a formidable problem. THEORIES OF SENESCENCE Numerous theories to explain senescence have been promulgated. Each of them offers some promise, at least, for understanding the heightened susceptibility of the older population to infections. Here, we briefly review the more

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Aging, Immunity, and Infection

substantive theories and later, consider more fully the theories most relevant to infections in the elderly. The theories in their various forms are presented below under four major subheadings. Nutrition and Body Composition To date, the only known practical method of extending longevity of mammals is dietary restriction (DR; ref. 18). This was first recognized in 1935 by the nutritionist, McCay (19). Actually, it is the restriction of caloric intake that results in prolongation of life in model studies with rodents (18,20). Within reasonable limits, the greater the restriction of calories the longer life expectancy is extended (20). Although in most studies DR has been initiated at the time of weaning of juvenile rodents, DR commenced in young adult or midlife ages also results in life extension (20,21). DR results in a significant retardation of the age-associated decline in immunological competence and a significant lowering of the incidence of tumors in rodents, both of which are well correlated with increased longevity (20,22). The reasons offered to explain this apparently beneficial effect of DR are discussed in Chapter 5. The application of DR to extending longevity in humans at present is out of the question. It is far from certain that it would succeed and, if it did, that exaggerated morbidity would not occur. Furthermore, there is considerable uncertainty about the optimum body composition associated with health and longevity. For example, there are unanswered questions about the optimum proportions of lean muscle and fat masses and the rates of change of those components at different physiological and chronological ages (23–25). Free Radical and Oxidative Damage Theory of Aging Reactive radicals of nitrogen (nitric oxide and derivatives such as peroxynitrite) and of oxygen (superoxide anion, hydrogen peroxide, hydroxyl radical) can inflict considerable damage on macromolecules (proteins, nucleic acids, complex lipids), give rise to carcinogens (e.g., nitrosamines), and trigger (or sometimes prevent) apoptotic death of cells such as macrophages and vascular epithelial cells. There are mechanisms for scavenging and antagonizing those highly reactive species of molecules and for repairing damage caused by them. However, unless such mechanisms are absolutely effective, damage inflicted by free radicals may accumulate, even in a self-potentiating or exponential manner. There is evidence that the efficiency of mitochondrial electron transport and energy-generating processes deteriorate with age, resulting in increased appearance of oxidizing free radicals (26,27). Moreover, antioxidant resistance declines with age (28,29). Thus, the free radical and nitric oxide theories of aging are topics of considerable significance and research (30–32).

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Postsynthetic Modifications and Molecular Crosslinking of Proteins Contribute to Aging Following translation, proteins are susceptible to several chemical modifications including oxidation (33), prenylation (34,35), homocysteinylation (36), glycation, and the formation of crosslinks or advanced glycosylated end products (37). It is presumed that the gradual accumulation of altered proteins such as crosslinked collagens, elastins, and other structural proteins will lead to morphological and functional alterations of cells and tissues and the manifestations of senescence. There is compelling evidence of an increasing pool of oxidized, defective enzymes during aging (33) that probably parallels the increase in oxygen free radicals. As Stadtman (33) expressed it: “substantial decreases in the amounts of important enzymes and the accumulation of massive amounts of damaged protein as occurs during aging seriously compromise cellular integrity.” The gradual enlargement of intracellular pools of defective proteins, especially enzymes, could partially explain the well-known senescent decline in reserve functional potential that is characteristics of major organ systems such as the immune system, the kidneys, and the liver. Genes Influence Aging: “Gerontogenes” and “Virtual Gerontogenes” Is there a small number of dedicated genes that control senescence and impose themselves on all other genes at some predetermined rate or express themselves at some predetermined time in the lives of individuals of a species? Is there a single gene, or perhaps a few, that control the average longevity of a species? These are difficult questions to answer at the present time. There are data, however, and a plethora of opinions and interpretations. There is reasonable agreement with the conclusion that senescence and life expectancy are not controlled in a simple manner by a few genes (38). Even within inbred lines of a given species such as mice or fruit flies there is considerable variation in the apparent rate of aging and life expectancy. Several lines of evidence lead to the conclusion that aging and longevity are controlled in complex fashion by both genes and environmental influences. There are several recent demonstrations of genes that influence longevity. These include genes in Drosophila (38,39), yeast (40), the nematode Caenorhabditis elegans (41–43), and the Mediterranean fruit fly, Ceratitis capitata (44). In related work, investigators have identified genes in human cell lines that are responsible for converting cells considered to have an immortal phenotype into a senescent phenotype (45). One such gene, termed MORF4, appears to encode a transcription factor that regulates expression of several other genes that are involved in the senescent phenotype (45). The term gerontogenes has been applied to genetic factors that regulate aging (46).

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Although it is highly unlikely that a single gene, or even a very few genes, play a determining role in the rate of aging or the duration of life-span, it is possible for a single gene that affects the expression of a panel of other genes to play a considerable role. A particularly informative example is the age-1 gene in C. elegans that is involved in the resistance to various types of stress that affect life-span of the organism (47). Such genes, acting together in closely coordinated fashion, may resemble the action of a single gene and initially appear to be an “aging gene.” The several existing examples of such genes have led to the notion of “virtual gerontogenes” defined as “several genes whose functions are tightly coupled and whose combined action and interaction resemble the effect of one gene” (48). Referring to such genes as “virtual” implies that upon dissection and sequencing the individual component genes will be found to control some precise function concerned most likely with normal maintenance and repair. An immunological theory of aging was proposed (49) at a time when the only system that could be demonstrated to age in quantitative terms was the immune system. Because it was evident that the immune system plays such a central role in protection against infectious and neoplastic diseases, and because it appeared that diseases of the immune system, especially autoimmune, were associated with advanced age, the theory had merit at the time. In precise terms, the immune system plays no causal role in aging. However, as a factor in infuencing length of life in relation to disease it assumes major importance as is shown later. CHAPTER SUMMARY Aging may be viewed as a process that arose early in phylogeny in order to eliminate the competition of postreproductive individuals for limited resources. Because natural selection cannot operate on populations that have passed reproductive activity, those individuals who survived beyond reproductive competence were likely to accumulate random mutations. Although mutations frequently are deleterious, the expression of their adverse effects may require the influence of a hostile environment. Thus, aging may result from a combination of genetic and environmental influences. The remarkable extension of life expectancy in the human population over the last quarter century may reflect, especially, environmental changes coupled with advances in medicine. Recent trends in longevity may suggest that a limit on human life-span is about to be reached. During the next 50 years, there will be a major shift in demographics such that at least a third of the world’s population will require access to medical care. Such care and facilities may be available in the developed areas of the world but the sheer numbers of people who need them will create a heavy

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economic load. In the still-underdeveloped areas of the world, the medical care and facilities are likely to be inadequate and possibly prohibitively expensive. For those reasons, vigorous, adequately supported research focused on understanding the causes of senescence and the pathogenesis of diseases that afflict the elderly is essential. The diseases that are particularly debilitating in the elderly include cancer, cardiovascular and other degenerative disorders, diabetes, and infections. In many areas of the world that are experiencing a rapid increase in an aging population, infectious diseases are the foremost health problem. Those infections include tuberculosis, a variety of bacterial and viral diseases, and numerous parasitic diseases such as malaria and leishmaniasis. At present, methods for treatment of those infections are relatively unsatisfactory and expensive, and approaches to prevention are still under development. It is impossible to estimate how devastating those infections might be on populations of aging humans who lack vigorous immune systems, are difficult to immunize, and may already suffer from some other disorder. REFERENCES 1. United Nations Population Division. World Population Prospects: The 1998 Revision. New York: United Nations, 1998. 2. Oshima S. Japan: Feeling the strains of an aging population. Science 1996;273: 44–45. 3. Holden C. New populations of old add to poor nations’ burdens. Science 1996; 273:46–48. 4. Schneider EL. Aging in the third millenium. Science 1999;283:796–797. 5. US Census Bureau. US Population Estimates by Age, Sex, Race and Hispanic Origin: 1990 to 1994. Report no. PPL-21. Washington, DC: US Census Bureau, 1995. 6. US Census Bureau. Population Projections of the United States by Age, Sex, Race and Hispanic Origin: 1995 to 2050. Report no. P25-1130. Washington, DC: US Census Bureau, 1996. 7. Holliday R. Understanding Ageing. Cambridge: Cambridge University Press, 1995. 8. Finch CE, Tanzi RE. Genetics of aging. Science 1997;278:407–411. 9. Finch CE. Variations in senescence and longevity include the possibility of negligible senescence. J Gerontol Biol Sci 1998;53A:B235–B239. 10. Williams GC. Pleiotropy, natural selection, and the evolution of senescence. Evolution 1957;11:398–411. 11. Kirkwood TBL, Holliday R. The evolution of ageing and longevity. Proc Roy Soc London [Biol] 1979;205:531–546. 12. Holliday R. Causes of aging. Ann NY Acad Sci 1998;854:61–71. 13. Vaupel J. Trajectories of mortality at advanced ages. In: Wachter K, Finch CE, (eds.) Biodemography of Aging. Washington, DC: National Academy, 1977: 17–34. 14. Manton KG. Dynamic paradigms for human mortality and aging. J Gerontol Biol Sci 1999;54A:B247–B254.

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15. Harman D. Ageing: phenomena and theories. Ann NY Acad Sci 1998;854:1–7. 16. Olshansky SJ, Carnes BA, Cassel C. In search of Methuselah: Estimating the upper limits to human longevity. Science 1990;250:634–640. 17. Wilmoth JR. The future of human longevity: A demographer’s perspective. Science 1998;280:395–397. 18. Masoro ET Possible mechanisms underlying the antiaging actions of caloric restriction. Toxicol Pathol 1996;24:738–741. 19. McCay CM, Crowell MF, Maynard LA. The effect of retarded growth upon the length of the lifespan and upon the ultimate body size. J Nutr 1935;10:63–79. 20. Weindruch R. Immunogerontologic outcomes of dietary restriction started in adulthood. Nutr Rev 1995;53:S66–S71. 21. Yu BP, Maeda H, Murata I, Masoro EJ. Nutritional modulation of longevity and age-related disease. Fed Proc 1994;43:858 (abs. 3349). 22. Ross MH, Bras G. Tumor incidence patterns and nutrition in the rat. J Nutr 1965;87:245–260. 23. Losconczy KG, Harris TB, Cornoni-Huntley J, et al. Does weight loss from middle age to old age explain the inverse weight-mortality relation in old age? Am J Epidemiol 1995;141:213–221. 24. Allison DB, Gallagher D, Heo M, et al. Body mass index and all-cause mortality among people age 70 and over: The longitudinal study of aging. Internat J Obesity 1997;21:424–431. 25. Roubenoff R, Harris TB. Failure to thrive, sarcopenia, and functional decline in the elderly. Clin Geriatr Med 1997;13:613–621. 26. Sohal RS, Weindruch R. Oxidative stress, caloric restriction and aging. Science 1996;273:59–63. 27. Hagen TM, Yowe DL, Bartholomew JC, et al. Mitochondrial decay in hepatocytes from old rats: Membrane potential declines, heterogeneity and oxidants increase. Proc Natl Acad Sci USA 1997:94:3064–3069. 28. Erdineler DS, Seven A, Inci F, et al. Lipid peroxidation and antioxidant status in experimental animals: Effects of aging and hypercholesterolemic diet. Clin Chem Acta 1997;265:77–84. 29. Sanz N, Diez-Fernandez C, Alvarez A, Cascales M. Age-dependent modifications in rat hepatocyte antioxidant defense systems. J Hepatol 1997;27:524–534. 30. Harman D. Free-radical theory of aging: Increasing the functional life span. Ann NY Acad Sci 1994;717:1–15. 31. Beckman KB, Ames BN. The free radical theory of aging matures. Physiol Rev 1998;78:547–581. 32. McCann SM, Licinio J, Wang ML, et al. The nitric oxide hypothesis of ageing. Exp Gerontol 1998;33:813–826. 33. Stadtman ER. Protein oxidation and aging. Science 1992;257:1220–1224. 34. Zhang FL, Casey PJ. Protein prenylation: Molecular mechanisms and functional consequences. Ann Rev Biochem 1996;65:241–269. 35. Gelb MH. Protein prenylation, et cetera: Signal transduction in two dimensions. Science 1995;275:1750–1751. 36. Jakubowski H. Protein homocysteinylation: Possible mechanism underlying pathological consequences of elevated homocysteine levels. FASEB J 1999;13:2277–2283.

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37. Bucala R, Cerami A. Advanced glycosylation: Chemistry, biology and implications for diabetes and aging. Adv Pharmacol 1992;23:1–34. 38. Curtsinger JW, Fukui HH, Khazaeli AA, et al. Genetic variation and aging. Ann Rev Genet 1995;29:553–575. 39. Arking R, Force AG, Dudas SP, et al. Factors contributing to the plasticity of the extended longevity phenotypes of Drosophila. Exp Gerontol 1996;31:623–643. 40. Shama S, Lai CY, Antoniazzi JM, et al. Heat stress-induced life span extension in yeast. Exp Cell Res 1998;245:379–388. 41. Duhon SA, Murakami S, Johnson TE. Direct isolation of longevity mutants in the nematode, Caenorhabditis elegans. Development Genetics 1996;18:144–153. 42. Kenyon C. 1996 Ponce d’elegans: Genetic quest for the fountain of youth. Cell 1996;84:501–504. 43. Lakowski B, Hekimi S. Determination of life-span in Caenorhabditis elegans by four clock genes. Science 1996;272:1010–1013. 44. Carey JR, Liedo P, Muller H-G, et al. Relationship of age patterns of fecundity to mortality, longevity, and lifetime reproduction in a large cohort of Mediterranean fruit fly females. J Gerontol Biol Sci 1998;53A:B245–B251. 45. Bertram MT, Berube NG, Hang-Swanson X, et al. Identification of a gene that reverses the immortal phenotype of a subset of cells and is a member of a novel family of transcription factor-lilke genes. Molec Cell Biol 1999;19:1479–1485. 46. Rattan SIS. Beyond the present crisis in gerontology. Bio Essays 1985;2:226–228. 47. Lithgow GJ, Kirkwood TBL. Mechanisms and evolution of aging. Science 1996; 273:80. 48. Rattan SIS. Gerontogenes: Real or virtual? FASEB J 1995;9:284–286. 49. Walford RL. The Immunologic Theory of Ageing. Copenhagen, Munksgaard,1969.

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2 Aging and Altered Resistance to Infection All in all, my fellow pathogens, Homo is the opportunity that ultimately can benefit us all. Aside from their prevalence in numbers, they show all the weaknesses that maximize our effective potential. Although they themselves deny that there is such a thing as a free lunch, we know better. There is a free lunch, and it is them. —Thomas Eisner and Paul R. Ehrlich, New world pathogen strategy disclosed, Science 2000; 292, Editorial.

Altogether, microbial and parasitic diseases constitute the leading cause of morbidity and mortality worldwide. They affect preferentially the very young and the elderly, the two age groups that are deficient in immunological competence. This chapter is a review of some of the organisms that are particularly devastating to the elderly. A portion of the chapter deals with the remarkable variability that those microorganisms are capable of manifesting in order to ensure their adequacy to reproduce in their hosts. Optimally, a pathogen should be sufficiently virulent to thwart the defenses of its host without overwhelming it. A host that is quickly ravaged is unsuitable for the pathogen, which has the single objective of perpetuating itself. Upon infection, a struggle develops between host and pathogen with the advantage going first to one adversary and then to the other. Microbial pathogens are, of course, capable of much more rapid variation than are their hosts. Therefore, it is in the pathogen’s self-interest to utilize sparingly the weapons of virulence in its arsenal so that there is opportunity to reproduce and allow progeny to move on to new hosts. When a microbial pathogen (or any parasite) quickly overwhelms its host, it probably indicates that an adaptive equilibrium has not been achieved. That is sure to be the case when hosts that are immunodeficient are involved, i.e., hosts that are very young, those suffering from immunodeficiency diseases or being treated with immunosuppressive agents, and the elderly. From: Aging, Immunity, and Infection By J. F. Albright and J. W. Albright © Humana Press Inc., Totowa, NJ

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Aging, Immunity, and Infection Table 2-1 Some Important Geriatric Infectious Diseases and Their Relative Mortality Rates Infection

Relative mortality rate (compared with young adults)

Pneumonia Urinary tract infection Bacterial meningitis Tuberculosis Sepsis

3 5–10 3 10a 3

aExcluding HIV-infected young adults. Adapted from ref. 1.

RELATIVELY COMMON BACTERIAL INFECTIONS OF AGING HUMANS Some important infectious diseases and their relative mortality in elderly subjects are shown in Table 2-1 (1). As expected, that list reflects the fact that there are three principal routes of infection: respiratory, urinary, and gastrointestinal (GI). The most compelling explanations of the prevalence of those diseases in the elderly are: 1) age-associated changes in the structure and function of the respiratory, urinary, and gastrointestinal organs; 2) underlying pathological changes resulting from existing disorders (comorbidity); and 3) age-associated decline (dysregulation) in innate (natural) and acquired (adaptive) imunological competence. Respiratory and Urinary Tract Infection Table 2-2 provides a list of organisms found most often in respiratory and urinary tract infections of the elderly. The most common respiratory infection is bacterial pneumonia. In about half of the community-acquired pneumonia (CAP) cases, the etiologic agent remains unidentified (2). It is estimated that 20%–30% of all CAP infections are caused by Streptococcus pneumoniae and most of the remaining cases by the other bacteria listed in Table 2-2. Upper respiratory viral infections were studied in a group of 533 subjects ages 60 to 90 years living in England (3). In that group of patients, 52% of the infections were associated with rhinoviruses, 26% with coronaviruses, 9.5% with influenza A or B, and 7% with respiratory syncytial virus, and the remainder with other agents. In the case of urinary tract infections (UTIs) in the elderly, two independent studies, separated by an interval of 12 yr, gave very similar results. One study was performed in Sweden in 1986 on a group of 1966 subjects having a mean

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Table 2-2 Pathogens Found Frequently in Elderly Subjects with Respiratory or Urinary Tract Infections Organ system Respiratory tract (upper and lower)

Pathogen found frequently Bacteria Streptococcus pneumoniae Hemophilus influenza Legionella pneumophila Chlamidia pneumoniae Viruses Rhinoviruses Coronaviruses Influenza Respiratory syncytial

Urinary tract

Bacteria Escherichia coli Proteus Klebsiella Pseudomonas aeruginosa Enterococci

age of 70 years (4). The majority of those subjects were not in hospitals or institutions. The other study occurred in England in 1998 on a group of 3119 subjects all of age greater than 65 years (5). The results of the two studies agreed that Escherichia coli was the most common organism in UTIs. Klebsiella, Proteus, Pseudomonas aeruginosa, and enterococci were found less frequently but in significant numbers of subjects. The study performed in 1998 (5) included comparisons of the organisms found in bacteremic patients with respect to: 1) where the infection was acquired, i.e., in the community or in the hospital; and 2) the patients’ ages, over 65 years or in the range 18–64 years. In both age groups, E. coli was the dominant organism in more than 70% of the community-acquired infections. In the case of hospital-acquired infections, E. coli was the principal organism in approx 40% of the patients, regardless of age. Various other organisms (Klebsiella, Proteus, P. aeruginosa) were dominant in about 60% of the hospital-acquired infections. The list of organisms associated with UTIs reflects the fact that a major portion of UTIs is caused by pathogens derived from the patients’ colonic flora that enter the bladder by the “ascending route,” i.e., via the perineum, urethra, vagina, or prostate. Viral infections of the bladder are rare.

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The purpose of presenting the lists in Tables 2-1 and 2-2 is to provide a general indication of the types of microorganisms with which the elderly must contend. Geriatric infectious diseases per se are not discussed here; they are the topic of a major, recent publication edited by Yoshikawa and Norman (6). Gastrointestinal Infections Many factors can influence the GI flora; e.g., diet, medications, malabsorption, deficient intestinal motility, lumenal pH. Several of these may be altered with age as is discussed below. There are important reasons for giving special attention to intestinal microbial flora in the elderly. First, the gut is a likely source of pathogens that cause illnesses of high mortality in older patients (e.g., infective endocarditis, cholecystitis, sepsis); second, the importance of diet (caloric restriction) on longevity (discussed in a later chapter); third, the rather common problems of malnourishment, malnutrition, and dietary deficiencies (e.g., vitamins) in the elderly; and fourth, the translocation of microbial components and products from the gut to the circulation and the adverse effects on the health of the elderly. There is no known intestinal microbial pattern that distinguishes young adult from elderly. Given that there are more than 400 bacterial species in the colonic flora of a single individual (7), it is unlikely that a catalog of intestinal flora would be a useful biomarker of senescence. However, it is possible that one or a few species might be characteristically different in the young adult and the elderly. Apparently, this possibility has not been explored. The number of bacteria and the spectrum of species in normal adults varies with the segment of the intestine, as displayed in Table 2-3 (8). There are relatively few bacteria in the stomach and jejunum; those that are present are predominantly aerobes or facultative aerobes. In contrast, the colon is lushly endowed with bacteria, as revealed by fecal examination, the majority of which are anaerobes. Only small numbers of fungi, or protozoa, are present, even in the colon. The number and variety of bacteria in the gut remain rather constant in the healthy individual and are controlled primarily by gastric acid secretion and normal intestinal motility. In the healthy, well-nourished, elderly subject, the intestinal flora appears to be similar to that of the young adult. However, there is much more variation among the elderly for reasons that are considered below. As far as is known, there are no microbial pathogens that uniquely infect the elderly. Rather, the heightened susceptibility to infections associated with aging may be viewed in the following way (9): “Diminished physiologic reserve secondary to both biologic changes of aging and coexisting chronic diseases contributes to the higher mortality and morbidity rates observed for serious infection in older compared with younger persons.”

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Table 2-3 The Normal Gastrointestinal Flora of Humans Stomach 0–103

Jejunum 0–105

Ileum 103–107

Feces 1010–1012

Aerobic or facultative anaerobic bacteria Enterobacteria Streptococci Staphylococci Lactobacilli Fungi

0–102 0–103 0–102 0–103 0–102

0–103 0–104 0–103 0–104 0–102

102–106 102–106 102–105 102–105 102–103

104–1010 105–1010 104–107 106–1010 102–106

Anaerobic bacteria Bacteroides Bifidobacteria Gram-positive coccia Clostridia Eubacteria

Rare Rare Rare Rare Rare

0–102 0–103 0–103 Rare Rare

103–107 103–105 102–105 102–104 Rare

1010–1012 108–1012 108–1011 106–1011 109–1012

Total bacterial count

aIncludes Peptostreptococcus and Peptococcus. From ref. 8.

Age-Associated Changes in Anatomical–Functional Relationships The “diminished physiologic reserve” referred to in the preceding quotation includes anatomical and functional changes associated with aging of the respiratory, urinary, and gastrointestinal systems. In the case of the respiratory system, it is well established that pulmonary function deteriorates with age (2). Some of the anatomical changes that contribute to loss of function include: (a) decreased mean broncheolar diameter; (b) increased diameter of the alveolar sacs, which become shallower; (c) decrease in elastic fibers and increase in type III collagen. Those anatomical changes contribute to the following functional changes: (a) decrease in elastic recoil; (b) decrease in oxygen diffusion capacity; (c) small airway closure resulting in air trapping; (d) decreased expiratory flow rates. Spirometric changes include decreased inspiratory reserve volume, decreased expiratory reserve volume, and decreased vital capacity. In addition, the mucociliary clearance is substantially decreased in older subjects. The net effect of these changes is an increased probability of being unable to expire or clear infectious organisms that enter the lungs. The normal oropharygeal flora is a mixture of aerobic and anaerobic bacteria and may account for a significant number of cases of CAP. In fact, it has been estimated that aspiration of oral flora is second only to S. pneumoniae in causing CAP (10).

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The healthy bladder is quite resistant to colonization by bacteria. Emptying of the bladder is the most effective way of preventing bacteria from colonizing. The elasticity of the bladder diminishes with age, which makes effective emptying more difficult. Whereas among mature adults the incidence of bacteria is much greater in females, in males and females over age 65 the incidence of bacteria is almost equal. The principal contributing factors are 1) obstructive uropathy from prostatic disease in males, 2) impaired emptying of the bladder with residual urine in females, and 3) urethral catheters and associated paraphernalia in both (5). As long as the physiological condition of the individual remains good, there are no changes in the GI system that become threatening. That is not to say that there are no changes in the GI system, rather that what changes may occur are of no serious consequence. This point was made by Saltzman and Russell (11), who wrote: “The multiorgan system that composes the gastrointestinal tract has a large reserve capacity, and thus there is little change in gastrointestinal function because of aging in the absence of disease.” That can accurately be said about many organ systems with respect to aging. Consider the large functional reserve of the liver, the necessity for only one kidney, the reserve capacity of the lungs, or the large excess capacity of the bone marrow for hematopoiesis. Certainly, there is a large excess of immunological potential in the young adult that gradually diminishes with advancing age as is discussed in Chapters 3 and 4. Indeed, it can be argued that the gradual diminution in potential declines to a point approaching the level that must be expressed to deal with an acute need or an emergency; beyond that point the effects of aging are manifested. There are functional changes that occur in the GI system with age, beginning with the fact that gastric acid secretion diminishes resulting in an increase in pH in the proximal small intestine and the potential for bacterial overgrowth. In addition, normal intestinal motility may not be maintained, a factor that also disposes to bacterial overgrowth. The latter condition can cause histological changes in the mucosa of the small bowel such as hypertrophy of villi and crypts, vesiculation of the cytoplasm of mucosal cells, swollen mitochondria, and dilated cisternae of the endoplasmic reticulum (12–14). SELECTED EXAMPLES OF AGE-ASSOCIATED SUSCEPTIBILITY TO BACTERIAL INFECTIONS Mycobacterium tuberculosis Worldwide, tuberculosis (TB) is a major cause of morbidity and mortality. In the more-developed countries, during the early 20th century, TB gradually declined and by midcentury was not considered a significant public health problem. That changed in the 1970s with the onslaught of HIV-1 infections and the associated immunodeficiency. The incidence of TB rose significantly over the next 20 years or more. Prior to 1970, it was already recognized that there was a

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clear association between advancing age and the susceptibility to TB. For example, in 1970 the incidence in the United States among persons 65 and under was approx 7 per 100,000 population and among persons over 65 about 35 per 100,000. In 1992, it was reported (15) that slightly over half of all TB cases in the United States were found in people over 65 who, at that time, constituted about 14% of the population. Research concerned with TB was at a low ebb during much of the 20th century. In the 1980s there was a resurgence of research prompted by the recognition that (a) TB was a prominent opportunistic infection among AIDS victims and (b) many cases of TB were caused by antibiotic-resistant organisms. Much has been learned in the last decade. There has been some debate concerning which experimental animal serves as a suitable model of human TB; and, further, as to whether or not aging experimental animals are more susceptible to Mycobacterium infections than young adults. It was reported that old mice were no more susceptible than young adults to M. tuberculosis (16). The levels of bacteria in target organs and the frequency of death from infection were reported to be essentially the same in young and aged mice. Systematic studies by Orme and associates have shown that there is a difference in the way mice (young and old) cope with M. tuberculosis infection depending on the route of infection and the dose (number) of bacteria provided to the animals (17–19). Aged mice were definitely more susceptible than young when a relatively high number of bacteria was given intravenously. However, when a much smaller number of bacteria was provided aerogenically (modeling a realistic human exposure) the course of infection in the lungs of young and aged mice was similar. Nevertheless, there remained important differences between young and aged mice with respect to elements of the immune system involvement in the infection. For example, T cells collected from infected aged mice failed to confer adoptive immunity on recipient mice whereas T cells from infected young mice did. In the lungs, the levels of mRNA specific for several cytokines, especially IL-12 and IFN-γ, were severalfold lower in aged than in young adult mice. In this regard, it was found that M. tuberculosis infections progressed unabated in interferon (IFN)-γ knockout mice (18). Recent work has shown that components of M. tuberculosis can block IFN-γ-induced, STAT-1 mediated gene transcription in macrophages (20). (STAT is the acronym for “signal transducer and activator of transcription.”) The dissemination of live bacteria from the lungs to form granulomas in livers of aged mice was much greater than in young mice. Orme and associates concluded that there exist (unidentified) mechanisms in the aged animals that can compensate for the impaired immune control of M. tuberculosis infection. Their finding suggested that CD4+ T cells, which play a pivotal role in the control of infection, are affected by aging.

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Recent work has revealed that T cells other than the CD4+ subset can afford protection against M. tuberculosis and probably other intracellular infections (21). T cells were isolated and cell lines generated that were CD4–CD8– (double negative) or CD4–CD8+ and were CD1-restricted. Those T-cell lines possessed αβ T-cell receptors and responded to M. tuberculosis lipid and lipoglycan antigens when the latter were presented by CD1+ macrophages. Both the double negative and the CD8+ lines could affect lysis of M. tuberculosis-infected macrophages. However, the mechanisms of lysis by the two types of T cells were entirely different. Lysis achieved by the double-negative cells was mediated by way of interaction of Fas on the infected target cells and Fas ligand on the T cells and, therefore, was an apoptotic event. Lysis by the CD8+ cells involved exocytosis of granules containing the lytic factors, perforin and granzymes, in typical cytotoxic T lymphocyte (CTL) fashion. Only the CD8+ cells were able to destroy the intracellular M. tuberculosis organisms. Thus, CD1-restricted, CD8+ T cells are candidates for the mechanism postulated by Orme and associates that compensates in the old mice for senescent CD4+ T cells. Of course, there are other candidates. It should be informative to analyze the effects, if any, of senescence on CD1-restricted T cells. Listeria monocytogenes This bacterium is a Gram-positive, human pathogen. The natural portal of entry is oral, leading to invasion of mucosal surfaces of the small intestine. However, L. monocytogenes, which is a facultative intracellular organism, can invade and replicate inside a variety of mammalian cells including those that are, and are not, typical phagocytes. Once ingested, the bacteria are incorporated into phagosomes from which they escape by lysing the phagosomal membrane. The bacteria replicate in the cytoplasm and spread from cell to cell often without becoming extracellular. Thus, they become sheltered from the humoral immune response of the host. Immune defense against L. monocytogenes is cell mediated and involves both activated phagocytic cells, especially IFN-γactivated macrophages, and cytotoxic T cells (see Chapters 3 and 4). Foci of infection may be seen in various organs, such as the liver and spleen where they appear as granulomas. One of the early reports that aged animals are more susceptible to infections than young adults, was a study of L. monocytogenes in mice (22). When mice were inoculated intravenously with a moderate number of L. monocytogenes, the course of infection was similar in young and old animals as judged by the numbers of bacteria in livers and spleens. However, when a larger inoculum was used, the numbers and persistence of bacteria in livers and spleens were substantially greater in the case of the aged mice. Moreover, adoptive immunity conferred on recipients by transfer of either spleen cells or enriched T cells

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from immunized donors was much more effective with cells from young compared to old donors. The results of this very interesting study were challenged by a report of a very similar investigation performed in the same location utilizing the same strain and ages of mice and the same strain of Listeria (23). The conclusion was drawn that aging was without detriment on the ability of mice to generate T-cell immunity to L. monocytogenes. It was found that the numbers of bacteria surviving in the livers and spleens of aged mice were considerably lower than in young mice over the first day following intravenous inoculation of the same number of bacteria. Therefore, some nonimmunological mechanism that destroyed the bacteria in aged mice prevented an optimum dose of antigen from reaching immunological tissues. When a significantly larger number of bacteria was provided to the old than to the young mice (to compensate for those destroyed), it was now found that the T-cell responses in aged mice were equivalent to those in the young. Thus, the apparent defect in T-cell responses in aged mice was in reality a matter of inadequate antigen reaching sites of immune response. It was argued that destruction/sequestration of bacteria by the more-active monocytes/macrophages of aged mice prevented antigens from stimulating the immune response. The discrepancies between the findings in the two reports (see refs. 22 and 23) remain unexplained. Whatever the explanation may be, it is clear that aged mice in the experiments of Lovik and North (23) required a larger inoculum of L. monocytogenes to generate a T-cell response equal that in the young mice. Considerably more bacteria were retained in the livers of old than of young mice. The condition of the bacteria in the livers of aged mice was not determined. It is now well-known that macrophages vary in the way ingested L. monocytogenes are handled; they may be killed or they may be retained in viable condition (24). They may not have been killed but, rather, retained alive in the Kupffer cells as occurs, for example, for 24–48 hours after intravenous inoculation of the parasite, Leishmania donovani (25). If those entrapped bacteria were subsequently released by the Kupffer cells, a large bolus of antigen might arrive at sites of immune response just in time to drive an anamnestic response. Thus, the response reported in ref. 23 might not have been an assessment of the competence of aged mice for a true primary immune response. The question of why the livers of aged mice retained bacteria more effectively than livers of young mice is a separate matter. The effects of senescence on macrophages and their ability to deal with microorganisms are discussed in Chapter 3. The need to provide aged mice with 10- to 50-fold more L. monocytogenes to obtain a T-cell response equal to that of the young, as found by Lovik and North, could be a reflection of inefficient antigen processing by dendritic cells of old mice, or a reflection of a requirement for more intense processed-anti-

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gen stimulation of senescence-altered T cells. The effects of aging on dendritic cells (DCs) and T cells are discussed in Chapter 4. At this point, it need only be mentioned that dendritic cells are the critical antigen-presenting cells that prepare microbial antigens for triggering immune responses. It appears that more attention to the effects of aging on immune responses to L. monocytogenes could be rewarding. Much is now known about the mechanisms of natural and acquired immunological resistance to this organism (26) but that knowledge has not been applied to understanding the possible effects of senescence. Salmonella typhimurium In the preceding paragraphs, the finding (23) that intravenous L. monocytogenes are trapped effectively by livers (and spleens) of aged compared to young mice was discussed. That is also true of liver (and spleen) of aged rats inoculated with S. typhimurium (27). Perhaps that is the case generally for intracellular microorganisms. If so, it is important to determine why this is so and investigate the influence of macrophage entrapment of the microbes on the immune response to their antigens. Macrophages themselves are not efficient microbial antigen-presenting cells. However, after ingestion of bacteria, macrophages may undergo apoptosis, and components of bacteria picked up by immature dendritic cells. The latter may thus acquire the bacterial antigens, which they then present to T cells (28). Uptake of apoptotic material can induce maturation of the dendritic cells and expression of new surface molecules that allow the cells to migrate to lymphoid sites where they interact with T cells (29). Studies of these events in aged mice and other animals is likely to provide much new insight into the effects of senescence on immune responses. Before leaving this discussion of S. typhimurium infections, it should be mentioned that this pathogen typically enters the body by the oropharyngeal route. It traverses the intestinal barrier by invading epithelial cells and membranous epithelial (M) cells, which overlie the lymphoid follicles (see Chapter 4). After passing through the M cells, the bacteria encounter a network of macrophages and dendritic cells where the events described in the preceding paragraph can occur. However, there is an alternative process, which involves transmigration of the macrophages bearing live S. typhimurium from the intestine into the circulation and subsequent dissemination to sites where humoral antibodies can be generated (30). This is discussed in more detail in Chapters 3 and 4. BACTERIAL INTERACTIONS WITH MUCOSAL SURFACES Whether it be in the lungs, the urinary bladder, or the intestine, the flourishing of bacteria depends upon their attachment to, and successful interac-

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tions with, mucosal surfaces. The interactive processes in which various types of bacteria engage include: attachment and effacement, translocation across epithelial or endothelial barriers either between cells (pericellular route) or through cells (transcellular route), and invasion of host cells. Only in the last decade have these various events been elucidated. Most of the studies have been done in model, in vitro systems or in young, experimental animals. At present, little is known about how the various interactive events might differ in the case of mucosal surfaces of aged hosts. There follows a series of brief descriptions of the interactive events as currently understood. Bacterial Attachment The attachment to host cells is required for bacterial proliferation, colony formation, invasion of host cells, or translocation across endothelial or epithelial host cell layers. Both the bacteria and the host cells may be altered as a consequence of activation of genes in both. Adherence allows the bacteria to resist host defensive processes such as mucociliary sweeping. There is a clear correlation between the ability of a pathogen to adhere to host cells and the susceptibility of the host to that pathogen. For example, among individuals who experience recurring UTIs, adherence of E. coli to epithelial cells of the subjects may be as much as five times greater than in the case of subjects who remain free of infections (31). Pathogens, including bacteria, employ a variety of mechanisms for adhering to host cells. In several, well-studied cases, known adhesion molecules are involved (32). For example, outer membrane molecules of several bacteria (Yersinia spp., Bordetella pertussis), protozoa (Leishmania mexicana), and even viruses (echovirus 1, adenovirus) have been found to bind directly to integrins present on model host cells in vitro. Either β1or β2 integrins may be utilized. Several studies have revealed that in some cases bacteria such as Streptococcus spp., P. aeruginosa, and Staphylococcus aureus bind first to host cell molecules such as laminin, collagen, and fibronectin, which then associate with integrin receptors. Other pathogens such as Legionella pneumophila may bind selectively to the complement component, C3bi, which is a ligand for αmac β2 integrin. B. pertussis display several adhesive molecules of which two have been fairly well studied, viz., the filamentous hemagglutinin (FHA) and the pertussis toxin (PT) (reviewed in ref. 33). Although not a pathogen of elderly humans, what has been learned about adherence of B. pertussis to human cells is broadly instructive. Ciliated cells and macrophages are the host cells to which B. pertussis binds. Mutant strains of B. pertussis have been prepared that lack either FHA or PT or both (34). When tested in normal rabbits, wildtype strains localized to cilia in the respiratory tract and produced lesions.

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Mutants lacking both FHA and PT were cleared without inducing pathology. Mutants lacking either adhesin failed to attach to cilia but managed to pass into the alveoli where they caused pathological changes (33). FHA is a functionally complex molecule that displays several domains capable of interacting with complementary sites on host cells. These domains include: an N-terminal lectin domain that binds sialic acid and is required for hemagglutination; a lectin domain for ciliated host cells; a domain containing an arginine-glycine-asparagine (RGD) sequence that binds to the leukocyte integrin CR3 (CD11b/CD18) and two regions that resemble sites on factor X of the coagulation mechanism that also bind to leukocyte CR3 (33). This complexity allows FHA to interact with a variety of receptors on host ciliated cells, erythrocytes, and leukocytes. The PT protein is a hexamer. One monomeric subunit bears the catalytic site that affects adenosine 5'-diphosphate (ADP) ribosylation of guanine nucleotide proteins involved in host cell signal transduction and thus exerts the toxic effect of PT. The pentameric region of PT displays the binding sites that promote binding to host cells and intracellular delivery of the toxin. Those binding sites include lectin subunits (S2 and S3) of the pentamer, which are responsible for PT binding to glycoconjugates on cilia (S2) and the association of B. pertussis with macrophages (S3). Studies on the binding specificities of the S2 and S3 subunits, especially their recognition of the Lewis “a” and “x” blood group determinants, suggested that they are selectins. They possess significant sequence homology to stretches of known selectins (35) and structural studies have revealed that those stretches of homology are superimposable in the crystal structures of S3 and E selectin (36). It has been demonstrated that FHA and PT resemble natural ligands to the extent that they can act as competitive inhibitors of integrins and selectins, respectively. As a consequence these components of B. pertussis can interfere with neutrophil adherence and endothelial transmigration. This is an example of how bacterial components might exacerbate infection by interfering with a host defense mechanism. Although whooping cough is not a problem in the elderly, the preceding discussion of B. pertussis adherence to host cells highlights an important point; namely, that very little work has been done to understand whether or not adhesion receptors and ligands change with age and, if certain of them do, the consequences of such changes. That is an important consideration in the following discussion of infections with S. pneumoniae. S. pneumoniae is responsible for several localized and systemic infections such as otitis media, meningitis, sepsis, and pneumonia. There is a wellestablished relationship between pneumococcal bacteremia associated with pneumonia and mortality of aging subjects (38). Among patients with bacteremia, the fatality rate has been related to age as follows: 17–18% among

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young adults, 43% among those of age 60–69, 48% of those aged 70–79, and 60% among patients 80 years or older. S. pneumoniae enters the body by the nasopharyngeal route and pneumonia results from inspiration of the bacteria into the lower respiratory tract. Bacteria may be found in the alveoli from which they gain access to the circulation by crossing the endothelium of the alveolar capillaries. S. pneumoniae attaches primarily to cells of the nasopharynx, vascular endothelium, and other cells of the lung. The local inflammation that they induce is triggered by components of the cell wall. In fact, the pathogenesis of inflammation can be induced experimentally by mixtures of cell wall components (39,40). Those components are able to activate the complement system via the alternative pathway giving rise to leukocytosis, vascular permeability, secretion of interleukin-1 (IL-1) by macrophages, and other effects (33). The pneumococci bind to glycoconjugate moieties on host cells. Cells of the nasopharynx display glycoconjugate receptors of the neolactose type containing GlcNAc β1-3 Gal. The latter is also a component of the ABH, Lewis, and Ii blood group antigens and is present in human colostrum. In fact, colostrum can inhibit pneumococcal adherence to nasopharyngeal cells (41). The receptors on type II pneumocytes and vascular endothelial cells responsible for attachment of pneumococci are of two types; both of them differ from the receptor on nasopharyngeal cells. Saccharides that can competitively inhibit the adherence of S. pneumoniae to pneumocytes and vascular endothelial cells help to define those receptors. They include mannose, GalNAc, Gal, the glycoconjugates asialo-GM1 and GM2, and the Gal NAcβ1-3 Gal-containing Forssman glycolipid (33). It should be mentioned here that the exposure of type II pneumocytes and vascular endothelial cells to the inflammatory cytokines TNFα and IL-1 significantly elevate the glycoconjugate receptors for pneumococci (33,42). As a consequence, adherence of S. pneumoniae is markedly enhanced. Enhanced adherence entails a new receptor, viz., that for the platelet-activating factor (PAF). As pointed out (33), this is an example of an important principle of bacterial adherence; viz., the initial attachment of bacteria to resting host cells may involve one set of sugar specificities leading to activation of the cells and the expression of a new or altered receptor specificity. A virulent organism must parallel this change in the host cell by expressing a new cognate adhesion molecule. It has been emphasized that: “This action-and-reaction scenario underlies the success of virulent pathogens and illustrates the dynamic nature of the response of both partners in an adherence interaction” (33). It appears that little, if anything, is known about the possible changes that might occur in, for example, pneumocytes or endothelial cells with age that would affect attachment and transmigration of pneumococci or other pneumonia-causing pathogens.

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The ability to adhere to host enterocytes is a major determinant of virulence in the case of enteric pathogens. Most of those pathogens express adhesins that function as lectins (43). The adhesins may or may not be present on bacterial fimbriae. Adhesins present on rigid fimbriae are maintained at a distance from bacterial surface components that might interfere with adherence to host cells. Adhesins present on flexible fimbriae are allowed spatial freedom in binding to cognate receptors. Many enteric bacterial adhesins interact with and agglutinate erythrocytes from various species. They can be characterized by the spectrum of species of erythrocytes that they agglutinate. By this test, families of lectins have been identified (43); these include galactoside-specific, sialic acid-specific, and N-acetylglucosamine-specific lectins. Some adhesins fail, however, as hemagglutinins (perhaps because the saccharide moieties that they recognize on erythrocytes are inaccessible) but do interact with cells of the intestinal tract. Intestinal mucous contains numerous, potentially inhibitory saccharide moieties that may block bacterial adherence. Whether or not this occurs depends on a number of factors including the quantity and rate of formation of mucous. The wide variety of glycoconjugates with which bacterial lectins interact is shown in Table 2-4. That variety ensures the ability of many enteric bacteria to adhere at optimum sites and niches within the intestine. Intestinal E. coli strains can be categorized into several types depending on their attachment and invasive properties. Those categorized as enteropathogenic E. coli (EPEC) adhere to gut epithelial cells through intimin molecules. The latter are ligands for the bacterial complementary receptor known as Tir (translocated intimin receptor) (44,45). Following the initial adherence of EPEC to host cells, the bacteria introduce Tir, along with several other proteins, into the host cells via a type III secretion mechanism. The expression of Tir significantly enhances the adherence and, thus, promotes the secretion of effacing factors into the host cells. This is presumably a key event in the initiation of diarrhea. What is particularly interesting about this example of adherence is the fact that the bacterium provides to the host cell the receptor (Tir) to which it strongly binds. Additional interest in this interaction arises from the recent finding (46) that the ensuing inflammatory reaction that leads to pathology of the colon is dependent on the involvement of type 1 T helper (Th1) cells (CD4+) and the IFN-γ that they secrete. It appears that intimin not only interacts with Tir but also with β1 integrin molecules on T cells. The resulting hyperplastic changes in the gut provide further opportunities for EPEC colonization. This is an excellent example of the diversion of host immune defenses (T cells and their cytokines) into paths that promote the welfare of the bacterial pathogen. There are other outstanding cases such as that of S. typhimurium (47). In this case, the bacterium secretes proteins via a type III mechanism that

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Table 2-4 Some Interactions of Bacterial Lectins-glycoconjugates Bacteria Klebsiella pneumoniae Mycobacteria Salmonella Serratia marcescens Shigella flexneri Vibrio cholerae Streptococcus pyogenes Escherichia coli (urinary pathogens)

Lectin type Type 1 & 3 Mycotin

P-related (F7–F16) G-I G-II G-III

Clostridium difficile Escherichia coli Streptococcus pneumoniae Escherichia coli (sepsis pathogen)

Escherichia coli (urinary pathogen) Vibrio cholerae Helicobacter pylori

Mycoplasma galliseptum; M. pneumoniae Candida albicans Haemophilus influenzae Staphylococcus aureus Bordetella pertusis Borrelia burgdorferi (Lyme disease)

AFA (enterotoxin) G S

(enterotoxin)

PHA

Carbohydrate specificity mannose mannose, mannan mannose mannose α-mannoside fucoside galactose Galα1-4Gal in globotriaosylceramide globotetraosylceramide globopentaosylceramide (Forssman antigen) Dr blood group antigen Galα1-3Galβ1-4GlcNAc N-acetylglucosamine GlcNAcβ1-3Gal NeuAc α2-3Gal β1-3GalNAc & NeuAcα2-3Galβ1-3(NeuAcα2-6) GalNAc NeuAcα2-3Galβ1 GM 1 GM3 (NeuAcα2-3 Gal β1– 4Glc-cer)So3-GM3; Leb-group blood antigen NeuAc α2– Lewis (a) antigen Lewis (a) antigen Lewis (a) antigen Sulfated glycolipids, heparin Gal-Cer, Lac-cer; GD1a, GD1b, GM2, GM3

Modified from ref. 43.

activates signaling pathways in the host epithelial cells leading to production of IL-8 and other proinflammatory cytokines, e.g., TNF (tumor necrosis factor) α, and GM-CSF (48–50). Bacterial Type III Secretion Mechanism There are four different mechanisms utilized by bacteria to export synthesized products, especially virulence factors (51). Types I and II lead to secre-

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tion of materials directly into the surrounding milieu. In type I secretion, the mechanism employs three proteins that form a channel through the inner and outer bacterial membranes. The type II mechanism has been studied extensively in Vibrio cholerae (52). There are at least 12 proteins that appear to create a pore that bridges the inner and outer membranes and through which bacterial products are secreted. The type IV system was discovered fairly recently and the mechanism is under scrutiny (53). Among other products, the secretion of immunoglobulin A (IgA) proteases occurs via the type IV system. The type III secretion system is of particular interest owing to its complexity and the fact that it is employed by a number of Gram-negative pathogens to introduce virulence- related substances directly into the cytosol of host cells (54). Some substances are secreted into the extracellular environment rather than translocated into a host cell. The type III system is employed by both animal and plant pathogens. Among the animal pathogens are Shigella spp., Yersinia spp., Chlamydia spp., P. aeruginosa, EPEC, enterohemorrhagic E. coli, and S. typhimurium. The type III secretion mechanism involves a structure termed the needle complex, which has been isolated from interacting S. typhimurium and host cells (55). Electron microscopy showed that the structure resembles a long hollow tube attached to a cylindrical base that anchors the structure to the bacterial inner and outer membranes. Other bacteria may induce the formation of pedestals or cuplike structures between themselves and host cells. The formation of such structures is initiated upon contact and association of bacterium and host cells facilitated by adhesion molecules. Some bacteria, e.g., EPEC, induce extensive cytoskeletal reorganization in host cells leading to pedestals that contain cytoskeletal proteins (e.g., actin, α-actinin, talin). Those cytoskeletal rearrangements result in effacement, which includes loss of microvilli and the resulting diarrhea. Bacterial Invasion of Host Cells Some bacteria are obligate intracellular pathogens (e.g., Chlamydia spp.), many important pathogens are facultative intracellular organisms (e.g., Salmonella spp., L. pneumophila, Mycobacterium spp., L. monocytogenes), and others are predominantly extracellular (e.g., enterotoxigenic E. coli, Haemophilus influenzae, V. cholerae). Pathogens that penetrate epithelial barriers survive by invading and replicating in host cells. Tight junctions (zona occludens) that normally prevent penetration of epithelial cell layers also divide the epithelial cells into apical (lumenal) and basolateral surfaces. Some pathogenic bacteria such as Salmonella invade host cells from the apical surface whereas others (Yersinia, Shigella) interact with and invade through the basolateral surface. The invasion of host cells by S. typhimurium has attracted the attention of several groups of researchers (55–57), whose findings are quite significant.

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The adherence of the bacteria triggers some pronounced cytoskeletal and membrane modifications of the host cells. Those modifications are triggered by the introduction (via a type III secretion mechanism) of bacterial proteins including Sop E, which activates GTPases of the Rho subfamily, and Sip A, which binds to actin and prevents depolymerization of actin filaments. The formation of actin filaments is required for bacterial internalization. Other proteins involved in cytoskeletal changes include α-actinin, talin, and ezrin. Associated with those changes is an intracellular Ca2+ shift and activation of “mitogenactivated protein” (MAP) kinase. These and other events, still to be elucidated, in a signal transduction pathway lead to membrane ruffling, macropinocytosis, and thus to internalization of the bacteria by a host cell. The preceding descriptions of attachment, type III secretion, and invasion of bacteria ,vis-à-vis, epithelial cells are fundamental to future studies of epithelial cells that have been altered by senescence. To underscore the importance of such studies, it is worth stressing the growing evidence (discussed later) that aging results in some considerable changes in the composition and structure of membranes. As noted above, pathogens such as Yersinia and Shigella invade epithelial cells via the basolateral surface. Salmonella can also take this as well as the apical approach. How do bacteria that can only penetrate the basolateral surface gain access to that surface? This question is particularly relevant to enteric pathogens. The answer appears to be that they are transported indiscriminately through specialized M cells overlying Peyer’s patches that are scattered throughout the small intestine (56). M cells lack all but a thin layer of mucous and are nearly devoid of villi. It is agreed that the transport of macromolecules and particulate matter from the intestinal lumen through the M cell brings the transported material into proximity with macrophages and lymphocytes located in “pockets” on the antilumenal side of M cells. In this manner bacteria can reach the basolateral surfaces of epithelial cells. Shigella employ an alternate method of crossing the barrier. They can induce chemotaxis of phagocytic cells, especially polymorphonuclear leukocytes. As the latter migrate toward the bacteria they open spaces in the tight junctions through which the bacteria pass across the epithelial surface (58). It is interesting to note that S. typhimurium is representative of several bacteria that can cross the intestinal barrier either via M cells or by traversing enterocytes (56). The entrance and fate of bacteria that enter into host macrophages and dendritic cells are discussed in some detail in Chapter 3. As mentioned previously, the case of L. monocytogenes may be considered prototypic. Some macrophages readily destroy ingested L. monocytogenes whereas others lack that ability (24). Bacteria replicate in the latter and may be passed from cell to cell without becoming accessible to the host’s immune system.

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Biofilms and Quorum Sensing Individual bacteria that lodge in alveoli or attach to the epithelium of the intestine or urinary bladder are in a precarious situation. The hostility of the environment makes it unlikely they will survive for long. Therefore, in order to outmaneuver host defenses, the bacteria replicate rapidly and form microcolonies. Many species of bacteria, including a number of pathogens, form organized communities termed “biofilms.” Only in the last decade has a clear understanding of the mechanisms and significance of biofilm formation begun to emerge (59). For example, it was realized that particles of biofilm formed by L. pneumoniae and circulating in building air ducts were responsible for the notorious outbreak of Legionnaires disease in Philadelphia in 1976. In 1993–1994, hundreds of asthmatic individuals received albuterol that, although drawn from a disinfected processing tank, was contaminated with particles of P. aeruginosa biofilm (60). It is now well-known that the lungs of cystic fibrosis patients are sites where biofilms of P. aeruginosa are formed. The importance of biofilms in infectious diseases associated with aging is suggested by the contents of Table 2-5. In addition to the infections shown in that table, there is growing evidence that biofilms may develop and complicate bacterial pneumonia and intestinal infections (bacterial overgrowth) and may perpetuate the durable infections of M. tuberculosis often associated with the recurrence of tuberculosis in the elderly. The recent realization that bacteria present in biofilms are notoriously resistant to antibiotics and are protected from both humoral and cell-mediated immunity of the host are of major concern in treating infections of the elderly. The community of bacteria in biofilms is protected by an extracellular matrix of polysaccharide (termed “glycocalyx”). The chemical nature of the latter varies with the species of bacteria and whether the community is mixed or of a single species. Moreover, the community organization allows functional heterogeneity and regional specialization much like an organized tissue. Individual bacteria (called “planktonic”) may leave the biofilm and disperse to other sites somewhat resembling metastasis of tumor cells. Planktonic bacteria are susceptible to antibiotics and host immune response. The formation of biofilms by P. aeruginosa has received considerable attention because it is a critical event in the devastating disease, cystic fibrosis (59,61). This same bacterium is found in most healthy individuals in whom it causes no disease. It is an opportunistic organism that only becomes pathogenic in compromised individuals. A brief account of biofilm formation by P. aeruginosa on the epithelial linings of the lungs of cystic fibrosis patients provides a concept of the process. Attachment factors are present on hairlike appendages of the bacteria called type IV pili (62). Those pili allow a twitching

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Table 2-5 Some Human Infections Involving Biofilm Formation Infection or disease

Common biofilm bacterial species

Dental caries Periodontitis Biliary tract infection Osteomyelitis Bacterial prostatitis Native valve endocarditis

Gram-positive cocci (e.g., Streptococcus) Gram-negative anaerobic oral bacteria Enteric bacteria (e.g., E. coli) Several bacterial and fungal species E. coli and other Gram-negative bacteria Viridans group Streptococci

Nosocomial infections ICU Pneumonia Sutures Contact lens Urinary catheter cystitis Hickman catheters Vascular grafts Biliary stent blockage Orthopedic devices

Gram-negative rods Staphylococcus epidermidis and S. aureus P. aeruginosa and Gram-positive cocci E. coli and other Gram-negative rods S. epidermidis and C. albicans Gram-positive cocci Various enteric bacteria and fungi S. aureus and S. epidermidis

Modified from ref. 59.

motion that aids the bacteria in assembling into colonies. P. aeruginosa utilizes a type III secretion system to secrete toxin into epithelial cells, which among other effects interferes with ciliary sweeping thus further aiding microcolony formation. The attached bacteria proliferate, and as their number reaches a certain minimum density, sets of genes become activated the products of which include both virulence factors (e.g., toxin A, exoenzyme S) and other substances that promote bacterial cell wall remodeling and glycocalyx formation. In the case of P. aeruginosa, the principal glycocalyx material is the polysaccharide, alginate. At this point, the bacteria embedded in the glycocalyx are protected from antibodies, cell-mediated immunity, and antibiotics. Antigens are released by bacteria in the biofilm as well as planktonic forms and high levels of immunity may prevail in the host, but to no avail. As noted in the preceding paragraph, when bacteria in the developing colony reach a certain number (density) activation of a new set of genes occurs. This event reflects the recognition of a threshold concentration of organisms termed “quorum sensing.” It was first noted in bioluminescent marine bacteria in which the intensity of luminescence increased dramatically at a certain bacterial density. What is the mechanism of quorum sensing? There appear to be several mechanisms employed by different species of bacteria as well as certain fungi

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and possibly protozoa. The best understood mechanism, at present, operates in several Gram-negative human pathogens and has been analyzed extensively, again utilizing P. aeruginosa (61,63). As the density of the proliferating bacteria increases, so does the local concentration of small molecules that the bacteria synthesize known as acylhomoserine lactones (acyl-HSL) [e.g., N-(3oxododecanoyl)-L-homoserine lactone (3OC12-HSL) and N-butyrylL-homoserine lactone (but-HSL)]. The acyl-HSL molecules are the quorumsensing signals. Two quorum-sensing systems have been identified in P. aeruginosa: one is the lasR-lasI system and the other the rhlI-rhlR system. The product of the lasI gene directs the synthesis of 3OC12-HSL and the product of lasR, in the presence of a sufficient concentration of 3OC12-HSL, activates a set of virulence genes that includes rhlI and rhlR. The gene, rhlI, is responsible for a product that directs the synthesis of but-HSL, which is involved in the activation of virulence genes. Progress in understanding quorum sensing and the variety of systems that participate in the phenomenon has been rapid (see, e.g., ref. 64). Some biofilms comprise a single species of bacteria in which case the signals utilized by that species must be distinguished from those of other species. Other biofilms are composed of mixed species and in this case organisms may need to recognize several signals but obviously not all. Signal sensing among various species may help a given species, or group of related species, to avoid competition, ascertain an appropriate niche, or perhaps produce an antimicrobial substance to minimize or eliminate local competitors. From the perspective of bacterial infections in aging subjects, biofilms would appear to be of great importance. First and perhaps foremost, biofilms render many pathogens safe from antibiotics and immune attack. Second, it is likely that biofilm formation by various bacteria that are nonpathogenic in healthy, young adults may lead to serious infections in immunocompromised elderly or those already afflicted with some disorder. Third, the widespread use of urinary catheters, the high prevalence of prostatic disease among elderly males, and the frequency of bone and joint repair and replacement in the elderly offer to microbial pathogens a range of opportunities for clinical biofilm formation. Finally, it seems important to stress that biofilms is a subject that has received very little attention in relation to the susceptibility of the elderly to infections. Are conditions in aging tissues more or less favorable for the formation of biofilms? Or unchanged? Consider the mounting evidence that changes in the cytoplasmic membranes accompany senescence of various cells; does such a change occur in epithelial cells and might that influence bacterial adherence and biofilm formation? Do senescing tissues offer more and better-sheltered niches for bacterial colonization? An opportunity seems to exist for research that could have quite significant consequences.

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ANTIBIOTIC RESISTANCE AND BACTERIAL VARIATION Resistance to antibiotics is increasing rapidly among human pathogens as pointed out by many authors (e.g., refs. 65 and 66). The reasons center around one problem: the failure to control the human use of antibiotics. Numerous studies have shown that in medicine antibiotics are frequently prescribed unnecessarily or inappropriately. For example, it was estimated that in 1992, 12 million adults who presented bronchitis or upper respiratory infections received prescriptions for antibiotics that offered little or no benefit (67). Similar studies of inappropriate antibiotics usage have focused on Canada, Europe, and Japan. In developing countries, antibiotics usage has been poorly regulated, patient compliance has been poorly monitored, and much of the supply of antibiotics is of low quality. The common use of antibiotics in veterinary medicine and in agriculture has contributed to the problem to an extent that is difficult to determine but likely to be considerable. Geriatric medicine has certainly contributed to the growing problem of microbial antibiotic resistance. As a group, elderly patients in hospitals and long-term care facilities (LTCFs) are the major recipients of antibiotics. It has been estimated that among residents of LTCFs approx 40% of prescribed drugs for systemic use are antibiotics (68). A distressing result is the promotion and spread of antibiotic-resistant microorganisms (69). The types and origins of antibiotic-resistant pathogens in LTCFs have been carefully reviewed in a recent publication (70). Information about antibiotic resistance in the population of elderly who reside in LTCFs is probably the most reliable available. As stated in the review article (70), the antibiotic resistant bacteria of greatest concern to geriatricians are: 1) β-lactam resistant organisms, especially penicillin-resistant pneumococci and aerobic Gram-negative bacilli resistant to third-generation cephalosporins; 2) vancomycin-resistant enterococci; and 3) quinolone-resistant Gram-negative and Gram-positive bacteria. As recently as 1997, it was reported that more than one-third of S. pneumoniae isolates analyzed in broad survey were resistant to penicillin and more than 13% of them were highly resistant (71). Relatively resistant isolates have been found in many regions of the world. Altered penicillin-binding proteins (enzymes involved in the final stage of bacterial cell wall formation), which have low affinity for penicillin, are responsible for resistance to that antibiotic. Other organisms noted for β-lactam antibiotic resistance are certain species of Staphylococcus (some resistant to all penicillins, cephalosporins, and carbapenems) and Enterococci, which are resistant to all cephalosporins because they lack significant penicillin-binding proteins. Enzymes known as β-lactamases are largely responsible for the ability of bacteria to resist the cephalosporins. Considerable effort to produce enzyme-

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resistant derivatives of those antibiotics has been expended leading to so-called third-generation cephalosporins that showed considerable promise. However, a number of enzymes have now been reported (called “extended spectrum βlactamases”) that can cleave advanced-design cephalosporins and penicillins. Many of the extended spectrum β-lactamases (ESBLs) are encoded by genes borne on plasmids such as those encoding TEM-1 associated with enteric bacteria. TEM-1 is responsible for close to three-fourths of plasmid-borne β-lactamase resistance world wide (72). The TEM group of enzymes is encoded by the TnA transposon, which probably accounts for the β-lactamases present in more than one-third of H. influenzae isolates in the United States. Several pathogenic Gram-negative bacilli (Enterobacter, Citrobacter, Pseudomonas) produce βlactamases that are encoded by a chromosomal gene. Those AmpC enzymes are able to inactivate all cephalosporins. Several AmpC β-lactamases are now known to be conveyed by mobile, conjugative plasmids in E. coli and in Klebsiella species. The presence of those enzymes can result in resistance to penicillins, cephalosporins, cephamycins, and β-lactamase inhibitors (73). It should be noted that resistance to β-lactam antibiotics can occur as a result of restricted entry of the antibiotics as well as low binding affinity to the penicillin-binding proteins and destruction by β-lactamases. Drug efflux pumps, which restrict antibiotic entrance into bacteria, have become a major problem in antibiotic therapy. They result in inadequate accumulation of antibiotics inside bacterial cells to be effective. The formation of transport proteins, which bind and inactivate antibiotics or escort them out of the bacterial cell, prevents the antibiotics from reaching critical targets. Some of these efflux pumps are drug-specific such as Tet B in enteric bacteria and H. influenzae; others act in a broad, “multidrug” pattern. Tet B is plasmid encoded, although chromosomally mediated tetracycline resistance occurs in some bacteria such as Proteus. Tetracycline resistance conveyed by plasmids is situated near insertion sites and as a consequence those plasmids rather readily acquire other genetic information, which results in broadening the specificity of the resistance. It is likely that the widespread use of tetracyclines in animal feed is responsible, in part, for the existence in many regions of the world of resistant Enterobacteriaceae. The quinolone antibiotics such as nalidixic acid and ciprofloxacin bind to DNA gyrase (type II topoisomerase) in Gram-negative bacteria, and to topoisomerase IV in Gram-positive bacteria thus interfering with DNA replication. Some of the later quinolones have other actions in addition to their effects on topoisomerases and display a broad spectrum of antimicrobial activity (74). Resistance to these antibiotics may emerge as a result of mutations in bacterial topoisomerases, diminished membrane penetrability in Gram-negative bacteria, or active efflux transporter proteins. Such changes are generally

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caused by alterations in the chromosomal DNA such as point mutations in the A subunit of DNA gyrase (74). Several species of Staphylococci and Enterococci are notorious for being nosocomial infections. The antibiotic, vancomycin, has been widely used to combat these infections because for many years no resistance to this substance was reported. Resistance of entercocci was first reported in 1986, which resulted in considerable effort to elucidate the causes (75,76). Vancomycin is a glycopeptide that interferes with cell wall formation of Gram-negative bacteria. It interacts with D-alanine at the C-terminus of precursors of peptidoglycans. This creates a complex from which the precursor substances cannot be transferred by transglycosidases to the growing peptidoglycan cell wall. Resistance to vancomycin appears as a consequence of expression in bacteria of transposable genes, which encode cell-wall-synthesizing enzymes that alter the C-terminus of the peptidoglycan precursors from D-alanine to D-lactate. This change allows cell wall construction to continue even in the presence of vancomycin. There are four known phenotypes of vancomycin-resistant enterococci of which two (Van A and Van B) are associated with moderate to high resistance to the antibiotic (70). Both of those phenotypes are readily transferred on plasmid and transposon elements. The Van B phenotype is also associated with a chromosomal complex that closely resembles the organization of the Van A transposable element. The importance of vancomycin resistance to the geriatrician is the common usage of that antibiotic to treat UTIs and infected pressure ulcers, which are relatively common in LTCFs (70). Vancomycin-resistant enterocci are introduced most often into LTCFs by accepting patients who have acquired resistant organisms in hospitals. To complete this brief discussion of antibiotic resistance, a word about transposons is in order. Those genetic elements, conveyed by resistance plasmids (R-plasmids), are responsible for much of the current microbial resistance to antibiotics. There are collections of bacteria, assembled in the preantibiotic era, that display recognizable plasmids; but most of those plasmids lack antibiotic-resistance elements. This must mean that current bacterial pathogens displaying antibiotic resistance harbor familiar plasmids that have become R-plasmids by acquiring resistance transposons. That is a consequence of the excessive use of antibiotics, which has given selective advantage to bacteria possessing R-plasmids. Among the latter, those that convey multiple antibiotic resistance vary with respect to the transposons they contain. Some have a single transposon composed of multiple resistance determinants. Some have several transposons located in different sites. In some cases, there is present a complex element in which one transposon has become integrated into another. Apparently, there has been no effort to evaluate the R-plasmids present in bacterial

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isolates from older persons, especially those residing in LTCFs, to determine whether or not the plasmids and the resistance transposons differ from isolates obtained from young adults. If a significant difference exists, such information should be useful in planning judicious antibiotic therapy. The extreme variability, versatility, and adaptability of bacteria arise from two processes that are not found in eukaryotic organisms: (a) horizontal (lateral) transfer of genetic material (77); and (b) hypermutability associated with “mutator” strains (78,79). There are three mechanisms for delivering exogenous DNA into recipient bacteria: (a) transformation, which involves bacterial uptake of naked, ambient DNA; (b) transduction, in which new DNA is delivered by bacteriophages; and (c) conjugation, which requires physical contact between donor and recipient cells and, most frequently, transfer of a plasmid. Once inside the recipient cell, the DNA must become assimilated either as a stable episome or by integration into the recipient’s genome if it is to be expressed. Hypermutation, exemplified by mutator strains, appears to arise from mutations in genes that affect the synthesis, modification, or repair of DNA. A recent study (78) of the presence of mutator strains of P. aeruginosa isolates from cystic fibrosis patients has provided strong evidence for the concept that the emergence of mutator strains is a mechanism employed by bacteria for rapid adaptation to nonoptimal, even hostile conditions in their hosts. This concept (see comments in ref. 79) could be of major significance with regard to infections in the elderly in whom microenvironmental conditions in organs such as the lung, urinary bladder, and GI system may differ considerably from those in younger subjects. VIRAL INFECTIONS IN AGING HUMANS Viruses are small, composite particles of nucleic acid and protein, and are obligate, intracellular parasites; i.e., they cannot replicate outside a host cell. An individual virus contains only one type of nucleic acid, either RNA or DNA, which is protected by the associated protein from destruction by hostile substances, such as nucleases, present in its environment. Viral proteins serve two other crucial functions; first, they are responsible for attachment of virions to host cells and, second, they include a minimal array of enzymes that are necessary to cajole the host cell machinery into synthesizing new virions. Together, the nucleic acid and associated protein form the nucleocapsid. Some viruses are encased in a lipid bilayer, derived from host cell membrane, termed the “envelope.” It is often studded with outward-protruding, complex molecules of glycoprotein. Viral nucleic acid (genome) may be either RNA or DNA arranged in linear or circular fashion. The nucleic acid may occur in singlestranded or double-stranded form. If the genome is single-stranded RNA, it is considered to be in the positive (plus) sense orientation if it can serve as its

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own mRNA and in negative (minus) sense orientation if a copy must first be made by a viral RNA transcriptase, which then serves as mRNA. Most DNA viruses display a linear, double-stranded genome although a few families are characterized by linear single-stranded (parvoviruses), circular double-stranded (papovaviruses), or even circular single-stranded (circoviruses) DNA. Comprehensive introductions to viral genomes and virus replication are provided in many excellent texts (e.g., ref. 80 and 81). Viral infection begins with attachment of virions to host cells. Specific attachment that leads to virion penetration generally depends upon complementary interaction between viral protein (counter-receptor or anti-receptor) and specific receptors on host cells. Viruses may display a single species of protein counter-receptor or multiple species of counter-receptors in the case of some complex viruses such as herpes simplex. Whether or not a host cell is susceptible to a given virus depends upon the cell having receptors. Cells lacking receptors are not susceptible. If a host cell supports the complete reproduction of a given virus, it is termed “permissive.” Some host cells can be shown to be permissive but not susceptible because they lack the appropriate receptors. The cellular receptors for viruses are generally glycoproteins. Some of the receptors are familiar molecules known to be involved in other functions. Table 2-6 is a list of a few of those receptors. The ability of viruses to usurp surface molecules designed for some other purpose as receptors for themselves is well illustrated by human and simian immunodeficiency viruses. Those viruses utilize members of the chemokine superfamily (CXCR4, CCR5) along with CD4 as coreceptors for entrance into T cells and monocytes (reviewed in ref. 82). Chemokine receptors have been appropriated also by other viruses; e.g., myxoma virus can utilize CCR1, CCR5, CXCR4 for entrance into host cells (83). Myxoma virus is a poxvirus the receptors for which have been difficult to identify. The epidermal growth factor receptor is utilized by vaccinia virus another poxvirus. A poxvirus that has the human as primary host is the molluscum contagiosum virus (MCV), which causes persistent, benign, skin neoplasms in children and severe opportunistic infections in AIDS victims. Both children and AIDS victims are immunodeficient (to much different degrees, of course). Elderly individuals are immunodeficient. It seems natural, therefore, to wonder whether or not the elderly are also susceptible to MCV. If so, it has not been reported (to our knowledge). Perhaps factors other than immunodeficiency are involved in rendering subjects susceptible to MCV. It would be useful to know. The complexity of viruses is exemplified by their nomenclature, which comprises some 8 major families of DNA viruses and 14 families of RNA viruses (with more to come, no doubt). Of those 22 families, 20 include members that have medical importance (see ref. 84 for a concise overview). The first five of

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Table 2-6 Some Familiar Cell Membrane Receptors for Viruses Virus Adenovirus Epstein-Barr Herpes simplex Influenza A, B Respiratory syncytial Rhinoviruses

HIV-1, -2

Receptor Integrin (α5β3) Complement type 2 receptor (CD 21) Proteoglycans (heparin sulfate moieties) Glycoproteins of 5Ac Neu Hemagglutinin glycoprotein Intercellular adhesion molecule (ICAM) CD4, galactosyl ceramide, chemokine receptors

Representative important cell infected Respiratory epithelium B lymphocytes Oral and genital epithelium Oropharyngeal cells Respiratory epithelium Nasal epithelium

T lymphocytes

six families listed in Table 2-7 contains members that cause respiratory disorders any of which can progress to pneumonia in the elderly. The influenza viruses of family Orthomyxaviridae are of the greatest concern because the elderly are so susceptible and because each new flu season may bring an antigenic variant arising from the “drift” and “shift” in antigenic types that are so typical of influenza viruses. The far-right column of Table 2-7 is headed “Persistence.” Two families (four types) of viruses are listed to illustrate persistence. There are three types of persistence, termed “chronic” (diffuse or focal), “latent,” and “slow.” Here, we are interested in latent persistence. Latency refers to the fact that some viruses may integrate into the genomes of host cells where they persist for extended periods without replicating or killing host cells and without causing disease. An outstanding example of latency in a bacterial infection is that of the Mycobacteria, which cause tuberculosis. The four viruses listed in the table are representative of persistence. This phenomenon may be of unrecognized importance in aging humans. It should be stressed that (to our knowledge) there is no irrefutable evidence that persistent viruses afflict the elderly inordinately. However, it should be stressed equally that (to our knowledge) there have been no systematic studies of that possibility. In the following brief paragraph, evidence is marshalled to support the idea that more attention should be focused on assessing persistent viral infections in aging humans.

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Table 2-7 Significant Viruses of Aging Humans Family

Virus

Coronaviridae

Coronaviruses (two major types) Orthomyxoviridae Influenza (A,B,C) Paramyxoviridae Respiratory syncytial virus Picornoviridae Rhinovirus (>100 serotypes) Adenoviridae Adenovirus (numerous serotypes) gastroenteritis Herpesviridae Herpes simplex

Cytomegalovirus

Varicella-zoster Virus

Disorder

Persistence

Common cold (sinusitis) Influenza (pneumonia) Respiratory infections (upper and lower) Respiratory infections (common cold) Respiratory infections (colds, pneumonia) Gingivostomatitis, genital herpes, herpetic keratitis, encephalitis Mononucleosis (multiple organ infection in immunocompromised) Herpes zoster (shingles)

No No No No Yes

Yes

Yes

Yes

Consider, first, the adenoviruses. There are at least six subgenera and numerous serotypes of human adenoviruses. The principal targets of the viruses are the respiratory tract, ocular tissues and, less frequently, the GI system. The ability of a few types of human adenoviruses to induce tumors in hamsters and transform human and animal cell lines has attracted attention for many years although there is little evidence that they are oncogenic in humans. The adenoviruses present a classical example of latency. The viruses or their genomes are found in tonsils. Cells of the tonsils of individuals who have experienced infections but have been symptom-free for extended times may have whole or partial virus genomes integrated in their own genomes. It is uncertain how long the virus genomes may continue to replicate in individuals who remain symptom-free. Whether or not latent adenoviruses may be reactivated under certain conditions in aging subjects is a question that seems not to have been addressed. The establishment of latency generally involves integration of viral genomes into the host cell genome or occasionally an episome. Integration of adenovirus DNA has been demonstrated in transformed human cells and in virusinduced tumors in hamsters; and integrated viral DNA may persist for long

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periods in human tonsil cells. What restricts the viral replication in those cells, and the events or factors that trigger reactivation, are unknown. Clearly, this emergence from latency deserves careful study on the hypothesis that adenovirus and other viruses may be reactivated in the immunodeficient elderly and precipitate disease. Three of the viruses in the family of Herpesviridae that are well known for their latency are listed in Table 2-7. First, there is herpes simplex, which exists as two closely related types (HSV-1 and -2). The former is primarily responsible for gingivostomatitis in young children, the latter for genital herpes in adults. HSV-1 is the principal cause of focal, sporadic encephalitis, which in the United States occurs in approximately 1 in 150,000 population. Second is the cytomegalovirus (CMV), which when acquired congenitally (approx 1% of live births in the United States) causes severe disease in infants and young children. In adults and older children, CMV may cause a mononucleosis which resembles that caused by Epstein-Barr virus. The third herpes virus known for latency is varicella-zoster virus (VZV), the causative agent of chickenpox, which may occur in children or adults. Reactivation of VZV may produce herpes zoster (“shingles”), which appears in about 1% of individuals over age 50. HSV-1 and -2 infections occur preferentially at mucocutaneous sites. As the infection and accompanying inflammation progress, the viruses ascend peripheral sensory nerves to reach dorsal root ganglia. The viruses replicate in nervous tissue and then migrate in retrograde fashion along axons to reach other mucosal and epithelial surfaces thus spreading the infection. Latency is established in cells of the dorsal root ganglia. Herpes simplex encephalitis affects preferentially the temporal lobe of the brain and can be initiated by reactivated viruses in addition to viruses of the primary infection. Primary CMV infections occur most efficiently in salivary glands and kidneys. Persistent infections are found in those tissues and in breast, endocervix, seminal vesicle tissues, and peripheral blood leukocytes. Patients with deficient immune systems, such as bone marrow transplant recipients and those with immunodeficiency diseases, are at risk of primary or reactivated CMV infections. In those patients, infection may involve the lungs, GI system, liver, and other organs/tissues, and often becomes life-threatening. It would be interesting, and probably quite significant, to determine whether, and how frequently, CMV-induced respiratory and GI disorders occur in the aging population as a consequence of reactivation of host cell-integrated viral genomes. Similar to HSV-1 and -2, VZV assumes latency in the dorsal root ganglion. Herpes zoster appears as a result of reactivation of latent virus. An important fact about herpes zoster stands out; viz., acute neuritis is characteristic in most patients whereas the frequency of postherpetic neuralgia occurs in about half of the adults, but not in juveniles, and the frequency seems to increase in older

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patients. As noted above, herpes zoster occurs with a frequency of about 1% in adults over 50. All four of the described latent viral infections are serious problems in immunocompromised individuals in whom multiple organ sites are involved. It is well established that aging humans (and laboratory animals) are deficient in one or more aspects of immunity and that T-lymphocyte-dependent antiviral immunity is one such aspect (Chapters 3 and 4). We are not so foolish as to suggest that an elderly subject is similar to an AIDS victim or an individual under treatment with an immunosuppressive drug; however, we do suggest that lessons learned from those patients may be applicable to the elderly. Under conditions of good health and environmental circumstances, most elderly persons retain sufficient immunological potential to cope effectively with acute infections. However, when the immune potential is further reduced by illness, injury, stress, or severe xenobiotic (pollutant) exposure, many elderly subjects may become vulnerable to microbial pathogens. Those are precisely the insults and injuries that are known to activate latent viruses. The need for effective, safe antiviral drugs will continue with increasing urgency in the years ahead. One reason it has been difficult to find or develop antivirals is because viruses utilize so much of the host cell machinery for their own fabrication. Another reason is the extreme ingenuity displayed by viruses to defend and protect themselves as shown in Table 2-8 (85). A new, promising direction toward antivirals is that of interfering with, or redirecting, viral association with cellular receptors and is based on detailed structural knowledge. For example, the counter-receptor site (“knob domain”) in association with the binding domain of the cognate receptor (the Coxsackie and adenovirus receptor, or CAR) has been crystallized and analyzed to the 2.6 A resolution level (86). Whether or not the extensive viral use of receptors involved in key host cell functions will allow the development of discriminating antivirals remains to be discovered. PROTOZOAN PARASITES IN AGING SUBJECTS There are no reliable data on the relative susceptibility of aging humans to animal parasites (i.e., parasites other than microbial) or on the relative severity of parasitic infections in aged compared to young or middle-aged subjects. The principal reasons for this dearth of information are (a) parasitic infections are largely restricted to tropical climates in underdeveloped regions of the world where public health records are limited, and (b) where parasites abound, the majority of the population carries chronic infections acquired in childhood or young adulthood. Similarly, there are few data concerned with parasitic infections relative to age in natural animal populations. An example of the few published studies in animals reported an analysis of cattle infected with the parasite,

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Table 2-8 Intracellular Defense Strategies Used by Viruses Type of Virus Epstein-Barr

Host cell antivirus mechanism Apoptosis (cell death)

Virus counter strategies Homologs of bcl-2

Rabbit pox

Serpins

Simian virus 40

p 53 binding protein

Herpes virus

Intracellular signaling

Myxoma virus

Tyrosine kinase modulation Receptor mimicry

Adenovirus Cytomegalovirus

Viral antigen presentation

MHC Class I suppression

Molluscum contagiosum

Oxidative stress response

Antioxidant selenoprotein

Modified from ref. 85.

Onchocerca ochengi (87). Those cattle lived in an area of high endemicity in the Cameroon and 71% of those studied were infected. Although there was no difference in the prevalence of infection among the three age groups studied (1.5–2.5 years, 3–5 years, >8 years of age), the parasite burden (“worm load”) was significantly greater in the group >8 years of age. In contrast, there was a significantly lower number of the immature forms (microfilariae) in the older compared to the younger cattle. Whether or not this latter finding reflected more effective immunity or some other, inimical physiological change with age could not be determined. It is necessary, therefore, to extrapolate from experimental studies in laboratory animals to gain insight concerning the abilities of elderly humans to cope with parasites. The earliest study (of which we are aware) was of infections of rats of different ages with the nematode, Trichinella spiralis (88). The data suggested that the severity of infection (parasite burden) was significantly greater in the oldest animals. Apparently, there have been no other studies with parasites other than protozoa. The work of Gardner and Remington has shown clearly that aged mice develop significantly worse infections with Toxoplasma gondii than do younger mice (89,90). T. gondii is not a natural human parasite but can infect normal infants and young children in whom it may cause serious central nervous system disorders. T. gondii is one of the major opportunistic, protozoan infections in AIDS victims. The susceptibility of aged mice was shown to be, in part, the

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result of decreased antibody production against the parasite in both the acute and chronic phases of the infection. However, of greater importance was the finding of a pronounced difference in the activation of macrophages of young and aged mice during the acute phase of infection. T. gondii is an intracellular parasite and, therefore, immunological resistance is primarily a cell-mediated process. The depressed activity of macrophages, which play key roles in natural/innate resistance and immunity to intracellular parasites, was considered responsible for the heightened infections found in aged mice. The effects of senescence on macrophages are discussed in Chapters 3 and 4. However, T cells (both CD4+ and CD8+) play roles in immunity to T. gondii (91) and those cells are significantly altered by senescence, as is discussed later. The protozoan, Trypanosoma musculi, is a natural parasite of mice. It infects all of a number of inbred strains of mice; however, the severity of infection, judged by the parasite burden, varies over a 20-fold range (approximately) among different strains (92). Regardless of the strain, however, aged mice develop significantly worse infections. This is illustrated in Figure 2-1, where the course of infection in young and aged mice of strain A is depicted. T. musculi organisms live extracellularly in the bloodstream of mice and the parasite burden can be assessed by determining the level of parasitemia, i.e., counting the numbers of parasites in blood samples. T. musculi infections are self-limiting; i.e., after a prolonged period of about 3 weeks in young adult mice the infections terminate. Thereafter the cured mice are permanently immune to reinfection. As Figure 2-1 shows, both the parasite burden (parasitemia) and the duration of infection (time before the cure) are markedly extended in aged compared to young adult mice. To demonstrate that the elevated parasitemia in aged mice was a reflection of a deficient immune response, the technique of adoptive conferral (“transfer”) of immunity was employed. The conferral of immunity to T. musculi on irradiated, immunologically incompetent mice by the transfer of a predetermined, optimum number of spleen cells from normal, infected, or cured donor mice was evaluated. After receiving the donor spleen cells, the irradiated recipient mice were inoculated with viable T. musculi and the course of infection monitored. The results of such a study, in which equivalent numbers of spleen cells were transferred from young or aged infected donor mice into irradiated young-adult recipients, are depicted in Figure 2-2. Irradiated mice, lacking a competent immune system, that were inoculated with T. musculi but given no donor spleen cells died from overwhelming T. musculi infection (Fig. 22A).The transfer of spleen cells from young donor mice on day 7 of their infection was able to protect irradiated recipients and cure their infection in about three weeks (Fig. 2-2B). In contrast, the same number of spleen cells from aged donors on day 7 of infection conferred no protection on the irradiated

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recipients (Fig. 2-2B). On day 14 of infection, spleen cells from both young and aged donor mice were able to protect irradiated, young recipients from lethal T. musculi infection. However, the cells from young donors were much more efficient than those from aged donors as shown by the marked differences in levels of parasitemia and duration of infection in the recipient mice (Fig. 2-2C). Finally 21 days after initial infection, cells from aged donors were able to protect aged recipients but only after a prolonged infection (Fig. 2-2D). The two preceding examples of the relative inability of aged mice to cope with protozoan infections provide compelling evidence that senescence cripples the immune system. In both cases, there is considerable understanding of the nature of the immune response against the parasites in young adults as is discussed in Chapters 3 and 4. It should be stressed here that the two parasites, T. gondii and T. musculi, are quite different in their life cycles and in other aspects. T. gondii are intracellular parasites whereas T. musculi are extracellular. Immunity to T. gondii is a cell-mediated process whereas immunity to T. musculi is dependent on specific antibodies, probably of IgG2a isotype (mouse) (93). T. gondii will establish infection in several hosts (cats, mice, humans) whereas T. musculi is strictly a mouse-specific parasite. Considered together, studies of these two protozoa suggest that the ability of aged mice to generate both humoral and cell-mediated immunity to pathogens is impaired. FUNGAL INFECTIONS IN AGING SUBJECTS There have been few attempts to evaluate the frequency or severity of fungal infections in aging subjects. On the other hand, it is well-established that fungal infections are rather common in other immunodeficient individuals such as AIDS victims, persons being treated with immunosuppressive drugs, patients on antibiotic therapy or suffering from burns, diabetes, or malnutrition (94). One study in particular, strongly indicates that more attention should be given to fungal infections in the aging (95). In that study, the frequency of mortality as a consequence of systemic infections with bacteria (bacteremia) or fungi (fungemia) was assessed from the medical records of 500 patients identified as having true bacteremia or fungemia. The parameters relevant to the present discussion that were considered included: 1) mortality associated with both bacteremia and fungemia; 2) the primary site of the infection; 3) body temperature; and 4) the degree of leukopenia. There was a substantial increase in the risk of death of subjects over age 50 and deaths were more frequent in males than females. The risk of death was significantly greater when the primary site of infection was a surgical wound, a burn or even untraumatized skin, an abscess, or the respiratory tract compared to other sites. There was a markedly greater (about fivefold) frequency of mortality of patients whose body temperature was less than 36°C compared to those whose temperature was over 40°C. A peripheral leukocyte count

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Fig. 2-1. Course of parasitemia in young (䊉) and aged (䊊) A/He mice following inoculation with T. musculi. Four or five samples per point. Bars represent 1 S.E.M. (From Albright JW, Albright JF. Mech Ageing Dev 1982;20:315–330.)

of less than 4000/µL, or a granulocyte count of less than 1000/µL, both correlated with substantially higher mortality. Given that (a) injury and trauma (i.e., stress) significantly alter immune responses in the elderly (see later), (b) the skin and respiratory systems of the elderly are sites of common fungi that are benign in younger individuals (e.g., ref. 96), and (c) that elderly humans are less disposed to run fevers (97), it is difficult to avoid the conclusion that fungal infections are significant problems in the elderly. Emerging, opportunistic fungi (98) and drug-resistant fungi (99) will begin to compound the problems for the elderly in the near future. CHAPTER SUMMARY There are no known microorganisms that uniquely infect elderly humans. Overt diseases caused by some pathogens (e.g., tuberculosis, pneumonia, influenza, UTIs, sepsis) are clearly more common in the elderly. The reasons

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Fig. 2-2. Course of parasitemia in irradiated A/He recipients of 3 × 107 spleen cells from : (A) uninfected donors; (B) donors on day 7 after trypanosome inoculation (*indicates parasites at this time largely transferred as contaminants of donor spleen cell preparations); (C) donors on day 14 after trypanosome inoculation; (D) donors on day 21 after trypanosome inoculation. Donor-recipient combinations: (䊊) young donor-young recipients; (䊉) young donors-aged recipients; (䊐) aged donors-young recipients; (䊏) aged donors-aged recipients. Data from one of two replicated experiments, 5–6 samples per point. Bars represent 1 SEM. (D means all were dead). (From Albright JW, Albright JF. Mech Ageing Dev 1982;20:315–330.)

for this include (a) functional and anatomical changes in organs (e.g., respiratory, urinary), (b) comorbidity, and (c) decline in immunological competence. With regard to the latter, research with experimental animals has provided compelling evidence that immunological resistance to infection declines with age. Such is the case of infections with M. tuberculosis, S. typhimurium, T. gondii, T. musculi, and probably L. monocytogenes. Bacterial infections begin with attachment of the microorganisms to host cells (epithelial, endothelial) followed by secretion of toxins and other virulence factors both outside and inside (type III secretion) host cells. Attachment involves a number of well-known cell adhesion receptors and counter-receptors as well as some that are restricted to microorganisms. In some cases, bacteria may secrete their own receptors into host cells. In order to survive in the hostile environment of the host, microorganisms often engage in colonial orga-

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nization. To form organized colonies, called biofilms, bacteria reproduce rapidly and upon reaching a certain density, lay down a matrix that protects them from host defenses, including both humoral and cell-mediated immunity. Those bacteria that can form biofilms (many, perhaps most, species can) are able to sense population density and to distinguish between self and other species, an ability termed quorum sensing. The formation of biofilms also renders colonized bacteria impervious to antibiotics. Is it possible that biofilm formation by bacteria (or fungi that also appear capable) that have been selected for resistance in the elderly is a major factor in the elevated morbidity and mortality that characterize the aging? At present, very little is known about bacterial adherence and the efficiency of secretion of virulence factors into host cells of aging subjects. There are limited data that suggest that intercellular adhesion is altered in aging humans, perhaps as a consequence of the effect of senescence on cytoplasmic membranes. If so, it is likely that changes occur in bacterial adherence to host cells. Similarly, very little attention has been given to date to evalulating the relationships between susceptibility of aging humans to infection and the formation of biofilms in aging subjects. Both persistent (latent) bacterial and persistent viral infections in the elderly require attention. Tuberculosis appearing as a result of reactivated Mycobacteria is a classical example of bacterial latency. It is probable that bacterial latency is much more important in the pathogenesis of infectious diseases than has been realized (see ref. 100). Herpes zoster (shingles) is one of several well-known examples of viral latency that result in diseases in the elderly. It is likely that the decline in immunological potential contributes in a major way to the reactivation of latent infections in aging subjects. However, this has not been afforded rigorous proof. If it is true, it becomes important to ask which of numerous other latent infections reappear in the elderly. Has sufficient attention been given to this question? To what extent are the well-known opportunistic infections in immunocompromised individuals paralleled in less severe degree in the elderly? Has this question received adequate attention? Finally, it should be pointed out that both parasitic (especially protozoan) and fungal infections in the elderly deserve much more concern than they have received to date. Both environmental pollution and global warming are likely to precipitate a significant increase in the prevalence of infections by those organisms in the years ahead. And in the case of both protozoa (e.g., malaria) and fungi (e.g., Candida spp.) drug-resistant forms are already with us. REFERENCES 1. Yoshikawa TT. “Perspective”: Aging and infectious diseases; past, present and future. J Infect Dis 1997;176:1053–1057.

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2. Chan ED, Welsh CH. Geriatric respiratory medicine. Chest 1998;114:1704–1733. 3. Nicholson KG, Kent J, Hammersley V, Cancio E. Acute viral infections of upper respiratory tract in elderly people living in the community: Comparative, prospective, population based study of disease burden. Brit Med J 1997;315:1060–1064. 4. Nordenstam GR, Brandberg CA, Oden AS, et al. Bacteruria and mortality in an elderly population. New Engl J Med 1986;314:1152–1156. 5. Eykyn SJ. Urinary tract infections in the elderly. Brit J Urol 1998;82(S1):79–84. 6. Yoshikawa TT, Norman DC, eds. Infections in the Aging: A Clinical Handbook. 2000. Humana, Totowa, NJ. 7. Moore WEC, Holdeman LV. Discussion of current bacteriologic investigations of the relationships between intestinal flora, diet and colon cancer. Cancer Res 1975;35:3418–3420. 8. Simon GL, Gorbach SL. Intestinal flora in health and disease. Gastroenterology 1984;86:174–193. 9. Norman DC, Yoshikawa TT. Infection and fever in the elderly. In: Cunha BS, ed. Infectious Diseases in the Elderly. Littleton, MA: PSG Publishing, 1988:18–23. 10. Bartlett JG. Anaerobic bacterial infections in the lung. Chest 1987;91:901–909. 11. Saltzman JR, Tussell RM. The aging gut: nutritional issues. Gastroenterol Clin North Am 1998;27:309–324. 12. Toskes PP, Gianella RA, Jervis HR, et al. Small intestinal mucosal injury in the experimental blind loop syndrome. Gastroenterology 1975;68:1193–1203. 13. Gianella RA, Rout WR, Toskes PP. Jejunal brush border injury and impaired sugar and amino acid uptake in the blind loop syndrome. Gastroenterology 1974;67:965–974. 14. Gracey M, Papadimitriou J, Bower G. Ultrastructural changes in the small intestines of rats with self-filling blind loops. Gastroenterology 1974;67:646–651. 15. Dutt AK, Stead WW. Tuberculosis in the elderly. Med Clin North Am 1993;77:1353–1368. 16. North RJ. Minimal effect of advanced aging on susceptibility of mice to infection with Mycobacterium tuberculosis. J Infect Dis 1993;168:1059–1062. 17. Orme IM. Responsiveness of macrophages from old mice to Mycobacterium tuberculosis and its products. Aging: Immunol Infect Dis 1993;4:187–195. 18. Orme IM. Mechanisms underlying the increased susceptibility of aged mice to tuberculosis. Nutr Rev 1995;53:S35–S40. 19. Cooper AM, Callahan JE, Griffin JP, et al. Old mice are able to control low dose aerogenic infections with Mycobacterium tuberculosis. Infect Immun 1995;63:3259–3265. 20. Ting LM, Kim AC, Cattamanchi A, Ernst JD. Mycobacterium tuberculosis inhibits IFN-gamma transcriptional responses without inhibiting STAT 1. J Immunol 1999;163:398–406. 21. Stenger S, Mazzaccaro RJ, Uyemura K, et al. Differential effects of cytolytic T cell subsets on intracellular infection. Science 1997;276:1684–1687. 22. Patel PJ. Aging and antimicrobial immunity: Impaired production of mediator T cells as a basis for the decreased resistance of senescent mice to listeriosis. J Exp Med 1981;154:821–831.

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3 Senescence of Natural/Innate Resistance to Infection If an experiment does not hold out the possibility of causing one to revise one’s views, it is hard to see why it should be done at all. —Peter Medawar, Advice to a Young Scientist

It has been realized for decades that in humans and higher vertebrates there are, in addition to the adaptive immune system, accessory mechanisms and systems that contribute to overall immune defense against infectious organisms. They have been grouped under the heading “natural” or “innate” immunity. They include the reticuloendothelial system comprising “fixed” and “mobile” (circulating) monocytes (Mo’s) and macrophages (MPs), polymorphonuclear (PMN) cells (especially neutrophils and eosinophils that can discharge antimicrobial peptides such as defensins), natural killer (NK) and other “naturally cytotoxic” (NC) cells, and the complement (C) system; all of which have been preserved and handed down during the evolution of higher vertebrates from primitive vertebrates and invertebrates. The significance and importance of the innate system (sometimes called the “constitutive system”) and its complementary relationship to the adaptive (acquired) immune system have been clearly recognized only in the last decade (1,2). In the vertebrates, it is the job of the rapid-response, innate system to prevent infections from overwhelming the host before the more powerful, but more slowly developing, adaptive response is manifested. In addition, the innate response facilitates the development of the adaptive response by way of C components, cytokines and chemokines, and overlapping receptors and signaling pathways.

From: Aging, Immunity, and Infection By J. F. Albright and J. W. Albright © Humana Press Inc., Totowa, NJ

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PATTERN RECOGNIZING RECEPTORS OF INNATE IMMUNITY Certain conceptual difficulties that arose with the recognition of the roles played by the innate system in vertebrates are framed by the following questions: (a) how does the innate system, which possesses limited recognitive diversity, detect microbial substances and distinguish them from self? and (b) how can a sufficient number of reactive host cells be mobilized in a very short time (roughly, 12 h or less)? The answer to the first question lies in the use of pattern-recognizing receptors (PRRs) by the innate system to recognize microbial constituents. Those constituents comprise conserved structural cores that cannot be significantly altered by mutations without destroying the pathogenicity of the microorganism. Those constituents that form common patterns on microbes include lipopolysaccharides of Gram-negative bacteria, lipoteichoic acids of Gram-positive bacteria, lipoproteins of bacteria and parasites, glycolipids of mycobacteria, mannans of yeast, and double-stranded RNAs of viruses. It is probable that “receptors for these structures have been selected over evolutionary time to provide broad-spectrum recognition of harmful foreign materials” (1). The answer to the second question also invokes the use of PRRs. The latter are not clonally restricted; that would limit their distribution to relatively few cells. Rather they are likely to be present on the majority of certain types of cells. If all the cells that possess a given, broad-spectrum receptor can be activated into effectors quickly, rapid control of a pathogenic infection can occur. That response to infection will be quite different from the adaptive response, which begins with the activation of a small number of lymphocytes of a clone displaying cognate receptors for a precise epitope of a pathogen. The ensuing development of a controlling response will be delayed by the time required for proliferative expansion and maturation of the effector cells. There are several receptors of broad specificity for common microbial components. Four that have been most thoroughly studied are: (a) mannose-binding protein (MBP), (b) the mannose receptor (MR), (c) a group of homologous proteins termed Toll-like receptors (TLRs), and (d) a family of related proteins known as scavenger receptors (SRs). Mannose-Binding Protein (3,4) MBP is produced by the liver and released as an acute phase reactant. It belongs to a family of related proteins termed “collectins”; other members include lung surfactant proteins, SP-A and SP-D, bovine conglutinin, and bovine collectin-43. The collectins exhibit a broad range of target binding. For example, they associate with Gram-negative bacteria, yeasts, Pneumocystis carinii, influenza, and HIV viruses. SP-A and SP-D interact with airborne particulate material including pollen grains. The common feature of the target substances is their accessible carbohydrates (oligosaccharides).

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Structural information about MBP is informative with regard to pattern recognition. The seemingly broad range of target specificity does not include the carbohydrates associated with self-glycoproteins. The explanation for this important restriction lies in the particular spatial orientation of the hydroxyl groups situated at positions 3 and 4 of the hexose moiety (e.g., N-acetylglucosamine, glucose, fucose, mannose) common to many microorganisms (5,6). In comparison, the hydroxyl groups of those sugars commonly found at the termini of mammalian glycoproteins (viz., sialic acid and galactose ) are not recognized by MBP. Additional information about the structure of MBP aids in understanding its function of eliminating pathogenic organisms (7). It is particularly important that the assembled polymers of MBP molecules can activate the C system thus promoting the destruction of pathogens. This is a consequence of the structural similarity of MBP to the first component, C1q, of the C system. The individual molecules of MBP are trimers composed of a collagen domain, a neck region, and a globular carboxy-terminal, C-type lectin-binding domain. The three polypeptide chains in the neck region are properly aligned and trimerized as a result of interactions between three parallel α-helical coiled coils (8,9). Molecules of MBP, like those of SP-A, assemble as hexamers of trimers and resemble a “bunch of tulips” (10,11) (Fig. 3-1). Until recently, it was believed that MBP is able to interact with the two serine proteases, C1r and C1s, and thus to activate the classical complement pathway through C4 (12). It is now clear that two novel serine proteases, MASP-1 and MASP-2 (i.e., “mannan-binding-lectin-associated serine protease”), act similarly to C1r and C1s to cleave molecules of C4 and C2 and to effect cleavage of the pivotal component C3; or a complex of MBL-MASP-1 and another factor, Map 19, may act directly to activate C3 (7). In addition to its ability to effect complement activation, MBP can directly mediate phagocytosis of microorganisms (13), i.e., serve as an opsonin not involving complement components. Presumably, this is achieved by way of an MBP receptor that has not been clearly identified but may be complement receptor 1 (CR 1). Both SP-A and SP-D are surfactants and are particularly important in defense against infections in the lungs. Both interact with Gram-negative bacteria. SP-A has been shown to promote phagocytosis in the aveoli whereas SPD probably does not (14). Mannose Receptor The MR facilitates the phagocytic and endocytic ingestion of both particulate and soluble glycoconjugates. It comprises eight C-type, CRDs through which glylcoconjugates are bound providing they display accessible mannose, fucose, or N-acetylglucosamine residues (15) (see Table 3-1). Only one of the

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Fig. 3-1. Superimposed molecules of human (black) and rat (white) trimeric mannose binding proteins (MBPs). (A) View down the threefold axis of superimposed α-helical coiled coils of human and rat trimers. (B) Sideview of (a) rotated 90° showing resemblance to a “bunch of tulips.” (From Epstein J et al. Curr Opin Immunol 1996;8:29–35.) Table 3-1 Binding of Mannose Receptor Fragments Expressed In Vitro to Immobilized Monosaccharides Relative bindingb

Immobilized Monosaccharidea Man Fuc Glc Nac Glc Gal

CRDs 1–3 <0.02 <0.04 0.08 0.16 0.16

CRDs 4–5

CRDs 6–8

1.00 1.42 1.14 1.05 0.17

0.05 <0.02 0.05 <0.04 0.04

a Man,

mannose; Fuc, fucose; GlcNAc, N-acetylglucosamine; Glc, glucosamine; Gal, galactose. bBinding is expressed relative to the retention of CRDs 4-5 on mannosesepharose. Data adapted from ref. 15.

eight domains, viz. CRD4, has significant binding energy and specificity on its own; the remaining domains collectively contribute additional binding energy. There is a soluble form of the MR (15,16), which has been found capable of binding selectively to metallophilic MPs in the marginal zone of the spleen and subcapsular sinus of lymph nodes. There, the binding appears to involve inter-

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action between the cysteine-rich, N-terminal end of the MR and sialoadhesin present on the surface of MP. Toll and Toll-Like Receptors The protein termed “Toll” was discovered to be involved in the establishment of dorsal-ventral organization during embryonic development in Drosophila (see refs. 17, 18, and 19 for a review). At about the same time, it was recognized that Toll and the Toll-signaling pathways are instrumental in the resistance of Drosophila to infection (2,19,20–22). Immunity in Drosophila is, of course, of the innate variety involving a series of antimicrobial peptides each having specificity for a particular set of pathogenic microorganisms (22). Seven or more distinct peptides are responsible for the resistance of Drosophila to infection. One of these, drosomycin, is a powerful antifungal agent. Another peptide, attacin, is selectively toxic for Gram-negative bacteria. The Toll protein serves as the receptor to promote production of drosomycin whereas another related receptor, “18-wheeler,” is involved in attacin production in response to Gram-negative bacteria. Sequences upstream of each of the seven peptide-encoding genes represent sites that engage transcription factors belonging to the Rel and nuclear factor kappa B (NFκ B) family of inducible transactivators (2,22). Receptors and signaling pathways quite similar to the Toll family in Drosophila have been discovered in a variety of other organisms ranging from plants to humans (2). In humans, nine Toll-related receptors have been reported, the TLRs (23,24). To date, TLR 2 and TLR 4 have received the most attention. An analysis of the distribution of TLR 4 among human cells of the immune system was performed by isolation and identification of TLR 4 mRNA (23). It was found in DCs, γδ T cells, Th1 and Th2 T cells, and B cells. In addition, an active form of the receptor was expressed in monocytic cell lines and was capable of triggering NFκB activation and cytokine production. The Toll and TLRs in Drosophila, plants, and humans are of a structure that enables them to perform similar functions in different organisms. The intracellular, cytoplasmic domains of the receptors show a significant degree of homology with the interleukin-1 receptor (IL-1R) signaling domain; this is termed the Toll/IL-1R homology region. It is this region that transduces signals upon ligand occupancy of the external domain of the receptor. The outstanding characteristics of the external domain are the unusual frequency of leucine residues (“leucine-rich”) and a modest degree of amino acid sequence diversity. Several studies have led to the conclusion that the diversity of the extracellular, leucine-rich domains is critical in determining host recognition of, and responses to, “pathogen associated molecular patterns” (PAMPs) (25). In mammals, TLR 2 (but not TLR 4) is involved in responsiveness to microbial lipoproteins such as found, for example, on mycobacteria (26), certain

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spirochaetes (27), and Borrelia burgdorferi (28). TLR 2 appears to be the primary mediator of responses to Gram-positive bacteria (29,30). On the other hand, TLR 4 is the principal form of the receptor that mediates responses to lipopolysaccharides (LPs) (31–34). Reports that TLR 2 also mediates LPS responsiveness (35,36) probably can be explained by the contamination of LPS preparations by highly active endotoxin-associated proteins (37) or by crossconnections of TLR 4 and TLR 2 cytoplasmic signaling pathways (38). Antigen-processing and -presenting cells (APCs) may be considered pivotal in that they are instrumental in both innate and adaptive immune responses. Mo/MPs and immature and mature DCs are the key APCs and it is, therefore, essential to understand the roles played by TLRs in the mechanisms employed by APCs to recognize and respond to PAMPs. Those roles include the following: (a) mediating the uptake of pathogens by phagocytes, (b) promoting the intracellular destruction of ingested pathogens, and (c) triggering signaling pathways that lead to transcription of appropriate genes such as antimicrobial peptides, inflammatory cytokines, and enzymes involved in formation of reactive oxygen and nitrogen radicals. Considerable effort has been made to elucidate the involvement of TLR 4 in the activation of MPs by LPS, which is an endotoxin found on the outer membrane of Gram-negative bacteria. Current evidence indicates that TLR 4 is one of several components of a receptor and signaling complex that binds LPS at the surface of MPs (see Fig. 3-2). An important discovery was the involvement of the LPS binding protein, CD14, in the attachment of LPS to the cell surface (39,40). However, CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein (40) and incapable of transducing signals to pathways in the cytoplasm. TLR with the IL-1R signaling domain became an obvious candidate as a co-receptor with CD14 when it was found that certain strains of mice that respond weakly to LPS (C3H/He J and C57BL/10 ScCR) suffer mutations at key positions in the tlr 4 gene (31,32). Recent work (34) has contributed importantly to understanding the LPS receptor complex on macrophages and to clarifying the significance of LPS dosage on activating the expression of various genes in macrophages. A chemical analog of Taxol, which is an LPS mimetic, was employed for photoaffinity crosslinking of molecules near to the LPS receptor. In this way, it was learned that Taxol binds to the common CD18 subunit of β2 integrins, especially to CD11b/CD18 (Mac-1 or CR 3). An extensive series of studies was performed to investigate the involvement of CD14, TLR 4, and CD11b/CD18 in the expression of various LPS/Taxol-activated genes in macrophages. The results of those studies led to the conception of the LPS/Taxol receptor complex depicted in Fig. 3-2. In that conception, interaction of all three components of the receptor complex, CD14, TLR 4, and CD11b/CD18, with the ligand LPS or

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Fig. 3-2. Model of the interactions between CD14, TLR 4, MD 2, and CD11b/ CD18 to form a receptor/signaling complex on the surface of macrophages that is capable of transducing signals following LPS or Taxol binding. (From Perera, P.Y., et al. J. Immunol, 2001;166:574, with permission.)

Taxol results in expression of the full set of inducible genes (TNFα, IL-12 p35, IL-12 p40), cyclooxygenase-2 (COX-2), IFN-inducible protein-10 ( IP-10 ) and IFN consensus sequence binding protein (ICSBP). The cytoplasmic domains of both TLR 4 and CD11b/CD18 are envisioned as substrates for kinases and phosphatases involved in the initial steps of signaling. In addition, it was concluded that there is a CD11b/CD18-independent pathway for activating the expression of genes that encode TNFα, IP-10, and ICSBP. An interesting finding was the apparent ability of high doses of LPS or Taxol to activate expression of TNFα by way of TLR 4 (and the essential cofactor MD 2) but independent of both CD14 and CD11b/CD18. The results of this seminal investigation led to the following general conclusion regarding macrophage recognition of and stimulation by microbial lipopolysaccharide: “...LPS or Taxol leads to the formation of multimeric receptor complexes that elicit complex patterns of signaling, which, in turn, dictate which genes are activated.” It was also noted as being likely that “these individual receptors are brought together following ligand binding and that each contributes toward the production of general signaling molecules, such that the overall effect of these individual receptor associations determines the threshold, strength, and the specificity of each response and the genes induced” (34). The latter statement points to the possibility that the formation of “these individual receptor associations” occurs in lipid rafts as is the case in the assemblage of the B- and Tcell receptor complexes (see Chapter 4). As is discussed later, it is quite likely

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that the formation of lipid rafts in cell membranes may be deranged as a consequence of senescence and that the impaired functions of macrophages (and other types of cells) may reflect the improper assemblage of key receptors. Both TLR 2 and TLR 4 are expressed on macrophages. It appears they provide those cells with the ability to assess the types of organisms that have been ingested and to make appropriate responses. This is achieved by the ingestion of the receptors in association with the phagosomes whereupon they “sample” the organism contained in the phagosome and initiate appropriate signaling (41). Chinese hamster ovary (CHO) cells, which lack normal (wild-type) TLR 2, were transfected with a wild-type TLR 2 gene. In addition, they were provided with the gene for CD14 that facilitates activation of TLR 2 by bacterial components. The ingestion of zymosan particles, which requires the mannose receptor, resulted in strong activation and nuclear translocation of NFκB. The same was true when the cotransfected CHO cells were exposed to S. aureus. A further study on the role of TLR 2 in signaling leading to the production of the proinflammatory cytokine, TNFα, was performed with the mouse macrophage cell line RAW-TT10. Cells of this line were transfected with a mutant form of TLR 2, designated P681H, in which histidine rather than proline was present at position 681. It was found that this dominant negative mutation resulted in failure to produce TNFα as well as failure of NFκB translocation when the cells ingested zymosan. Similar cells not endowed with the mutant TLR 2 were not affected in their ability to produce TNFα or translocate NFκB. The mutant form of TLR 2 did not affect zymosan binding to the macrophages or the ingestion of particles inside phagosomes that displayed the TLR 2. Thus, the mutant receptor appeared to be unable to initiate signaling leading to NFκB nuclear translocation and TNFα gene expression. Further studies with this ingenious system revealed that exposure of the macrophages endowed with the TLR2P681 H mutant failed to respond to Gram-positive bacteria (S. aureus) by producing TNFα. In contrast, Gram-negative bacteria as well as LPS elicited normal TNFα production by the TLR 2-mutant macrophages. Conversely, expression of a TLR 4-mutant gene in RAW-TT10 macrophages strongly interfered with TNF production elicited by LPS or Gram-negative bacteria but had no effect on the response to Gram-positive bacteria. Two significant conclusions drawn from the results of this study are: (a) Toll family receptors endow specificity on cells with respect to their proinflammatory responses to different pathogens, and (b) the ingested, phagosome-associated receptors sample the ingested microbes and initiate appropriate signaling. Finally, with regard to TLRs, it is important to note that bacterial stimulation of IL-12 production by human and mouse monocyte/macrophage cell lines involves TLRs (26). The interaction of cell wall components of M. tuberculosis, especially a 19 kD lipoprotein, with TLRs on Mo/MPs not only elicited

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production of IL-12 but, also, expression of the enzyme, inducible nitric oxide synthase, and production of nitric oxide. These results place TLRs at a pivotal point in the intersection of innate and adaptive immunity and suggest a key role for TLRs in the destruction of microbial pathogens. As excellent discussion of TLR involvement in immune responses can be found in ref. 42. SR Family There are three classes, designated SR-A, SR-B, and SR-C, in the SR family. The SR-Cs are found in Drosophila and are not discussed. The proteins of class A and class B receptors share a considerable degree of amino acid homology and certain domains but also differ significantly in structure. Class A receptors of which three are known (designated SR-AI, SR-AII, and MARCO [macrophage receptor with collagenous structure]) are trimeric. They display significant cytoplasmic tails, transmembrane, and spacer domains. SR-AI and A-II possess extramembrane domains of α-helical coiled coils, followed by collagenous domains and, in the case of SR-A1, an N-terminal cysteine-rich domain. MARCO differs from SR-AI and II particularly in lacking the coiled coil domain and having a much longer collagen-like domain. SR-AI and SR-AII are located mainly on monocytes and macrophages. They are found also on some dendritic cells and certain specialized endothelial cells. They may act as adhesion molecules in directing the tissue localization of macrophages (43). Expression of MARCO appears to be limited to macrophages located in the marginal zone of the spleen and the medullary cords of lymph nodes (44). SRs of class B are SR-B1 and CD36. They consist of a single protein chain having a short to moderate cytoplasmic tail (10–45 residues) and an amino terminal domain relatively rich in proline, glycine, and cysteine residues. The class B receptors are found on monocytes, macrophages, B lymphocytes, capillary endothelial cells, platelets, and adipocytes (45–47). SRs attracted attention initially owing to their capability of binding low-density lipoproteins (LDLs) and modified LDLs (e.g., oxidized LDL, “Ox LDL,” and acetylated LDL, “AcLDL”) and their involvement in atherosclerosis. Three of the numerous lines of active investigation are: (a) LDL-SR activation of Mo/ MPs that affects cytokine production (46,47), (b) the nature of Ox LDL binding to SRs (48), and (c) the SR-mediated accumulation of lipids during the pathogenesis of foam cells from MPs (49). The effects of LDL binding on MP production of cytokines are of particular interest. Ox LDL and Ac LDL are rapidly incorporated into MPs via SRs. It was shown that the uptake of Ox LDL resulted in significant reduction in the amounts of LPS-stimulated, specific mRNAs for several cytokines including macrophage chemotactic protein-1, TNFα, and IL-1 (46). This did not occur when Ac LDL was incorporated into MPs. However, depressed levels of the same mRNAs were found in MPs exposed to a modified

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protein, maleylated bovine serum albumin. Maleylation of proteins renders them ligands for SRs (50,51) and enhances protein antigenicity. An important effect of ligating SRs is the reported inhibition of IL-12 production by MPs in response to LPS stimulation (52). Binding of ligands to SRs, Fc gamma receptors, and complement receptors all resulted in similar inhibition of LPS-stimulated IL-12 production. Based on these reports, it appears that ligands that interact with certain of the SRs could severely depress both innate and adaptive immune responses, especially those of the inflammatory, pathogen-resistant type. The SRs are PRRs for components of various pathogens. The depression of inflammatory cytokines upon association of the pathogens with MP-SR could significantly cripple the immune resistance to pathogens. SRs of both class A and class B have received attention with respect to the components of pathogens that may act as ligands (53). SR-AI and -AII recognize various polyanions, especially bacterial LPS and lipoteichoic acid. The most compelling evidence of the importance of SR-A in innate control of infections comes from studies using SR-A knockout mice. SR-AI/II–/– mice were found to be significantly more susceptible to infection with Gram-positive S. aureus than were wildtype mice (54). The knockout mice died of disseminated infections. Phagocytosis, by MPs from knockout mice, in the absence of opsonins, was significantly impaired compared to normal controls. Similar studies with SR-A–/– mice revealed that they were noticeably more susceptible to L. monocytogenes infection than were normal controls (55). Moreover, MPs derived from bone marrow cultures of SR-A–/– mice ingested significantly fewer E. coli in comparison to wild-type cells whereas MPs from knockout and wild-type mice were equally effective at ingesting antibody-opsonized E. coli. The third type of class A SR, known as MARCO, has a limited distributionn on MPs of the splenic marginal zone and in the lymphatic medullary sinuses of lymph nodes. Several agents, LPS, zymosan, Bacillus Calmette-Guerin, and L. monocytogenes, have been found to up-regulate the expression of MARCO on MPs in other sites such as liver and different areas of the spleen (56,57). Although it was not clear that the MARCO induced on MPs in liver and spleen played a significant role in bacterial clearance in vivo, MARCObearing MPs in the splenic marginal zone and lymph nodes clearly became engaged with bacteria, suggesting a role for such MPs in defense against bacterial pathogens. Class B-SR involvement in pathogen pattern recognition is less certain than in the case of class A-SR. Evidence has been presented that CD36 can bind highdensity lipoprotein and promote the intracellular accumulation of lipoproteinderived cholesterol and lipid (58). The presence of SR-B on steroidogenic tissues and liver cells may indicate their involvement in steroid biosynthesis.

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There is increasing evidence that SRs are involved in the well-known phagocytic removal of apoptotic cells that occurs without inflammation. It is likely that phagocytic recognition of effete cells involves recognition of molecular patterns similar to those of microorganisms but distinct from normal self (59,60). The removal of damaged and dying erythrocytes is a related process that is largely a function of liver Kupffer cell phygocytosis. The SRs have not been identified but were shown not to be SR-I and -II (61). Peroxisome Proliferator-Activated Receptors This discussion of PRRs would be incomplete without at least mentioning a group of nuclear receptors/transcription factors, termed “peroxisome proliferator-activated receptors” (PPARs), that appear to moderate, even counteract, certain effects of SRs and possibly other PRRs. In the case of MPs, stimulation via PPARs may moderate or inhibit the expression of antimicrobial mechanisms set in motion by microbial pattern recognition. First, a word about the physiological significance of PPARs. Peroxisomes are small, cytoplasmic organelles, bounded by a single limiting membrane, within which at least two important functions occur: viz., the generation and the destruction of H2O2. The former is a result of β-oxidation of fatty acids and the latter a consequence of the catalase enzyme that the peroxisomes house. Peroxisomes are devoid of nucleic acids and therefore proliferate as a result of growth and subsequent binary fission (62). All of the various proteins present in peroxisomes are synthesized on free ribosomes and subsequently transported into the organelles, guided by a specific leader sequence. Various stimuli such as carcinogens, hypolipidemic drugs, and diverse xenobiotics (63,64), as well as a naturally occurring arachidonic acid metabolite, 15-deoxy-∆12,14-prostaglandin J2 (15d-PGJ2) (65,66), can induce extensive proliferation of peroxisomes. These stimuli exert their effects by way of the PPARs. The PPARs are frequent on the surfaces of cells of adipose tissue, spleen, and adrenal gland as well as on stimulated MPs. PPARs serve a critical role in regulating adipogenesis and the expression of adipocyte genes. Recently, it has been concluded that the stimulation of PPARs by agonists such as 15d-PGJ2 can result in inhibition of the expression of NOS2 (iNOS), a matrix metalloproteinase, and SR-A in MPs stimulated with TNFγ (67). Similarly, it was reported that PPARγ agonists can effect inhibition of production of inflammatory cytokines by MPs stimulated with phorbol ester (68). The results of these studies were interpreted as indicating that the inhibition of the MP inflammatory response resulted from effects on transcription of genes such as those encoding iNOS, TNFα, and IL-2. It should be stressed that a note of caution has been sounded; a study of a panel of PPARγ agonists led to the conclusion that the one agonist that clearly suppressed the MP inflammatory

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response, viz., 15d-PGJ2, exerted its effect by a pathway not involving PPARγ (69). Several other representative agonists were ineffective in interfering with inflammatory responses. Because the previous studies relied primarily on the use of 15d-PGJ2, the conclusions drawn from those studies must remain in doubt for the present. There is a fascinating recent report (70) that treatment of aged mice with the compound, dehydroepiandrosterone (DHEAS), a PPARα activator, resulted in reducing the tissue levels of active NFκB and reduced the levels of mRNA as well as the secreted amounts of several inflammatory cytokines. It will be interesting to learn whether DHEAS and 15d-PGJ2 exert this effect via a common pathway or separate pathways. If the two compounds act synergistically, the combination could be quite useful in correcting what appears to be a common, moderate chronic inflammatory condition in elderly subjects (71). It is apparent that significant changes in the capability of aged individuals to resist and control infections are likely to stem from changes in the receptors associated with innate immunity. Such changes can readily be envisioned to arise from lipid peroxidation, protein modifications, and damage to nucleic acids, all of which are associated with senescence (72–74). As is discussed later, there is substantial evidence that cellular membranes become altered during aging, including cells of both the innate and adaptive immune response. Changes in the density, orientation, conformation, or associations of membrane molecules could readily alter the energy of association between receptors and counter-receptors resulting in altered recognition patterns and/or signaling efficiency. There is a dearth of information concerning this broad topic. The results of the study of the reversal of oxidative stress of cells by treatment with the PPARα agonist DHEAS (70) reveal how potentially rewarding research on this topic is likely to be. Given that the MBPs, MRs, TLRs, and PPARs are all associated with phagocytic cells, it is appropriate to turn next to a discussion of Mo’s and MPs. After that, neutrophils and NK cells, both of which are key elements of innate immunity, are considered. PHAGOCYTIC CELLS: MONOCYTES/MACROPHAGES The functions of Mo’s and MPs are highly complex. They, along with neutrophils, have been called “professional phagocytes” (75). The functions of MPs, for example, are necessary, on the one hand, for removal and destruction of pathogenic microorganisms by way of inflammatory responses. On the other hand, the MPs are required to remove effete and apoptotic cells in a way that completely avoids inflammation. Under other circumstances, MPs provide food and shelter to pathogens that have learned to avoid their defensive capabilities. This discussion focuses on the manner in which Mo/MPs cope with pathogens

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and, especially, the growing list of modifications and defects that they evince as a reflection of senescence. Ingestion and Intracytoplasmic Fate of Microorganisms (Bacterial and Protozoa) Consider first the events that result in the phagocytic ingestion and intracellular destruction of microorganisms. This process is initiated upon mutual interaction between a microorganism and a phagocytic cell. Here we limit the discussion to phagocytic Mo/MPs. The interaction between phagocyte and microbe may occur via one or more of the PRRs described above and the microbial PAMPs. Alternatively, the microbes may be opsonized as a consequence of activating the C system or binding specific antibody; in those cases, their association with phagocytic cells involves complement receptors (CRs) and Fc receptors (FcRs), respectively. Binding of the microbe to the phagocyte surface triggers the internalization process. In the case of CR, binding may involve CR1, CR3, and CR4. Internalization of particulate matter, however, requires CR3 or CR4. The latter are members of the integrin family of adhesion molecules and bind selectively to C3bi. An additional stimulus to the phagocyte is required to effect CR-mediated internalization (76). That stimulus may be provided by activators such as phorbol esters, by the cytokines TNFα or GM-CSF, or by attachment to a suitable substrate. In the case of FcR-mediated internalization, no additional stimulus beyond crosslinking of adjacent receptors is necessary (77). Changes in the membrane of the phagocytic cell leading to microbial ingestion and formation of a vacuole surrounding the microbe result from signals transduced via the receptors. For both CRs and FcRs, the details of signaling have been reasonably well elucidated (77,78). At present, less is known about signaling via MRs, TLRs, and SRs. The outcome of the signaling cascade is quite different in the case of the FcR and the CR (78). For example, activation of the MP via the FcR results in the extension of veils of cytoplasm that surround the particulate. Then, in coordination with the sequential interaction between MPs, FcRs, and the IgG molecules bound to the particle (a “zippering” effect), ingestion of the particle (bacterium, yeast, protozoan, or other) occurs (79,80) to form a phagosome. The microfilaments of the phagocyte are involved in the process as demonstrated by treatment with colchicine, which disrupts microfilaments and prevents FcR-mediated phagocytosis. CR-mediated phagocytosis, in contrast, occurs without the zippering process by what appears a “sinking into the cell” (80) without the aid of prominent MP pseudopodia. Furthermore, cytoplasmic microtubules, rather than microfilaments, of the MPs are involved. There is another method used by certain bacteria such as S. typhimurium to enter MPs. Those organisms induce macropinocytosis at the surface of the phagocyte resulting in the ingestion of the organisms into relatively large endocytic vesicles.

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In addition to the differences between the FcR- and CR-mediated formation of phagosomes, there is a considerable difference in the types of inflammatory mediators released by MPs that have been stimulated via CRs and FcRs. When MPs are stimulated via the latter, reactive oxygen intermediates and arachidonic acid metabolites are produced. The same is true of MPs activated via the mannose receptor (81,82). Stimulation via CRs, however, results in neither being produced by MPs (83,84). How the two signaling pathways resemble or differ from each other has not as yet been elucidated. Considerable effort has been devoted to gaining an understanding of signal transduction via the FcR (85,86). In brief, the crosslinking of surface FcγRs results in phosphorylation of the ITAMs (immunoreceptor tyrosine activation motifs) present on the γ subunits of the FcγRs. The phosphorylated ITAMs provide high-affinity binding sites for elements of the Syk family of tyrosine kinases. Syk plays a key role in further transmitting the signal that leads ultimately to the formation of actin nucleation sites and the polymerization of actin that is required for the formation of the phagosome (see refs. 85 and 86 for details). Binding and internalization of microorganisms by the MP probably activate signaling pathways in addition to the one leading to cytoskeletal rearrangements, for example, signaling that results in production of reactive oxygen radicals and arachidonic acid metabolites. It has been suggested (85) that both of the latter lead to products that aid in phagosome formation and they are involved in other functions such as the destruction of ingested organisms or apoptotic cells. At some point in the processes of internalization and breakdown of ingested matter, signaling occurs that results in production of several cytokines, IL-12 in particular. The timing and coordinated manifestations of all that signaling are topics for futher investigations. The next major step in the pathway of destruction of ingested microorganisms is the formation of the phagolysosome (87). This step involves phagosome maturation and, until recently, was thought to include phagosome fusion with lysosomes. It is now known that the phagosome metamorphoses to become a phagolysosome. Once internalized, the phagosome fuses with endosomes, as well as with transport vesicles, some of which may be pinched off from inverted plasma membrane whereas others are derived from the Golgi apparatus. The phagosome itself may bud and give rise to vesicles capable of interacting with other cytoplasmic vesicles. In this manner, the phagosome may gain and lose components and experience changes that are termed “maturation” (87). For example, over a period of 20 min after internalization, phagosomes may lose 50% of their contingent of FcRs and 75% of their MRs. During the same interval, they may acquire substantial quantities of enzymes such as cathepsin D and β-glucuronidase (87). The mature

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phagosome is now a phagolysosome able to fuse with secondary lysosomes. Thus, the interior of the phagolysosome becomes progressively more hostile toward the enclosed microbe. Reactive Oxygen and Nitrogen Intermediates Some of the most injurious of the hostile substances produced inside the phagolysosome are toxic oxygen metabolites, frequently referred to as “reactive oxygen intermediates” (ROIs) (88,89). These are largely derived through various biochemical reactions from the superoxide anion (O 2–). The latter is produced by the action of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase present in the phagosome membrane that reduces O 2 to O2–. The NADPH oxidase system is susceptible to activation by bacterial components such as LPS and lipoproteins, by IgG and C components bound to their receptors, and by certain cytokines such as IFNγ and IL-8 (see Fig. 3-3). The transfer of an electron from NADPH to O2 is catalyzed by a complex of at least four proteins including subunits of cytochrome b558, a 91kDa glycoprotein, and a smaller 22 kDa protein, together with p47 phox and p67 phox, which are cytoplasmic phagocyte oxidases (89). The energy for the transfer is derived for GTP and involves a small GTPbinding protein, generally Rac 2. The details can be found in (89). The ROIs that phagocytic cells are able to derive from O2– include hydrogen peroxide (H2O2), a reaction catalyzed by superoxide dismutase (SOD), and the hydroxyl radical (OH) and hydroxyl anions (OH–) produced through the Haber–Weiss reaction (89). Another powerful oxidant, hypochlorous acid (HOCl), is formed from H 2 O 2 and Cl – ions, a reaction catalyzed by myeloperoxidase, which is present in granulocytes and macrophages (90). All of those ROIs may be present in the lumen of the phagolysosome. HOCl may interact with nitrite ions (NO2–) generated in the nitric oxide (NO) pathway, to form nitryl chloride (NO2Cl). All of those intermediates, H2O2, •OH, OH–, HOCl, NO2–, NO2Cl, are capable of reacting with the macromolecular components of microorganisms thus leading to their destruction (see Fig. 3-4). Equally important in the destruction of phagocytosed microorganisms are the products of the nitric oxide pathway called reactive nitrogen intermediates (RNIs) (88,91). These are derived from the actions of nitric oxide synthases on the substrate L-arginine resulting in the products L-citrulline and NO; the latter is formed by the combination of the N from L-arginine with molecular oxygen. The principal isoform of nitric oxide synthase in phagocytic cells is NOS2 (iNOS). The radical form of NO (•NO) can react readily with O2– to form the strong oxidant, peroxynitrite (ONOO–). The latter is capable of modifying macromolecular components of microorganisms through reactions such as oxidation of thiol groups and nitration of tyrosines. •NO can also react with transition metals (Fe, Cu, Zn) to form metal-nitrosyl complexes. The latter are known

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Fig. 3-3. Mechanism of the oxidative burst in phagocytes triggered by binding of a bacterium and formation of a phagolysosome. The formation of O2– is catalyzed by NADPH oxidase. Myeloperoxidase (and defensins in the case of neutrophils), secreted by the phagocyte, utilizes H2O2 to generate the powerful oxidant HOC1. (From ref. 90, with permission.)

to regulate the functions of certain enzymes, transcription factors, and signaling cascades, thus influencing various biological activities (92). The production of NO by the action of NOS2 (iNOS) in macrophages has been studied in considerable detail (93–95). NOS2 expression can be induced by various agents including IL-1β, INFγ, TNFα, and bacterial LPS. Sustained expression of NOS2 can result in high output of NO, which is beneficial for combatting infection, reducing thrombosis, and facilitating blood flow to injured tissues. But NO can be detrimental if produced in excess by contributing to conditions such as septic shock, rheumatoid arthritis, and tissue injury. Therefore, it is not surprising that tissues vary in their responses to substances that affect NO production, some responding by increasing production while others respond negatively. Macrophage production of NO catalyzed by NOS2 may be enhanced or depressed depending on the stimulus and the conditions. In general, it appears that the level of NO produced is a reflection of rates of transcription although the stability of NOS2 mRNA may vary under certain conditions. In rats and mice, expression of the NOS2 gene is controlled predominantly by the tran-

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Fig. 3-4. Reactions catalyzed by myeloperoxidase that result in damage to cellular macromolecules. (From ref. 90, with permission.)

scription factor NFκB during inflammation and related immune responses (see ref. 95 for review). The production of NO by MPs is, in part, self-regulating (95,96). There is compelling evidence that local tissue concentrations of NO autoregulate NOS2 expression. This feedback mechanism appears to operate through regulation of the state of NFκB (95–97). Thus, evidence suggests that NO produced by nos 2 in sufficient concentrations acts negatively to inhibit transcription of the nos 2 gene mediated by NFκB. Conversely, at low concentrations of NO as is the case, for example, at the time of initiation of MP activation, NFκB-directed transcription of the nos 2 gene is promoted. It is well established that nucleotide sequences upstream from the nos 2 gene, in both mouse and human, comprise both promoter and enhancer sites for NFκB binding (98,99). Related to NO regulation of nos 2 gene expression is the broader question concerning the effects of NO on the expression of other genes by phagocytic cells, in particular those encoding proinflammatory cytokines. NO is known to influence the actions of various mammalian transcription factors in addition to NFκB (92,94) and to affect the production of cytokines and other substances by phagocytic cells. For example, NO has been reported to inhibit the production of COX-2 (100,101), TNFα (102), IL-1β (103), macrophage inflammatory protein (MIP)-1α (104), and IL-6 (105). However, there are numerous reports that NO stimulates enhanced production of many of the same factors including IL-1 and IL-6 (106), COX-2 (106), and TNFα (102). The probable resolution of this paradox lies in the evidence that NFκB activity (and other transcription factors, e.g., AP-1) is dependent on local concentrations of NO. Thus, NO may either enhance or depress the activation of proinflammatory genes according to its concentration in the MP relative to the time the MP was

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activated. It is highly likely, therefore, that the intensity of proinflammatory gene expression is closely related to the level of transcription of the nos 2 gene, which, in turn, reflects the intracellular concentration of NO. Information concerning the mechanisms through which NO exerts effects on NFκB activity is accumulating rapidly (see ref. 95). That information, together with the detailed understanding of the structure and functions of the NFκB/Rel family of transciption factors and of the IκB family of inhibitory proteins (107), makes possible a provisional explanation of the biphasic effects of NO on the expression by macrophages of proinflammatory genes. Inhibition of the dissociation of NFκB from its inhibitor, IκBα, may explain the inhibition of proinflammatory gene transcription by relatively high concentrations of NO. In this model, NO interferes with the phosphorylation and/or proteasome degradation of IκBα, thus preventing release of NFκB (97,108). Alternatively, inhibition of binding of NFκB to a promoter site may occur as a consequence of nitrosylation of cysteine 62 of the p50 protein of κB (109). The most likely explanations for the activation of the genes at low concentrations of NO involve nitrosation and activation of the redox-sensing, signaling protein, p21ras (110,111). The effects of such a biphasic process can be envisioned as: (a) activation of genes encoding proinflammatory factors, including the nos 2 gene, at relatively low NO concentrations following interaction of MPs with pathogens; and (b) depression of expression of those genes by relatively high NO concentrations at later times after MP stimulation. Thus, the proinflammatory factors would be produced early to marshal defenses against the pathogen, but shut down later to minimize tissue damage and abnormal physiological events. Finally, it should be mentioned that there is considerable interest in the probability that the pathways for generating ROIs and RNIs may intersect at one or more points to yield antagonistic or synergistic effects. To date this difficult subject has yielded only grudgingly to investigation. The tantalizing findings include the observation that various stimulants of nos 2 expression also stimulate sod expression (e.g., ref. 112). As pointed out in ref. 95, the efficient scavenging of O2– by SOD will spare NO from interacting with O 2– to form peroxynitrite (ONOO–). Thus, when cells such as MPs begin to upregulate the formation of SOD, the high concentration of NO required for negative feedback inhibition of proinflammatory gene expression may result. IL-12: A Key Cytokine Probably the most important of the proinflammatory substances generated by activated MPs is IL-12. IL-12 exerts a pivotal influence on NK cells to produce IFNγ and on Th1 cells to produce several proinflammatory cytokines (see Chapter 4). A variety of substances have been reported to activate IL-12

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production by Mo/MPs: LPS, lipoteichoic acid, intracellular bacteria, and protozoa in particular. However, the actions of those activators is distinctly modulated by other factors such as certain cytokines, prostaglandins, complement components, and others. There have been numerous studies of the effects of NO on IL-12 production by MPs. The findings range from enhancement of IL-12 production (113), to no effect (114), to significant inhibition (115). Several important studies have employed LPS as a representative activator in order to analyze inhibition of IL-12 production. The results of those studies have shown that the binding of appropriate ligands to MP receptors for Fcγ, C, or SR selectively inhibited MP production of IL-12 upon stimulation with LPS (116). Another study of C-mediated inhibition of IL-12 production (117) identified the C component, anaphylatoxin C5a, as rendering Mo’s unresponsive to LPS; this was true even when the Mo’s were first primed with IFNγ before exposure to C5a followed by LPS. The results of studies such as those cited indicate that by activating Mo/MPs through receptors that facilitate phagocytosis, microorganisms shut down one of the principal cytokines that triggers both the innate and adaptive immune responses. For example, failure of IL-12 secretion by the MP prevents vigorous IFNγ production by both NK cells and Th1 cells as well as other proinflammatory factors by the T cells. Other substances that are known to suppress IL-12 production by Mo/MPs are IL-4, IL-10, PGE2, and TGFβ when provided together with bacterial activators such as LPS. However, if IL-4 is used to pretreat the cells, their subsequent production of IL-12 in response to LPS may be enhanced. Pretreatment of MPs with low doses of LPS may induce suppressed responses to subsequent exposure to LPS in combination with costimulants such as IFNγ or GM-CSF (118). The pathways through which the production of IL-12 is suppressed by this range of substances remain to be discovered. One important process appears to be the influx of extracellular Ca2+ resulting from changes in the cytoplasmic membranes (116,119). Support for this interpretation was found in a study of regulation of IL-12 and TFNα production by calcineurin (Cn) and vacuolar adenosine triphosphatase (V-ATPase; 119). Cn was found to act as a negative regulator of cytokine gene expression subsequent to Ca2+ flux. V-ATPase, a major proton extrusion enzyme, appeared to prevent acidic intracellular pH from activating cytokine gene expression. Together, Cn and V-ATPase exerted negative regulation of cytokine gene expression by restricting NFκB activation. From the preceding discussion of IL-12 suppression mediated through phagocyte receptors with which microorganisms and their components interact, we see one of several mechanisms employed by those organisms to evade phagocytic host defenses. Several other mechanisms are described in the following section.

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MICROBIAL EVASION OF PHAGOCYTIC DESTRUCTION Microorganisms have developed ways to avoid almost every process employed by phagocytic cells to destroy them (120). These evasive mechanisms have been most thoroughly investigated with respect to bacteria but at least some of them are used by fungi and protozoa. Several of the betterunderstood mechanisms are reviewed in brief. A widely used stratagem to evade phagocytic destruction is to avoid professional phagocytes altogether. A number of pathogenic bacteria choose to take residence in host cells such as fibroblasts and epithelial cells. Frequently, known adhesion molecules are exploited to gain entry. A particularly interesting case is found in enteropathogenic E. coli, which utilize a type III secretion mechanism to inject their own protein into host cells where it triggers cytoskeletal rearrangements to form the typical pedestal to which the bacteria like to adhere (121). Other bacteria such as Salmonella and Shigella inject nonphagocytic host cells with several substances that collaborate to induce cytoskeletal changes, membrane ruffling, and facilitated internalization of the bacteria (122,123). L. monocytogenes enters both phagocytic and nonphagocytic cells. Entrance into the latter involves recognition of two bacterial adherence proteins, internalins-A and -B, by host cell E-cadherin (124) and complement receptor C1q (125), respectively. Another evasive stratagem adopted by bacteria such as S. aureus and S. pyogenes is to gain entrance via macrophage CRs. Internalization via that route usually does not trigger vigorous, microbicidal responses by the phagocyte (84,126). The difference between CR-mediated and FcR-mediated triggering of the oxidative burst appears to reside in the differential use of Rho family GTPases (guanosine triphosphatases), which are involved in cytoskeletal rearrangements and phagosome formation. Cdc 42 and Rac are involved in FcRmediated bacterial internalization whereas only Rho are involved in CR-mediated internalization (127). Bacteria that enter the phagocyte via the CR thus avoid creating a hostile environment in the phagolysosome. A fascinating variation of that stratagem occurs in pathogenic, but not in nonpathogenic, mycobacteria. The former can appropriate complement component C2a from blood plasma to form at their surface a C3 convertase. The resulting complex is able to cleave C3 resulting in opsonization of the bacteria and CRmediated phagocytosis (128). A third mechanism to evade phagocytic destruction also is employed by mycobacteria. This mechanism involves the retention on the cytoplasmic face of the phagosome of a host cell protein, viz., tryptophan-aspartate-containing coat protein (TACO). TACO typically is released from the developing phagosome. In the case of mycobacteria ingestion, TACO is retained and blocks the fusion of phagosomes with vesicles to form phagolysosomes (120). The expla-

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nation for the retention of TACO in the mycobacterial phagosome lies in the findings that (a) the entry of these bacteria into macrophages depends upon interaction between bacterial surface components and macrophage membrane cholesterol, and (b) the association of TACO with the phagosome surface is cholesterol dependent (129). Certain bacteria such as Listeria, Rickettsia, and Shigella avoid destruction by escaping from the phagosome. For example, once internalized Listeria express the pore-forming protein, listeriolysin O, which allows them to exit the phagosome. The cytosol is a much friendlier place to live (130). Evasive mechanisms become even more complex in the case of intracellular protozoan parasites. Similar to Mycobacteria, Leishmania persist within phagocytic vacuoles. However, in the case of L. mexicana and Leishmania amazonensis the amastigote forms reside inside phagolysosomes, which contain the typical hydrolases and in which the pH is between 4.7 and 5.2 (131). The amastigotes are able to resist those hostile conditions. The protozoan T. gondii does most of the work to enter phagocytic cells (132). Cells of this protozoan are distinctly polarized displaying a complex of secretory organelles located in the apical region. This region contacts the host cell and a circumferential attachment ring is formed through which the parasite enters the cell enrobed in a rather tight-fitting vacuole. That the parasite does most of the work is attested by the finding that invasion occurs even of macrophages that have been lightly aldehyde fixed. Entrance of Trypanosoma cruzi into macrophages is arguably the most bizarre process of all (133). It is far from a typical phagocytic process. Contact with the MP occurs at the posterior end of the parasite where the flagellum originates (the “flagellar pocket”). In the vicinity of this contact point, macrophage lysosomes congregate. They are lysosomes as indicated by their content of lysosomal markers. There is an absence of pseudopodia as is typical of actively phagocytosing MPs. The parasites enter the MP encased in membrane much of which is derived from the lysosomes. In two hours or less after entering the MP, the trypanosomes are found to have been released into the cytosol. This results from the gradual breakdown of the lysosomal membranes. It should be emphasized that the preceding descriptions of protozoan entry into phagocytic cells applies in the absence of immune manifestations and prior to the initiation of immune responses. In the presence of opsonins, even those of “natural antibody,” Leishmania are ingested by phagocytes via CRs (134). AGE-RELATED CHANGES IN MACROPHAGES When the field of immunogerontology was still quite young, it was generally concluded that MPs display no effects of senescence. Now that the field is older, more wisdom has accrued. MPs do, in fact, show signs of aging. The changes in

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MPs for which the evidence seems most compelling are: (a) diminished ability to generate ROI upon stimulation; (b) enhanced potential for producing NO upon stimulation; (c) altered arachidonic acid metabolism associated, in particular, with elevated intracellular activity of inducible COX-2; (d) depressed potential for production of certain cytokines, especially of the proinflammatory type; (e) changes in pteridine metabolism and/or secretion; (f) changes in the circulating levels of the soluble forms of certain receptors and other regulatory proteins released by MPs; and (g) changes in levels of certain hormones that affect MPs. Studies concerned with the effects of aging on MPs must be conditioned by the fact that MPs are quite heterogeneous, both in form and function (135–137). For example, both their state of maturation and their tissue location (spleen, liver, lungs, blood, peritoneal space, and other) may influence and/or reflect their functional characteristics. In the alveolar and peritoneal locations, resident MPs differ significantly from elicited MPs. Both the eliciting agent and the time after its administration affect the functions of elicited MPs. Given the complexity of the overall population and lacking the ability to achieve reproducible separations of stable subsets of MPs, most investigators have studied aging of mixtures of subsets of MPs. Macrophages and ROIs With regard to the ability of MPs to genrate ROIs, most of the evidence indicates a significant decline with advancing age. MPs from rats (138), mice (139), and human (140,141) have been analyzed for their ability to produce O2– in response to stimulation. The diminished ability of the MP to produce ROIs is apparent regardless of the stimulant employed: LPS, IFNγ, fMLP, or phorbol ester (PMA). Results from utilizing PMA as the stimulant suggest that the agemodified step(s) in the signaling cascade occur beyond the surface receptors. For example, inefficient tyrosine phosphorylation in response to IFNγ was characteristic of MPs from aged mice (139). In the case of MPs from aged individuals, it is difficult to evaluate the causes of the reported 50–70% loss of O2– production. Binding of various ligands to the corresponding receptors on phagocytes results in organization of the NADPH oxidase complex (142,143), which is capable of secreting superoxide radicals into the extracellular space or the interior of the phagosome. Some ROIs are involved in damaging/killing the internalized microorganisms; the ROIs may be O2–, H2O2 or the highly reactive hydroxyl radical (143). Studies of NADPH oxidase (144,145) have revealed no changes in the activity of that enzyme. A reduction in the prevailing ratio of NADPH/NADP + was found both in unstimulated and PMA-stimulated MPs from aged rats suggesting age-related changes in NADPH-producing enzymes such as glucose-6-phosphate dehydro-

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genase and 6-phosphogluconate dehydrogenase (144,146). A particularly informative study was conducted on thioglycolate-elicited peritoneal MPs of young and aged mice (145). The design of that study took into consideration the stressful conditions associated with inflammation, viz., elevated body temperature, and repeated exposure of MPs to stimulants (zymosan and oxidized zymosan were employed). The results revealed that 1) the oxidative burst to a second exposure of stimulant was significantly less in MPs of aged compared to young animals, and 2) the ability to generate an oxidative burst by MPs recovering from heat stress was substantially reduced in the case of aged mouse MPs. The integrity of NADPH oxidase and superoxide dismutase was essentially unchanged in the aged MPs. The investigators suggested that aging might affect signal transduction or surface receptors in aged MPs. As there seems to be reasonably good agreement among investigators that the respiratory burst is deficient in aging MPs, the reasons for that deficiency need to be understood. Attention should be given to changes in the redox status of aging MPs including studies on levels and turnover of reduced and oxidized glutathione, the enzymes glutathione reductase and glutathione S-transferase, and interactions with the nitric oxide metabolic pathways. For example, as is discussed in the following section, NO production appears to be enhanced in aging MPs, thus raising the possibility that the production of ROIs may be reduced in aging MPs as a consequence of the elevated NO production. Alternatively, ROI levels may be reduced by reaction between O2– and NO to form peroxynitrite (ONOO–) and (perhaps) thence to the highly destructive hydroxyl radical (·HO) (see ref. 143). ROIs such as O2– are highly diffusible, readily traverse cell membranes, and may transit from one cell to contiguous cells where they might interfere with signaling pathways (147). One of the most important elements of the intracellular redox system, which has received virtually no attention vis-à-vis aging of cells of the innate immune system, is the thioredoxin system of proteins including thioredoxin reductase (148,149). Macrophages, NO, and RNIs Although there is some disagreement, the conclusion that NO production is elevated in aging, compared to young-adult, MPs is supported by compelling evidence. For example, thioglycolate-elicited MPs from aged mice were found to produce substantially greater amounts of NO in response to LPS, zymosan, or heat-killed S. aureus than MPs from young mice (150). The levels of NOS2 mRNA were significantly greater in aged compared to young mouse MPs after LPS stimulation. Analysis of the responses of young and aged mouse spleen cells to stimulation in vitro with LPS, or a combination of LPS and IFNγ, revealed the loss of control over NO production with age (71). The significantly greater production

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of NO by MPs from aged mice was attributable to elevated levels of iNOS (NOS2). Stimulation with LPS alone resulted in significant release of NO by cells of aged mice but little or none by cells of young-adult animals. The combination of LPS and IFNγ stimulated vigorous output of NO by young mouse cells and moderately enhanced the output by aged cells compared to stimulation with LPS alone. It was shown that the substantial production of NO by aged cells in response to LPS alone reflected concomitant, synergistic action of IFNγ present endogenously in the aged MPs. The presence of endogenous IFNγ in aged MPs was associated with elevated nuclear translocation of the transcription factor STAT 1 and enhanced expression of the nos 2 gene. Very interesting results were obtained from efforts to restore regulatory control of NO production to cells of aged mice (71). Treatment of mice with an antioxidant (vitamin E) or agonists of the peroxisome proliferator-activated receptor α (dehydroepiandrosterone sulfate and compound WY-14,363) significantly reduced the production of NO by cells of aged mice. The effect of vitamin E was attributed in part to alteration of the intracellular redox status and the resulting decrease in translocation of transcription factor NFκB, which is well known to be redox sensitive. LPS and IFNγ act synergistically to motivate signaling via several pathways involving STAT 1 and interferon regulatory factor (IRF)-1 as well as NFκB. The restoration of the control of NO synthesis in aged MPs by use of PPARα activators could be explained in several ways. The two most likely explanations seemed to be: (a) PPARα-induced elevation of certain enzymes involved in elimination of lipid-derived inflammatory signaling molecules such as the leukotrienes, or (b) PPAR-mediated antagonism of transcription factors (but see previous discussion of PPAR and ref. 69). Aged mice have been found to succumb to much lower concentrations of LPS than is the case of young mice (151). Thus, the LD50 dose of LPS given intraperitoneally to young mice was approximately 18 mg/kg body weight but only 1.8 mg/kg in the case of aged mice. The plasma concentrations of NO and of TNFα were substantially higher in aged compared to young mice given equivalent doses of LPS. Both of these substances appeared to be involved in the LPS toxicity for old (and young) animals. The results of this study demonstrated an apparent loss in the ability of aged mice to regulate NO production. The preceding review of three publications (71,150,151), which agree well with each other, seems to build a strong case for the conclusion that MPs of aged mice can, upon stimulation, produce elevated levels of NO compared to young adult animals owing to loss of control of nos 2 gene transcription. However, some investigations have produced different results. For example, a careful study of the ability of MPs from young and aged mice to inhibit tumor growth revealed that cells from aged animals were significantly less capable (152). Stimulation in vitro of resident MPs from mice of the two ages with a combination of IFNγ and

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LPS resulted in significantly less output of TNF, IL-1, and NO by cells of the aged mice. It was concluded that peritoneal MPs of aged mice suffer from an intrinsic defect in their ability to destroy tumor cells that reflects the defective production of TNF, IL-1, and NO. The difference between that conclusion and the conclusions of the three studies reviewed previously probably arise from (a) the difference in experimental approach, the latter study having been performed entirely in vitro, and (b) the source/type of MP employed. The importance of the source of MP was emphasized in ref. 152 by the statement: “Because MP isolated from different anatomical sites display a diversity of phenotypes and capabilities, peritoneal MP may not be representative of every MPs involved in tumor cell killing.” A similar statement can be made about the difference between resident and elicited or activated MPs. Mechanisms and Consequences of Dysregulation of ROI and RNI Formation Associated with Senescence The weight of the available evidence favors the conclusion that aging is accompanied by impaired regulation of O2– and NO production in MPs. There is abundant literature to support the argument that such senescent changes in MPs reflect oxidative damage to the mechanisms of redox homeostasis in aging Mo/MPs. Analyses of several types of cells of aging mammals and insects have revealed that changes occur in the redox status of aging cells, shifting the balance between oxidative and reductive potential toward the oxidative (153,154). In that regard, cells such as MPs and neutrophils are extraordinary because generating destructive oxidants is one of their principal functions. Presumably it is the confinement of oxidants to vacuoles and the presence of active antioxidant enzymes such as superoxide dismutase and catalase that protect those cells from immediate oxidative destruction. As is true of most if not all types of cells, the consequences of the altered redox status and the dysregulated control of ROI and RNI formation in aging MPs are considerable. Foremost are: (a) the damage done to macromolecules, especially enzymatic proteins and membrane lipids; and (b) alterations of transcription factors and the loss of control of gene transcription. In healthy, nonstressed, young-adult cells the ratio of oxidative to reductive potential heavily favors the latter. Senescence results in an increasingly oxidative state and the consequent modifications of structural and enzymatic proteins, altered bases in nucleic acids, and peroxidation of complex membrane lipids. Some, perhaps a substantial portion, of those oxidative changes can be attributed to leakage of O2– and H2O2 from mitochondria, which may become “leakier” as cells senesce (155). In the case of MPs (and other phagocytes such as neutrophils) leakage of ROIs from phagosomes is likely. Much has been written about the effects of O2–, H2O2, NO, and other ROIs and RNIs on signaling and gene transcription (156–159). In some signaling

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pathways, certain RNIs and ROIs may serve as co-signaling factors. Generally, however, ROIs and RNIs, especially in aging cells, alter normal signaling and affect gene transcription—either accentuating or depressing it. An abbreviated list of genes and factors that are known to be susceptible to the intracellular redox status is given in Table 3-2 (see refs. 156–158). Alterations in gene transcription caused by ROIs and RNIs have been studied extensively in the case of genes controlled by NFκB. It has been emphasized that a variety of factors influence the effects manifested via the NFκB pathway, for example, the nature of the pathway activator, the type of ROI and/ or RNI that is enlisted, and the type of cell activated (95). The NFκB pathway is dysregulated in cells of the aging immune system, including the MPs (160). In aging mice, NFκB is present in an elevated, constitutively activated state apparently as a consequence of the altered redox status. The elevated NFκB is associated with altered production of several cytokines that are produced by MPs, viz., IL-6, TNFα, and IL-12 (160,161). Inducible COX-2 is also elevated. Treatment of aged mice with antioxidants (α-tocopherol or DHEAS) is partially successful in reversing the age-associated traits to resemble those more characteristic of young adults. A transcription factor that deserves more attention for its actions in aging MPs having elevated levels of NO is Sp 1. This factor binds to a sequence in the promoter of TNFα (and various other genes) and activates TNFα gene transcription. The presence of NO increases the activity of the TNFα promoter as a result of direct or indirect interaction with Sp 1 (162). Sp 1 may be part of a larger, more complex, NO response region. Because Sp 1 associates with promoters of other genes, it may be that those genes, too, are affected by elevated NO levels. It has long been realized that MPs have a tendency to accumulate at sites of wounds, infections, tumors, and other pathological states where oxygen tension is low. MPs function under such hypoxic conditions by changing their metabolism (adapting). This means that there is a change in the genes that they express. What changes are evident in adapted MPs? How do MPs sense the ambient oxygen tension and initiate the changes that allow them to function under hypoxic conditions? Some of the more dramatic changes are (a) a shift toward glycolysis for maintaining energy adenosine triphosphate [ATP] production, (b) elevated NOS 2 levels (and NO production upon return of oxygen), and (c) significant changes in cytokine production (e.g., elevated vascular endothelial growth factor; increased TNFα, MIP-1α, and IL-8; moderate or no changes in IL-1 and IL-6) (162). Those and other changes are illustrated in Figure 3-5. Recent publications (163–165) provide considerable insight concerning the ability of various types of cells to assess oxygen tension in tissues. The key is

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Table 3-2 Some Genes and Factors That Are Sensitive to Cellular Redox Conditionsa Gene or factor

Cell type

Redox modulator

Effect

CD3 (zeta chain gene)

T-cell line

H2O2

Repression

Cyclooxygenase-2

Rat mesangial cells

H2O2

Increased mRNA

CYP1A1

Hepatocyted cell line

H2O2

Repression

Erythropoietin

Rat kidney Rat kidney

H2O2 Hypoxia

Repression Increased mRNA

Extracellular regulated kinase (ERK)

Jurkat T cells

NO

Activation

Hypoxia-inducible factor-1 (HIF-1)

Hep 3B cell line

H2O2, diamide

Impaired DNA binding

Interleukin-2

T cell line

H2O2, xanthine oxidase

Repression

Janus kinase-1 (JAK-1)

Rat-1 cells

H2O2

Increased phosphorylation

Mitogen-activated protein kinase (MAPK)

Jurkat T cells HeLa cells

H2O2, NO Hypoxia

Activation Inhibition

Macrophage inflammatory protein-1 (MIP-1)

Rat macrophage cell line

H2O2, menedione

Elevated mRNA

Mn-dependent superoxide dismutase (Mn SOD)

Pulmonary adenocarcinoma cells

H2O2, TNFα

Elevated activity, mRNA

Nitric oxide synthase (endothelial)

Aortic endothelial cells

Glucose oxidase

Increased mRNA

Nuclear factor-κB (NFκB)

Jurkat T cells Mouse macrophages

H2O2 H2O2

Activation Activation

Protein kinase C

COS-7 cells

H2O 2

Stimulation of substrate phosphorylation

SP-1

Rat liver

H2O2, aging

Decreased DNA binding

ZAP-70 tyrosine kinase

T lymphocytes Jurkat T cells

H2O2, UV radiation H2O2

Activation Activation

aFor

a more detailed listing, see refs. 159–161.

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Fig. 3-5. Illustration of the variety of effects of hypoxia on the functions of macrophages. (From ref. 163, with permission.)

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the hypoxia-responsive factor known as “hypoxia-inducible factor” (HIF). This is a heterodimeric molecule, composed of two subunits (HIF-1α and HIF-1β), which functions as a transcription factor. HIF binds selectively to a tetranucleotide response element (5'-CGTG-3') present in the promoter of a number of genes which, when activated, permit the cell to adapt to low pO2. Among the important genes are several that encode: enzymes of the glycolytic cycle, erythropoietin, vascular endothelial growth factor, and TNFα. The presence of stimulants such as LPS and IFNγ at hypoxic sites results in additional gene activation, of nos 2 in particular. Tissue hypoxia frequently is associated with infection and the presence of LPS or other bacterial components could serve to promote MP responses aimed at the infection. The elevation of NOS 2 should be such a response. Unfortunately, there is not a corresponding elevation in NO formation because: (a) the enzyme, arginase, is also elevated and depletes the pool of arginine substrate that is required by NOS 2; and (b) the presence of oxygen is required to combine chemically with the nitrogen derived from L-arginine. The subunit, HIF-1β, of the HIF is also termed the arylhydrocarbon receptor nuclear translocator (ARNT) and is well known for its role as a component of the transciption factor that activates genes of the cytochrome P450 system. HIF1α is the hypoxia-sensitive component of the HIF heterodimer. Under normoxic conditions HIF-1α is rapidly ubiquitinated and degraded by proteasomes. When hypoxia prevails, a prolyl hydroxylase enzyme adds an hydroxyl group to a proline residue located in the highly conserved, oxygendependent, degradation domain of HIF-1α, thus interfering with its attachment to ubiquitin and favoring its association with HIF-1β to form the transcription factor (164,165). The preceding is a brief description of the role played by HIF-1 in detecting hypoxia; for details, refs. 164 and 165 should be consulted. Also, it should be cautioned that MP sensing of low tissue pO2 might not depend, at least not entirely, on the HIF-1 mechanism. Some evidence supports a role for NFκB in activating MP genes under hypoxic conditions (163). No matter what the precise mechanisms may be, the structural and functional responses of aging MPs to hypoxia is a topic worthy of serious attention, especially in aging tissue environments. Concerted research devoted to this topic, always keeping in mind the considerable diversity of Mo’s and MPs, could be quite rewarding from at least two perspectives, viz., infections and cancers of the aging population. With thorough understanding of the effects of hypoxia on various genes and their promoters, and better understanding of the effects of aging on various cells and tissues, the design of therapeutic methods tailored to meet the needs of aging patients should become possible. Recent progress in transfecting MP with genes such as IFNγ under the control of hypoxia-inducible promoters (163,166) illus-

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trate the promise of this approach for treatment of certain infections and cancers of the elderly. Prostaglandins and COX-2 in Aging MP One prominent change in MPs associated with aging is the increased activity and intracellular level of the enzyme COX-2 (167,168). This enzyme is barely detectable in resting, unstimulated MPs but the activity rises rapidly after stimulation of MPs with substances such as Ca-ionophore (A23187), IL1β, or LPS. The enzyme activity and abundance of specific mRNA of COX-2 and the formation of the product, prostaglandin E2 (PGE2), are four- to fivefold higher in MPs of aged than in young-adult mice. Furthermore, breis of tissue such as whole spleen, kidney, and lung also generate more PGE2 when prepared from aged compared to young rodents (see ref. 168). Prostaglandins, especially PGE2, have long been of interest to students of immunosenescence because they are broadly immunosuppressive; e.g., they inhibit proliferation of lymphocytes and cytokine production by T cells and MPs. Prostaglandins are derived from arachidonic acid which, in turn, arises from membrane phospholipids (see ref. 169). Free arachidonate serves as the substrate for a bifunctional enzyme, prostaglandin H2 synthase (PGH2 synthase), which has both cyclooxygenase and peroxidase activity. PGH2 is a single-chain, heme-containing protein. In addition to PGH 2 synthase, arachidonate is the substrate for other enzymes known as lipoxygenases, which convert arachidonate to several leukotrienes and hydroxyeicosatetraenoic acids. This pathway does not concern us here. It is important to try to understand the elevated COX-2 activity and PGE2 production by MPs of aged subjects for at least two compelling reasons. First, the PGE2-mediated inhibition of T-cell and MP functions may account, in part, for the diminished ability of aged subjects to control various infections. Second, evidence is accumulating that suppression of COX-2 inhibits the development of certain cancers. It appears that elevated COX-2 activity is closely associated with the elevated NOS 2 activity and NO content of aging MPs. Recent work requires that the apparently coordinated activities of NOS 2 and COX-2 and their products NO and PGE2 be considered at two levels: (a) transcriptional and (b) posttranslational. Let us consider the latter first. Recent studies in the Meydani laboratory (168) have shown that the administration of vitamin E (α-tocopherol) to aged mice results in a significant reduction of COX-2 activity. Further study of that effect of vitamin E led to the fascinating conclusion that it is the peroxynitrite radical (ONOO –), formed by the interaction of O2– and NO in aged MPs, that leads to ONOO – enhancement of COX-2 activity as a result of posttranslational modification of the enzyme. The ONOO – radi-

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cal was considered to appear as a result of the age-associated change in MP redox balance. Restoration of the proper redox balance by treatment with vitamin E is thought to eliminate the excessive oxidative potential of the aging MPs. That conclusion should be evaluated in a rather broad and complex context, as outlined below, the principal value of which, at present, is heuristic. Now, it is well known that NOS 2, like COX-2, is a heme-Fe2+-containing enzyme (170). NOS 2 requires for its enzymatic activity a pteridine co-factor, tetrahydrobiopterin (H4B) (171), which probably interacts with the heme-Fe2+ to facilitate dimerization of the subunits of NOS 2 (170). Murine (mouse, rat) MPs readily synthesize H4B starting from guanosine triphosphate (GTP). Three enzymes are involved (172): GTP-cyclohydrolase, which converts GTP to dihydroneopterin 3'-triphosphate; 6-pyruvoyltetrahydropterin synthase, which catalyzes the formation of 6-pyruvoyltetrahydropterin; and sepiapterin reductase, which catalyses the third step, which yields H4B. Human MPs are unable to synthesize H4B because they possess little or no 6-pyruvoyltetrahydropterin synthase (173); instead, they generate the derivative neopterin (174), which cannot serve as a co-factor for NOS 2. Stimulation of murine MPs with IFNγ only moderately elevates the synthesis of H4 biopterin because it is synthesized in significant amount in a spontaneous (i.e., unstimulated) manner. Stimulation of human MPs with IFNγ markedly enhances the production of neopterin. The effect of IFNγ is to dramatically elevate the activity of the enzyme, GTPcyclohydrolase (174). The addition of sepiapterin to cultures of mouse MPs prior to stimulation with IFNγ, LPS, or both, results in marked enhancement of H4B synthesis and NO formation (173). Sepiapterin can readily permeate cell membranes and, once intracellular, serves as substrate for the enzyme sepiapterin reductase, which catalyses H4B synthesis. There was no effect of sepiapterin on human MP production of NO even though H4B was produced in them (173). Clearly, the inability of human MPs to generate NO involves more than their inability to generate H4B. There exists, then, a curious puzzle concerning the significance of elevated NO and COX-2 activities in aging MPs. In the case of aged mouse MPs there exists a significant level of H4B synthesis, which can be enhanced by stimulation of the cells with IFNγ and/or LPS, especially when adequate sepiapterin is available. The production of NO is related to the amount of available H4B. Because human MPs appear to generate little or no NO (and not only because they are unable to synthesize H4B), it seems unlikely that any elevation in COX-2 activity in those MPs can be attributed to posttranslational modification of the enzyme by ONOO- radicals. It should be noted that: (a) evidence of COX-2 activity in human MPs has been reported by some, but by no means all, investigators; and (b) stimulated human MPs can generate NOS2, as detected by a specific monoclonal antibody, but in an apparently inactive form (173). It

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Fig. 3-6. Interaction between NO and COX-2 in the regulation of PGE2 production by macrophages. In COX-2 deficient cells, NO-enhanced PGE2 production in a dosedependent manner. (From ref. 182.)

may be that a proportion of human subjects can generate sufficient NO to facilitate the activity of COX-2. The elevated activity of COX-2 in aging mouse MPs reflects not only the effect of NO or ONOO– on the existing enzyme but, in addition, the synthesis of new COX-2 is elevated (167,168). This finding introduces an additional element of complexity to the puzzle concerned with NOS 2 and COX-2 interactions in aging MPs. Particularly noteworthy is the fact that mouse MP cell lines simultaneously express elevated amounts of both NOS 2 and COX-2 induced by LPS or other stimulants (175–177) along with substantial amounts of NO and PGE2. This appears to be a reflection of the mutual regulatory influences of these two systems on one another (“cross-talk”; see Fig. 3-6). Thus, the production of PGE2 by COX-2 modulates NOS 2 formation; PGE2 may either inhibit (178,179) or stimulate (180,181) NOS 2, and the production of NO, depending on its concentration in the MPs (the same occurs in certain other cells ). Conversely, evidence has been adduced supporting an effect of NO on COX-2 expression in MPs (182,183).

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The simultaneous stimulation of MPs with LPS and uridine triphosphate (UTP) results in elevated levels of both NOS 2 and COX-2 above the levels induced by LPS alone (181). The effect of UTP is manifested through a pyrimidine receptor. Analysis of the UTP potentiation of NOS 2 and COX-2 expression by use of a panel of selective inhibitors indicated that the principal effect of UTP was to enhance the activity of Ca2+/calmodulin-dependent protein kinase which, in turn, potentiated the action of NFκB on the genes encoding NOS 2 and COX-2. Further analysis led to the interpretation that the enhancement of NOS 2 production by PGE2 was mediated by way of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA). It appears that PGE2, generated by COX2 activity in response to stimulants such as LPS, modulates the activity of adenylate cyclase and, thus, the level of cAMP (181,184). Cyclic AMP is distinctly involved in the control of transcription of the genes encoding both NOS 2 (184,185) and COX-2 (186) in MPs. In the case of NOS2, available evidence strongly suggests that it is the degree or extent of activation of the cAMP-dependent transcription pathways that determines whether NO production will be elevated or depressed (184,185). In the case of cox-2 expression, a cAMP responsive element (CRE) has been identified in the cox-2 gene promoter that overlaps an E-box (186). Stimulation with endotoxin (LPS) resulted in the binding of transcription factors (cjun, CREB, USF-1) to those elements. Those two elements, together with at least two other, more-distal, enhancer sequences control the expression of the COX-2 encoding gene. Thus, there is the potential for graded expression of COX-2 and either a potential or dampened response depending upon which combination of transcription factors associate with the promoter. Diagrams and descriptions of the promoters for the NOS2 and COX-2 encoding genes are provided in refs. 184 and 186, respectively. Both promoters embrace cAMP- and NFκB-responsive elements. Thus, it is apparent why the production of both NO and PGE2 are dependent on NFκB- and cAMPfacilitated transcription. The actual transcription factors that can associate with CRE are termed CRE-binding (CREB) proteins. Their binding to CRE requires that they become phosphorylated through the action of PKA. The latter exists in inactive form in the cytoplasm and is activated upon phosphorylation by cAMP (see ref. 187 for review). Although the details remain to be clarified, it is highly likely that the amplification or inhibition of expression of the nos-2 and cox-2 genes is determined by the specificities, composition, and relative binding affinities of the various transcription factors that can interact with enhancer sites in the promoters of those genes. Before leaving the topic of COX-2 in aging phagocytic cells, there are several other mechanisms/processes that may influence its presence and activity that should be mentioned. First, there is recent evidence that the level of COX-2 in

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human mammary epithelial cells is influenced by ceramide (188). The latter is a component of cell membrane sphingomyelin and glycolipids that are most abundant in the Golgi apparatus. Ceramide induction of COX-2 was accompanied by elevated levels of specific mRNA and elevated synthesis of PGE2. It was further demonstrated that the effect of ceramide was manifested via mitogen-activated protein kinase (MAPK) activation of c-jun which, in turn, associated with a CRE in the COX-2 promoter. This finding raises the interesting possibility that ceramide levels may be altered as a consequence of oxidative modification of membranes in aging MP; this might, in part, account for the elevated COX-2 protein and mRNA levels. A second item of information regarding COX-2 levels in phagocytes is the fact that the cytokines, IL-4 and IL-10, strongly inhibit LPS-induced cox-2 expression both in monocytes/macrophages and in neutrophils (189). This fact raises the interesting question as to why those cytokines, which in aged mice and humans are produced at higher levels than in young adults (161,190–194), fail to control cox-2 expression in aged phagocytes. In the case of IL-4, at least, it is possible that the presence of soluble IL-4 receptors, the level of which is influenced by the level of IL-4 secreted by Th2 cells (195), may interfere with an influence of IL-4 on cox-2 expression. Another possibility is the age-associated uncoupling of interlocking regulatory feedback circuits that control the levels of cytokines (TNFα, IFNγ, and IL-10), cAMP, NO, and COX-2. For example, it has been reasoned that the relative levels of the cytokines, TNFα and IFNγ on the one hand, and of IL-10 on the other, regulate the expression of nos-2 (e.g., ref. 196). The levels of those cytokines, in turn, reflect the concentrations of cAMP, which is known to modulate those cytokines in opposite directions, driving up the expression of TNFα and IFNγ while depressing the expression of IL-10, or vice versa. Similar, interlocking circuitry probably exists to regulate expression of COX-2. Uncoupling of such circuits associated with aging may explain why the relaatively high levels of IL-4 and IL-10 fail to dampen the expression of COX-2 and NOS 2 and even account, in part, for the high levels of those cytokines. Even greater complexity is added by the ageassociated changes in neuroendocrine effects on MPs and other phagocytes. Endocrinological Influences on Aging MPs Too often the obvious fact that Mo/MP functions occur in specific tissue environments is overlooked or ignored during the design of experiments. This becomes an even greater problem in the formulation of experiments concerned with aging owing to the inordinately high costs of obtaining and maintaining aged animals. Enlisting suitable aged human subjects for research studies also can be a challenge. Nevertheless, it will not be possible to fully develop therapeutic and prophylactic strategy without the knowledge of MPs, form and func-

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tion in situ. A clear example of this comes from studies of the influences of the endocrine and neurological systems on MP function. One publication (138), which provided compelling evidence that MPs of aging rats were markedly deficient in the ability to generate O2– in response to opsonized zymosan and rat IFNγ, also demonstrated that the respiratory burst could be restored to MPs of aged rats by providing them with syngeneic pituitary grafts from young donors. Deficient TNFα production was also restored. Restoration of O2– and TNFα production was not complete in most cases but was nevertheless dramatic. The effect of the pituitary graft was attributed to its secretion of growth hormone and prolactin both of which can prime MP for response to IFNγ. An analysis of elicited peritoneal MPs of young and aged mice revealed that those from aged mice strongly inhibited proliferative responses of young mouse lymphocytes (stimulated with Con A) (197). This effect was demonstrated to be caused by a higher proportion of mature MPs bearing a high surface density of Fcγ receptors II/III (FcγRII/IIIbright by flow cytometry) among the elicited cells from old mice. It was further demonstrated that the MPs of aged mice expressed receptors for glucocorticoids and that the circulating levels of corticosterone were substantially higher in aged mice, even those that had been protected from stress. The suppression exerted by FcγRII/IIIbright MPs was attributed to their release of NO. MP-mediated suppression under similar experimental conditions has also been attributed to H2O2 and prostaglandins (198). Compelling evidence that the endogenous glucocorticoid was responsible for elevating the suppressor MP in vivo was produced by treating the aged mice with the glucocorticoid antagonist RU 38486, which appeared to restore the peritoneal population to the young-adult condition. The results of this study point clearly to the likelihood that elevated glucocorticoid in aged subjects contribute to the dysregulated MP function; indeed, this could be a primary cause. The production of NO by splenic MPs is influenced by events in the central nervous system. It has been demonstrated that central opioid receptors play a role in regulation of splenic MP NO production (199). Rats were injected with LPS and at intervals spleen cells collected and analyzed for NOS 2 protein and mRNA. Other rats were given naltrexone, an opioid receptor antagonist, along with LPS. The levels of nitrite/nitrate (an index of NO production) were assessed in samples of plasma collected from the rats. Rats given graded doses of LPS alone developed significant levels of NOS 2 and mRNA in the spleens and of nitrite/nitrate in their plasma. Naltrexone strongly reduced those responses to optimum doses of LPS. Similar experiments were performed with the N-methyl derivative of naltrexone (N-methylnaltrexone), which does not traverse the blood brain barrier. When this antagonist was provided by intracerebroventricular route, the splenic NOS 2 responses to LPS were strongly inhibited, but not when

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the drug was given by the subcutaneous route. It was clear that splenocyte (more precisely, MP) production of NO can be regulated via central nervous system mu-opioid receptors. In a previous study (200) the same group of investigators showed that microoinjection of a mu-opioid receptor agonist, a modified enkephalin, stimulated elevated NO production by splenic MPs. The results of this last study raise key questions, viz.: (a) how does LPS communicate with opioid receptors in the central nervous system ? and (b) how do those opioid receptors communicate with splenic MPs? Add to those questions a further question: how does aging affect opioid receptors and the production of endogenous opioids? It is clear that there is a great deal yet to be learned about the in vivo regulation of MP antimicrobial defenses and how it all is affected by the aging process. PHAGOCYTIC CELLS: NEUTROPHILS As a rule, the neutrophil is the first type of defensive host cell to reach a site of infection. Neutrophils respond to chemoattractants, are activated by microbial products (e.g., LPS and N-formyl-methionyl-leucyl-phenylalanine or fMLP), adhere to and traverse endothelial capillary walls, and ingest and kill pathogenic organisms. They produce both ROIs and RNIs in relatively copious amounts and, in addition, possess intracellular granules containing antimicrobial peptides and enzymes. There are relatively large numbers of them in the blood, produced by progenitor cells located primarily in the bone marrow. There are significant effects of aging on neutrophils. Ingestion of pathogens and killing of those that are ingested are the most obvious features of neutrophils that are affected by aging of animals and humans. Before entering into a discussion of those age-related changes, a brief consideration of neutrophil functions may be helpful. Antimicrobial Functions of Neutrophils The rolling and tethering of neutrophils in association with the wall of capillaries in the vicinity of sites of infection has attracted the attention of many researchers. Those events precede the movement of the neutrophils out of the capillaries and into extravascular spaces, a process termed “margination.” Any current textbook of immunology or microbiology will present a discussion of neutrophil extravasation (e.g., ref. 201). The process of extravasation is quite similar for both neutrophils and Mo/MPs. Microbial infection of a tissue site elicits an activation response from vascular endothelial cells (VECs) in the vicinity. This activation of VECs is in response to a variety of mediator substances produced both by the invading microorganisms and host cells. The activated VECs express cell-adhesion molecules known as selectins, which interact with complementary mucin-like molecules present on neutrophils. As

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a consequence, the latter are momentarily bound to the VECs; but, owing to low binding affinity, the neutrophils are swept along by the shearing action of the moving blood only to be momentarily bound again. In this manner, the neutrophils roll along the surface of the endothelium. Soon the neutrophils become activated by locally produced chemokines that function as chemoattractants. IL-8 and macrophage inflammatory protein (MIP-1) are examples. Complement cleavage products, C3a, C5a, and C5b67, may also serve as chemoattractants. The activated neutrophils (or Mo’s) experience a change in conformation of their surface β2 integrin molecules such that they can now interact strongly with cell adhesion molecules (CAMs, members of the immunoglobulin superfamily) that are displayed by the VECs. This time, the tethering is strong and the neutrophils no longer roll. Rather, they proceed to traverse the capillary wall by moving between the endothelial cells and enter the extravascular space. The details of this process are not yet known. The neutrophils (or Mo/MPs) now encounter the invading microorganisms, which they prepare to eliminate. The destruction of microorganisms begins when they are ingested by neutrophils. The integrin, Mac-1 (CD11b/CD18, also known as CR3), is a major receptor for C3bi and thus facilitates ingestion of organisms that have activated the complement system through the alternative pathway. Similarly, CR4 (CD11c/CD18) serves as a receptor for C3bi and facilitates microbial ingestion by neutrophils. Many microbial pathogens trigger the alternative pathway of complement activation including both Gram-negative and Gram-positive bacteria, fungal and yeast cells (via zymosan), various protozoan parasites, and a few viruses. In addition, neutrophils display receptors for LPS including the CD14/TLR-4 complex and a neutrophil-specific, LPS-binding receptor termed bactericidal/permeability-increasing (BPI) protein (202). Apparently, neutrophils do not to any significant extent rely on mannose receptors or scavenger receptors for phagocytosis of pathogens. Overall, the formation of phagosomes and their fusion with vesicles to form phagolysosomes in neutrophils resembles that process in MPs. However, there are differences that arise from the process of fusion of neutrophilic granules with phagosomes and with the discharge of granules into the extracellular milieu. For example, at least two proteins, abbreviated VAMP-2 and -3 (vesicleassociated membrane protein) are required to facilitate phagosome formation (203). Other, related proteins (dubbed SNARE for “soluble N-ethylmaleimidesensitive factor attachment receptor”) are involved in attaching granules to the plasma membrane during granule exocytosis (see ref. 204). Both ROIs and RNIs are generated by activated neutrophils (see ref. 90 for review). However, whether or not human neutrophils generate significant amount of NO is uncertain (205,206). Mouse neutrophils produce substantial

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levels of NO, and the derivative RNIs play important antimicrobial roles. Neutrophils possess the highly active enzyme, myeloperoxidase, which can cause considerable oxidative damage of macromolecules (90). Myeloperoxidase does not act directly on macromolecules, rather it produces low-molecularweight intermediates that convey oxidative damage. One intermediate is the tyrosyl radical, which is produced by the enzyme’s action on the amino acid tyrosine. The tyrosyl radical can facilitate the crosslinking of protein tyrosine residues, cause peroxidation of lipids, and participate in the formation of the highly reactive aldehyde, p-hydroxyphenylacetaldehyde (90). Other aldehydes derived from lipid peroxidation are capable of modifying LDL such that it becomes a ligand for MP SRs (207) leading to the formation of foam cells. The latter are associated with atherosclerosis. In addition to the powerful oxidants derived from O2–, NO and the intermediates produced by myeloperoxidase, the neutrophils are endowed with a variety of hydrolytic enzymes, microbicidal peptides, and other proteins that are located in granules (208,209). There are four recognized types of granules that appear at different stages of neutrophil maturation (210). Certain of the enzymes, peptides, and other proteins tend to be found in association with a particular type of granule; for example, myeloperoxidase, BPI, neutrophil elastase, and defensins are associated with azurophilic granules. Others such as lysozyme and serglycin are found in all four types of granules. This heterogeneity is a reflection of the stage of neutrophil maturation when mRNA transcripts are expressed and the subsequent persistence of those transcripts (211). The preceding discussion concerning the variety of reactive radicals formed in netrophils and the properties of neutrophil cytoplasmic granules is fundamental to understanding the antimicrobial defenses fashioned by neutrophils. Both the reactive radicals and the microbicidal peptide and protein constituents of the granules may be introduced into microbe-containing phagolysosomes. In addition, some of those substances, including O 2–, H2O2, myeloperoxidase, cathepsin G, defensins, and properdin, are exocytosed into the extracellular millieu where they can attack ambient pathogens. The release of properdin is noteworthy because it provokes activation of the complement system via the alternative pathway. The defensive capabilities of neutrophils are not limited to elaboration of reactive radicals and granular constituents. Neutrophils also produce several key cytokines that are instrumental in other processes of innate as well as adaptive immunity (reviewed in ref. 212). A listing of some of those cytokines that have been demonstrated to be produced both in vitro and in vivo is presented in Table 3-3. A variety of stimuli can trigger the production of cytokines and chemokines by neutrophils such as LPS, fMLP, C5a, and a range of microorganisms that includes bacteria, fungi, and certain viruses. But the contingent of cytokines/

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Table 3-3 Cytokines Produced by Neutrophils Produced by neutrophils both in vitro and in vivo GROα IL-1α IL-1β IL-6 IL-8 IL-12 MIP-1α MIP-1β TNF-α TGFβ1 Modified from ref. 215.

chemokines produced may vary from one stimulus to another. For example, fMLP appears to trigger a transient discharge of the chemokines, IL-8 (CXCL 8) and GROα (CXCL 1) but not cytokines; LPS, on the other hand, stimulates release of proinflammatory cytokines including IL-12, TNFα, IL-8, and others (212). The presence of IFNγ potentiates LPS-induced release of IL-10 and IL12. The production of LPS-induced IL-8 is enhanced by adherence of the neutrophils to a substratum coated with fibrinogen or fibronectin (a counter-receptor of integrin α4β1, VLA-4). This is an example of the fact that cross-binding of surface integrins activates several functions of neutrophils including production of IL-8, IL-1β, and MIP-2 (213). The list of microorganisms and microbial components that can stimulate IL-8 production continues to lengthen and now includes a variety of bacteria, yeasts, and even Plasmodium-infected erythrocytes (214). As described previously, IL-8 is a key chemokine in the chemotaxis, activation, and adherence of neutrophils to vascular endothelial cells. Thus, IL-8 is a neutrophilic autocrine. Another important autocrine (another chemokine) produced by neutrophils is known as “growth-related oncogene” or, better, “growth-related gene product-α” (GROα, CXCL1). LPS stimulation results in substantial release of GROα whereas fMLP stimulates very little. A number of bacteria and yeasts and certain of their constituents are capable of stimulating neutrophil production of GROα. The significance of that chemokine in infections is its potency for attracting and activating neutrophils including effects such as degranulation and elevated expression of adhesion molecules. PMNs, particularly neutrophils, are the first of the defensive types of cells to be mobilized to a site of injury or infection. A brief time later Mo/MPs begin to

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infiltrate the site. It is no surprise, therefore, to learn that neutrophils produce macrophage inflammatory proteins (MIP-1α, CCL3 and MIP-1β, CCl4). Those chemokines are potent factors for the chemotaxis and activation of Mo’s, eosinophils, and certain subsets of T cells (215,216). The ability of mature neutrophils to produce IL-12 is particularly significant in light of the fact that the appearance of IL-12 early in the course of infection stimulates both NK cells and the Th1 subset of T lymphocytes to produce IFNγ. The production of the latter is critical for activating and enhancing the functions of MPs and dendritic cells and for driving the adaptive response along the proinflammatory path. Thus, the ability of neutrophils to produce IL-12 along with TNFα, IL-1α, and IL-1β places them in the pivotal role of evoking fullblown innate responses and initiating adaptive responses to pathogens. It is important to note that the variety of stimulants that can induce IL-12 production by neutrophils is restricted and may be limited to microbial constituents such as LPS (217). Moreover, IFNγ is required as a costimulant for IL-12 production. The regulation of the output of cytokines by neutrophils appears to be a function of cytokines IFNγ, IL-4, and IL-10. There is considerable evidence that IL-10 inhibits LPS-induced production of IL-1α and β, and IL-12 (reviewed in ref. 212). In general, IFNγ exerts opposing effects. The levels at which NK, Th1, and Th2 cells, which are sources of IFNγ, IL-4, and IL-10, are involved in regulating neutrophil production of other cytokines remains undecided. Neutrophil Mobilization to Sites of Infection As we have seen, neutrophils are inflammatory cells that transport an arsenal of microbicidal weapons to sites of invading pathogens. Their ability to exit the bloodstream and enter infected tissue sites is crucial to the efficient elimination of bacterial, fungal, and parasitic infections. Upon ingesting and degrading pathogens, neutrophils generally succumb to apoptotic death (218). Neutrophils have the shortest life-span of any of the leukocytes (around 12– 18 h) and a very large number of new cells are produced each day (1–2 × 1011 per day in the normal human) by the bone marrow. An example of the dynamics of infiltration of a site of infection is shown in Figure 3-7, which illustrates the dramatic shift of neutrophils from the blood to the peritoneal space in response to an intraperitoneal injection of mice with LPS (219). An equally dramatic shift of neutrophils from blood to peritoneal space occurs in mice inoculated with the protozoan parasite, T. musculi (220; Albright JW and Albright JF, unpublished) as shown in Figure 3-8. In the response to a persisting infection with T. musculi, the loss of mature and immature neutrophils from the blood is mirrored by their appearance in the peritoneal cavity. Subsequently, there is a steady influx of both mature and immature neutrophils, which continues until the infection is cured (see ref. 221). A similar response has been

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Fig. 3-7. Dynamics of the redistribution and generation of WBC and PMN in the blood, peritoneal space, and bronchoalveolar fluid after injection of LPS intraperitoneally. (A) Rapid decline and rapid rebound in the numbers of PMN (䊉), rapid decline and gradual rebound in the numbers of WBC (䊊). (B) Rapid increase in the numbers of PMN in the peritoneal space (䊊) but not in the bronchoalveolar fluid (䊉) following intraperitoneal of LPS. (From ref. 219, with permission.)

reported of mice inoculated intraperitoneally with live tachyzoites of T. gondii (222). Granulocyte-depleted mice were unable to control the T. gondii infection and the mice soon died. Neutrophils recovered from the peritoneal cavity produced both IL-12 and TNFα in response to soluble tachyzoite extract. The inability of the neutrophil-depleted mice to control the infection was associated closely with the failure to produce significant quantities of TNFγ, IL-12, and TFNα during the first several days of infection. This latter finding was interpreted as being evidence that T cells were not activated in a timely manner by cytokines from neutrophils of the innate system and that inadequate T-mediated

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Fig. 3-8. Dynamics of redistribution and generation of mature (䊊) and immature (䊉) neutrophils in the blood (A) and peritoneal space (B) of mice following intraperitoneal inoculation of the mouse-specific parasite, Trypanosoma musculi.

responses explained the development of lethal parasite infections. The key role of neutrophils in driving a Th1-type of adaptive immune response was demonstrated in the case of L. pneumophila-induced pneumonia (223). The requirement for neutrophils early (24–48 h) in the course of L. monocytogenes infections has been reviewed by Unanue (224), who points out that they are involved both in the early control as well as, later, combatting the dissemination of the organisms among hepatocytes. The preceding are examples of the rapid mobilization of neutrophils to sites of infection and the fatal consequences when that process fails. Failure of that process could result from (a) inadequate generation and maturation of neutrophils in the bone marrow, (b) ineffecient margination of neutrophils at sites of infection, (c) defective ability of neutrophils to phagocytose and destroy microorganisms, or (d) defective ability to produce cytokines (e.g., IL-12) or respond to cytokines and chemokines (e.g., G-CSF or IL-8). In the next section, we explore the effects of senescence on those processes.

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The Effects of Aging on Neutrophils Although the effects of aging on most key aspects of the generation and functions of neutrophils have been investigated, there frequently have been differences in the results obtained and the interpretations of the results. However, one aspect of neutrophil physiology about which there is agreement is that of the circulating numbers and production of new neutrophils by the bone marrow of young and aged subjects (225,226). The responses of marrow of young and aged subjects to progenitor cell stimulants, GM-CSF and IL-3, were essentially equivalent. It was noted, however, that an age-related, slightly less vigorous response of lineage-committed precursors to G-CSF occurred (225). Chemotaxis, rolling, adhesion to vascular endothelium and margination of neutrophils appear to be unimpaired in the aged (226–228). The amount of CD15 (Lewis X or SSEA-1) expressed on the plasma membrane was slightly increased on neutrophils from aged compared to young adults; CD15 interacts with E-selectin and participates in neutrophil rolling. The integrin α chains, CD11a and CD11b, were unchanged or slightly increased on neutrophils of elderly subjects. There have been several reports concerning phagocytosis by neutrophils of the aged. Most of those studies have been focused on ingestion of opsonized particulates (bacteria, yeast, zymosan) and the results have been reasonably consistent in showing defective phagocytosis (e.g., refs. 229–232). As an example, a strong negative correlation was found between the number of complement-opsonized E. coli ingested and the age of neutrophil donors (232). The same was true for opsonized S. aureus. Cells from 70-year-old subjects phagocytosed 30%–40% fewer bacteria than subjects of age 20. Thus, it is clear that senescence alters the efficiency of neutrophil phagocytosis that is dependent on Fc receptors (FcRs) and/or CRs. There have been few studies on ingestion of microorganisms by neutrophils in the absence of opsonins. One study (231) revealed that the ingestion of unopsonized bacteria was about the same for neutrophils of young and aged subjects. Furthermore, the number of bacteria ingested was low in the absence of opsonins for neutrophils of both young and aged subjects. Clearly, there is a need for much more data regarding the phagocytic capabilities of aged neutrophils given the current abundant evidence that those cells play a critical defensive role in the early course of infection. There are numerous reports of defective microbial killing by neutrophils of the aged. A critical, first step is the fusion of the phagosome with vesicles to form the phagolysosome. A shift to higher concentrations of cytosolic free Ca2+ accompanies the process of phagocytosis and the elevated level of Ca2+ promotes fusion of the phagosomes with lysosomal vesicles (233,234). There is significant, age-associated reduction in the magnitude of Ca2+ flux in neutro-

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phils challenged with agonists such as fMLP (232,235). That diminished flux probably reflects reduced Ca2+ mobilization as well as higher resting, cytosolic concentrations in aged neutrophils. In addition, Ca2+ extrusion is impaired in fMLP-stimulated neutrophils (235). The diminished Ca2+ flux is probably, in large part, a reflection of aberrant signaling via the inositol phospholipid pathway. Thus, in aged neutrophils there is a pronounced deficiency in the generation of diacylglycerol (DAG) and inositol triphosphate (IP3) in the immediate response to fMLP stimulation (236,237). Systematic studies of the underlying causes of that age-related deficiency in second messengers showed that the problem is not in the fMLP receptors or in the enzyme phospholipase-C-β that cleaves the substrate phosphoinositol biphosphate (PIP2) to produce DAG and IP3. Rather, the evidence points to diminished amounts of metabolically active membrane phosphoinositides (PIP2, PIP, and PI) which compose the pool of precursors of DAG and IP3. A fascinating conclusion has been drawn from the results of these studies on aged neutrophils resulting in the hypothesis that: “the prime cellular change occurring with age is an alteration in membrane lipid composition that is associated with reductions in critically important phosphoinositide pools” (236). There is considerable evidence to support the idea that cell membranes undergo rather major changes associated with aging; this topic is discussed in Chapter 4. Most investigators have concluded that neutrophils of aging subjects show impaired generation of ROIs and defective microbicidal ability (232,236,238– 241). For example, the burst of O2– production by aged human neutrophils in response to fMLP stimulation was significantly less than by young adult neutrophils (236). In contrast, neutrophils of both ages generated equivalent amounts when stimulated by phorbol myristate, again showing the effect of senescence on early, postreceptor, membrane events. Treatment of the cells with ionomycin to elevate cytosolic Ca2+ also resulted in equivalent bursts of O2– by young and aged neutrophils in response to fMLP. Exposure of young and aged donor neutrophils to S. aureus revealed a considerable difference in their ability to generate O2– (232). Significantly, that difference was not found when the young and aged neutrophils were exposed to E. coli. In this study, it was also shown that there is a strong correlation between age of the neutrophil donors and the resting, intracellular concentrations of Ca2+. The investigators suggested that it may be the flux of Ca2+ upon stimulation that correlates with the magnitude of O2– production; thus, a higher resting level would diminish the flux upon stimulation. That interpretation would seem to contradict the enhanced production of O2– by aged neutrophils treated with ionomycin demonstrated in (236). The reports reviewed above are consistent in finding that O2– production by aged neutrophils is significantly less than by those from young subjects. How-

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ever, the extent of the reduction is not dramatic, being in the range of 15%– 30%. That degree of reduction in O2– production coupled with the demonstrated loss of phagocytic ability of neutrophils could, presumably, account for substantial impairment of resistance of the elderly to infection. In order to gain further insight as to the cause of the reduced formation of O2–, a study of individual, aged neutrophils was initiated to determine whether or not only a proportion of them is defective (239). The individual neutrophil content of H2O2 was assessed as an index of the respiratory burst. The surprising result was that the level of H2O2 was decidedly higher in aged compared to young neutrophils and this was true of the majority if not all of the neutrophils. The resolution of that paradox came from analyses of various enzymes that affect the intracellular levels of H2O2. There were no significant differences between young and old, resting or fMLP-activated, neutrophils in the activities of superoxide dismutase or catalase. The activity of glutathione peroxidase, however, was markedly impaired in aged, stimulated neutrophils although the amounts of the enzyme were equivalent in cells of the different age groups. Glutathione peroxidase plays a major role in removing peroxide, including H2O2, to protect cells from excess oxidative damage. It is an unusual enzyme in that selenium is required in its active site. The investigators studied the levels of selenium and found no differences in the amounts present in young and aged neutrophils. What does the significantly elevated level of H2O2 mean with regard to the microbicidal ability of aged neutrophils? The most effective antimicrobial utilization of H2O2 is in the formation of hypochlorous acid (HOCl), which is extremely powerful against bacteria, viruses, fungi, mycoplasmas, and protozoans. The utilization of H2O2 to form HOCl involves the enzyme myeloperoxidase (see ref. 90). Though myeloperoxidase plays an active role in the microbicidal activity of several species that have been studied, rodents in particular, it does not appear to be essential for microbicidal activity in normal humans (90,242). Therefore, because the studies on defective glutathione peroxidase were done with human neutrophils, it would be most interesting to determine whether or not the same is true in neutrophils of aged rodents. Perhaps a defective glutathione peroxidase would favor the utilization of H2O2 by myeloperoxidase to generate microbicidal HOCl in neutrophils of aged mice. NATURAL KILLER/LYMPHOKINE-ACTIVATED KILLER CELLS The discovery of NK cells emerged from observations of “natural” or spontaneous cytotoxicity directed against certain tumor cells (243,244). For many years, NK cells remained of interest primarily to specialists in tumor immunity (see, e.g., ref. 245). Gradually, evidence accumulated in support of NK cell involvement in resistance to certain viruses (246) and fungi (247), and in the phenomenon of “hybrid resistance” (248). Then, in close order, several impor-

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tant advances changed the study of NK cells from a backwater of immunology to a mainstream topic of intense interest. Some of those advances were: 1) compelling theory to explain how receptors can control the seemingly erratic killing of prospective targets by NK cells (reviewed in ref. 249); 2) the discovery of several sets of receptors involved in NK-cell functions, prompting one author to title his review article, “...From No Receptors to Too Many” (250); and 3) the critical role of NK cells in the early development of innate responses to pathogens, especially the secretion of pivotal cytokines such as IFNγ (e.g., refs. 251 and 252) and of chemokines that promote leukocyte margination (253). It is now firmly established that NK cells, their lymphokine-activated killer (LAK) forms, and their relatives known as NK-T cells, are essential for the early control of infection and for alerting the more slowly developing adaptive responses. In some cases, NK/LAK cells may act directly to eliminate pathogens, as in killing of virus-infected cells, whereas in other (probably most) cases, NK and NK-T cells activate other cells (especially MPs or DCs) that actually kill the pathogens. These activities are accomplished, of course, through a network of cytokines, chemokines, and their receptors. A Plethora of NK Receptors For some years, a dominating concern among those interested in NK cells was the lack of understanding about NK-cell recognition of the target cells they could kill (mostly tumor and virus-infected targets), and the related question of why NK cells seemed oblivious of normal self. Knowledge in that area of concern advanced rapidly with the publication of the “missing self” hypothesis, which postulated that targets for NK cells are deficient or devoid of major histocompatibility complex (MHC) class I molecules whereas those molecules on normal self cells inhibit NK cytotoxicity (see ref. 249). A spate of publications in recent years has provided considerable insight into the receptor regulation of NK/LAK functions and activities (reviewed in refs. 254–256). Antibody-assisted NK cytotoxicity (known as antibody-dependent-cellmediated cytotoxicity, or ADCC) has been known for some time although, until recently, it was considered to be an in vitro curiosity of no particular physiological significance. The ADCC mechanism involves interaction of target-bound antibody with the Fcγ receptor III (CD16) on NK cells, the anti-body imparting recognition specificity to the NK cells and promoting their cytotoxicity. In addition to target killing, the ADCC effector (NK) cells secrete cytokines (257). There are three sets of genes that appear to determine the functional activities of NK cells. One set, known as ly 49 and found in mice, comprises at least nine isoforms (Ly 49A–Ly 49I). Homologs of ly 49 have been demonstrated in rats and recently in humans; the human homolog is a pseudogene. A second set of genes, encoding the CD 94/NKG2 protein complex, have been most thoroughly

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studied on human NK cells. However, homologs of both cd 94 and nkg 2 have been identified in rats and mice. The third set of genes, designated kir (KIR, for “killer-cell immunoglobulin-like receptor”), have been demonstrated in humans and primates but not in rodents. The receptors encoded by all three sets of genes interact with selected MHC molecules (generally of class I) on prospective target cells. Within each set of genes there are those that encode inhibitory receptors and a few that can activate NK cells. The net effect on NK-cell function (whether activatory or inhibitory and the intensity of either) probably is determined by the balance of stimulatory and inhibitory signals. The Ly 49 receptors of mouse NK cells are type II membrane glycoproteins and belong to the family of C-type lectins. The following isoforms transduce inhibitory signals that block the cytotoxicity and cytokine production by NK cells: Ly 49A upon interaction with Class I molecules of H-2Dd or Dk; Ly 49G2 upon interaction with H-2kb or Dk. The mechanism of inhibition involves an immunoreceptor tyrosine-based inhibitory motif (ITIM) located in the cytoplasmic portion of the receptor. The ITIM becomes phosphorylated upon engagement of the receptor and then recruits a phosphatase, SHP-1 (Src homology 2 domain-containing protein tyrosine phosphatase); the latter dephosphorylates and, thus, inactivates certain intermediates in the signaling cascade that lead to target cell lysis (258,259). Isoform Ly 49D is an NK activator; target cells expressing H2-Dd or Dr are selectively lysed (260). Ly 49D, as well as Ly 49H, another NK activator, lack ITIM motifs in their cytoplasmic domains; they also lack ITAMs (immunoreceptor tyrosine-based activation motif). However, they are able to transduce activating signals by associating with an ITAM-bearing molecule, DAP 12 (261). More than one inhibitory isoform of Ly 49 can be expressed on a given NK cell. As a consequence, NK cells fall into overlapping subsets according to Ly 49 isoform expression. If a given NK cell expresses two inhibitory isoforms, engagement of either can result in inhibition of the cells functions (cytolysis, cytokine secretion). The inhibitory effect of engaging two or more isoforms on a given NK cell simultaneously is cumulative (262). Thus, in general, the degree of NK-cell inhibition is a quantitative reflection of the number of different inhibitory isoforms engaged simultaneously. It should be mentioned that inhibition is a transient matter and is reversible; it does not lead to permanent inhibition or NK-cell death. In order to better understand the functional significance of NK activation via Ly 49D receptors, highly enriched preparations of IL-2-treated LAK cells were stimulated by crosslinking the Ly 49D receptors with a specific mAb. The mRNA from those cells was extracted, cDNA prepared, and subjected to microarray analysis of gene expression (263). The majority of the genes activated were in two categories: (a) cytokines and chemokines or (b) apoptosis

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related. Among the former were genes encoding IFNγ, MIP 1α, MIP 1β, and lymphotactin. Among the latter were caspase II, apoptosis receptor TDAG 8, and metallothionein I. Also, some genes were suppressed such as mouse TCRγ chain, mouse MEL 91, mouse diacylglycerol acyltransferase, and several others. Overall, it could be concluded that substances directly involved in regulating immune responses are produced at the site of receptor activation and that “NK cells and their activating Ly 49 receptors [are] important initiators of microbial immunity and key elements of the innate immune system” (263). The two major human sets of NK receptors, known as CD94 /NKG 2 and KIR, include both inhibitory and activatory receptors. The CD94/NKG 2 complex is composed of CD94 invariant chain and one of four isoforms of NKG 2 (-A, -C, -E, -F). In order to be expressed in the NK-cell membrane, NKG 2 must be disulfide-bonded to CD94. Isoforms NKG 2-A and -B have intracytoplasmic tails bearing ITIMs and upon interacting with Class I molecules on target cells inactivate the cytotoxic and cytokine-generating activities of the NK cells. In contrast, isoforms NKG 2-C and -D have short cytoplasmic tails and lack ITIMs. NKG 2-C and -D can deliver activating signals to the NK cells by associating with, respectively, DAP 12 or DAP 10; both of the latter display ITAMs in their intracytoplasmic regions. The KIR are members of the immunoglobulin superfamily of receptors. Their extracellular regions may consist of one to four immunoglobulin domains each of about 110 amino acids. Their intracytoplasmic tails may be either short or long. Those with short tails are capable of activating the NK cells that bear them by associating with ITAM-bearing molecules, DAP 12, or the zeta chain of the CD3 complex (the other members of the CD3 complex are not expressed on NK cells). Inhibitory KIR recruit SHP-1 phosphatase to associate with their ITIMs similar to the inhibitory Ly 49 receptors discussed above. The molecular bases of the specificities evinced in the interactions between the NK receptors discussed above and the Class I HMC molecules on prospective target cells have been carefully reviewed (254–256) and are not discussed here. It should be mentioned that the KIR receptors and Ly 49 receptors interact with classical (HLA or H-2) Class I molecules. The peptide present in the cognate cleft of these Class I molecules seems to have some influence on the specificity of interaction with various NK receptors but nothing like the specificity of T-cell receptor (TCR) interactions with Class I-peptide conjugates. The CD94/NKG 2 receptors interact selectively with nonclassical, nonpolymorphic HLA-E molecules of MHC Class I. In this case, too, it appears that the peptide associated with the Class I molecule has influence on the specificity of interaction with CD49/NKG 2 receptors but the details have not been elucidated. Nonamer peptides found in association with HLA-E molecules are generally derived from the leader sequences of other class I molecules.

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Cells with NK and T-Lymphocyte Properties (NK-T cells) NK and NK-T cells share certain antigenic markers (e.g., Ly 49A, and NK 1.1 in C57BL strains of mice). In addition, both types of cells are active in lysing appropriate target cells and in secreting IFNγ upon stimulation. They differ, however, in the ability of NK-T, but not NK, to produce IL-4. NK-T, but not NK, cells express a version of TCR: in mice, Vα Jα281, generally associated with Vβ 8.2; and in human Vα 24 Jα Q paired with Vβ 11. Unlike conventional T cells, NK-T-cell receptors interact selectively with a nonclassical class Ib molecule known as CD 1d (264). The ligand is not a peptide; rather, it is a glycolipid. Glycosylceramides, specifically, α-galactosylceramide (265,266) and α-glucosylceramide (265), have been studied in considerable detail. It appears that those two glycosylceramides, with which CD 1d interacts specifically, associate with the CD 1d molecule through two, rather large, hydrophobic pockets. Thus, longer, rather than shorter, fatty acyl and sphingosine bases associated with the ceramide moiety are required for optimum hydrophobic bonding to CD 1d and optimum activation of NK-T cells. For a time, research concerned with NK-T cells was hampered by the lack of precise markers for their identification. This led to some conflicting claims in the published literature. A recent, comprehensive study has identified those markers that can be relied upon (267). The same study also showed that, by far, the richest source (in mice) of NK-T cells is the liver. NK-T cells, like NK cells, are enlisted early in the course of many infections. Cytokines such as IL-2 and IL-12 stimulate both types of cells to produce IFNγ and display cytotoxicity. Activators such as α-galactosylceramide stimulate rapid activation of NK-T cells. Evidence has been provided that NKT activation leads quickly to NK activation and IFNγ secretion (268). Those activated NK-T cells also produce IL-4, which is likely to bias development of the subsequent, conventional T-cell response toward the Th2 response. Thus, available information frames an interesting picture in which the type of immune response that develops in response to an infectious organism depends on the early interaction of the NK and NK-T cells and the emergent dominance of one of them. DCs, especially those of the liver where NK-T cells are most abundant, are likely to be a central subject in that picture. DCs perform two key functions: they (a) efficiently activate NK-T cells through presentation of α-glycosylceramide, and (b) secrete substantial amounts of IL-12 (269). Glycolipids are commonly present on the outer surfaces of microbiota and, therefore, early NK-T responses to invading pathogens are expected to occur. Recently, it has been demonstrated that NK-T cells respond in CD 1d-restricted fashion to the GPI moieties of GPI-anchored proteins (270). GPI-proteins of two protozoan parasites, Plasmodium falciparum and Trypanosoma brucei, were included in the study. It was shown further that the activated NK-T cells

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were competent to condition a humoral antibody response when allowed to interact with B lymphocytes; presumably, the IL-4 produced by the NK-T cells played a major role in facilitating the production of IgG antibody by the B cells. This study provided an excellent example of the role that NK-T cells, activated early in the course of infection, are likely to play in directing the types of later adaptive responses to pathogens. Cytokines, Receptors, and Signaling in Innate NK and NK-T Responses to Microorganisms The cytokines that have major influence on the early activation of NK and NK-T cells are those produced by phagocytic cells, i.e., neutrophils, DCs and MPs. Both proliferation and maturation of NK cells are triggered by IL-2 in relatively high concentration. Maturation leads to a heightened preparedness for target cell destruction. IL-2-activated NK cells are referred to as LAK cells. IL-2 binds to complementary receptors (IL-2R) present on NK cells. The majority of NK cells display IL-2R of intermediate affinity for IL-2; i.e., the receptors comprise the β and common γ subunits but lack the α subunit, which would endow them with high affinity. However, in humans, the subset of NK cells that express higher surface density of CD56 (CD56 bright) express the high-affinity IL-2R. Therefore, the majority of NK cells are not functionally activated by low concentrations of IL-2 alone but the addition of other cytokines does lead to functional activation. For example, the combination of a submitogenic concentration of IL-2 with IL-12 or IL-15 triggers NK cell proliferation (271). Either of the latter two interleukins alone is without effect. IL-15 combined with either IL-10 or IL-12 is effective in stimulating NK proliferation. IL-2 and IL-15 have been used as stimulants in several studies concerned with NK cell proliferation. The receptor for IL-15 utilizes β and common γ chains characteristic of the IL-2R. Whereas the transduction of signals through IL-2R and IL-15R may engage several pathways (see ref. 272), proliferation of NK cells appears to involve phosphorylation of tyrosines of the Janus kinases (JAKs) 1 and 3. The phosphorylated tyrosines then serve as docking sites for proteins known as STATs 5a and 5b. The latter, once phosphorylated, form homodimers, which are translocated to the nucleus of the cell where they bind to the promoters of selected genes, such as genes involved in cell cycle progression (273,274). NK-cell activation includes heightened ability to kill targets and to secrete IFNγ. A major element in target cell killing is the protein perforin, which closely resembles complement component 9 (C9). The NK-cell content of perforin in substantially elevated in IL-2-generated LAK cells. Transcription of the perforin gene appears to involve STATs 1, 3, and 5, which recognize enhancer elements in the promoter regions of perforin genes (275).

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IL-12 together with IL-18 is, by far, the most effective combination of cytokines for stimulating NK cells to secrete IFNγ (253,276). STAT 4, which is translocated to the NK-cell nucleus as a result of signaling via the IL-12R, plays a major role in activating transcription of the gene encoding IFNγ. Although the combination of IL-12 with IL-15 can enhance IFNγ production, the amount is a small fraction of that elicited by IL-12 along with IL-18. Crosslinking of the coreceptor, CD28, with monoclonal Ab, or binding of the B7.2 (CD86) natural ligand to CD28, concurrent with IL-12 stimulation, is another effective method to stimulate IFNγ production (277–279). Another costimulant of IFNγ production is IL-1β. The latter along with IL-12 appears to trigger IFNγ release by a subset of human NK cells, viz., about 40% of resting (CD56 bright) NK cells (280). Because IL-2 appears to be produced only by T cells of the Th1 type, and because T cells do not appear to be activated during the early course of the innate response, it seems unlikely that IL-2 plays a role in the early activation of NK and NK-T cells. The fact that IL-2 can activate cells of the NK type may be a reflection of the fortuitous sharing of the β and common γ chain by the IL-2R and IL-15R. Those cytokines that are likely to be involved in activating NK cells shortly after the initiation of infection are IL-12, IL-15, IL-18, IL-1β, IFNα/β, and TNFα. All of them are produced by DCs, MPs, or neutrophils and may arise early in the course of infection as a consequence of microbial stimulation. Those cytokines, in appropriate combinations, acting alone or quite likely in synergy with microbial components such as glycolipids or endotoxins, effect the activation of NK and NK-T cells. B7.2 (CD86), which is present on activated MPs and DCs, may well play a significant, costimulating role in NK/NK-T activation. In humans CD94, a C-type lectin, plays a costimulatory role when ligated with a specific mAb (281). There are three important ways in which NK and NK-T cells can influence the course of infection: 1. First, by producing adequate amounts of IFNγ to (a) enhance the microbicidal activity of Mo/MPs and immature DCs, and (b) to aid in channeling the development of T cells into the Th1 stream (see Chapter 4). 2. Second, by killing (lysing) target cells harboring intracellular pathogens, especially viruses but also bacteria and fungi (224,251,252,282,283). The infected cells become susceptible to NK cytolysis as a result of the ingested microbeinduced down regulation of surface class I MHC molecules (e.g., ref. 284). 3. Third, by participating as helpers in the maturation of CD8+ immediate precursors into fully functional cytotoxic T cells (285). With regard to this last NK cell attribute, it is one way that NK cells may serve to link innate and adaptive immunity.

NK cells also play critical roles in defense against protozoan parasites (286). At one time, it appeared NK cells might kill the parasites, or parasite-infected

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cells, directly but subsequent studies have provided little support for that idea (287–290). Rather, there is overwhelming evidence that the role played by NK cells is indirect, manifested through the cytokines and chemokines they secrete (reviewed in ref. 286). Thus, resting NK and NK-T cells become activated by the cytokines (IL-12, TNFα, IL-1β, IL-15) released by Mo/MPs, DCs, and neutrophils that have encountered the parasites (or components of the parasites). Costimulation of the excited NK cells may be provided by B7 molecules, which are upregulated on activated MPs, DCs, or perhaps B cells, and/or by components of the parasites that are recognized by pattern recognition. The release of IFNγ and TNFα results in marked enhancement of the phagocytic cell destruction of the parasites. In addition, the activated NK cells may participate in the arousal of cytotoxic T cells capable of killing host cells that harbor intracellular parasites. Age-Related Impairment of NK-Cell Function The question of whether or not NK cells suffer from the effects of aging has, in the past, raised considerable controversy. In part, the controversy reflected the fact that NK cells from two quite different sources were being studied, viz., human peripheral blood and mouse spleen. Even more confusing were the reports from different laboratories involved in studies of human peripheral blood NK cells that the functions of those cells were depressed, enhanced or unaffected by aging (see refs. 291 and 292 for reviews). At least three variables accounted for the disparate results of studies performed up until recently: (a) the index used to evaluate NK status, i.e., numbers, proliferation, cytotoxicity, or cytokine production; (b) complexity of the system of cells under study (e.g., entire leukocyte population or partially enriched or highly enriched NK preparations); and (c) the health and/or nutritional condition of the blood cell donors. Better attention to those and other variables have produced recent data that are less confusing. It is now generally accepted that in healthy humans the number of NK cells increases with age in the peripheral blood. That increase results primarily from elevated numbers of the CD56dim subset. CD56 is a characteristic marker of circulating NK cells in humans. The relative surface density is high in cells that have not completed maturation (CD56bright) and relatively low in fully matured NK cells. The individual NK cells present in the blood of healthy aged humans appear to be defective in the ability to lyse target cells. This is true of both their spontaneous target cell lysis and their Ab-facilitated (ADCC) cytotoxicity (293,294). Binding of NK to their targets (cell contact is essential for target cytolysis) is not impaired. Although the perforin content of resting NK cells appears to be significantly reduced (295), once the cells are activated the perforin content of young and aged cells appears to be about the same (296).

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At present, there is only limited information about the precise, age-associated defect in NK cells. A recent study (297) provided evidence of defective phosphoinositide turnover in aged NK cells challenged with target cells. There were substantially reduced levels of inositol triphosphate (IP3). That change would be expected to lead to weak Ca2+ mobilization, reduced protein kinase C activation, and defective translocation of transcription factors (e.g., NFκB, NFAT). Aged NK cells display alteractions in the cytokines they produce upon stimulation. Aging reduces the ability of NK cells to produce IFNγ (298,299). But after several days of culture in IL-2, the LAK cells generated from young and aged NK can produce equivalent amounts of IFNγ (299). This aspect of NK-cell aging, viz., the production of, and responses to, cytokines and alterations of the associated signaling processes, requires much more attention. For example, it is well known that the production of IL-2 is markedly depressed in aged individuals and that the majority of NK cells express constitutively the IL-2R of intermediate affinity. However, the subset of NK cells characterized as CD56bright express the high-affinity IL-2R and can be activated to LAK-cell status by low concentrations of IL-2. Perhaps those IL-2-activated LAK cells play a significant role in the responses to infection in aged individuals. They might be sources of soluble IL-2R (sIL-2R) that further deplete available IL-2 in the aged (see ref. 300). It is important to note the evidence (300) that NK cells in aged humans appear to be in an activated state (i.e., CD 69+). A provocative recent report (301) has revealed that resting, but not activated, human NK cells can be stimulated in such a way as to selectively activate IFNγ production but not target cell killing. This selective activation is manifested through a receptor (KIR 2DL4, CD 158d) that belongs to the killer cell Ig-like family. That receptor is expressed on all NK cells rather than being differentially distributed. The ligand for KIR 2DL4 has not been identified. Activation of IFNγ production via KIR 2DL4 utilizes the p38 mitogen-activated kinase pathway unlike activation via the IL-2R, which engages the ERK mitogen-activated kinase pathway. The selective activation of IFNγ production may represent a way of stimulating and maintaining IFNγ production but without potentially pathological (i.e., self-destructive) cytotoxic activation. IFNγ from that source could be critical in driving both innate and T-cell-mediated defenses. It will be important to determine whether or not selective IFNγ production is possible by aged NK cells. Purified NK cells from healthy aged humans are not as capable of IL-2induced proliferation as are those from young subjects (302). That deficiency is associated with the selective expansion of the CD56dim subpopulation of NK cells (fully mature, IL-2R of intermediate affinity) in elderly subjects. Moreover, deficient proliferation in response to IL-2 is paralleled by diminished

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expression of the activation marker, CD69, by the aged NK cells. It should be pointed out that in this study of proliferation (302) experiments in which costimulation was provided were not performed. Therefore, it will be interesting to learn whether or not proliferative competence can be restored to aged NK cells by appropriate costimulation or whether some or all of the aged cells are terminally differentiated. It appears that there are several possible costimulatory routes including CD28 and 2B4 (CD244) (303) in mice and humans and KIR 2DL4 and CD94 in humans. There is the fascinating possibility that the age-associated defect in NK-cell proliferation reflects the gradual loss of intracellular reducing potential and that restoration of redox balance might be accomplished by appropriate stimulation. The deprivation of thiol compounds in an NK-cell line was carefully analyzed and found to inhibit proliferation but not the activation of the IFNγencoding gene (304). The effect on proliferation was found to reflect dysregulation of cyclin-dependent kinases and premature and prolonged phosphorylation of the retinoblastoma (RB) gene product. This suggests that two functions of NK cells, proliferation and IFNγ production, are independently regulated, as are cytotoxicity and IFNγ production in resting NK cells (301). Some of the early evidence that NK-cell function declines with age, and that the decline might be associated with heightened severity of infections, was generated by studies of aging mice (305–307). For example, Table 3-4 presents data from some of our early studies (305) that show the significant age-related impairment of splenic NK-cell cytotoxicity. Utilizing the best methodology available at the time, we showed that the decline in NK-cell cytotoxicity was not paralleled by a depletion of NK cells; rather, the numbers of NK cells present in the spleens of young and aged mice were about equal. Recent studies (e.g., ref. 308) have confirmed that the loss of cytotoxic potential of NK cells in aged mice is not accompanied by a decline in number of NK cells but is correlated with an increase in the proportion of NK1.1+ CD8+ cells. Thus, in both mouse and man aging does not cause a decrease in the numbers of NK cells (including LAK cells) but does reduce the functional competence of the cells. In the last few years, it has been recognized that NK cells are souces of chemokines including lymphotactin (XCL 1), RANTES (CCL5), MIP 1α (CCL 3), MIP 1β (CCL 4), IP-10 (CXC 10), IL-8 (CXCL 8), and others. Recent studies (292,309) have revealed that NK cells from aged humans and mice are quite defective in the ability to produce chemokines. It has been reported that the output of several chemokines by aged human NK cells is about half that of young human NK cells (292). The situation in mice is striking (309). The biosynthesis of XCL 1 by aged NK/LAK cells in response to IL-2 or IL-15 was a small fraction of that synthesized by young NK/LAK cells as judged by mRNA ribonuclease protection assay. Even more dramatic, aged NK/LAK cells generated few,

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Table 3-4 Effect of Aging on the Cytotoxic Ability of Mouse NK Cellsa 51Cr

release by YAC-1 targets (percentage of maximum) Young mice Old mice

Treatment

E:T ratio

E:T ratio

In vitro

20:1

10:1

5:1

20:1

10:1

5:1

None IL-2 poly I : poly C

32.5 60.1 39.7

18.9 – –

13.9 21.7 17.8

16.3 19.1 16.1

9.8 – –

4.4 5.3 8.9

aData obtained in previous studies (Albright JW and Albright JF, unpublished); similar data may be found in publications by others (e.g., Saxena RK et al. Immunology. 1984;51:719). bMouse spleen cells placed in culture and not further stimulated or stimulated either with IL-2 (200 U/mL for 3 d) or poly I : poly C (20 µg for 3 d).

if any, transcripts of CCL 5, CCL 3, CCL 4, CXCL 8, and CXC 10. This ageassociated deficiency in chemokine production appears to be one of the most clear-cut effects of senescence on immune responses that has been found so far. CHAPTER SUMMARY The innate immune system, or subsystem to be more precise, comprises the cellular elements (neutrophils, Mo/MPs, DCs, NK cells, and NK-T cells) and soluble or humoral elements (the C system, acute phase reactants, and some of the cytokines and chemokines). The innate subsystem constitutes the rapidresponse, first line of defense against pathogenic microorganisms. The elements of the innate subsystem recognize and respond to complex molecular entities or patterns of molecules associated with microorganisms. The cellular elements of the innate subsystem lack the highly specific, epitope-combining receptors that characterize the B and T lymphocytes of the adaptive subsystem. Rather, the PRRs of the neutrophils, Mo/MPs, and DCs have broader specificities for complex molecules such as lipopolysaccharides of Gram-negative organisms, lipoteichoic acids of Gram-positive organisms, lipoproteins, glycolipids, mannans, and double-stranded RNA of viruses. There are four types of PRR associated with cells of the innate subsystem: (a) MBP, which is produced as an acute phase reactant by the liver and associates with cells as an opsonin; (b) MR, a cell-surface receptor that has broad specificity for glycoconjugates that display accessible mannose, fucose, or N-acetylglucosamine residues; (c) TLRs, exemplified by TLR 2 and TLR 4, which mediate responses to Gram-positive and Gram-negative pathogens, respectively, TLR 4 having received the greatest attention owing to its being

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a component of the complex lipopolysaccharide/Taxol receptor of Mo/MP; and (d) the family of SRs composed of Class A found on Mo/MPs and some DCs that function as adhesion molecules, and Class B found on Mo/MPs, B cells, platelets, and adipocytes that promote uptake of LDL and modified LDL. A fifth family of receptor, the PPAR, is gaining considerable importance because of its roles in regulating the MP burst of reactive oxygen species (ROS) formation (310) and regulating cytokine formation by splenocytes, both Mo/MPs and lymphocytes (311). Together, neutrophils and Mo/MPs constitute the first cellular elements to be alerted to infection. They are drawn to infection sites by chemoattractants (cytokines and chemokines) that are released from vascular endothelial cells and other leukocytes in the vicinity of the infection. NK cells, LAK cells, and lymphocytes in the vicinity contribute chemokines, which add to the intensity of the chemoattraction. Marginated neutrophils and MPs are activated via their PRRs and their receptors for proinflammatory cytokines, chemokines, CRs, and FcγRs, and mount a formidable attack on the microbial invaders. Their arsenal includes opsonins, ROIs, RNIs, hydrolytic enzymes, and a variety of toxic peptides. NK and LAK cells are enlisted in the defense early during the course of combatting infection. They are activated by cytokines released by MPs and DCs, especially IL-12, and contribute IFNγ, which is a powerful Mo/ MP activator, and chemoattractants such as lymphotactin (XCL1), RANTES (CCL5), MCPs (CCL 2, 8), and other chemokines. In addition, LAK cells may kill virus-infected cells. Aging exerts significant deteriorative effects on the cellular elements of innate immunity. It causes impaired regulation of ROI and RNI production by MPs along with dysregulated expression of transcription factors and inducible enzymes such as NOS 2 and COX-2. Phagocytosis and/or intracellular killing of ingested pathogens have been found to be deficient in both aged MPs and aged neutrophils, especially in the case of opsonized bacteria. Both Mo/MPs and neutrophils release several important cytokines. The effects of aging on cytokine (and chemokine) production by those cells remain to be adequately studied. That is particularly true of IL-12, IL-15, and IL-18, which play important roles in stimulating and regulating the activities of other cells such as NK/LAK cells and T cells of the adaptive subsystem. One aspect of innate immunity, as it is concerned with infections, that needs much more attention is that of redox conditions both intracellularly and microenvironmental. Apart from their abilities to generate ROIs and RNIs, very little is know about the age-associated changes in redox balance in Mo/MPs and neutrophils. The same is true of NK and LAK cells. It seems highly likely that there occurs significant oxidative damage to proteins and membrane lipids in MPs and neutrophils of aging individuals. One probable example of that is the defec-

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tive activity of the enzyme, glutathione peroxidase, in aged human neutrophils. It has been suggested that the accumulation of oxidative damage, even in neutrophils of the young adult, contributes to the short life-span and rapid turnover of those cells. As there appears to be no change in the turnover rate of neutrophils in the elderly, it might be supposed that more oxidative damage accumulates during the lifetime of neutrophils in the aged. The fact that hypoxic conditions tend to prevail at sites of infection further complicates the formulation of questions about the competence of redoximbalanced MPs and neutrophils to cope with pathogenic microorganisms in elderly individuals. Consider, for example, the possibility that oxidative damage to proteins and lipids might compromise the hypoxia-serving mechanisms that signal the expression of genes needed by MPs and neutrophils (and NK/ LAK cells) to combat infections in the elderly. There have been few investigations dealing with that complex matter (see ref. 163 and 166). REFERENCES 1. Janeway CA Jr. The immune system evolved to discriminate infectious nonself from noninfectious self. Immunol Today 1992;13:11–16. 2. Hoffmann JA, Kafatos FC, Janeway CA Jr., Ezekowitz RAB. Phylogenetic perspectives in innate immunity. Science 1999;284:1313–1318. 3. Epstein J, Eichbaum Q, Sheriff S, Ezekowitz RAB. The collectins in innate immunity. Curr Opin Immunol 1996;8:29–35. 4. Pearson AM. Scavenger receptors in innate immunity. Curr Opin Immunol 1996;8:20–28. 5. Weis WI, Drickamer K, Hendrickson WA. Structure of a C-type mannose-binding protein complexed with an oligosaccharide. Nature 1992;360:127–134. 6. Weis WI, Taylor ME, Drickamer K. The C-type lectin superfamily in the immune system. Immunol Rev 1998;163:19–34. 7. Gadjeva M, Thiel S, Jensenius JC. The mannan-binding-lectin pathway of the innate immune response. Curr Opin Immunol 2001;13:774–778. 8. Hoppe HJ, Barlow PN, Reid KB. A parallel three stranded alpha helical bundle at the nucleation site of collagen triple-helix formation. FEBS Lett 1994;344: 191–195. 9. Sheriff S, Chang CY, Ezekowitz RAB. Human mannose-binding protein carbohydrate recognition domain trimerizes through a triple α-helical coiled coil. Nature Struct Biol 1994;1:789–794. 10. Reid KB, Turner MW. Mammalian lectins in activation and clearance mechanisms involving the complement system. Springer Semin Immunopathol 1994;15:307–336. 11. Malhotra R, Lu J, Holmskov U, Sim RB. Collectins, collectin receptors and the lectin pathway of complement activation. Clin Exp Immunol 1994;97(S2):4–9. 12. Ikeda K, Sannoh T, Kawasaki N, Kawasaki T, et al. Serum lectin with known structure activates complement through the classical pathway. J Biol Chem 1987;262:7451–7454.

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284. Lieberman N, Mandelboim O. The role of NK cells in innate immunity. In: Keisari Y, Ofek I, eds.The Biology and Pathology of Innate Immunity Mechanisms. New York: Kluwer Academic/Plenum, 2000:137–145. 285. Kos FJ, Engleman EG. Requirement for natural killer cells in the induction of cytotoxic T cells. J Immunol 1995;155:578–584. 286. Scharton-Kersten TM, Sher A. Role of natural killer cells in innate resistance to protozoan infections. Curr Opinion Immunol 1997;9:44–51. 287. Albright JW, Hatcher FM, Albright JF. Interaction between murine natural killer cells and trypanosomes of different species. Infect Immun 1994;44:315–319. 288. Albright JW, Munger, WE, Henkart PA, et al. The toxicity of rat large granular lymphocyte tumor cells and their cytoplasmic granules for rodent and African trypanosomes. J Immunol 1988;140:2774–2778. 289. Rose ME, Hesketh P, Wakelin D. Cytotoxic effects of natural killer cells have no significant role in controlling infection with the intracellular protozoan Eimeria veriformis. Infect Immun 1995;63:3711–3714. 290. Johnson LL, Sayles PC. Strong cytolytic activity of natural killer cells is neither necessary or sufficient for preimmune resistance to Toxoplasma gondii infection. Nat Immun 1995;14:209–215. 291. Pawelec G, Solana R, Remarque E, Mariani E. Impact of aging on innate immunity. J Leukoc Biol 1998;64:703–712. 292. Solana R, Mariani E. NK and NK/T cells in human senescence. Vaccine 2000;18: 1613–1620. 293. Vitale M, Zamai L, Neri LM, et al. The impairment of natural killer function in the healthy aged is due to a post binding deficient mechanism. Cell Immunol 1992;145:1–10. 294. Mariani E, Roda P, Mariani AR, et al. Age-associated changes in CD8+ and CD 16+ cell reactivity: Clonal analysis. Clin Exp Immunol 1990;81:479–484. 295. Rukavina D, Laskarin G, Rubesa G, et al. Age-related decline of perforin expression in human cytotoxic T lymphocytes and natural killer cells. Blood 1998;92: 2410–2420. 296. Mariani E, Sgobbi S, Meneghetti A, et al. Perforins in human cytolytic cells: the effect of age. Mech Ageing Dev 1996;92:195–209. 297. Mariani E, Mariani AR, Meneghett A, et al. Age-dependent decreases of NK cell phosphoinositide turnover during spontaneous but not Fc-mediated cytolytic activity. Internat Immunol 1998;10:981–989. 298. Hsueh C-M, Chen S-F, Ghanta VK, Hiramoto RN. Involvement of cytokine gene expression in the age-dependent decline of NK cell response. Cell Immunol 1996;173:221–229. 299. Krishnaraj R, Bhooma T. Cytokine sensitivity of human NK cells during 1996;50: 59–63. 300. McNerlan SE, Rea IM, Alexander HD, Morris TCM. Changes in natural killer cells, the CD 57 CD 8 subset, and related cytokines in healthy aging. J Clin Immunol 1998;18:31–38. 301. Rajagopalan S, Fu J, Long EO. Cutting edge: Induction of IFN-γ production but not cytotoxicity by the killer cell Ig-like receptor KIR2DL4 (CD 158d) in resting NK cells. J Immunol 2001;167:1877–1881.

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302. Borrego F, Alonso MC, Galani MD, et al. NK phenotypic markers and IL-2 response in NK cells from elderly people. Exp Gerontol 1999;34:253–265. 303. Boles KS, Stepp SE, Bennett M, et al. 2B4 (CD244) and CS1: Novel members of the CD2 subset of the immunoglobulin superfamily molecules expressed on natural killer cells and other leukocytes. Immunol Rev 2001;181:234–249. 304. Yamauchi A, Bloom ET. Control of cell cycle progression in human natural killer cells through redox regulation of expression and phosphorylation of retinoblastoma gene product protein. Blood 1997;89:4092–4099. 305. Albright JW, Albright JF. Age-associated decline in natural killer (NK) activity reflects primarily a defect in function of NK cells. Mech Ageing Dev 1985;31: 295–306. 306. Saxena RK, Saxena QB, Adler WH. Interleukin-2-induced activation of natural killer activity in spleen cells from old and young mice. Immunology 1984;51: 719–726. 307. Albright, JW, Albright JF. Age-associated impairment of murine natural killer activity. Proc Natl Acad Sci USA 1983;80:6371–6375. 308. Plett A, Murasko DM. Genetic differences in the age-associated decrease in inducibility of natural killer cells by interferon-alpha/beta. Mech Ageing Dev 2000;112:197–215. 309. Albright JW, Bream J, Bere W, et al. Aging of innate immunity: Functional comparisons of NK/LAK cells obtained from bulk cultures of young and aged mouse spleen cells in high concentrations of interleukin-2. J Immunol (submitted). 310. Fischer B, von Knethen A, Brune B. Dualism of oxidized lipoproteins in provoking and attenuating the oxidative burst in macrophages: Role of peroxisome proliferator-activated receptor-γ. J Immunol 2002;168:2828–2834. 311. Cunard R, Ricote M, DiCampli D, et al. Regulation of cytokine expression by ligands of peroxisome proliferator-activated receptors. J Immunol 2002;168: 2795–2802.

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4 Aging of Adaptive/Acquired Immunity It seems inconceivable that future generations will be able to discard B lymphocytes and immunoglobulin as absurd interpretations of reality, in much the same way as we today discard the four humours of Hippocratic medicine. —John W. Fabre, “The Last Unknown Fact,” Nature Immunology 2002;3:3

It is appropriate to begin this chapter by quoting from an article (1) written by Philippa Marrack and John Kappler: Spurred on by the great immunochemists of the early twentieth century, immunologists acted for many years as though one antigen was as good as another. Antigens were used because they were cheap or convenient, so we learned a lot about the properties of immunity to materials such as sheep red blood cells, egg albumin, dinitrophenol, and so on. What immunologists found out, of course, was tremendously important, and most of the principles that are the foundation of modern immunology were learned with these models. This course of action, however, was to some extent misleading, because the fact of the matter is that, in real life, most infectious organisms have spent their millions of years of coevolution with the immune system developing mechanisms of manipulating the system. The upshot is that no invading organism behaves exactly like a sheep red blood cell and, if immunologists really want to understand how infectious diseases interact with their hosts, they have to study the disease and host themselves. Artificial substitutes simply will not do.

Most of what has been learned about the effects of aging on adaptive immunity derives from studies that have employed “artificial substitutes.” Admittedly, much has been learned. The challenge that now must be faced is to learn how capable the aging immune system is at dealing with real-life infectious organisms. Coevolution of hosts and the organisms that infect them has provided the opportunity for hosts of reproductive ages to select and transmit changes that offer advantages in coping with pathogens. But the aging immune From: Aging, Immunity, and Infection By J. F. Albright and J. W. Albright © Humana Press Inc., Totowa, NJ

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system lacks the opportunity to coevolve with infectious organisms and, therefore, must rely on the persistence into old age of adaptations suited to young adults. When aging alters those adaptations of the immune system, susceptibility to disease may and often does result. The foundation and principles of adaptive immunity are well presented in several excellent textbooks (e.g., refs. 2–5) and the wealth of information is far too voluminous even to summarize here. Therefore, this chapter focuses on the aspects of adaptive immunity that aging seems to affect. Recent information that has not yet been incorporated into the textbooks is included. We begin with a discussion of aging of the thymus, a central organ of the immune system, and thymus-derived (“T”) cells in the periphery of the body (spleen, lymph nodes, respiratory-, and gut-associated lymphoid tissues [RALT and GALT, respectively, sometimes referred to as sites of “regional immunity”]). Next, a discussion of aging of another central tissue, the bone marrow, and especially of the peripheral B cells derived from it is presented. Along the way, other cells that are largely derived from the marrow are discussed including Mo/MPs, and DCs, which (together with activated B cells) constitute the category of APCs. AGING OF THE THYMUS AND THYMUS-DERIVED (T) CELLS The origin of all of the peripheral T cells (CD4+ and CD8+) can be traced back to the thymus. There is very little decline in the numbers of peripheral T cells with advancing age except in the very old. Yet, involution of the thymus begins early in life and with increasing age there is a progressive decrease in the export of new naive T cells (6). There is considerable loss of thymic mass and cellularity during the second decade of life in humans and between two and three months of age in mice. The importance of extensive thymic export of cells to the periphery early in life is underscored by the findings that there is only a modest deficiency of T cells in mice that were thymectomized after five days of neonatal life or in humans thymectomized after six months of age (see ref. 7). One of the first studies on the rate of export of cells by the thymus compared to the rate of peripheral T-cell turnover (8,9) indicated that in adult mice far too few cells were released by the thymus to maintain the peripheral T-cell pool (10-fold to 300-fold too few). It seemed likely, therefore, that new T cells could be generated by T cells already in the peripheral tissues. There are several lines of evidence indicating that the constancy of numbers of T cells is achieved by both export from the thymus and generation from existing (immature) peripheral T cells; and that the contribution from the thymus gradually declines with age whereas the production by existing T cells gradually increases (9–11). That is the case both in mice and humans. Compelling evidence that the adult mouse thymus can continue to produce T cells as well as evidence of extrathymic generation of new T cells from

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existing T cells was obtained from studies of chimeric mice either having or lacking a thymus (7,12). Irradiated mice with or without a thymus were provided with syngeneic bone marrow and marked (Thy congenic) lymph node cells. The regeneration of the T-cell pool in those chimeras was studied. The results were clear: chimeric mice having an intact thymus rapidly regenerated T cells and the majority of them came from precursor cells derived from bone marrow. Chimeric mice lacking a thymus suffered a prolonged T-cell deficiency and the majority of those that were generated displayed the Thy marker of the donor lymph node cells. Moreover, the T cells regenerated from bone marrow precursors in euthymic mice were largely of the naive phenotype whereas those derived from lymph node precursors in athymic mice were mostly of memory phenotype. Additional evidence that the adult thymus, including the thymus of aged mice, is capable of producing T cells from bone marrow precursors is illustrated in Fig. 4-1 taken from ref. 13. Bone marrow cells from young B 10. Thy1.1 donor mice were injected directly into the thymus of C57BL/6 Thy-1.2 recipients, the latter ranging in age from 1 day to 18 months. Six weeks later, the numbers of donor cells present in the thymus and spleen of the recipients were determined. It is apparent that there was a precipitate decline in the export of T cells between 1 day and 1 week of age. However, donor cells did persist in the thymus even of 18-month-old mice and in 6-month-old mice exported detectable numbers of T cells to the spleen. Studies on T-cell regeneration in humans of varying ages who have been treated by chemotherapy have provided insight concerning loss of thymic function with age (7,14). Cancer patients ranging from 1 to 25 years of age were given intensive chemotherapy, which severely depleted peripheral blood T cells. Those T cells that remained were predominantly of memory phenotype. The restoration of T cells was followed in those patients for 6 months after cessation of therapy. As shown in Fig. 4-2, there was a strong inverse relationship between age of the patient and restoration of CD4+ T cells. Moreover, the more complete the T-cell restoration, the greater the proportion of naive (presumably thymus-derived) cells among those T-cells. The conclusions drawn from this study were that T-cell regeneration after intensive chemotherapy was largely a function of the thymus but that such regeneration became limited in early adulthood. From the preceding groups of investigations, it can be concluded that: 1) involution of the thymus and declining output of T cells occurs rather rapidly from the time of birth on into adolescence; and 2) the constancy of the number of peripheral T cells reflects both the decreasing input from the thymus and the increasing generation of T cells by existing, peripheral, thymus-derived T cells. If so, then there must exist in the peripheral tissues, relatively immature T

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Fig. 4-1. Presence of donor-type T cells in the thymus (shaded bar) or spleen (open bar) of recipient mice following direct injection of young donor bone marrow cells into the thymus of recipients ranging in age from 1 to 18 mo. The number of donor-type T cells was determined 6 weeks after the injection. (From ref. 13, with permission.)

cells, originally derived from precursors in the thymus, that have not been stimulated to proliferate and complete their maturation. Is there any evidence for that? Indeed, there is. First, it is by now well established that consonant with the decline in thymic output of T cells the proportion of the peripheral T cells that are naive also declines. The naive T cells display characteristic surface molecules: high surface density of CD45 (CD45hi) and low density of CD44 (CD44lo) in the mouse; the CD45 RB isoform in the human. In contrast, memory T cells are CD45loCD44hi in the mouse and CD45RO or CD29+ in the human. The point to be made here is that although the proportion of naive cells steadily declines with advancing age, there remains a detectable number of those cells well into old age (90 years or older) as shown in Fig. 4-3 (13). There is a marker that offers considerable promise for identifying peripheral T cells that either have recently emigrated from the thymus or have been in the periphery for some time but have not been activated. That marker is the DNA that was excised during the process of T-cell receptor gene rearrangements,

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Fig. 4-2. Decline with age in the reappearance of CD4+ T cells in the blood of patients following intensive chemotherapy (left-hand graph). Gradual return of naive CD4+ T cells following chemotherapy as reflectecd by the CD45RA+/CD45RO+ ratio (right-hand graph). (From ref. 14, with permission.)

which remains in cells in episomal form. It is termed “T-cell receptor excision circles” (TRECs). The circles include the recombination signal sequences; therefore, the TRECs are known as “signal joint TRECs” or sjTRECs. Upon stimulation of cells containing them to proliferate, the sjTRECs are diluted out among the progeny. SjTREC+ cells have been identified among peripheral T cells in both chickens (15) and humans (16). In humans, it was found that sjTREC+ cells were located predominantly in the CD45 RA+ (naive) population of T cells and that the number of sjTREC+ cells was almost 10-fold less in thymectomized compared to non-thymectomized subjects. In some subjects there persisted a subpopulation of CD45 RA+ sjTREC+ cells for as long as 10 years postthymectomy; those cells had not undergone mitotic division and presumably were competent to generate other T cells upon activation. The discussion, thus far, has provided evidence for a decline with age in thymic export of T cells. Contributions both from the thymus and precursor cells in peripheral tissue maintain a constant number of T cells at least until very old age. However, there is a progressive increase with age in the proportion of memory T cells and a corresponding decrease in naive cells of recent thymic origin. In the absence of thymus, generation of T cells from peripheral sites is insufficient to maintain the normal total number of T cells. Now, we should ask whether or not the population of T cells composed predominantly of memory cells presents the full range of antigenic recognition characteristic of the young adult population. Apparently, it does not.

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Fig. 4-3. Numbers of naive (CD4+ CD45RA+) and memory (CD4+ CD29+) T cells in the blood of humans ranging in age from 6 to >90 years. (From ref. 13, with permission.)

Virtually all of the T cells (CD3+ cells) in the blood and peripheral tissues are either CD4+ “helper” T cells (Th cells) or CD8+ (CTLs). Naive cells of both types are largely recent thymus emigrants whereas memory cells of both types are largely generated from other peripheral T cells. In mice, the ratio of CD4+/CD8+ cells declines steadily from birth into very old age, largely reflecting the decline in CD4+ T cells (9,17); that appears to be true in humans as well. Although the total number of CD8+ cells remains nearly constant over the life-span, there are important and somewhat surprising changes within the population of CD8+ T cells. Namely, there are disproportionate increases with age in a restricted few clones of CD8+ cells. This occurs in both mice and humans (17–19). In some individuals, expansion of a few clones can contribute up to 70% of the entire CD8+ T cell population. As stated in ref. 19: “Thus, in man nearly 1011 and in mouse nearly 5 × 107 T cells can be descended from the same founder.” Evidence that cells of a limited, few clones, occasionally a single clone, comprise the expanded CD8+ T cell population was provided by the finding of identical Vβgene segments in the rearranged TCR β chains of the large number of cells of individual CD8+ T-cell clones (17). Compelling

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evidence has been provided against the notions that the expanded clones are transformed cells or virus-infected cells or that they expand in response to superantigen. Rather, evidence has been adduced that the clones represent expansions of cells that, in aged mice, are particularly susceptible to control by two opposing cytokines, IL-2 which inhibits and IL-15 which promotes the clones (20). It is important to stress that no comparable expansion of CD4+ Tcell clones has been found in mice or man. The reasons why CD8+ T cells expand but CD4+ T-cells do not remain obscure. However, it is worth noting that the studies of T-cell regeneration in patients exposed to intense chemotherapy revealed that the CD8+ population was regenerated considerably quicker than the CD4+ population (21). The reason for this was the presumed, greater thymus independence of CD8+ T-cell regeneration, perhaps including the more stringent signaling requirement for CD4+ T-cell activation (22). Studies on diversity among T cells regenerated in irradiated, thymectomized mice were performed by transferring inocula of lymph node cells from TCRtransgenic donor mice. Those studies revealed that, in the absence of antigen specific for the TCRs encoded by the transgene, the inoculated lymph node cells generated neither CD4+ nor CD8+ progeny (23). This led to experiments in which graded numbers of lymph node cells were transferred into athymic recipients. It was found that when moderate numbers of lymph node cells were transferred, the derived progeny in the recipient mice expressed the expected broad range of Vβ genes in relatively typical proportions (12). Presumably, there was general memory clone expansion in response to indigenous antigens. However, when small numbers of lymph node cells were transferred, there was skewed expansion of limited numbers of Vβ clones. Thus, there was limited Tcell repertoire diversity in the recipients of limiting lymph node inocula which, presumably, reflected the frequency of precursor cells that were stimulated by the available indigenous antigen. Additional insight concerning the role of antigens both in generating T cells of memory phenotype and in skewing the repertoire of expanded T-cell clones was provided by two groups of investigators. The experiments of one group (23) involved transfer of lymph node cells from transgenic donor mice carrying the rearranged gene-encoding T-cell receptors for pigeon cytochrome c. When antigen was provided to the recipient mice, there was a marked skewing of the expressed TCR repertoire; up to 65% of the recipients’ T-cell population expressed the transgenic TCR whereas very little expression occurred in recipients not provided with the specific antigen. The experiments of the second group (24) were performed with mice bearing a transgene for a peptide of pigeon cytochrome c. The phenotype of the CD4+ T cells was determined as those mice aged; the mice were not provided with the specific antigen. Most of the transgene-bearing CD4+ T cells remained of the naive phenotype (CD45

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RBhi CD44lo) even in aged mice, indicating that the preponderance of memory T cells in aging individuals reflects an antigen-driven process. The reports from the four groups of researchers reviewed just above (17–24) are amenable to a reasonable synthesis, as follows: 1. Old mice may be considered to be close to athymic and to contain predominantly memory T cells, many of which are functionally defective. Therefore, for the purpose of studying T-cell expansion/generation (regeneration), aged mice may not be very different from adult, thymectomized, irradiated mice that received a limited inoculum of lymph node cells. 2. The apparent requirement for antigen to drive the regeneration (polyclonal expansion) of T cells in the thymectomized, irradiated recipients of donor lymph node cells, including the skewed clonal expansion in recipients of limited numbers of donor cells, suggests that indigenous antigen (viral, environmental) may be necessary for the restricted clonal expansion of T cells in aged mice. The finding that different clones appear to be expanded in different aged mice may mean that either (a) different antigens persist in immunogenic quantity in different aged individuals or (b) aging renders different T-cell clones sensitive to antigen, or insensitive to control by IL-2 and IL-15, in different aged individuals. It has been suggested (25) that the clones that emerge in aging individuals are expanded T-cell clones that have lost the ability to stop dividing or to be deleted by apoptosis. 3. And, finally, memory T cells develop in limited numbers, if at all, in the absence of antigen activation.

Additional information concerning regulation of T-cell numbers and subsets has derived from studies focused on CD8+ T cells. For example, a comprehensive investigation of CD8+ naive and memory T cells in mice (26) showed that: 1) the thymus of adult mice does not generate a sufficient number of naive T cells to repopulate both the naive and memory T-cell pools; 2) similarly, expansion of peripheral T cells is insufficient to repopulate the naive and memory T-cell pools; 3) naive T cells in the periphery, in the absence of antigenic stimulation, are nondividing and long-lived; and 4) peripheral expansion of naive cells to generate memory cells requires antigenic stimulation. Of particular significance was the compelling evidence that the population sizes of naive CD8+ and memory CD8+ T cells are under homeostatic control and that the number of cells in each population is regulated independently. That arrangement seems to ensure that 1) a pool of naive cells representing repertoire diversitiy is always present regardless of encounters with antigens, and 2) clones of rapidly responsive memory cells, capable of extensive proliferation and clonal expansion, are prepared to respond to critical antigens. Aging affects the CD8+ T-cell population. In particular, there is a significant change in the dynamics of CD8+ T-cell turnover, aging mice having a much-reduced rate of turnover of the memory subset compared to young adults (27). That change may explain, in part, the increasing proportion of memory

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cells in the CD8+ population with age. Evidence was adduced that suggested that the reduced rate of turnover in aged mice resulted from both excessive expression of the anti-apoptotic factor, Bcl-2, and an anti-proliferative influence of type I interferon (IFN-I). Causes of Age-Associated Dysfunction of the Thymus Five explanations have been advanced concerning the age-associated decline in export of new, naive T cells and involution of the thymus. First, studies on the maturation of T cells in the thymus of aged mice (28) suggested that a block of further maturation occurs at about the stage of TCR β-chain gene rearrangement. Mice carrying an already rearranged TCRαβ transgene showed much less evidence of thymic atrophy compared to similarly aged normal mice. The defective β-chain rearrangement in aged mice was attributed to changes in the local spectrum of cytokines (29) such as the deficiency in IL-7. The latter cytokine, generated by thymic stroma, can influence the expression of recombinase-activating genes (rag 1 and rag 2) in differentiating thymic stem cells in old mice (30). The second explanation, somewhat related to the first, holds that the production of growth-stimulating and -inhibiting factors by thymic epithelial cells changes with age (13,31). This explanation has received support from studies on cell lines of cortical epithelial cells derived from thymus of newborn mice (31). Some of those lines were found to produce a stimulant of developing T cells in the thymus that acted in synergy with IL-1 and IL-7. That stimulant was identified as the proenzyme form of cathepsin L. The potential protease activity of that substance was unrelated to its action as a growth stimulant of the developing T cells. It was demonstrated that the intrathymic frequency of epithelial cells containing cathepsin L proenzyme as well as the levels of intracellular mRNA declined significantly in the thymuses of young adult and aging mice. In contrast, other cortical epithelial cell lines were found to produce TGFβ in levels sufficient to induce death of thymocytes. The production of TGFβ was found to peak in the thymuses of young adult mice suggesting that it might be one of the factors contributing to thymus dysfunction. Although the above-described study on cathepsisn L in cortical epithelial cells provided results that suggested no role for its enzymatic activity in stimulating growth of T cells, it would seem worthwhile to renew and extend that line of investigation. Recent investigations (32,33) of the role of cathepsin L in positive selection of CD4+ T cells in the thymus raised the fascinating possibility that age-related changes in that protease, such as the decline in its biosynthesis or persistence in cortical epithelial cells of the aged (31), might account for age-related thymic dysfunction. Analyses of cathepsin L gene-targeted (knockout) mice revealed that at seven weeks of age both the thymus and

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spleen were markedly deficient in CD4+ T cells (reduced by as much as 60– 70%). Furthermore, the loss of CD4+ T cells was compensated by an increased proportion of CD8+ T cells. No such effect was found in mice bearing targeted cathepsin B genes. Experiments were performed to demonsrate conclusively that the defect in cathepsin L of the cortical epithelial cells was responsible for failure to positively select CD4+ T cells in the thymus. A third possibility, viz., that age-associated thymic dysfunction and involution reflect changes in the intrathymic cytokines, has received attention. A comprehensive survey of the levels of mRNA transcripts of genes encoding a variety of cytokines in the thymuses of humans of varying ages has been reported (11,34). The effects of aging on the cytokines were categorized as: (a) those that displayed decreasing mRNA concentrations with increasing age (IL-2, IL-9, IL-10, IL-13, IL-14); (b) those that displayed increasing mRNA transcripts with age (LIF, OSM, SCF, IL-6, M-CSF); and (c) those having unchanged levels of mRNA with advancing age (IL-7, IL-15, G-CSF). It was shown that administration of excessive amounts of each of those cytokines, which increased with age to young mice, induced at least some degree of thymic atrophy. The fourth explanation of thymus involution is based on the considerable evidence that the T cells generated in the thymus are descendants of stem cells that migrate into the thymus from the bone marrow. It has been proposed that aging affects the generative potential of those stem cells (35,36). However, a well-designed experimental test of that hypothesis failed to provide evidence of age-related defects in the stem cells (13). The fifth explanation that has been offered to account for thymus dysfunction and atrophy with age is endocrinological. That explanation seemed logical given that thymic involution is associated chronologically with puberty and given the strong thymus-suppressive effect of glucocorticoids. However, compelling evidence of a decisive role for any hormone in thymus involution is not available, to date. One of the more interesting observations, that growth hormone could increase the size of the thymus in aged rats (37), stirred considerable interest. However, no evidence of improved immune responses attributable to growth hormone has been obtained (38,39). An analysis of the proposed prevention of thymic involution by growth-hormone treatment of growth hormonedeficient mice provided no positive evidence for such an effect (40). Moreover, hypophysectomy of aged mice improves immunity in aged mice (41). That there is an endocrinological influence on the thymus is indicated by the effects of destroying the anterior portion of the hypothalamus. The consequence of such treatment is a two- to threefold increase in thymus weight, an effect that is longlasting (13). The hyperplastic effect on the thymus, but not other immunological organs, has been demontrated both in aged and young mice.

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THE FUNCTIONS AND DIVERSITY OF PERIPHERAL T CELLS Elaboration and Maintenance of Memory T Cells The complex processes of TCR α and β chain gene rearrangements, positive and negative selection of TCR-displaying thymocytes, and differentiation into CD4+ and CD8+ T cells prior to being exported to the periphery all occur in the thymus. Those events have been studied in detail (see refs. 42 and 43 for reviews). The consequence of normal thymopoiesis in neonatal and adolescent individuals is the production of a peripheral T-cell population that in the human comprises a TCR β-chain diversity of about 106 and an α-chain diversity of about 25 for a total TCR diversity in the range of 2.5 × 107 among naive T cells (44). Direct estimation of diversity among memory T cells revealed the involvement of 1–2 × 105 β chains, each of which was found to pair with a single α chain (44). Thus, the diversity displayed by the memory T-cell population was roughly 1% of that displayed by the naive T-cell population. At this point it is appropriate to consider the questions: What are memory T cells? How do they arise? Do they persist and, if so, how? And, finally, what is the effect of aging on the development and persistence of memory T cells? The concept of immunological memory was suggested by the observed anamnestic (“recall”) responses that resulted from re-exposure to certain pathogens. Individuals who had recovered from a primary infection, or who had been vaccinated, were resistant to subsequent exposures to the same pathogen. Experiments then revealed that secondary immune responses (especially humoral) were greater, developed faster, differed qualitatively from primary responses, and were initiated by significantly greater numbers (10-fold or more) of cells (“memory cells”) as indicated by limiting dilution analysis (45,46). Additional characteristics of memory cells have been elucidated in recent years (47–50). For example, the magnitude of clonal expansion in microbial infections may be surprisingly large. In the case of responses to certain viral antigens, the frequency of specific, CD8+ T cells generated in response may increase 100,000-fold in a matter of a few days (51,52). Such a vigorous response requires a CD8+ T-cell doubling time of about 6 h. The subsequent decline in number of specific CD8+ cells after peak response is equally dramatic leaving, after a few days, a pool of memory cells some 10- to 100-fold greater than the original frequency of corresponding naive cells. The clonal expansion of CD4+ helper T cells is also substantial although less than in the case of CD8+ cells. One study (53) of CD4+ T-cell response to pigeon cytochrome c revealed a 1,200-fold clonal expansion. Again, following peak T-cell proliferation there was a period of rapid cell death leaving, as a remnant, the pool of CD4+ memory cells. That pool of CD4+ memory T cells is many times greater (10- to 100-fold) than the number of corresponding, specific naive cells.

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A series of interrelated reports (see refs. 49 and 50) have clarified several critical aspects of naive and memory T cells. Of particular importance is the recent finding that memory T cells (both CD4 and CD8) can be separated into an early, or “central,” and a late/terminal, or “effector,” subset based on the presence or absence of the chemokine receptor, CCR 7. Thus, central memory (TCM) cells have the phenotype (in human) of CD45 RA–CCR 7+, effector memory (TEM) are CD45 RA–CCR7– and both can be distinguished from naive (CD4 or CD8), which are CD45 RA+ CCR7+. In addition to the differential expression of CCR7, the two types of memory cells differ in the display of α and β integrins (much higher in TEM), production of cytokines in response to activation (IL-2 by TCM, IFNγ by TEM), and the level of expression of CD 62L (L-selectin, significantly higher on TCM). Furthermore, activation of TEM required significantly lower doses of stimulant (substrate-bound anti-CD3ε) than TCM; both types of memory cells required less intense stimulation than did naive T cells, further substantiating the conclusion that activation of memory cells involves less stringent conditions than activation of naive cells (54). Finally, a dynamic and functional analysis of the descendants of activated, naive CD4+ T cells provided compelling evidence in support of linear maturation of cells from CD45 RA+ CCR7+ (naive) to CD45RA– CCR 7+ (TCM) to CD45RA– CCR7– (TEM). Although the linear maturation model that terminates with TEM as discussed just above is appealing and supported by evidence, there is other, puissant evidence that suggests that memory CD8+ T cells are derived in linear fashion, from activated, effector cells (55,56). In one set of experiments (55), CD8+ memory T cells were shown to be derived from perforin-expressing effector cells. The other, ingenious, set of experiments (56) provided evidence that a reporter gene, permanently turned on, strictly as a result of activation, was expressed in activated, effector cells as well as in memory CD8 T cells presumably derived from the effector cells. Perhaps the most important conclusion to emerge from the studies of Sallusto and Lanzavecchia (49,50) concerns the patterns of distribution of the two types of memory cells (see Fig. 4-4). It was concluded that TCM cells, similar to naive T cells, circulate through, or temporarily reside in, lymphoid tissues (spleen, lymph nodes). In contrast, TEM cells are distributed peripherally to sites such as lung, liver, kidney, and lamina propria of the intestine. That division of responsibility seems to make good sense. The distribution of TEM cells puts them at the forefront of resistance to invading pathogens where they are capable of rapid, potent response. On the other hand, the TCM remain in lymphoid tissue where they are positioned to be stimulated by antigens brought in by DCs (and other APCs) in response to which they rapidly proliferate and mature into TEM cells to replace those lost in the periphery.

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Fig. 4-4. Two distinguishable types of memory T cells, which differ in their choice of homing sites. The memory T cells are termed “central memory” (TCM), which express CCR7, and “effector memory” (TEM), which lack CCR7. Naive T cells also express CCR7 as well as the CD45RA isoform. (From Mackay CR, Nature 1999;401:659– 660, with permission.)

Conclusive evidence for the peripheral distribution in nonlymphoid tissues of both CD8+ and CD4+ memory T cells has been published recently (57,58). CD8+ T cells were isolated from lymphoid and nonlymphoid tissues of mice at intervals following a primary exposure to virus (57). The CD8+ cells, which were specific for the virus and expressed the integrin CD11a, were enumerated by flow cytometry. Such cells appeared within a few days in both lymphoid and nonlymphoid tissues and then declined rapidly in lymphoid tissues but more gradually in nonlymphoid tissues (lung, lamina propria, kidney, and the peritoneal cavity). After the initial decline, cells persisted in the lymphoid tissues in small numbers, as expected of memory cells. The numbers of cells that persisted in nonlymphoid sites were substantially greater than those in the spleen and lymph nodes and they were present for a long time (at least 296 days). Moreover, those memory cells isolated from lung and lamina propria (as well as liver and spleen) rapidly responded to stimulation with viral antigen by producing IFNγ and by killing virus-infected target

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cells. Comparable results were obtained from studies of memory CD8+ cells directed against L. monocytogenes. The analysis of CD4+ T-cell memory (58) was performed by employing sophisticated immunohistological methods. Recipient mice were provided with naive, CD4+ T cells from donors of a line bearing a T-cell antigen receptor (TCR) transgene specific for an ovalbumin peptide. The donor cells were carrying a marker, Thy1.1, to distinguish them from recipient T cells (Thy 1.2). Recipient mice were exposed to the specific antigen and, after an appropriate interval, they were sacrificed and whole body sections prepared and analyzed for the tissue distribution of CD4+ memory cells. In response to antigen, the T cells proliferated and migrated out of the lymphoid tissues to peripheral sites, especially lungs, liver, gut, and salivary glands. In control mice not exposed to antigen, the donor CD4+ cells remained in lymphoid tissues. When the ovalbumin antigen was given along with a bacterial product, LPS, the memory cells that redistributed to nonlymphoid sites persisted for months. Furthermore, when restimulated with antigen, the memory cells from nonlymphoid tissues generated IFNγ whereas those obtained from spleen produced IL-2 but not IFNγ. Those two publications (57,58) extend and substantiate the concept that there exists a subset of memory cells (TEM) that reside in nonlymphoid sites associated with portals of entry for pathogenic microorganisms. Those memory cells are poised for rapid response and are at the forefront in the defense against pathogens. The persistence of long-lived memory cells for months (mice) or years (humans) has been discussed above. Questions concerning the life-spans of memory cells are legion. For example: Are individual memory cells capable of living for, say, 40–50 years in the human, or do they turn over at some slow but perceptible rate? To survive, must memory cells be continuously stimulated or otherwise engaged with antigen even in very low concentrations? Must memory cells be supported by MHC molecules identical to those that were involved in the positive selection of their progenitor, naive T cells in order to survive? What niches in the lymphoid tissues do they occupy in order to survive? Information is accruing that, at the least, allows those questions to be formulated more precisely. The question of whether or not the presence of antigen is necessary for the persistence of memory was raised many years ago and was addressed in some classical studies by Nossal and others (see refs. 59 and 60). In those studies it was shown that antigen could persist for extended periods of time by attaching to the membranes of follicular dendritic cells located in spleen and lymph nodes. Antigen persisted in the form of antigen-antibody complexes and might either stimulate T cells directly or be transferred to other APCs. More recently several attempts to demonstrate that memory does not require persistent anti-

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gen have been performed by transferring memory cells from immunized donors into antigen-free (presumed), syngeneic recipients. Two studies yielded particularly convincing results against the need for antigen in long-term maintenance of memory (61,62). To illustrate, one study (61) involved transfer of sorted, highly enriched, CD8 memory cells from lymphocytic choriomeningitis (LCM) virus-immunized donors. The transferred cells were first analyzed for any trace of the virus before transfer. The recipients were analyzed for persistence of memory cells in the absence of viral antigen and found to retain memory cells for more than two years. The preceding, along with several other studies, have made it appear unlikely that either B cells or antibody they might produce to form antigen-antibody complexes are involved in the persistence of CD8 T-cell memory. If persisting antigen is not required for long-term maintenance of memory, it might, then, seem possible that the antigen-presenting MHC molecules provide support for maintenance of memory cells. One test of that possibility (63) involved the use of recipient mice lacking T cells of their own as well as being unable to express Class I MHC molecules (β2-microglobulin defective). CD8+ memory cells from LCM virus-immunized mice were transferred into those recipients. Thus, the transferred CD8+ cells were unable to encounter antigen associated with Class I molecules, or even the Class I molecules themselves, on the surface of APCs. When the recipient mice were examined 10 months after transfer of CD8 memory T cells, it was apparent that memory cells had persisted as well in the β2-microglobulin-defective mice as they had in control mice expressing Class I molecules normally. Concerning the persistence of antigen, and the support of Class II molecules, as being requisite for maintenance of CD4+ T-cell memory, a study involving transfer of transgene-expressing T cells into Class II-deficient recipients was performed (64). Naive, CD4+ T cells obtained from TCR transgenic mice, were activated in vitro under conditions in which no detectable APCs survived. After four days, the activated CD4+ T cells, more than 99% of which bore the transgene, were transferred in graded numbers into two groups of recipients, one group possessing normal Class II molecules and the other (knockout mice) lacking Class II molecules. Both groups supported the generation and persistence of CD4+ memory T cells equally well, and in numbers proportional to the number of activated CD4+ T cells transferred. Due to the fact that the memory cells responded vigorously by producing IL-4 (associated with the Th2 type of effector cell) upon restimulation with the cognate antigen recognized by their transgene, the memory cells were considered to be of the effector variety. Based on the two reports (63,64) discussed above, it seems highly probable that the maintenance and long-term persistence of memory require neither con-

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tinuous nor intermittent antigen stimulation. It seems equally probable that survival of the memory cells does not require the support or aid of MHC molecules. Those conclusions apply to both CD4+ and CD8+ memory cells. However, the maintenance of certain complex functional capabilities by memory cells may be another matter. As assessed by sensitive, in vivo, functional tests, CD4+ memory T cells require the presence of MHC substances in order to perform immunological functions such as providing help for immunoglobulin class switching (IgM to IgG) and cell-mediated rejection of skin grafts (65,66). Certain cytokines are involved in the persistence and homeostatic regulation of memory cells (67,68). Because many viral infections result in strong proliferation of CD8+ memory cells, and because virus infections commonly induce production of type I IFN (IFN-I), a study of the effects of IFN-I on CD8+ memory was conducted (67). Injection into mice of poly I: poly C, a strong inducer of IFN-I, resulted in rapid, marked proliferation of CD8+ memory cells, an effect that was negated by concurrent injection of neutralizing antibody against IFN-I. Subsequent studies showed that several cytokines might be induced in addition to IFN-I by agents that simulated virus infection (e.g., injection of LPS) but of those cytokines only IL-15 was able to induce proliferation of CD8+ memory cells. The effect of IL-15 was attributed to the presence on CD8+ cells of the IL-2R, which shares a common β chain (CD122) with the IL-15R. And, finally, it was concluded that the stimulation of proliferation of CD8 memory T cells by IFN-I probably is indirect, the effect being mediated by IL-15 produced by macrophages (67). However, that conclusion may need to be modified in the case of aged mice in which homeostasis of memory CD8+ cells is dysregulated (27). There are several recent publications that strengthen the conclusion that macrophageproduced IL-15 is directly involved in maintaining CD8+ memory T cells and that both IL-15 and the presence on the cells of CD122 are required (69– 73). The apparent selective action of IL-15 on CD8+ memory T cells is explained by the low surface density of β chain (CD122) in the receptors (mostly IL-2R) on CD4+ memory as well as naive CD8+ T cells. A particularly interesting, recent finding is the apparent role of IL-4 in the generation of long-lived CD8+ memory cells (74). When naive T cells were stimulated with cognate antigen in vitro, and then transferred into recipient mice, the duration of survival in the host was determined by the cytokine that was present during in vitro antigen stimulation. Thus, the presence of IL-2 or IL12 resulted in the generation of relatively short-lived CD8 memory whereas IL-4 supported long-lived memory in the recipient mice. Further analysis of this phenomenon revealed that the presence of IL-4 during naive cell stimulation enticed the progeny cells to express significantly higher surface densities of IL-2Rβ chain than did IL-2 or IL-12.

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The positive regulatory effect of IL-15 appears to be counteracted by a negative regulatory effect of IL-2 (71). Thus, the opposing effects of those cytokines may maintain long-term homeostasis of CD8+ memory cells. Cytokines that may control homeostasis of CD4+ T cells have yet to be elucidated. IL-15 is not one of them (72,73). In addition to IL-2 and IL-15, a role for IL-7 in homeostatic regulation of both naive and memory CD8+ T cells has been conjectured (75). Evidence was obtained that indicated IL-7 helped to support proliferation of naive CD4+ and CD8+ T cells in T-depleted hosts (in the absence of cognate antigen stimulation). Moreover, IL-7 was required to support the development of CD8 memory in response to viral antigens. IL-7 was probably produced by macrophages and exerted an effect on maintenance and expansion of T cells by upregulating Bcl-2 thus diminishing apoptotic cell death. Memory T Cells and Aging From the review presented in the preceding paragraphs, several conclusions about memory and memory T cells in adult subjects can be drawn with confidence. First, the relative proportion of T-cells displaying the memory phenotype increases with subject age, both CD4+ and CD8+. Second, the diversity of the memory T-cell population is substantially less than that of the naive T-cell population. Third, when the memory T-cell population is intensely stimulated with a multispecific antigen or oligoclonal activator, the responsive memory cells react quickly and proliferate extensively generating a large population of effectors; when the stimulus ends, there is an equally dramatic elimination of those effector cells. Fourth, the activation threshold is substantially lower in the case of memory compared to naive T cells; i.e., activation requires much lower antigen concentration and/or shorter exposure times and less or no costimulation. Fifth, both CD4+ and CD8+ memory cells persist for extended times, perhaps for a lifetime, and turn over at slow rates; the turnover of CD4+ cells is demonstrably slower than that of CD8+ cells. Sixth, maintenance and the slow proliferation of memory cells do not require interaction with either cognate antigen or MHC substances; however, to perform their immune functions effectively, memory cells (at least CD4+ cells) do require contact with MHC molecules. Seventh, in the case of CD8+ memory T cells at least, they are distributed as two distinguishable populations: the TCM set that circulates through or resides in the central lymphoid tissue (spleen, nodes), and the TEM set that resides at regional sites (lung, liver, intestinal mucosa, etc.). The two sets are readily distinguishable by the presence of the CCR 7 and certain integrins on TCM but not on TEM, which account for their different homing proclivities. Eighth, the cytokines IL-15 and IL-7 (especially the former) are

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required for long-term maintenance of CD8+, but not CD4+, memory cells and the homeostasis of CD8+ memory cells is regulated by the opposing influences of IL-15 and IL-2. Given the rather substantial fund of information concerning naive and memory T cells, it is well to ask whether or not the available information reveals how well immunological memory serves to protect the elderly from infectious diseases. In addition, we should ask what might be done safely to improve the protective capabilities of memory. A synthesis of information about CD8+ memory T cells leads to the conclusion that, although the presence of antigen is not required for long-term persistence of memory cells, the continuous presence of antigens during normal aging shapes the immunological response repertoire. That repertoire represents a mere fraction (perhaps 1%?) of the repertoire represented in naive T cells. The diversity represented in the memory T cells probably reflects the most commonly encountered antigens over the course of the adult life-span. For each of those antigens there is, presumably, a small set of memory cells, quite sensitive to antigen, requiring little if any stimulation, capable of prodigious expansion and rapid, targeted deployment of effector cells to sites of infection. In many cases, memory cells (TEM) may already be present at prospective sites of infection and prepared for immediate response. As indicated, the diversity of the CD8 memory T cells is restricted and may not include memory for antigens infrequently or rarely encountered during adulthood, or encountered in subthreshold amounts. Thus, the absence of memory clones for key antigens of infrequently encountered pathogens may account for some infections seen in the elderly. Furthermore, memory clones may be lost in the elderly owing to proliferative failure. CD8+ memory T cells turn over at a slow rate in young adults and at a significantly reduced rate in aged mice (27). That age-associated change in proliferation reflects as yet unidentified changes in the internal milieu of the aged subject; possibly changes in the cytokine milieu or other elements that result in combined anti-proliferative effects of elevated macrophage production of type I interferons and CD8+ T-cell levels of Bcl-2, the latter an inhibitor of apoptosis (27). Another consideration derives from the demonstrations of enormous clonal expansion that may accompany antigen restimulation of CD8 memory clones (20,51,52), especially viral antigens. An extremely robust expansion of clones in response to antigens of one virus might well dampen or delay the response to antigens of a second pathogen in the manner of antigen competition. This might be particularly apparent in aged subjects in which CD8 memory cells (but not CD4+ memory cells) turn over at a significantly slower rate. Finally, it is likely that studies on the separate populations of TCM and TEM in relation to aging will be performed in the near future and provide new insight concerning aging of CD8 T-cell memory and resis-

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tance to infection. For example, it will be important to determine the turnover rates of TEM, their life-spans, and what factors (cytokines? chemokines?) support their survival. In the case of CD4+ memory T cells, they, too, perform less well in the aged compared to the young-adult milieu (24). Naive CD4+ cells turn over very slowly, if at all, unless challenged by antigen or some other stimulant (e.g., superantigen). Moreover, in the absence of antigen challenge they remain naive. When challenged in the aged milieu, they proliferate less well, and produce lesser amounts of cytokines such as IL-2 and IL-3, than in the youngadult milieu. However, they give rise to memory CD4+ cells that have the characteristic phenotype (CD 44hi CD 45RBlo in the mouse). The development of those memory cells from their naive precursors appears to be accompanied by remodeling, and probably restriction, of the response repertoire (24). There is much yet to be learned about CD4 T-cell memory. Before closing this discussion on memory T cells, the role played by DCs and other professional APCs in the development and maintenance of memory cells and their naive T-cell precursors should be considered. APCs are also seen as important in the next section concerned with subsets of CD4+ and CD8+ T cells (Th1/Th2 and Tc1/Tc2, respectively). The requirements for antigenic activation of naive cells, both CD4+ and CD8+, are significantly more demanding than for memory cells. Activation of naive CD4+ T cells requires presentation of antigen by DC and concurrent costimulation whereas antigen presentation by B lymphocytes, without costimulation, can be sufficient to activate memory CD4+ memory cells (76). Similarly, DC loaded with appropriate peptides are required to activate naive CD8+ T cells both in vitro and in vivo (77). A major cytokine produced in the process is IL-12, which significantly enhances the proliferation (“expansion”) of the CD8+ population (78) but is not strictly required for the naive cell activation (79). Later in the chapter, evidence that aged T cells suffer changes in the ability to transduce and transmit signals is discussed. Such changes appear to be reflected by aberrant formation of the “immunological synapse” (the contact area between APCs and T cells). Senescent changes in the APCs themselves may affect the exchange of signals and other information via the immunological synapse. Such changes may radically alter the development and expression of memory as well as effector cells in both the CD4+ and CD8+ T-cell compartments. It is a fact that we know very little about the ability of the aged immune system to manifest T-cell memory and maintain the memory that is initiated late in life. The information that is available suggests that it differs both quantitatively and qualitatively from memory in the young adult. Therefore, it may be necessary to develop special protocols for immunizing (vaccinating) the elderly.

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Table 4-1 Phenotypic Characteristics of T-Helper Cells Characteristic Types of immune response Cell-mediated immunity (contact sensitivity, delayed hypersensitivity, intracellular parasites) Humoral immunity (immediate hypersensitivity, extracellular parasites, helminth expulsion) Cytokines produced IL-2, IFN-γ IL-4, IL-5, IL-10, IL-13 Chemokines produced Ltn (XCL 1), RANTES (CCL 5) T-cell activation gene 3 (TCA 3), MDC (CCL 22) Chemokine receptors expressed CCR 5, CCR 7, CXCR 3 CCR 3, CCR 4, CCR 8 aProduction

Th1

Th2

+a +

+ + + + + +

of antibody of IgG2a isotype (rodents).

Functionally Distinct Subsets of CD4+ Helper and CD8+ Cytotoxic T Cells When naive T cells are adequately stimulated with cognate antigen they give rise to functional, effector progeny. CD4 T cells produce two types of helper (Th) cells (Th1 and Th2) and CD8 T cells generate two types of cytotoxic (Tc) progeny (Tc1 and Tc2) (80–85). Cells of the Th1 type generally help to support cell-mediated immune responses such as contact sensitivity, delayed type hypersensitivity, and resistance to intracellular parasites. Their ability to provide help reflects the types of cytokines they secrete (IL-2, IL1β, IFNγ) as well as the chemokines they secrete and the chemokine receptors that they express (see Table 4-1). It is particularly interesting that some of the chemokines that they produce also are chemokines to which they can respond (86–89). A clever consequence is that T cells that respond to a chemokine call from a site of tissue trauma can add to the chorus and strengthen the call for recruitment of additional T cells. By virtue of the IFNγ that they secrete, Th1 cells also support selective production of a particular isotype of antibody (IgG2a in rodents). Cells of the Th2 type provide help for humoral immune responses. Thus, they are involved in responses that include IgE and other components of immediate hypersensitivity reactions, complement-mediated attacks on microorganisms, expulsion of helminths, and other events. Those roles played by Th2

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cells reflect the particular spectrum of cytokines and chemokines that they secrete and the chemokines to which they respond (80–89). Considerable effort has been expended by investigators in many laboratories around the world to elucidate the mechanisms involved in the selective differentiation of Th1, Th2, Tc1, and Tc2 from their precursors, naive CD4 or CD8 T cells. A major finding was that the development of Th1 cells is promoted by IL12, a product of activated APCs (DCs, MPs). Similarly, the differentiation of Th2 cells is promoted by IL-4 and IL-10, especially the former. The actions of those two sets of cytokines appeared to be mutually antagonistic; in particular it could be demonstrated conclusively that IL-10 inhibits the production of IL-12 by APCs. The situation is, of course, more complex than that, as shown by the scheme in Figure 4-5, which reflects current thought about Th1 and Th2 development from naive CD4+ precursors (85). The activation of naive CD4 cells by cognate peptide-class II MHC complexes presented by professional APCs (DCs) triggers clonal expansion. Concurrently, the expanding T cells are challenged by numerous substances in the surrounding milieu. One group of substances led by IL-12 and other cytokines released by DCs or MPs (the APCs may be activated by the same microbial material) exerts a strong influence on the naive CD4+ cells to differentiate along the Th1 pathway. IFNγ produced by other, previously differentiated Th1 cells in response to IL-12, or by NK cells, exerts additional pressure on the naive cell to proceed along the Th1 pathway (in part, by inhibiting the Th2 pathway). Continued stimulation by IL-12 and IL-18 (from activated DCs and MPs or other sources) produces a mature Th1 cell that secretes some IL-2, IL-10, and considerable amounts of IFNγ and selected chemokines. Differentiation along the Th1 pathway is antagonized directly by stimulation of the activated naive cells with IL-4 or indirectly by the action of IL-10, which shuts off IL-12 production by DCs and MPs. If the predominant influence on the expanding, naive T cells is IL-4 (and associated factors such as the chemokine MCP-1), the Th2 pathway of differentiation is the fate of the naive cells. As shown in Figure 4-5, a quartet of cytokines is produced by the mature Th2 cells along with a few chemokines as discussed above. The preceding is a description of the differentiation pathways open to naive CD4+ T cells. The questions, now, are: How does all this happen? And what determines the conditions/influences that will prevail on the naive CD4+ cells? The approach to answers to those questions has relied, to a considerable extent, on an in vitro system, which allows selective stimulation into the Th1 or Th2 pathway (90–92). In brief, the procedure involves cultivation of enriched T cells in the presence of IL-12 plus antibodies against IL-4 and IL10 to drive the cells into the Th1 path; or in the presence of IL-4 plus antibodies against IFNγ and IL-12 to coax the cells into the Th2 pathway. This

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Fig. 4-5. The complex interplay of cytokines form different sources determines the maturation of precursor T cells along the Th1 or Th2 pathways. (From ref. 85, with permission.)

procedure, together with the use of various gene targeted (“knockout”) mice in numerous laboratories, has produced results which support the conclusions that: (a) the differentiation of Th1 cells requires signals initiated via the IL12R (enhanced by signals via the IL-18R) and nuclear translocation of the transcription factor, Stat 4; and (b) Th2 differentiation involves signaling via the IL-4 receptor and the transcription factor, Stat 6 (see ref. 85 for review). Several other transcription factors have been invoked in one pathway or the other but the only ones for which definitive proof is available are GATA-3 and c-MAF, which are critical in the Th2 pathway (93–95). GATA-3 is one of a family of factors that recognizes the tetranucleotide, GATA. GATA-3 has been proposed to control a master switch that selectively directs naive CD4+ cells into the Th2 path (see ref. 85). Until recently, Stat 4 seemed the only transcription factor employed in differentiation of naive CD4+ T cells along the Th1 pathway. That changed, however, with the discovery of T-bet (T-box expressed in T cells) (91). T-bet is a transcription factor found only in thymocytes and Th1 cells. Various experiments have demonstrated that T-bet is expressed in Th1 but not Th2 cells, selectively transactivates the IFNγ gene, and represses expression of the IL-2 gene. Ectopic expression of T-bet in T cells can override the effects of stimuli that normally bias the differentiation of Th2 cells. And, perhaps most impressive of all, T-bet can, when introduced into stable

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Th2 cells, destabilize the differentiated state, significantly reduce the production of IL-4 and IL-5, and trigger production of IFNγ (91). How T-bet exerts its powerful bias on naive CD4+ cells into the Th1 path is uncertain. It has been perceived as a “master switch” that turns on the Th1, and turns off the Th2, mechanisms (see ref. 96). Recently, evidence has been presented (92) that strongly suggests that T-bet, along with transcription factor Stat 1, direct events that lead to Th1 differentiation, one prominent event being the induction and stabilization of the β2 chain of the IL-12 receptor, which is essential for IL-12-initiated signal transduction. IL-18 enhances the effects of IL-12 vis-à-vis differentiation of Th1 cells, especially the production of IFNγ. This effect is probably the result of sustained expression of mRNA encoding the IL-12Rβ2 chain (97). Thus, T-bet and IL-18R, the latter (like IL-12Rβ2) being presented only on Th1 cells (97), may have similar effects in upregulating and/ or stabilizing the β2 chain of the IL-12R. Although in the early phase of their differentiation Th1 and Th2 cells are rather plastic and (in the absence of adequate stimulation) reversible (ref. 98 presents what appears to be a good example of this), the mature cells are stable and can be analyzed as separate populations. The same is true of Tc1 and Tc2. Thus, it became important to compare the genes that were expressed by the four types of mature cells. This was achieved by the combined use of real-time polymerase chain reaction and analysis of the prepared cDNA by DNA microarray methodology (99). In excess of 100 genes were found to be differentially expressed in comparisons of Th1 and Th2 cells and in comparing Tc1 with Tc2 cells. Many of the same genes were expressed by Th1 and Tc1 but not by Th2 and Tc2 and vice versa. For example, Th2 and Tc2 differentially expressed genes encoding IL-3, IL-4, IL-5, IL-10, and GM-CSF whereas Th1 and Tc1 differentially expressed genes encoding IL-2, IFNγ, IL-12β2, and IL-18R. Th2 cells differentially expressed GATA-3, and Tc2 cells expressed genes encoding chemokine receptors CCR1, CCR5, and CXCR4. Th1 cells, by contrast, expressed genes for multidrug resistant protein and STAT 4 whereas Tc1 cells expressed CCR7. This analysis must be viewed as demonstrating the power and potential of the methodology, not as the “final answer” to the question of what genes are active in these mature T cells. Why is all of the above information concerning types of T helper and Tcytotoxic cells important in the context of aging of immunity? Because those cells are key to the immune defenses against various pathogens (and tumors) and because the generation and characteristic functions of those cells are affected by aging. The effects of aging on T cells are manifested clearly by the changes in cytokines (and chemokines) that they produce. The results published in a few (of many) reports concerned with aging of T-cell production of cytokines are summarized in Table 4-2. The table also shows the results of stud-

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Table 4-2 Effect of Aging on Production of Selected Cytokines

Cytokine

Aged species

Type of T cell

Stimulant

Change with age

Ref.

Human Mouse Mouse

PBMCa CD8+ memb CD4+ total

Mitogen PMA+ ionomycin anti-CD3 + anti-CD28

↓ ↓ ↓

99 100 101

IL-4

Human Mouse Mouse Mouse

PBMC CD8+ mem. CD4+ total CD4+ total

Mitogen PMA+ ionomycin anti-CD3 + anti-CD28 anti-CD3

↑ ↑ ↑ ↑

99 100 101 102

IL-3/IL-5

Mouse

CD4+ total

anti-CD3

↑

102

IL-10

Human Human Mouse Mouse Human

PMBC PBMC CD4+ total CD4+ total PBMC

Mitogen SEBc anti-CD3 + anti-CD28 anti-CD3 Mitogen

↑ ↑ ↑ ↑ ↑

99 103 101 104 105

IL-12 (p70)

Human

PBMC

SEB

↑

103

IFNγ

Human Mouse Mouse Mouse

PBMC CD8+ mem CD4+ total CD4+ total

Mitogen PMA + ionomycin anti-CD3 + anti-CD28 anti-CD3

↑ ↑ ↑ ↑

99 100 101 102

IL-2

aPBMC:

peripheral blood mononuclear cells. memory. cSEB: Staphylococcus enterotoxin. bmem:

ies on IL-12, a product of monocytes and B cells. The cytokines included in Table 4-2 are those that have direct relevance to the effects of aging on Th1 and Th2 cells (100–107). It is generally agreed, with certain noteworthy exceptions (e.g., ref. 103), that IL-2 production by T cells of aged mice is considerably less than by T cells of young-adult mice. Consider, for example, the CD8 T-cell population. The CD8+ memory cells, which increase relatively in aging animals, include a smaller proportion of cells that produce IL-2, and a greater proportion that generate both IFNγ and IL-4, than do CD8– memory cells of young mice (101). Thus, the shift with age toward an increased proportion of CD8– memory cells in the mouse spleen is accompanied by a shift within the memory population toward a lower proportion of cells that produce IL-2. The bulk of IL2 production is performed by CD4+ T cells. Here, too, the deficiency in produc-

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tion of IL-2 is associated with the increasing proportion of CD4+ memory cells (102). Although Table 4-2 indicates that IL-2 production by human T cells is depressed, there is considerable uncertainty about that conclusion (see ref. 25 for a thoughtful review). A significant problem with studies of cytokine and chemokine production by lymphocytes of the human is the necessity to study peripheral blood cells because there are many variables that can influence the results: e.g., subclinical infections, stress and other neuroendocrine variations, diet, exposure to xenobiotics, and so on. Aging further compounds the variables. It is often frustrating to compare the results from studies of mouse splenocytes with those from human peripheral blood leukocytes. The results of selected studies of IL-4, IL-5, IL-10, and IFNγ production by T cells of young and aged subjects, both mouse and man, are shown in Table 4-2. As in the case of IL-2, not all studies of those cytokines produced results that concur with those shown in Table 4-2. However, it seems probable that the availability of IL-4, IL-10, and IFNγ does increase with age. If it is correct that the production of IL-2 decreases whereas production of IL-4, IL-10, and IFNγ increases with age, it would appear that aging is accompanied by increasing dominance of Th2 cytokines. A number of investigators have concluded that the eminence of Th1 declines with age; thus, aged individuals suffer from a weakened ability to generate immune responses against infections and tumors, but a greater tendency toward antibody-mediated disorders such as IgE-mediated allergies (e.g., see ref. 108). One particularly interesting aspect of this age-associated shift is the appearance of subsets of splenic CD8+ cells, one of which expresses the phenotype CD45RBhi CD44hi and another that simultaneously produces IL-4 and IFNγ (101). Those subsets suggest that the aged spleen microenvironment is unable to support the complete maturation of memory cells or of the Th1 and Th2 subsets. Change in the aged splenic milieu has been noted in several reports (24,27), initially by Price and Makinodan (109). Such a change is manifested clearly by the failure of germinal center formation as is discussed below. It is possible that aging alters the structure and/or composition of the secondary lymphoid tissues in ways that account for the differences in T-cell differentiation and cytokine production between young and aged mice. And that discrepancies between the findings in aged mice and humans reflect the changes in the tissue milieu in mice that do not occur in the milieu of human peripheral blood cells (110). Nevertheless, in order to determine whether or not intrinsic aging affects cells of the immune system similarly in mouse and man, studies should be done with cells derived from, or conditioned by, similar microenvironments. More care should be given to other “technical” considerations in future research concerned with aging of T cells. In particular, the objectives of the research should dictate the choice of stimulants and activating substances.

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There are adequate reports that demonstrate significant differences between the results obtained from activation of cells with: mitogens; phorbol esters with and without inonomycin; anti-CD3ε, soluble or immobilized, with and without costimulation with anti-CD28, soluble or immobilized; superantigens; live or heat-killed microorganisms. Likewise, the objectives should dictate the type of T-cell preparation to use in the study; i.e., highly enriched, semienriched, with or without DCs, whole unfractionated cell mixtures. Given the large number of possible experimental variables, and the favorite conditions used in many laboratories, it is not surprising that it is impossible to draw unequivocal conclusions about the more sophisticated aspects of aging of T cells. Care in choosing stimulants, activators, and other conditions according to the objectives of the experiments will be become increasingly critical as research is expanded to interactions between ambient cytokines that drive responses of aged T cells. A recent study (111) is a good example. The objective of the study was to evaluate the influence of several cytokines on the production of Th1 (IL-2, IFNγ) and Th2 (IL-4) cytokines by whole spleen cell suspensions obtained from mice of different ages. Suspensions of spleen cells were stimulated with phorbol myristate and concanavalin A without or with individual cytokines. After appropriate intervals of incubation, the cells were harvested and used for analysis of mRNA transcripts of IL-2, IL-4, or IFNγ. Other cultures were maintained longer before collecting the medium for assays of corresponding cytokine proteins. The results obtained from control cultures (not exposed to exogenous cytokines) confirmed the poor ability of aged cells to generate IL-2 and IFNγ proteins. In this study, the production of IL-4 by aged mouse spleen cells was much lower than production by young or middle-aged (adult) mouse spleen cells. The next phase of the study was designed to assess the effect of exogenous cytokines (added to cultures in graded doses) on the production of IL-2, IL-4, and IFNγ by young, adult, and aged spleen cells in culture. Parallel, short-duration cultures were used for isolation of CD4+ cells and assays of mRNA transcripts of the three cytokine genes. The results, in brief, showed that: (a) production of IL-2 protein was enhanced in adult mouse cells, and even more enhanced in aged mouse cells, by IL-4 and IL-12 at even moderate doses; (b) stimulation with exogenous cytokines (IL-1β, IL-2, IL-3, IL-4, IL-12) even in low doses enhanced IFNγ production by aged mouse spleen cells but the same cytokines even in high doses had no effect on IFNγ production by aged mouse spleen cells, neither on protein nor on mRNA output; and (c) all five of the cytokines added to the spleen cell cultures enhanced IL-4 production by aged mouse spleen cells but not cells from young or adult ages. Thus, the results of this series of experiments suggest that elevated production of IL-2 by aged mouse Th1 cells can occur in response to stimulation with exogenous IFNγ or IL-12 provided that

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the cells have been strongly stimulated through other receptors (Con A and PMA in this case). On the other hand, aged Th1 cells do not increase their production of IFNγ regardless of the combination of stimulants employed whereas Th1 cells from adult mice do. The output of IL-4 by Th2 cells of aged mice can be enhanced by stimulation with a variety of cytokines (all five employed in this study). Overall, the results of this study seem to agree with many others in showing that aging results in depressed IL-2 production. However, the results seem to be at variance with at least some other studies in suggesting that the production of IFNγ by aged mouse spleen cannot be enhanced by exposure to exogenous IL-12. In the case of IL-4, the results are curious in suggesting that many cytokines can cause elevated production by aged mouse spleen, in a rather nonspecific fashion. The point in reviewing this paper (111) in some detail is not to criticize. It is a good paper given the current limitations and degree of confusion of methodology. That, of course, is the point—to promulgate an encouraging word for a careful review of methodology. Those culture conditions, activating stimulants and costimulants, exogenous cytokines, chemokines, and other factors that are likely to provide clear, reproducible and, especially, interpretable results should be identified and all the others discarded. For example, reagents, conditions, and so on. that are likely to be informative might be identified from the perspective of intracellular signaling. Is a condition, reagent (cytokine or other) likely to produce a result that can be understood with reference to a known signaling pathway that it activates or influences? Conditions commonly employed for in vitro studies may or may not be appropriate for analysis of the effects of aging. There is, for example, considerable evidence that the effects of aging often are attributable to oxidative damage, and that redox conditions in tissues such as the spleen tend to be biased toward oxidative in aged animals compared to young adult. Therefore, for example, it may not be appropriate to include 2-mercaptoethanol and unscreened (for glutathione, thioredoxin) fetal bovine serum in media used for cultivating aged animal cells and tissues. There are satisfactory methods for evaluating splenic pH, redox conditions, and relevant enzymes and reducing substances in situ. Those conditions should be thoroughly evaluated and then simulated for many/most in vitro experiments. If, for example, the intrasplenic (or, better, intracellular) redox balance is tilted toward oxidative and/or the intrasplenic/intracellular pH is significantly different from that of the blood (or RPMI culture medium), those conditions that prevail in situ should be duplicated ex vivo. It is rather sobering to consider the possibility that the majority of studies conducted with dispersed spleen cells under conditions optimized for robust responses by young adult mice have, in fact, provided data of limited reliability vis-à-vis the immunological capabilities of aged animals and humans, in general.

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Involvement of APCs (Especially DCs) in the Differentiation of Type 1 and Type 2 T Cells The presentation of antigenic peptides in complexes of MHC molecules to cognate T-cell receptors can be accomplished by three different, professional APCs: MPs, B cells, and DCs. For many years, DCs were not thought to be physiologically significant because there was no evidence that they could process antigens so as to produce the requisite peptides. That view changed rapidly once methods were developed to produce DCs in large numbers sufficient for cytological and biochemical studies. It is now clear that DCs are the critical APCs. The role played by DCs in the immune response is pivotal and complex (see review in ref. 112). DCs are widely distributed in the body, being most common in and around portals of microbial entry. The cells of Langerhans, for example, are scattered throughout the epidermis of the skin. They remain at those sites as sentinels of invasion by pathogens. They are relatively immature until stimulated by microbial components or other noxious material. In the immature state, DCs are quite proficient at capturing and processing foreign matter. As they perform that function, they undergo maturation and mobilization. They migrate to nearby draining lymph nodes or the spleen where they enter the lymphoid areas as though searching for cognate T cells to activate. Their journey to and into lymphoid tissues is, to a considerable extent, under the direction of chemokines (113). Once they have encountered cells bearing complementary TCRs, they proceed to interact with them by formation of a substantial area of mutual membrane contact termed “the immunological synapse.” There is now abundant evidence that the events associated with the DC–T-cell union determine the differentiative fate of naive CD4+ T cells into Th1 or Th2. As discussed above, key factors that determine Th1 and Th2 differentiation are the cytokines IL-4 and/or IL-10 (Th2) and IL-12 (Th1). Although that idea remains valid, it has been amplified by several recent discoveries, in particular the realization that the populations of DCs are critical in Th1 and Th2 specification. For example, in the case of mice there are three distinguishable subsets of splenic DCs characterized by their surface phenotypes as follows (114): approximately 50% are CD4+ CD8–; about 25% are CD4– CD8–, and the remaining 25% CD4– CD8+. The CD8 molecules on the DCs are ααchain homodimers rather than the αβ heterodimers of conventional T cells. Immunohistological analyses revealed that the CD4– CD8+ DCs are concentrated in the T-cell areas of the spleen whereas the other two subsets are largely restricted to the marginal zones. Experiments conducted both in vivo and in vitro indicated that those three subsets of DCs are not related in some precusor–product manner or maturational hierarchy; rather, they appear to be stable subsets derived from some earlier progenitors. Other experiments per-

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formed both in vivo and in vitro revealed that all three subsets of DCs have phagocytic activity, even after being stimulated to maturity. That finding is in disagreement with the results of some other investigators who have reported that only immature DCs are capable of antigen capture (but see ref. 112). Additional studies on DC subsets in lymph nodes (115) produced results similar to those from spleen except for the presence in nodes of two other subsets that clearly were derived from dermal and/or epidermal Langerhans cells. The relationship of subsets of DCs to the differentiation of Th1 and Th2 cells from naive CD4+ T cells is a subject of great interest at present (see, e.g., 116). Although there remains much to be clarified, especially differences between mouse and human DC–T-cell interactions, there is good evidence that distinct subsets of DCs promote the differentiation of naive T cells either along the Th1 or Th2 pathway as illustrated in Figure 4-6. The DC1 cells that produce IL-12 and drive Th1 differentiation are CD8α+ whereas CD8α– DCs promote Th2 differentiation (117–122). A common view at present is that activation of the DCs by encounter with antigen (especially microbial) results in IL-12 production by CD8+ DCs and both the IL-12 and other signals delivered through the cognate TCRs drive Th1 differentiation of naive CD4+ T cells. Similarly, activated CD8α– DCs generate IL-4 (at least in the mouse) which, upon interaction between DC and T cell, biases CD4 naive cell differentiation toward Th2 cells. The two types of DC, originally localized in different locales in the spleen or nodes, both congregate in the T-cell areas once they become activated. The mature T cells then produce their distinctive spectra of cytokines and chemokines, which direct the course of the immune response, primarily cell-mediated response in the case of Th1 cytokines (IFNγ, IL-2), which is particularly effective against viruses and intracellular bacteria and protozoa, and humoral in the case of Th2 cytokines (IL-3, IL-4, IL-5, etc.), the response most effective against extracellular microbes, helminths, and a variety of xenobiotics including, unfortunately, allergens. As is so frequently true, it is the “details that are devilish.” For example, there may not be cells in the human immune system that correspond precisely to the CD8+/DC1 and CD8–/DC2 of the mouse. Three subtypes of human DC have been described: myeloid DC (also termed interstitial or dermal); Langerhan’s cell–derived DC; and plasmacytoid DC (118,119,123,124). The monocyte-derived, myeloid DCs (CD11c+) promote Th1 responses whereas plasmacytoid DCs (CD11c–) induce Th2 responses. However, that generalization is weakened by several caveats (see ref. 124 for discussion) that arise, in part, from variations in the methods for generating the DCs from precursors. There is confusion about the precise cytokine required at the time of naive T-cell activation to drive Th1 or Th2 differentiation; and even the necessity for the cytokine to be presented in the milieu at the time of activation. In the case

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Fig. 4-6. Dendritic cells (DCs) of distinguishable subsets and that produce different cytokines (e.g., IL-12 in the case of DC1) play critical roles in directing the maturation of T cells in the Th1 or Th2 pathways. (From ref. 116.)

of Th1 differentiation, it seems reasonably certain that IL-12 is required, that it must be present in the milieu but need not be produced by CD8a+ (type 1) DCs (120,122). In the event IL-10 is present in moderate, or greater, amount the differentiative response is likely to be of the Th2 type owing to the inhibition of DC1 IL-12 production by the IL-10 (125). It is not clear whether Th2 differentiation under those circumstances occurs in the absence of IL-4 but it seems likely that IL-10 can substitute for IL-4. In any event, IL-4 has been demonstrated to drive Th2 differentiation of activated, naive T cells. In studies that have not demonstrated the need for IL-4, it is probable that the small amount needed to trigger Th2 differentiation has been overlooked.

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As discussed in the preceding section, two powerful transcription factors are involved in the determination of which pathway a differentiating naive T cell will follow: T-bet in the case of the Th1 pathway and GATA-3 in the Th2 pathway. However, those factors are not, as once was thought, master switches. Rather, they act in conjunction with other transcription factors. As is now clear (126,127), members of the NFAT (nuclear factor of activated T cells) family of transcription factors are critically involved in influencing the differentiation of naive CD4+ cells into Th1 or Th2 descendants. NFATs are not translocated from cytosol to nucleus unless they are dephosphorylated. Calcineurin is the phosphatase that activates NFAT translocation. Because calcineurin activity is highly dependent on free Ca2+, the entry of NFATs into the nucleus is considered Ca-dependent. Mice that were NFATc2 (=NFATp) and NFATc3 (=NFAT4) deficient (i.e., NFATc2–/– and NFATc3–/– double knockout or DKO) were used as donors of CD4+ naive cells. The latter were stimulated with immobilized mAb against CD3 with or without anti-CD28 costimulation. Cell proliferation as well as output of cytokines was assessed. Cells from DKO mice differentiated only into Th2 descendants even in the absence of CD28 costimulation (wild-type, naive cells require CD28 costimulation to generate Th2 daughters). However, in the presence of exogenous IL-12, the naive precursors from DKO mice activity generated Th1 descendants. Thus, it appears that NFAT transcription factors exert regulatory influence on the differentiation of naive CD4+ T cells by controlling differentiation along the Th2 pathway. As part of the process, NFATs appear to complete with GATA-3 for binding to regulatory elements in the IL-4/IL-5/IL-13 gene complex (126). It is noteworthy that IL-12 may also compete with GATA-3, which would explain why the presence of IL-12 influenced the cells from DKO mice to follow the Th1 pathway. One conclusion drawn from the study of DKO naive CD4 cell differentiation (126) was that, in the absence of NFATc2 and NFATc3, the activation threshold of the naive CD4 cells was significantly lowered. Another recent, related, study has shown that low-intensity (low-affinity) stimulation of naive CD4+ T cells can selectively induce IL-4 production and Th2 differentiation (128). Low-intensity signals resulted in relatively high concentrations of nuclear NFATc (=NFATc1) and low concentrations of NFATp (=NFATc2). IL-4 production induced by low-intensity signaling was vigorous within 48 h after naive cell activation by APCs pulsed with cognate peptide. This IL-4 response occurred before expression of GATA-3 in the nuclei of the daughter cells and appeared to drive Th2 differentiation. According to the results of this study, low-intensity (low-affinity) stimulation of naive T cells via the TCR can provide signals capable of activating IL-4 expression. Weak signals transduced via the TCR also trigger weak, transient Ca2+ mobilization

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and together, the two weak events are able to initiate IL-4 production, which drives Th2 differentiation (128). The activation of IL-4 expression results from unequal, high concentrations of nuclear NFATc and low levels of nuclear NFATp. All of this is set in motion prior to the appearance in the nucleus of GATA-3. Once the latter occurs, full differentiation of the Th2 effector cells ensues (128). NFAT transcription factors are also critical in Th1 differentiation from naive CD4+ cells (128). This was shown by introducing a mutant form of the NFATc gene into naive CD4+ precursor cells resulting in constitutive production of NFATc by progeny of the precursor cells. The result of stimulating/activating those precursor cells was strong Th1 differentiation and IFNγ secretion. Moreover, when those precursor cells were stimulated under conditions that, in the case of wild-type cells, favor Th2 differentiation, development of Th2 cells was attenuated and cells that were generated produced below-normal amounts of IL-4 along with IFNγ. Thus, strong, sustained stimulation of naive CD4+ cells favored Th1 differentiation, a result that supports the conclusion that a strong TCR signal promotes nuclear translocation of significant amounts of both NFATc and NFATp and, therefore, failure of Th2 differentiation (128). To conclude this section on DC involvement in naive T-cell differentiation the following points should be mentioned. First, the activation of naive cells requires not only DC presentation of antigen but also costimulation (via CD28 and CD80/CD86 or CD40 and CD40L) followed by the appropriate cytokine to drive differentiation along the Th1 or Th2 pathway. Costimulation alone, via CD28, may be sufficient to send memory cell differentiation down the Th2 pathway (129). Second, the requirements for selectively biasing development of Tc1 and Tc2 cells from naive CD8+ T cells have not been adequately explored but probably resemble those involved in naive CD4+ T-cell differentiation. Third, indications are that the temporary dominance of DC type 1 or DC type 2 following the entrance of a pathogenic organism is determined by the nature of the antigens, some microorganisms promoting DCs of type 1, others promoting DC2 (130). In this manner the infected host has an opportunity to generate the most effective type of response. What governs the biased activation of the DC is uncertain but most likely involves PRRs such as the Toll isoforms (see Chapter 2). And, finally, recent work (131) has demonstrated that macrophages, too, can influence the pathway of differentiation of naive CD4+ cells. When MPs displaying MHC-peptide complexes were employed as APCs, strong Th1 responses were observed. When the same antigenic material was also bound to Fc γ receptors on those MPs the differentiation of naive CD4 T cells progressed mainly along the Th2 pathway. This system deserves more attention.

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The Immunological Synapse and T-Cell Signaling It has been known for more than a quarter century that the fluidity of cellular membranes is reduced by the presence of cholesterol, more precisely the cholesterol-to-phospholipid ratio (132,133). That knowledge prompted several early studies concerned with the effects of aging on membranes and associated functional consequences. It was observed that: (a) the microviscosity of the membranes of human lymphocytes increased with age in concert with the increase in the rate of membrane cholesterol relative to phospholipid, (b) the mole ratio of cholesterol to phospholipid present in the blood serum also increased with age, and (c) the proliferative response of peripheral blood lymphocytes to mitogen decreased in concert with the increase in cholesterol/ phospholipid ratios (134). It was further demonstrated that the impaired proliferative response could be significantly improved by treating the lymphocytes of aged mice in vitro with lecithin (135) or in vivo by feeding the mice a mixture of lipid derived from hen egg yolk (136). The restorative action of the latter was shown to correlate with its ability to extract cholesterol from the lymphocyte membrane. Other, pioneering studies were designed to study the density and distribution of lymphocyte receptors and their mobility when induced to congregate to form “caps” or to recover from photobleaching (137,138). Those experiments were performed with lymphocytes, including T cells, from young and aged rats. The results were quite conclusive: the cells of aged rats differed from those of young rats in that they displayed fewer characteristic membrane-associated molecules and the surface molecules that were present congregated into caps or migrated into bleached spots at a significantly reduced velocity. Unfortunately, the opportunity afforded by that pioneering work to experimental gerontologists to make significant inroads in understanding membranes and the effects of aging was largely ignored. Only in the last few years has the enormous importance of the effects of aging on membrane structure and function been appreciated. Intense investigation in recent years has revealed that many of the activities of cells of the immune system involve physical contacts of the membranes of two or more cells. For example, NK cells and cytotoxic T cells abut their targets in order to kill. In order to become activated, NK cells make contact with the DCs that deliver the required stimuli; the reciprocal is also true: viz., NK cells activate DCs and, for that, direct contact of the cells is involved (139– 141). T cells and B cells interact through areas of membrane contact, a phenomenon that led to the description, “immunological synapse” (142). The apogee of research concerned with cell–cell contacts and synapses has been reached with the current explosion of studies on T-cell–DC interactions. To better appreciate the significance of the current research, a brief discussion of biological membranes and protein-lipid microdomains is in order.

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Fig. 4-7. An illustration of the coordinated assemblage of different proteins distributed on different lipoprotein microdomains to form an organized signaling platform on the membranes of activated T cells. (From Hoessli DC, et al. Glycoconjugate J 2000;17:191–197, with permission.)

The plasma membrane of cells comprises two layers or leaflets. The outer layer, exposed to a cell’s microenvironment, is an expanse of unsaturated glycerophospholipids arranged so that the head groups of the molecules face outward and the hydrocarbon-like tails face inward contacting the molecules of the inner leaflet. The molecules that comprise the latter are arranged such that their hydrophobic tails interact with the tails of the molecules of the outer leaflet while their headgroups face the cytosol. In this manner, the membrane assumes the appearance of a lipid bilayer. Dispersed in that rather monotonous expanse of lipid are islands (microdomains) composed of proteins and more complex lipids (see Fig. 4-7). The lipids in those islands are largely sphingolipids, glycosphingolipids (including GM1 and GM2), and cholesterol (143,144). The preceding description applies to both the outer and inner leaflets. Beyond that, much more is known about the former than the latter because so many receptors and signaling complexes through which the cell

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communicates with its environment and with other cells are located on the outer leaflet. The islands or microdomains may be considered as moreordered, less fluid phases that can move laterally with little restriction within the lipid expanse of the outer leaflet. For that reason, they are commonly known as “lipid rafts.” They exist in a dynamic state, at times being dispersed, at times coalescing with other microdomains. One compelling example that rafts differ significantly in composition from the ambient membrane is their insolubility in detergent, a property that allows them to be separated from the membrane, enriched and subjected to analyses of both the lipid and the protein components. The proteins are chemically modified in order to endow them with affinity for the lipid rafts. The modifications include coupling to glycosylphosphatidylinositol (GPI), which tethers them to the rafts (GPI-anchored proteins), double acylation, and coupling to cholesterol or palmitic acid. A few membrane proteins seem to lack modification and probably associate with rafts through uncommon amino acid motifs. In resting cells, the size of the microdomains/rafts is below the resolution limit of standard light microscopes. Thus, the number of protein molecules in individual rafts is modest, in the range 10–30. It remains uncertain whether some (most?) rafts are molecules of several different proteins or a single species. Proteins that are associated with rafts via acylated fatty acids generally are coupled to saturated fatty acids. Studies performed both in vivo and in vitro have revealed that the saturated fatty acids can be replaced with unsaturated ones, which may result in those proteins dissociating from the rafts. Similar results have been obtained by providing exogenous gangliosides. The importance of lipid rafts in the case of T cells is under intensive investigation. It is clear that many of the protein molecules that comprise the TCR complex and co-signaling molecules exist in rafts and that the orderly coalescence of the rafts and reassembly of the proteins is crucial for the activation and functioning of T cells. As illustrated in Figure 4-7, the α/β chains of the TCR and associated elements of the CD3 complex probably exist in microdomains separate from those supporting the CD28 co-receptor and accessory moelcules such as CD4. Similarly, the early signaling molecules, Lck and LAT, exist in separate rafts. The protein tyrosine phosphatase, CD45, appears to be raft-independent, which possibly allows it to maintain Lck in dephosphorylated condition, thus preventing aberrant signaling. Upon interaction with an APC, the distribution of those T-cell surface molecules undergoes rearrangement as a result of raft aggregation and coalescence (Fig. 4-7). This results in spatial proximity within the enlarged lipid raft of molecules that initiate and maintain signaling and T-cell activation (e.g., CD28 and CD4) but not of molecules such as CD45, which would interfere with the initiation of signaling. The signaling cascade can then proceed as shown in

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Figure 4-7 by the autophosphorylation of Lck, a protein tyrosine kinase that then phosphorylates the ITAMs (immunoreceptor tyrosine-based activation motif) on the cytoplasmic tails of the CD3 epsilon and CD3 zeta chains of CD3. The next step is the activation of the key protein tyrosine kinase, ZAP70, followed by stepwise transmission of signals via one or more of three cascades. Those cascades end in the translocation to the T-cell nucleus of the transcription factors NFκB, NFAT, and AP-1 (cfos/cjun complex). The crucial event in starting the signaling and activation processes in the T cell is the interaction of the α/β chains of the TCR with the cognate peptide presented by the class I or class II MHC molecules located on the APC. Important accessory molecules located on the APC are the B7 ligands (CD80 or CD86) and ICAM-1 (CD54), which are complementary to, respectively, CD28 and LFA-1 (CD11a/CD18) located on the T cell. Therefore, a similar process of aggregation of rafts into a platform of interactive molecules must occur on the APC. It is now clear that, indeed, molecular reorganization does occur on the surface of the APC. There appear to be two ways in which peptide-MHC complexes can be reorganized: in one case in association with lipid rafts and accessory molecules (CD80, CD86, CD54); in the second case (in human DCs), in association with members of the membrane tetraspan protein family (CD9, CD81, CD82), a peptide-editing factor, HLA-DM, and accessory substance CD86 (145). Fascinating evidence has been provided (145) that suggests that the tetraspan microdomains bear MHC class II molecules (CDw78, in particular), all of which are complexed with identical peptides (perhaps reflecting peptide editing) and are thus substantially more efficient at activating cognate T cells than are lipid rafts displaying the same peptide intermixed with other peptide-MHC complexes. In any case, two organized, relatively large microdomains, one on the T cell and one on the APC (DC), each bristling with interactive receptors and counterreceptors, unite to form an immunological synapse. The formation of the synapse has been visualized and analyzed both in vitro (146–149) and in vivo (150–152). Studies of T-cell–APC interactions as well as T-cell interactions with planar lipid bilayers bedecked with tagged, fluorescent MHC-peptide and ICAM-1 have strongly implicated the following sequence of events: 1) during the first few minutes after contact of T cell and APC a relatively wide zone develops approximately at the center of the area of contact interface representing accumulation of ICAM-1 (CD54) that has interacted with T-cell LFA-1 (CD11a); 2) CD4 and CD3 zeta cluster in the center of the interface; 3) the preceding events are accompanied by Ca2+ flux in the T cell; 4) at about the same time, a circumferential ring of complexes appears composed of MHCpeptide-cognate TCR interactants. Then, within the next few minutes, the complexes of MHC-peptide TCR migrate centrally and congregate tightly at the

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center of the area of contact while the CD45-CD11a interactants along with CD4 regroup as a circumferential ring. Thus, there is a switch in the topography of the two sets of interactants. Within an hour following T-cell–APC contact, the central core of molecules vanishes. The early events following TCR–APC contact define three phases in T-cell activation: (a) junction formation, (b) MHC-peptide-TCR transport, and (c) formation and stabilization of the central cluster (MHC-peptide-TCR-CD3 zeta). Stabilization is achieved when the binding of the peptide is sufficiently strong that there is no exchange of peptides between MHC molecules inside and outside the central cluster. The rate and degree to which stabilization is achieved reflects the density of molecules contributing to the cluster. The density of MHC-peptide and cognate TCR molecules that congregate in the central cluster determine whether or not the T cell will be fully activated. Once the density exceeds a certain threshold, the T cell becomes fully activated and additional increments of clustered molecules give no additional impetus to the T cell. Other studies of T-cell–APC interactions via the immunological synapse have provided evidence that CD4 and CD28 function as costimulants by facilitating the early kinase activity of Lck (153,154). As noted above, CD4 appears early in the region of the central core and then retreats to the periphery. This behavior can be attributed to its function of conducting the kinase, Lck, to the central core and facilitating the transient autophosphorylation of Lck. CD28 co-localizes with the TCR to the central core of the synapse. CD28 is known to promote autophosphorylation of Lck. It appears to act sequentially with CD4, the latter first facilitating the gathering of Lck and initiation of its autophosphorylation followed by the action of CD28 to sharply enhance Lck autophosphorylation. Thus, the two costimulants are primarily involved in the initiation of signaling in the T cell. Another recent “twist” in the story of the immunological synapse is the evidence that signaling via the TCR precedes formation of a mature, stable synapse (149). A careful study of the synapse formed between fresh T cells and fresh APCs was conducted in which the appearance of phosphorylated Lck and ZAP-70 were used as indices of T-cell activation. It was found that at an early time when the TCRs were still located at the periphery of the synapse (see above) phosphorylated forms both of Lck and ZAP-70 were present in those same locations. Subsequently, both signaling factors were found in the central core region but neither was in active, phosphorylated form. Thus, it was clear that signaling involving the TCRs occurred well in advance of the development of the mature immunological synapse and was not dependent on congregation of molecules in the central core. Another important conclusion drawn from this study contradicted the view accepted by many that long-term stability of the synapse and of T-cell–APC interaction is required in order to fully

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activate naive T cells. Data from this study showed that two hours of T-cell– APC union is sufficient to prime T cells for their first mitotic division Then what is the function of the mature synapse? No certain answer is possible at present. But this is a rapidly advancing area of research and we are likely to have answers before we grow much older. Current ideas include the possibility that the synapse is instrumental in polarization of secretion by the cells (cytokines, chemokines, etc.) and, thus, perhaps in the differentiation of naive T cells into Th1 and Th2 or Tc1 and Tc2. Another plausible idea is that the synapse facilitates TCR down-regulation and endocytosis perhaps as a step in some signaling cascade or as a TCR morgue useful in controlling the magnitude of T-cell response. Related to the latter idea are the observations that T cells may ingest peptide-MHC class I complexes removed from their APC partners (155–157) and in so doing become susceptible to attack by other, nearby T cells in a form of fratricide (155,156). The phenomenon of TCR high-affinity maturation has been explained recently as resulting from the removal of peptide-MHC complexes from the surfaces of APCs by high-affinity receptors on T-cell synaptic partners (158,159). Thus, under conditions of relatively high ratios of cognate T cells to peptide-MHCpresenting APCs such as obtains in secondary immune responses, T cells bearing high-affinity TCRs stripped peptide-MHC complexes from the surfaces of APCs. In this manner, those “avaricious T cells” prevented other T cells displaying lower affinity receptors from becoming activated. As a result of this unfair (159) competition the average TCR affinity of the participating T cells gradually increased (“matured”). And, as a consequence, “...it is easy to envision the scenario by which multiple challenges of a host with antigen favors the outgrowth of increasingly higher affinity T cells, leading the entire antigenspecific population toward oligo- or mono-clonality” (158). Whether or not some process akin to this action of avaricious T cells can explain the oligoclonality of T cells in aged subjects is a question that awaits an answer. SUMMARY: KNOWN AND COGNIZABLE EFFECTS OF AGING T CELLS In the preceding section of this chapter, we have reviewed a substantial amount of information about the genesis, properties, and functions of T cells. Much of the information is recent and not yet available in textbooks. At this juncture, it seems worthwhile to summarize and demonstrate that the information presented and reviewed illuminates a path of research that could be followed to a much deeper understanding of how aging affects the competence of T cells to function in immune responses. After summarizing the discussion thus far, we present a brief accounting of four lines of research that (in our view), if pursued vigorously, should not only expand current understanding of

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T-cell aging but, in addition, should suggest practical approaches to invigorating immune capabilities of the elderly. Following that enterprise we proceed to a review of the effects of aging on B cells and humoral antibody production. Summary of Recent Advances in Knowledge About T Cells The size and cellularity of the thymus begin to diminish in adolescence, a process that continues on into old age. Involution is reflected by the gradual decline in export of mature T cells although some new T cells are released even in advanced age. The number of T cells in peripheral tissues changes very little with advanced age and is maintained by rather considerable production of new T cells from immature precursor cells outside the thymus. Those immature, naive, precursor cells previously released by the thymus may live for extended periods, even the lifetime of the organism. There is no evidence of a primary source of T cells other than thymus. T cells are generated in the thymus from committed progenitor cells that immigrate from the bone marrow. Aging seems to have no effect on the potential of those progenitors. The production of new T cells from the progenitor is decidedly influenced by the actions of stromal cellular elements within the thymus. Those stromal elements may be affected by aging as reflected by declining ability to generate chemokines, cytokines (e.g., IL-7), and proenzymes such as that of cathepsin L. There is a prominent, age-related change in the T cells of peripheral tissues: spleen, lymph nodes, and blood. The ratio of immature, naive T cells to mature, memory T cells changes from predominance of the former in young individuals to dominance of the memory population in the aged. That is true both of the CD4+ and CD8+ major subsets of T cells. It is generally agreed that continuous, low-level exposure of T cells to antigenic stimulation over a period of many months (mice) to years (man) drives that population shift. (However, that may not be the only cause.) Evidence that strongly supports that conclusion is found in the CD8+ subset when that subset of young mice is compared with old mice. Experimental data suggest that the restricted TCR recognition diversity associated with oligoclonality of T cells in the aged resembles that seen among memory T cells persisting after secondary immune responses. Other processes that may contribute to the oligoclonality among memory T cells include clonal deletion (resulting, perhaps, from eventual death of the progeny of earlier-inlife, vigorous, proliferative T-cell responses to antigenic stimulations) and the actions of avaricious TCRs which remove peptide-MHC complexes from APCs thus preventing activation of low-affinity TCR. Whether memory CD4+ and CD8+ T cells are set aside during primary response that is characterized as linear, leading from naive to mature effector cells, or as a subset of effector cells is uncertain. Current evidence seems in favor of the former model. In either case, there are memory cells that reside

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in or near common sites of infection (lung, gastrointestinal tract, urogenital system) that respond very quickly (T effector memory) to infection or insult and other memory cells (T central memory) that reside in secondary lymphoid tissues and play a supportive, “back-up” role. For a considerable time it was widely accepted that the long-term persistence of memory cells required at least intermittent stimulation by cognate antigen or, at least, cognate MHC substances. That paradigm appears to be changing as a result of growing evidence that memory cells do not require such stimulation for long-term survival. They may require, however, exposure to certain cytokines such as IL-15 and IL-7 in the case of CD8+ memory cells. T cells come in two varieties: Th1 and Th2 differentiate from naive, CD4+ T cells whereas Tc1 and Tc2 differentiate from naive, CD8+ T cells. The two varieties of helper cells (Th) can be distinguished by the type of immune response in which they participate, inflammatory in the case of Th1, and humoral Ab production in the case of Th2. They also differ in the spectrum of cytokines they produce (IL-2 and IFNγ characterize Th1 and IL-3, IL-4, IL-13 characterize Th2). The factors that control the variety of Th or Tc that the naive progenitor will produce by differentiating are not entirely known. The presence of IL-12 appears to be necessary for Th1 differentiation and IL-4 (or IL-10) for Th2 differentiation. Key transcription factors have been identified. Thus, members of the NFAT family are crucial for Th1 differentiation whereas GATA-3 plays a central role in Th2 differentiation from naive CD4+ precursor cells. Under normal, in vivo conditions, Th1 and Th2 differentiation from naive CD4+ precursors (and most likely Tc1 and Tc2 from CD8+ precursors) is probably controlled by the species of DC with which the naive T cell interacts. DC1, which can generate IL-12, drive Th1 differentiation and DC2, which produce IL-10, facilitate Th2 differentiation. The type of DC (DC1 or DC2) that is activated in response to a given potential pathogen is determined by the particular array of antigenic determinants that the DC recognizes (i.e., the DC’s PRRs) (see Chapter 3). Presumably, the activated DC, now bearing immunogenic peptides of the pathogen in association with Class I -or II-MHC substances, interacts with a cognate, naive T cell to form an immunological synapse. The events that ensue between the formation of the synapse and the determination of the naive T cell to differentiate into mature Th1 or Tc1 or, alternatively, Th2 or Tc2 descendants are unknown, at present. The primary function of the immunological synapse is the activation of the T cell bearing a cognate receptor appropriate for the peptide presented by the MHC complex of the DC (or other APC). This is achieved through coalescence of lipid microdomains (rafts) on each of the uniting cells: TCR complex, CD4 or CD8, CD28, LFA-1, and other accessory molecules in the case of the T cell; peptide-MHC complex, B7, ICAM-1, and other accessory molecules in the case

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of the APC. T-cell activation occurs early in the developing synapse when the TCR and peptide-MHC are at the periphery of the synapse and prior to the formation of the mature, central core of TCR-peptide-MHC-CD28 complex of interactants. Peripheral interaction of TCR with peptide-MHC is followed quickly by the initial events in intracellular signaling including a Ca2+ flux and phosphorylation of Lck and ZAP 70. Within a matter of hours, the T cell enters into the first mitotic division and subsequent, IL-2-driven, divisions. Cognizable Effects of Aging on T Cells The word cognizable (or, better, “cognoscible”) is used to refer to those features and/or functions of T cells that are “capable of being known or perceived.” In other words, those aspects of T-cell feature and function about which clues already exist and that await unearthing. The first of these cognizable effects of aging concerns clonal restriction and clonal dominance of T cells in aged individuals. This phenomenon has been observed in both human and mouse, is pronounced in the population of CD8+ T cells, and somewhat less evident in the CD4+ population (18,20,160,161). The specificities of the TCR of the expanded clones differ from one individual to another, even in highly inbred strains of mice reared in the same environment, and for that reason the process is termed “idiosyncratic” (161). The use of that term, rather than stochastic (random), seems appropriate because it suggests that there is a cause even if unknown at present. If we could identify the cause and learn to control or guide it, we should have an important prophylactic tool for protecting the elderly against pathogens. Therefore, research concerned with the causes of idiosyncratic T-cell clonal expansion and dominance in the elderly should be intensified. It seems important to determine, for example, whether or not: (a) avaricious TCRs play a role in facilitating selective emergence of dominant clones; (b) the structural and functional relationships of avaricious TCRs to the immunological synapse change with senescence; (c) there are unusual features of the synapses that drive clonal expansion in the elderly, given that the membranes of aged lymphocytes have undergone significant changes (especially, decreased fluidity, see above); and (d) the emergent clones are more responsive to cytokines such as IL-15 as has been suggested (20), or have escaped the inhibitory effects of IL-2 because IL-2 production is so markedly depressed in the elderly. These and several other rather obvious possibilities represent cognizable effects of aging. A second effect of aging on T cells and their functions that is “capable of being known” (i.e., cognizable) concerns production of cytokines and chemokines and the ability of their receptors to initiate appropriate signaling within cells of aged subjects. Apropos of the reported maintenance of dominant CD8+ T-cell clones in the aged human or mouse (see above), relatively little is

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known about the prevailing levels and potential for production of several very important cytokines (e.g., IL-12, IL-15, IL-18) and chemokines in aged individuals. With regard to chemokines, there is evidence (162) that the ability of peripheral blood NK cells of human nonagenarians to produce RANTES (CCL5), MIP-1α (CCL3), and IL-8 (CXCL8) is substantially depressed (at the protein but possibly not at mRNA level). We have found recently that splenic NK cells of aged mice are severely impaired in the ability to produce RANTES, MIP-1α, MIP-1β (CCL4), lymphotactin (XCL1), and IP-10 (CXCL 10) (J. W. Albright et al., unpublished). If that were true of other cells, especially T cells, which are major producers of chemokines, the consequences vis-à-vis the immune defenses in the elderly could be substantial. Although the evidence that the cytokine IL-2 is generated in much lesser quantity in aged individuals, the effect on the aged immune response is not well understood. Can IL-15 substitute for IL-2 in most, or all, important functions? Is there adequate production of IL-15? What about the effect of senescence on the receptor for IL-2 and IL-15? It is important to note that the common β and γ chains of the receptor for IL-2 and IL-15 are located and interact in the relatively fluid phase of the T-cell membrane whereas the α chain of the IL-2R is most abundant in lipid rafts (163). In order to form the high-affinity IL-2R, the α chain must enter the more fluid phase of the membrane where it can associate with the β and γ chains. Reduced fluidity of the T-cell membrane resulting from changes such as membrane lipid peroxidation associated with aging, will, at the very least, retard the organization of the high-affinity receptor and may reduce the surface density of them. An estimate of the number of IL-2R by flow cytometry using a labeled antibody against the α chain (such as anti-Taq) will not disclose the number of functional receptors, much less the affinity of those receptors. It would appear that a systematic analysis of the numbers and affinity of mature IL-2R on sets of naive and memory CD4+ and CD8+ T cells and their Th1/Tc1 and Th2/Tc2 descendants in young and aged individuals should be most informative. A similar suggestion may apply to the cytokine, IL-10, because the apparent level of that cytokine increases dramatically, as much as threefold in aged humans (105) and 10-fold in aged mice (106). In the case of IL-10, the production by aged humans was assessed in response to the T-cell superantigen Staphylococcus enterotoxin B (SEB). The assessment of T-cell production of IL-10 by aged mouse T cells was in response to immobilized antibody vs CD3ε. When IL-2 or IL-4 were combined with anti-CD3ε to stimulate T cells, it was observed that IL-10 production by aged T cells was less than by youngadult mouse T cells (164). Also, it has been reported that memory CD4+ T cells from aged mice generate less IL-10 in response to antigen than do cells from young-adult mice (25), in part due to the increased frequency of a type

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of CD4+ cell that expresses the substance P-glycoprotein but generates little IL-10. In the case of all cytokines (except possibly IL-2) caution should be exercised before reaching conclusions. The reason is the extreme variability in published findings (see ref. 25 for thoughtful review). Let us assume for the sake of this discussion that IL-10 production by the bulk of the T cells in the aged subject is significantly greater than in the young adult. IL-10 strongly inhibits IL-12 production by MPs and DCs as well as the up-regulation of expression of CD80 and CD86 (165); moreover, it inhibits macrophage proliferation and the ability of macrophages to kill ingested bacteria (166–169). Given those broad effects of IL-10 on DCs and MPs which may include interference with peptide-MHC presentation to T cells, it seems possible that elevated potential for production of IL-10 in aged subjects is a crucial factor in the defective ability of the aged to generate responses of the Th1 (and Tc1?) type. Such responses, as previously discussed, are of primary importance in immune defenses against various pathogens. It has been reported (170) that DCs generated in vitro from precursors in aged human peripheral blood are equivalent to young-adult DCs in the ability to produce IL-12 and activate T cells. It could be argued that this “restored” ability reflects the removal of the DC from ambient IL-10. To continue this discussion of cognizable effects of aging on cytokine production, it is worthwhile to ask if there are clues to why IL-2 production is affected to profoundly. The answer is “yes”; an important clue was derived from an immunohistological study of CD4+ T-cell interaction with APCs in lymph nodes of mice. Previous studies in vitro had provided strong evidence that lymphokines produced by T cells are concentrated at the point of contact with the APC in a polarized fashion prior to being secreted (171,172). To demonstrate that the same general process occurs in situ, transgenic mice bearing a specific TCR gene were immunized and at intervals lymph nodes were collected and analyzed for the cellular distribution of TCR and IL-2 (173). Both the TCR and IL-2 co-localized at the juncture between T cells and APCs. Thus, the results of this study indicated that TCRs and IL-2 became distributed in a polarized fashion in association with the immunological synapse. The study also revealed that TCR redistribution occurred independently of costimulation via CD28. That may not be the case of IL-2 polarization and secretion because signaling via CD28 regulates IL-2 production (174), perhaps by influencing the accumulation of IL-2 at the T-cell–APC interface by a costimulationdependent, myosin motor-driven process (175). The preceding and related studies suggest effects of aging that might explain the deficiency of IL-2 in the immunological environment of aged individuals. First, oxidative changes in the composition and texture of the membranes of T cells and APCs may severely delay or derange synapse formation

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resulting in aberrant interactions of TCRs and peptide-MHC molecules, abnormal switching of TCR-peptide-MHC interactants from peripheral ring to the central core of the synapse, and defective T-cell signal transduction. Second, costimulation of T cells via CD28 in aged mice may be defective as sugested by the defective activation of the JNK signaling pathway, which is CD28dependent (176). For whatever reasons, there is an accumulation with age in CD28-null cells among both the CD4 and CD8 T-cell sets. This accumulation of CD28-null cells has been related to the phenomenon of telomere shortening reflective of extensive mitotic activity (178). Moreover, accumulation of those cells has been associated with the relatively high levels of TNFα in aged individuals (179,180). TNFα in elevated concentration represses gene expression, in particular disruption of CD28 gene transcription (181,182). As discussed above, failure of proper costimulation will depress IL-2 production. Thus, the emergence of CD28-null cells with age, which may reach 30%–40% of the T-cell population, can easily account for some of the IL-2 deficiency. Given the combined effect of IL-10 on depressing surface B7 molecules on APCs, and the effect of TNFα on depressing CD28 expression by T cells, the required costimulation of Th1 cells and, thus, IL-2 production are sure to be severely impaired in aged individuals. It should be noted that depressed IL-2 production and T-cell proliferation may not be evident when highly purified preparations of T cells are assessed following stimulation with powerful unnatural agents such as antibody vs CD3ε and CD28 or phorbol esters (see refs. 183 and 184). Results obtained from such studies are apt to reflect two nonphysiological conditions; viz., (a) isolation of the T cells from the aged tissue environment, and (b) activation of T cells by abnormally powerful stimuli. To illustrate, no evidence of an effect of aging on T-cell responses was apparent when aged, T cells were purified and separated into subsets prior to evaluation of their ability to produce IL-2 (183). However, when the T cells were not separated prior to stimulation, diminished IL-2 production by aged, compared to young, T cells was evident. In all experiments, the T cells were stimulated with a combination of antibodies vs CD3ε and CD28. Whether or not the subsets of T cells influenced each other in such a way that the average number of CD28 molecules per CD4+ T cell, or the signaling efficiency via CD28 receptors, was differentially affected in cultures of aged T cells was not determined but should be informative to study. A third, cognizable effect of aging on T cells occurs in the early steps of signal transduction via the TCR-CD3 complex. That was briefly discussed above in connection with signals emanating from the immunological synapse. The following, short discussion expands on the previous one in order to emphasize the complexity of those early events and the likely susceptibility of those events to age-induced changes in the membranes of the T cells and

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Fig. 4-8. Two-color, confocal microscopic analysis of NF-ATc nuclear translocation in T cells that are engaged in immunological synapses with APCs. The first pair in each row shows a T-cell–APC conjugate (identified by polarized location of a synaptic component) in which NF-ATc remained in the cytoplasm, and the second pair in each row shows a conjugate in which NF-ATc relocated in the T-cell nucleus. (From ref. 187.)

APCs. Reports of the pioneering studies of Miller and associates should be consulted (25,164,185–187). A particularly instructive research report (187) provided the results of a study of T cells obtained from aged, TCR-transgenic mice. The abilities of those cells to unite with APCs to form immunological synapses, assemble kinase proteins and substrates within the synapse, and transmit signals downstream to translocate NFAT transcription factor were evaluated (see Fig. 4-8). A concerted effort was made to ascertain the involvement of several key signaling components in the formation of the central core of the synapse (often referred to as the “center of the supramolecular activation complex,” or c-SMAC). Signaling factors that localized in c-SMAC were LAT (linker for

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activation of T cells), CD3ε (containing ITAMs), and PLCγ (an isomer of phospholipase C). Other factors that were drawn into the synapse, or SMAC, but were not localized in the c-SMAC included Lck (a kinase that phosphorylates ITAM motifs), c-cbl (homologue of a viral oncogene that functions as an adapter protein), ZAP-70 (a kinase that binds phosphotyrosine residues in ITAM motifs), and Fyn (an src-family kinase). A particularly significant aspect of this investigation was the effort to correlate the inclusion of signaling factors in the assembly of SMACs with the downstream consequence of signal transmission, viz., translocation of transcription factor NFAT to the T-cell nucleus. The results of that effort were compelling demonstrations that: (a) there is a high degree of correlation between SMAC localization of LAT, c-Cbl, and PLCγ and nuclear translocation of NFAT; and (b) those events occurred in 75% of the T-cell–APC conjugates prepared from young, TCR transgenic mice but less than 30% of the conjugates involving T cells from aged mice. The results of that investigation provide an almost palpable clue that during the course of aging the population of T cells (or subpopulations therein) experience detrimental changes in their surface membranes that interfere with receptor-initiated signaling. For the moment, however, there are a number of gaps and missing pieces of what is likely to become a significant advancement in understanding aging of the immune system. By blending together several, major findings that have been discussed above, a fascinating story can be told. First, there is the evidence that TCR-transduced signaling induces rapidly a flux in cytosolic [Ca]i (146,147). Second, there is strong evidence that [Ca]i mobilization, as a step in signaling, is deranged in T cells of aged subjects (185); however, that defect has been demonstrated, thus far, in the memory cell population rather than in naive T cells. Third, members of the NFAT family are exquisitely sensitive to changes in [Ca]i. This is the consequence of their dependence on dephosphorylation of key ser/thr residues by the phosphatase calcineurin. The latter is in relatively low concentration in lymphocytes. Dephosphorylated NFAT molecules can be rapidly translocated to the T-cell nucleus where there are several cytokine genes that are regulated by NFATbinding elements (126,127). However, retention of NFATs in the nucleus long enough to act on gene promoter, enhancer, and repressor elements involves other factors not yet well understood (188). Without stabilization by those factors, NFATs are immediately phosphorylated by the nuclear enzyme, glycogen synthase kinase 3 (GSK3), and expelled from the nucleus back into cytosol (189). Fourth, as discussed above, the NFATs are involved crucially in influencing the Th1 or Th2 differentiation of naive CD4+ cells (also in CD8+ naive T-cell differentiation into Tc1 or Tc2). There is compelling evidence that NFATc1 plays an influential role in Th2 differentiation whereas NFATc2 (and

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NFATc3 in an enhancing capacity) facilitates Th1 differentiation (126,127). Evidence of an apparently contradictory nature was interpreted to mean that sustained residence in the nucleus, even of NFATc1, facilitated Th1 differentiation as a consequence of robust [Ca]i flux (128). Moreover, it appears that Th1 differentiation proceeds when naive cells are provided with a strong stimulus via the TCR whereas a weak stimulus results primarily in Th2 differentiation. The strength of the TCR stimulation is reflected by the magnitude of intracellular [Ca]i flux (127). Fifth, it has now been demonstrated that the signal emanating from the TCR occurs before completion of formation of the mature immunological synapse (149). At an early stage in synapse formation, the accessory molecules, LFA-1 (T cell) and ICAM-1 (APC) are clustered at the center of the synapse while the TCR and peptide-MHC interactants are located circumferentially. It is at this stage when signaling components Lck (the first to be phosphorylated) and ZAP-70 become phosphorylated and, thus, signaling is initiated. Shortly thereafter, there occurs an exchange of locations within the synapse; TCR and peptide-MHC interactants move to the center to form, with other molecules such as CD28, the central core (c-SMAC), while LFA-1 and ICAM-1 and other accessory molecules form a peripheral, concentric ring (p-SMAC). The five, preceding sets of observations can be integrated into a scheme that infers that defective immunological synapse formation in aged T cells results in weak signaling, damped [Ca]i flux, reduced calcineurin dephosphorylation of NFAT molecules, depressed NFAT nuclear translocation, preferential activation of genes such as encode IL-4, IL-5, IL-13, and preferential differentiation of naive CD4+ cells along the Th2 pathway. This scheme may well explain why immune responses that produce vigorous expansion of Th1 cells, a necessary event for effective defense against many types of microbial pathogens, are impaired in the elderly. The fourth and final cognizable (cognoscible) effect of aging that we discuss here is one that affects T cells indirectly. Four recent publications describe explorations of the physical interactions between DCs and NK cells and the consequences of such interactions (139–141,190). NK cells and DCs exert influences on each other once they have conjoined. Activated NK cells at a relatively low ratio (1 NK cells:5 DCs) can induce maturation of immature DCs and trigger the production of IL-12. The presence of IFNγ and TNFα further promotes DC maturation. Activation of NK cells may be achieved by stimulating them with IL-2 or bacterial LPS. Resting NK cells can be activated by LPS even in the presence of immature DCs and drive maturation of the latter. At high ratios (5 NK cells:1 DC) activated (but not resting) NK cells exert an inhibitory effect on DCs, principally as a result of killing the DCs. This killing occurs even though the DCs express MHC Class I molecules and, therefore, requires further study. Thus, NK cells have the potential to regulate

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the maturation and mature functions of DCs. On the other hand, both immature and mature DCs can promote the proliferation and maturation of NK cells including stimulation of IFNγ by the latter. This contrasts somewhat to DCs acting as APCs, a function performed only by mature DCs. A scheme suggested by these recent findings is that initial encounters between NK cells and microbial invaders or between MPs and neutrophils and microbial invaders can activate the NK cells. The NK cells then conjoin with immature DCs and drive their maturation. Cytokine production by both types of cells (IFNγ by NK cells, IL-12 and TNFα by DCs) accelerates the process and also leads to heightened production of IFNγ and various chemokines by the NK cells. Maturation and proliferative expansion of DCs affects T cells in two ways: first, by selectively biasing new T-cell production along the Th1 pathway and, second, by expanding the population of APC-bearing peptides of the microbial invaders, which can seek out and activate T cells that display complementary TCRs. The uniting (contact) of NK cells and DCs may resemble the formation of the synapse between APCs and naive T cells and therefore will require further analysis. It will be interesting to learn whether or not aspects of NK-cell–DC interaction can influence the maturation of immature DCs along the DC1 or DC2 pathways, and, especially to what extent the current concepts concerning the DC1 or DC2 polarization of DCs by the molecular patterns of microbial molecules (antigens) will require revision in the light of the new information about NK-cell–immature-DC interaction. There is strong evidence that the molecular patterns presented by microbes to the PRR on DCs detemine whether or not maturation to DC1 or DC2 occurs (124,130,191,192). It is not yet known, however, whether the polarization of DCs resulting from exposure to a microbial patterns results from a determinative influence of the pattern on the DC1 or DC2 pathway of maturation (the Instruction Model) or from a selective effect of the pattern on preexisting immature DC1 or DC2 (the Selection Model). In either case, the family of TLRs (Chapter 3) appear to be crucial in the DC polarization process. The possible effects of aging on NK-cells–immature-DC interactions are not difficult to discern. Given the evidence that NK cells in aged subjects are functionally deficient, and using the effects of aging on T cells as a model, it can be suggested that the physical interactions and formation of contacts between aged NK cells and immature DCs will be a sluggish, inefficient process. Transduction of signals leading to activation of cytokine and chemokine production by either type of cell is likely to be impaired. It will be interesting to learn, for example, whether or not the impaired ability of aged subjects to produce IL-2, which activates NK cells, depresses the NK-cell–mediated maturation of immature DCs. Equally interesting will be the study of whether or not defective NK-cell–immature-DC interactions in the aged subject (assuming

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they will be found defective) are the consequence of aged-related imperfections in the NK cells, the DCs, or both. DIFFERENTIATION, FUNCTIONS, AND AGING OF B CELLS It is often stated that the effect of aging on B cells is not as pronounced as it is on T cells. That is a rather superficial evaluation that probably reflects the paucity of knowledge about B-cell aging that prevailed until recently. At present, the knowledge of aging of B cells, per se, coupled with an understanding of the effects of aging on germinal center (GC) formation and function reveal considerable changes in the B-cell domain. In order to understand more readily the import of age-associated modification of B cells and GCs, we review briefly their major features and functions (for more details, see refs. 2–5). Diversification of B Lymphocytes (B Cells) There are two major sets of B cells known as B1 (CD5+), which at times are considered elements of innate immunity, and B2, the traditional cellular elements of adaptive humoral immunity. B1 cells are characterized by their limited range of antibody diversity, a reflection, in part, of the near-absence of point mutations in the variable segments of the Ig molecules they produce and the paucity of N-region modifications at the V-D-J junctions of those molelcules. B1 cells respond well to polysaccharide antigens of microbial origin (type 2, T-independent responses) but weakly to most protein antigens. Thus, B1 cells do not require T-cell help and few if any memory cells arise from them during a primary response. The antibody molecules produced by B1 progeny are predominantly of the IgM isotype or, to a lesser extent, the IgG3 isotype (but see later). One of the most noteworthy features of B1 cells is their location in vivo. The great majority of them reside in the peritoneal and pleural cavities. This distribution suggests that they are responsible for protecting those cavities from infectious organisms. Among the T-independent, type 2 (TI-2) antigens that elicit B1 responses are pneumococcal polysaccharide, polymerized flagellin of Salmonella, the phosphorylcholine moiety formed on various Gram-positive bacteria, and other molecules found on commensal bacteria. The common feature of those substances is their polymer-like nature exemplified by the repeated recurrence of particular basic chemical units. The nature of those antigens, their microbial origins, and the fact that the normal serum Igs of IgM isotype contain “naturally occurring” antibodies against them have been interpreted as indicating that the B1-cell population is in an activated state most of the time and is producing most of the IgM found in the body. Further discussion of the B1 cells continues below. Cells of the B2 set belong to the classical humoral response system. They arise primarily from bone marrow progenitors and in the process of differenti-

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ating experience extensive rearrangements of their V, J, and D gene segments. As a consequence of those rearrangements and other modifications, the enormous diversity of antibody specificities is generated. Individual specific clones can then be triggered by antigenic determinants (haptens). Triggering/activation occurs via specific membrane-bound antibody molecules known as B-cell receptors (BCRs). In order for a B2 cell to become fully activated and to begin to proliferate and complete its maturation (including genetic events that determine the isotype of antibody it will produce, i.e., “isotype switching”), it must interact with the helper T cell. The BCR of the B-cell and the TCR of the T cell must recognize the same antigen, although not necessarily the same determinant of that antigen. The determinant peptide associated with B-cell Class II MHC molecules is presented to the TCR. At the same time CD40 ligands (CD40L) on the surface of the T cell engage cognate CD40 receptors on the paired B cell thus providing the required costimulation. Following this union of B and T cells, which involves coalescence of lipid rafts to form signaling “platforms” at the B-cell–T-cell junction (193,194), the T-cell cytoplasm is reorganized (“polarized”) in order to facilitate the transfer of cytokine molecules into the B cell (171). Thus, depending on whether the T cell is type Th1 or Th2, the ensuing isotype switching will occur that determines which isotype of antibody the B-cell progeny (antibody-forming cells or AFCs) will produce. For example, IL-4 provided by Th2 cells may facilitate switching to the epsilon heavy-chain gene resulting in mature B cells that produce IgE. On the other hand, IFNγ produced by Th1 cells may favor switching to the gamma-2a heavychain gene resulting in the production of IgG2a antibodies (rodents). Organization of Secondary Lymphoid Organs This is the point at which to ask how the relatively rare members of a given clone of T cells, of B cells, and of APCs bearing cognate peptide can possibly find each other in what is for them an enormous mass of tissue comprising the body of an adult individual. The problem has been solved in a rather complicated but highly ingenious way. The solution involves recirculation of lymphocytes through secondary lymphoid organs, homing and selective adherence of lymphocytes within defined areas of those organs, and the formation of structures known as germinal centers (GCs) (195–198). The organization of peripheral or secondary lymphoid tissue in the spleen and lymph nodes is illustrated in Figure 4-9. In the spleen (Fig. 4-9C), lymphoid cells are comfined to the “white pulp” areas within which lymphocytes are distributed along central arterioles (branches off trabecular arteries) forming the “periarteriolar lymphoid sheath” (PALS). The PALS consists mostly of T cells. Around it, the B cells are arranged, some of which are loosely organized to form distinguishable follicles (in stained sections). The remaining B

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Fig. 4-9. Stained sections of lymph node (A) and spleen (C). Diagram of structural details of germinal center (B) such as those marked on the section of the lymph node. (From Roitt IM. Essential Immunology, 9th ed. London: Blackwell, 1997, with permission.)

cells comprise the marginal zone, clearly distinguishable from the “red pulp,” which is primarily engaged in erythropoiesis. The structure of lymph nodes is shown in Figure 4-9A. They consist of four major regions arranged in roughly concentric fashion: the medullary sinus, medullary cords composed of macrophages and plasma cells (and other cells in lesser number), the paracortical region composed primarily of T cells and DCs, and, closest to the periphery, the lymphoid follicular region in which most B cells reside. The marginal zone of the spleen, composed of B cells and DCs, is not evident in lymph nodes. Studies of gene knockout mice have revealed the remarkable fact that the inability of mice to produce the cytokine, lymphotoxin (LT), during a critical stage in development results in failure of lymph node development (199–202). Although the spleen is present in such mice, its follicular structure is abnormal (203) and the marginal zone is poorly defined. There are two forms of LT.

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LT-α3, a homotrimer that binds to, and initiates signaling via TNFR-I (TNF receptor I; note that LTs are members of the TNF family). The absence of LTα3 leads to failure of cervical and mesenteric lymph node development. The other form of LT, LT-α2β1, a heterotrimer, binds only to the LT-βR. In the absence of LT-α2β1, the cervical and mesenteric nodes develop but other nodes do not. Also, Peyer’s patches in the small intestine fail to appear. Another member of the TNF family, TNFα, also plays a role in lymph node and spleen architecture. Mice without TNFR-I or that lack the ability to generate TNFα lack follicular dendritic cells (FDCs) in lymph nodes and Peyer’s patches. Similarly, mice lacking LT-α2β1 or its receptor (LT-βR) are devoid of splenic FDCs. A common view, at present, is that lymph node development begins with organization of a stromal cell/DC network, which then acquires T cells and B cells with further development. The lymph nodes and spleen are designed to facilitate the encounters of specific T cells and peptide-MHC-bearing APCs as well as encounters of T and B cells bearing TCRs and BCRs specific for the same antigen. In both spleen and lymph node, there are two distinguishable types of DC: follicular dendritic cells (FDCs) in the B-cell areas, i.e., the follicles, and interdigitating dendritic cells (IDCs) in the T-cell zones. The former are unusual in that they are not derived from bone marrow precursors, as are other cells of the lymphomyeloid series. They are not phagocytic and express, at most, only meager amounts of surface class II MHC molecules. They have a well-developed ability to acquire, bind, and retain antigen-antibody C complexes on the external surface of their convoluted plasma membranes. That property has for decades suggested that the FDCs serve as depots of antigen that continue to maintain the responsive lymphocytes in an alerted state. In contrast to the FDCs, the IDCs are derived from bone marrow progenitors and display class II MHC molecules. Their extensive processes allow contact and interaction with many T cells. The structural organization of nodes and spleen favor the entrance of blood and lymph (in the case of nodes) in such a way that circulating lymphocytes, antigens, and DCs first migrate or percolate through zones rich in T cells. Antigens that enter may be trapped by IDCs and readied for presentation to appropriate T cells or, possibly, to other antigen-processing and -presenting DCs (APCs). However, many of the DCs that enter the T-cell zones from outside are likely to be bearing processed peptides. In any case, this architectural arrangement, like a vessel of oppositely charged particles, provides the opportunity for T cell to meet peptide-bearing DCs. Consequently, T cells become activated, begin to proliferate, express CD40 ligand (and other accessory molecules), and produce cytokines and chemokines. They are then likely to migrate near to the junction of the T-cell zone with the B-cell follicular zone as if in search of a cognate B cell. The other partner of the T-cell–APC union, viz.,

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the now activated DCs, produce their particular variety of cytokines and chemokines (see below). Those T cells that fail to encounter a recognizable peptide exit into lymph or blood and either die or begin another round of recirculation and searching. In the meantime, events involving B cells are transpiring. B cells may encounter cognate antigen in the blood or lymph or on the surface of FDCs, at the junction of the follicles and T-cell zones, or by entering the T-cell zone from blood or lymph en route to the junction of T-cell zone and follicles. In those same locations they may encounter activated T cells of agreeable specificity. Upon interacting with a cognate T cell, the B cell becomes activated, prepares to begin proliferating, and settles down in a B-cell-rich area, especially in a lymphoid follicle where it organizes a GC. Activated B cells of a different specificity may also occupy that GC but evidence shows that only a small number of B cells of different specificities occupy a given GC. The preceding overview of GC formation is necessarily superficial—there remains much to be learned. However, at present, research progress is rapid, especially in the area of selective localization of B and T cells and DCs within specific regions of secondary lymphoid organs. To a large extent, selective localization or “homing” of cells to secondary lymphoid tissues is under the control of chemokines (198,204–207). Both naive T cells and maturing (activated) DCs are guided to the T-cell zones of secondary lymphoid tissues by virtue of the chemokine receptor, CCR7. They move along a gradient of CCR7 ligands, e.g., CCL19 (EB virus-induced molecule 1 ligand chemokine), and CCL21 (secondary lymphoid tissue chemokine) both of which are released by stromal cells in the T-cell zones. DCs are considerably more sensitive to CCL 21 than are T cells. However, newly activated, mature DCs in the T-cell zone may enhance the attraction of T cells by the chemokines they release, including CCL17 (TARC) and CCL22 (MDC/STCP-1), both of which are ligands for CCR4 expressed by activated T cells. As noted above, the origin of the FDCs that populate the GC is uncertain. However, new insight has been provided with the realization that certain types of DC have a penchant for the B-cell follicular microenvironment. Thus, skinderived, dermal-type DCs were found capable of homing to B-cell zones as well as T-cell zones of lymph nodes (208). Those DCs expressed CXCR 5 and responded to CXCL 13 (B lymphocyte chemoattractant, or BLC). That behavior contrasted with the homing preference of bone-marrow-derived DC, which was limited to T-cell zones (208,209). The bone-marrow-derived DC lacked CXCR 5 but, as described above, expressed CCR 7 and responded to CCL 19 and CCL 21. In a recent study (210), bone-marrow-derived DCs were transduced with the gene encoding CXCR5 whereupon they recognized CXCL 13 and homed in vivo to the B-cell follicular zone of lymph nodes.

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CXCL 13 is strongly expressed in B-cell follicles of spleen, lymph nodes, and Peyer’s patches. The interaction of CXCL 13 with its receptors (CXCR 5) on B cells is crucial for the follicular localization of B cells (204,211). In addition to promoting the follicular localization of B cells (and dermal-type DC, as discussed in the preceding paragraph), the interaction with CXCL 13 is essential for immigration of another type of DC into follicles, viz., DCs from the marginal zone in the case of spleen (207). Those unusual DCs, characterized as CR–Fc+ DC (212), express CXCR 5 and migrate toward and into B-cell follicles in response to CXCL 13 generated by follicular stromal cells (207). The regulation of stromal cell expression of CXCL 13 is strongly influenced by B-cell membrane-bound LT. Recall that membrane-bound LT (LTα2β) signals through its receptor, LTβR, and that the interaction between LT and LTβR is crucial for secondary lymphoid tissue development. It would seem that there is now sufficient information to diagram one chemokine circuit that directs the organization of GCs. Consider the following: The interaction of LTα2β1 with LTβR on stromal cells during development creates the microenvironment necessary for secondary lymphoid tissue formation. That interaction probably signals the production of several chemokines that further mold the microenvironment. Furthermore, under the influence of chemokines produced by stromal cells in the prospective T-cell zones, the latter accumulate various cells including the precursors of the IDCs, which together with stromal cells generate attractants for T cells and other DCs. This group of attractants includes CCL19 and CCL 21, which guide both DCs and T cells to the area, and CCL 22, which attracts activated T cells (via CCR4). Antigen percolating through the fabric of the T-cell zone is trapped and processed by resident DCs and also brought in by peptide-charged DCs. At the same time, a different type of DC (not bone-marrow-derived) is forming a different microenvironment in the follicular zone, one that will be suitable for B cells. Upon encounter with antigen, T cells from the T-cell zone migrate toward the follicular zone. CR–Fc+ DCs migrate from the marginal zone of the spleen and the subcapsular sinus of the node into the follicular zone drawn by CXCL 13 expressed by local stromal cells. Those T-cell movements and CR–Fc+ DC movements are controlled by chemokines not yet clearly identified but some of which are produced by B cells. The result of all that activity is the settling down in the follicles of activated B cells, at most a few B cells of different specificity per follicle. The activated B cells proceed to divide and expand the GCs. To further complicate the matter of chemokine regulation of T-cell and perhaps DC homing, evidence has been reported that Th1 cells and naive T cells, both of which display CCR 7, localize to the periarteriolar lymphoid sheath in the spleen; in contrast, Th2 cells (lack CCR 7) localize at the periphery of the T-cell zones near to the lymphoid follicles (213). No doubt

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this will influence the quality and course of immune responses in several ways including antibody isotype (“class”) switching. What Goes on in the GC? The answer is: (a) clonal expansion of B cells, maturation of progeny into either memory B cells or AFCs and plasma cells (PCs); (b) isotype switching; (c) affinity maturation; and (d) export of AFCs and PCs to other sites especially to the bone marrow. Thus, the formation and characteristics of GCs are reflected by the properties and activities of the B cells that reside in them. In order for B cells to become fully activated, they must, first, bind molecules of antigen that can be recognized by their receptors (i.e., BCRs) and, second, be additionally stimulated (costimulated) through some other receptor. The most common receptor for costimulation is the molecule CD 40 located on the surface membrane of the B cell. The corresponding ligand, CD 40L (CD 154), is located on activated T cells. At the time a B cell unites with an activated T cell, the CD 40 R and CD 40L interact to costimulate the B cell. At the same time, the B cell receives additional stimulation from cytokines, especially IL-4 from Th2 cells or IFNγ from Th1 cells. Those cytokines induce isotype switching in the B cells, a reorganizational process by which heavychain genes preceding the gene that is selected for expression are deleted. This usually involves deletion of the µ and δ chain genes and one or more of the γ chain genes preceding a selected γ, ε, or α chain gene located farther downstream. The fully activated B cells enter into intense proliferation, dividing at intervals of 6–8 h. During this stage they are termed “centroblasts.” The area that they occupy is called the “dark zone” of the GC because it is so tightly packed with the centroblasts and their progeny (Fig. 4-9B). As the intense proliferation subsides, the B cells, now called “centrocytes,” spread out into the adjacent area known as the “light zone” of the GC, which is occupied by the FDCs and those maturing centrocytes. Centrocytes continue to proliferate in the light zone but at a less rapid rate. As the maturing centrocytes spread out, the FDCs also spread and with time the differences in appearance of the dark and light zones become obscure. Critical events that occur during the proliferation and expansion of B cells in the GC are hypermutation of the Ig V segment genes and selection of the mutant cells with increased affinity for antigen. Those two events underlie the process known as “antibody affinity maturation.” Point mutations in those genes occur with phenomenal frequency of roughly one base pair change per thousand base pairs per cell division. The majority of those mutations is detrimental, i.e., produces an antigen-binding site of lesser binding affinity. Cells that experience that negative change are marked for apoptotic death. They are quickly removed by MPs in the GC, cells that are known

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classically as “tingible body macrophages.” Only the minority of B cells that display heightened affinity for antigen as a result of V gene mutation will be selected and allowed further expansion. Those cells will interact with available cognate T cells and be stimulated to proliferate and experience V gene hypermutation and repeated selection of their progeny. This becomes an iterative process in which most mutant cells die but, at each new generation, a few are positively selected. Over time and repeated generations, the population of B cells will progress toward higher average antigen-binding affinity. This is the process termed “affinity maturation.” Thus, the combined effects of isotype switching and affinity maturation produce a mature B-cell population. Some of those cells are destined to become memory B cells whereas the majority undergo terminal differentiation into PC that produce copious amounts of specific antibody (as much as 10,000 molecules per second; 214). The mature GCs export both PCs and memory B cells during their existence. The majority of the PCs journey by way of the bloodstream to the bone marrow where they take up residence. Many of them die after a short lifetime of producing specific antibody. Many more persist for long periods of time in the marrow, some surviving for the lifetime of the individual whom they inhabit (215,216). The PCs in the marrow produce a gradually increasing proportion of the Ig in the blood as individuals age such that in mice two years of age at least 75% of the serum Ig is derived from marrow PCs (217). One reason for the extended lifetime of adult marrow PC is their extremely low rate of proliferation (215,216). The production of antibodies by the PCs in the marrow (some remain in the spleen as well) is of great importance to aging individuals in whom new antibody production is declining. Those antibodies, many of which are specific for microbial antigens encountered much earlier in life, provide a significant degree of protection against reinfection. The longterm persistence of marrow PCs and their vigorous production of Ig/Ab molecules accounts for the fact that serum Ig concentrations decline very little with advancing age (217,218). B memory cells persist for extended periods and are distributed in lymphoid tissue sites, in particular the marginal zone of the spleen. Because they were generated during GC events, they have already experienced isotype switching and selection for high-affinity antibody. The factors that determine the fate of a surviving B cell in the GC, whether it will become a PC or memory B cell, remain obscure. It has been reported that the presence of IL-10 nudges the centrocyte in the direction of PCs (219) and that a molecule SM-8D6 produced by FDCs stimulates proliferation of those terminally differentiating PCs. That contention will have to be confirmed using a less artificial experimental system. The molecule, CD 27, a member of the TNF-receptor family found on a high proportion of human peripheral blood

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B cells, interacts with its ligand CD70 and may promote the maturation of memory B cells into PCs (220,221). However, a recent study of mouse B cells has shown that ligation of CD27 inhibits their terminal differentiation into PCs (222). In anticipation of the following section in which failure of GC formaation in the aged is discussed, it should be mentioned here that there are several known causes of GC failure in addition to absence of LT and its receptors. Two elements of the GC that have been considered mandatory for GC formation are T cells with α/β TCRs and FDCs. It has now been shown that α/β TCRs are not required for initial GC organization (223). However, B cells that develop in the T-cell-less GC fail to undergo V gene hypermutation. Presumably, the B cells are attracted to the lymphoid follicles by chemoattractants produced by the FDCs. A study by a different group of researchers demonstrated that GCs can form in lymph nodes lacking the FDC network (224). That study employed LTβ knockout (ltβ–/–) mice which, as the study showed, do develop mesenteric lymph nodes. In response to antigen administration, GC formation in the node was, at first, normal even though FDCs were not present. Later, the numbers of B cells in GCs declined rapidly. It was also discovered that some memory B cells developed in the ltβ–/– mice although the antibody they produced was of moderate affinity. Earlier studies (e.g., ref. 225) had shown that GCs can appear in the absence of antigen trapping by FDCs but GC formation in the absence of FDCs is a novel finding. The findings reviewed briefly in this paragraph indicate that either FDCs or T cells are necessary for GC formation to be initiated. Perhaps either can release chemoattractants to draw B cells into the follicles. However, both FDCs and T cells are required for B cells to sustain isotype switching and mutation and selection to generate mature B cells capable of producing high-affinity antibodies. Memory B cells of low antibody affinity can be generated in the absence of FDCs. The Effects of Aging on B Cells and T-Dependent GC Formation One of the first questions to ask concerning the effects of aging on humoral immunity is how well the B cells of aging individuals respond to test antigens compared to young adults. The answer is that it depends on the test antigen. For example, CD5+ (B1) cells, which can be directly activated by TI antigens, increase in aged mice but in oligoclonal fashion (226,227). Studies with phosphorylcholine, a critical antigenic component of several bacterial cell walls, revealed a twofold increase in the frequency of responsive B cells (B1) in aged compared to young-adult mice (228). The studies with phosphorylcholine, a TI-2 antigen, also revealed a change in VH gene usage by antibodyforming cells in aged compared to young mice and a consequent decline in the protective ability of the antibodies in aged mice (229–231). These results sug-

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gest that aging exerts a detrimental change in B1 cells that can negatively influence the ability of the aged to protect against bacterial infection. However, it is possible that the effect could be on T cells, which might influence B1-cell responses to TI-2 antigens. Evaluation of the ability of B cells of aged mice to respond to T-dependent antigens can be difficult to perform owing to the necessity to evaluate the B cells apart from T-cell and APC influences. Nevertheless, several studies have been performed that suggest that B-cell frequency decreases or remains unchanged with advancing age. Examples of such results are provided in ref. 228. B cells capable of primary response to the haptens 2,4-dinitrophenyl (DNP) and acetylnitrophenyl (NP) were two- to threefold less frequent in the spleens of aged compared to young mice. That difference was even more exaggerated in the case of B cells capable of secondary responses in young and aged mice (three- to sixfold). A dramatic effect of aging on B cells is the decline in the rate of new B-cell production in bone marrow. This has been well studied in mice (228,232–234), A truncated version of the scheme that depicts B-lymphopoiesis in the bone marrow (228,235) is provided in Figure 4-10. Prominent differences exist between young and aged mice with regard to progression of cells along that differentiative pathway. First, there is an impediment somewhere in the pro-B-cell phase of differentiation such that in aged mice there are significantly fewer pre-B cells in the marrow. It has been suggested that the block occurs at the point of pro-B to pre-B transition (234), perhaps involving membrane expression of the µ-heavy chain. This block, wherever it occurs, results in many fewer pre-B cells because in young adults the early pre-B cells engage in, roughly, a half dozen rounds of proliferation thus expanding the pre-B population some 60-fold. Second, as the pre-B cells in the marrow of mice continue their maturation they migrate to the spleen as immature B cells; in aged mice there are roughly fivefold fewer of them in the spleen. And more than 50% of those immature B cells that appear in the spleens of aged mice display very low levels of surface-bound IgM compared to about 20% of the immature cells in the spleens of young-adult mice (215,228). That suggests another block or “bottleneck” in the further maturation of the immature (IgMlo) B cells. Given those impediments to differentiation/ maturation of B cells in the aged mouse it is remarkable that there is, at most, a slight reduction in the actual numbers of B cells in the peripheral lymphoid tissues of aged mice. The principal reason why there is not a greater reduction is the much-prolonged life-span of the B cells in aged animals (215,228). Additional reason for the extended life-span of B cells in aged mice was provided by testing the hypothesis that endogenous “environmental” antigen provides continuous stimulation to those cells (236). That study involved comparing the properties of B cells from aged mice, bearing a transgene that con-

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Fig. 4-10. Diagramm of B-lymphocyte development and maturation showing distinguishable stages in the bone marrow and peripheral lymphoid organs. (From Goldsby RA, Kindt TJ, Osborne BA. Kuby Immunology, 4th ed. New York: Freeman, 2000, with permission.)

ferred specificity for antigen not found in the internal environment, with normal, wild-type, aged mice. In both the transgenic and wild-type aged mice the B-cell population had similar characteristics, composed of memory-like cells that appeared to have been antigen activated. Cells bearing the transgene were diluted out in the aged, transgenic animals. Many B cells had characteristics of splenic marginal zone cells and many splenic B cells were CD5+. The latter, however, expressed significant levels of IgM and lacked the marker CD43; they were not considered to be B1 cells. The results of this investigation provide compelling evidence that the reason for the maintenance of normal B-cell numbers in aged animals is, in part, due to continuous stimulation by endogenous antigens, presumably in low concentrations. The antigen receptors (BCRs) on those B cells are likely to be of relatively low affinity because, as discussed next, the aged suffer from very poor ability to generate GCs in their peripheral lymphoid organs. Moreover, many of the B cells, which resemble marginal zone cells, probably express a restricted repertoire of BCRs (see ref. 237) and may represent the dominant clones of B cells that emerge in aged mice, as noted previously (226,227).

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Aged humans and mice give “weak” humoral responses to antigen compared to young-adult individuals. The description, “weak,” includes reduced antibody production, deficient ancounts of antibodies of IgG isotypes, and antibodies of relatively low affinity that are not as protective against infectious organisms. Those changes suggest defective GC activity in the lymphoid organs of aged subjects. That is, indeed, the case (see review in ref. 238). Both the number of GCs in spleen and nodes and their size (i.e., numbers of cells included) decline strikingly in aged mice (238–240). One critical change in aged mice is the poor development of the FDC network and, associated with that change, a defective process of antigen transport into lymphoid follicles (241). As a consequence, the formation of the typical (in young-adult mice) meshwork of elongated FDC processes displaying bound complexes of antigen/antibody/complement components termed “iccosomes” is poorly developed in the GC of aged mice (241243). Those dramatic deviations from the GC of young mice appear to be responsible for several functional aberrations in aged animals. First, there is an absence of somatic mutations in the genes encoding Ig VH segments in the B cells of aged mice (244). This has been compared to the failure of V H hypermutation in mice deficient in the costimulator receptor CD28 and, therefore, might be a reflection of age-associated defects in T cells (238,245). The absence of Ig VH hypermutation precludes affinity maturation, a prominent activity in the GC of young animals. Although affinity maturation of antibodies does occur to a degree in aged mice, it results from competition for antigen among B cells that express unrearranged germ-line genes of different affinities. Second, aberrant GC formation in aged mice affects the disposition of memory B cells as well as mature PC. It appears that in aged mice, the number of very long-lived PC in the marrow is curtailed and the recently acquired PCs in the marrow of aged mice are relatively short-lived (<3 months). If those conclusions are correct, the marrow of aged mice becomes increasingly populated with relatively short-lived PCs that produce low-affinity antibody. Small wonder that the elderly are substantially more susceptible to a variety of infections! Furthermore, antigen priming for later anamnestic responses to T-dependent antigens is highly defective in the aged; it is the PCs that arise during vigorous secondary responses that constitute the highly effective population of marrow PCs (216–218). Small wonder that effective vaccination of the elderly is difficult! Only the humoral responses to T-independent antigens seem relatively unimpaired in the elderly. And that is probably the result of continuous, lowlevel stimulation by environmental antigens most of which are self-components that share a determinant or two with the foreign antigen that initiated response while the individual was younger. That cross-reaction of determinants probably explains the gradual increase with age in the variety of autoantibodies most of which, fortunately, cause no overt disease.

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CHAPTER SUMMARY One of the most fascinating rewards of this review of aging B-cell and Tcell immunity is the realization that there are many parallel effects of senescence on the two systems of cells. First, there is the fact that the generation of new, naive lymphocytes both in thymus and bone marrow deteriorates significantly. This is not the result of aging of primary hematopoietic stem cells (246,247). Rather, aging affects the generation of intermediate developmental stages from lineage-committed progenitors. Those progenitors may suffer no intrinsic defects; rather, they may be restricted by an inimical microenvironment that no longer provides the proper nutrients, growth factors, cytokines, chemokines, and other substances that they require for optimum functions. Second, there is a qualitative shift with age in the type of T cell and B cell that is dominant: memory-like CD4 and CD8 T cells, B1-type and plasma cells in the case of B cells. Naive, memory T cells and B cells may persist for long periods, even the lifetime of the individual in whom they exist. Third, with advancing age there is oligoclonality in both B- and T-cell populations (CD8+ T cells, CD4+ to a lesser degree). Oligoclonality is associated with diminishing repertoire versatility. Fourth, questions regarding long-term maintenance of memory and specific responsiveness and the requirement for persistence of antigen have been central issues in the case of both T and B cells. Fifth, both cell-mediated and humoral responses develop in the spleen and lymph nodes and the capture of antigens, encounters between B and T cells and APCs, and recirculation of cells through those organs are fundamental to both types of responses. Sixth, activation of T cells requires signaling initiated within an immunological synapse and there is a distinct negative effect of aging on the signaling process; similarly B cells and T cells interact via a synapse and although it has not yet been studied, it is a reasonable conjecture that aging affects that signaling process. Events that occur during T-cell–APC union involving cytokines determine whether the T-cell response will be of the Th1 or Th2 type; aging appears to depress the Th1 response. Similarly, events that involve cytokines during the B-cell–T-cell union determine the isotype of antibody that will be produced; aging inhibits the production of antibodies of IgG2a isotype (rodents). All of the preceding parallel effects of aging on the B and T lymphocyte systems strongly suggest that the effect of senescence is directed at events or processes that are common to both types of response. The most likely common effect of aging is on the cellular membranes and the signals that are transduced at the plasma membrane. The most likely common site for such effects to be manifested are within the secondary lymphoid tissues. And, in our opinion, the most likely causes of age-associated changes are changes in

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the tissue milieu, especially changes in redox and related homeostatic control mechanisms. Before concluding this chapter, we should acknowledge the absence of a discussion of mucosal immunity in the elderly and the possible effects of aging on it. The reason is the paucity of congruent information from deliberate investigations of the effects of aging on acquired regional immunity including GALT and RALT. On the other hand, there has been no lack of research concerned with mucosal immunity in neonatal, adolescent, and adult life (248) spurred by problems of infant nutrition, inflammatory bowel disease, and other disorders. One of the earliest investigations (249) produced no evidence of an effect of aging on the response to antigen given intragastrically. Mesenteric and mediastinal lymphocytes from young and aged mice given the antigen TNP-BGG were assayed for AFCs specific for the TNP hapten. Concurrently, AFC were assayed in the spleens and peripheral nodes of other mice of the same ages immunized with the same antigen via intraperitoneal route. The results indicated that the responses generated in the spleens and peripheral nodes were substanatially less in aged compared to young mice. In contrast, there was no difference between young and aged mice in the responses given by the mucosal-associated lymph nodes (mesenteric and mediastinal). In retrospect (see below) it seems possible that cells of the B1 variety dominated the responses given by the mucosal-associated nodes against an antigen that was not exclusively T-dependent. As discussed above, aging appears to spare the functions of B1-type B-cells. Studies on responses of aged rats to an established T-dependent antigen, cholera toxin, revealed a markedly diminished ability to produce antibodies compared to young rats (250). Rats of different ages were immunized by the intraintestinal route. Antibodies of the IgA isotype, present in the bile, were assayed and AFCs were assessed in the lamina propria of the jejunum and ileum. The ability of aged rats to generate an IgA response was some fivefold less than that of young animals. Another study of the effect of aging on mucosal IgA responses was performed with young, middle-aged, and old rats immunized intragastrically with either a T-dependent or -independent antigen (251). The amounts of specific IgA antibodies were assayed in the saliva. AFCs were evaluated in spleen and various lymph nodes. This study provided no evidence of an effect of aging on secretory/mucosal immunity. A recent study involved the parasite, Trichuris muris, as the test antigen (252). T. muris is an intestinal parasite that localizes in the cecum of the mouse. Immune expulsion of the parasite is similar to that of other helminths and requires a T-dependent, Th2 type of response accompanied by vigorous mast cell infiltration (mastocytosis). Aged mice experienced a considerable delay in their ability to expel the parasite. Histological analysis of the lamina propria

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revealed deficient mastocytosis. The production of Th2-type cytokines, which is required for parasite expulsion, was both delayed and deficient in the aged animals. CD4+ T cells from mesenteric nodes of aged mice failed to respond to costimulation via the CD28 receptors. The results of this careful investigation strongly suggest that there is an appreciable change in mucosal immunity with age. This study, together with our studies on the decline of immunity to T. musculi (253), demonstrates the usefulness of parasites as pathogenic organisms that display authentic antigens for analyzing the effects of aging on immunity, the type of antigen that immunologists have been urged to employ (1). Finally, two recent reports demonstrate that a revised approach to investigating the consequences of aging on mucosal immunity is necessary. One of those reports (254; reviewed in ref. 255) provided compelling evidence that IgA antibodies specific for commensal bacteria are present in the intestine and to a large extent are produced by B1 lymphocytes. Neither T cells nor the formation of GCs was necessary for the formation of those antibodies. However, the antibodies were absent from the intestines of germ-free mice indicating that they are induced by commensal bacteria and not naturally present. It was demonstrated that the IgA antibodies were produced by B1 cells associated with mucosal lymphoid tissues, viz., Peyer’s patches and mesenteric lymph node. Those B1 cells were largely derived from the peritoneal compartment, not from bone marrow. It appears that there is frequent migration of B1 cells from the peritoneal compartment into the GALT and that migration is directed by signaling mediated via a receptor belonging to the TNF receptor family (255). The production of IgA by the B1 cells requires their localization in GALT. The preceding paragraph leads to the suggestion that the population of B1 cells may serve a very important function as a major line of defense against pathogenic infections in the elderly. There appears to be little effect of aging on that population. The specificity of the IgA antibodies they produce is toward normally occurring commensal bacteria. It has been demonstrated that B1 cells producing IgA of a new specificity can be induced by proper immunization with a foreign antigen (254). Moreover, the specific IgA antibodies that are produced against commensals often react with bacteria of several different strains (256) presumably via shared determinants. Therefore, it should be worthwhile to explore methods for selective mucosal immunization of the elderly against potential pathogens. It should be worthwhile also to determine the specificities of the IgA antibodies produced by B1 cells in elderly study groups from an epidemiological perspective. REFERENCES 1. Marrack P, Kappler J. Subversion of the immune system by pathogens. Cell 1994;76:323–332.

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5 Nutrition, Longevity, and Integrity of the Immune System It is one thing to ask whether we should increase people’s life span, and to answer no; it is quite another to ask whether we should make people immune to heart disease, cancer, dementia, and to decide that we should not. It might thus be appropriate to think of ‘immortality’ as the, possibly unwanted, side effect of treating or preventing debilitating illness. —John Harris, “Intimations of Immortality,” Science 2000;288:59

In his masterpiece, Thales to Dewey (1), the philosopher, Gordon H. Clark, wrote the following in a discussion of epistemology: “knowledge is explanation, and to explain a matter is to state its cause.” If that be true (not relatively, of course), then we know very little about the fascinating effects of dietary manipulation on longevity and on immune capabilities. We are able to describe the effects but not to explain them. The most dramatic consequence of dietary manipulation is the extension of life expectancy, i.e., enhanced longevity, attributable to reduction of caloric intake. The extension of life-span by reduced food consumption was demonstrated in rats as early as 1935 (2). Since that time, numerous studies have explored the composition and administration of diets that engender longevity (reviewed in ref. 3). The only diets or regimens that are known to be effective are those that provide restricted (limited) caloric ingestion. Extension of life expectancy by reduced calorie intake (RCI) has been demonstrated in lower eukaryotes including C. elegans (see ref. 4), yeast (5), and flies (6) as well as in murine species, both rats and mice. Based on studies in progress, RCI probably is effective in monkeys and humans (7). It is important to understand that “dietary restriction” does not mean malnutrition. The latter shortens life expectancy. Experimentally, RCI imposed on weanlings is not so severe as to prevent some gain in weight with age. For example, reduction in From: Aging, Immunity, and Infection By J. F. Albright and J. W. Albright © Humana Press Inc., Totowa, NJ

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caloric intake to about one-third of the intake by ad libitum-fed mice allowed a doubling in body weight accompanied by an extension of survival of the mice by almost 60% (i.e., from about 28 months to about 44 months) (8). Those figures represent the survival of approximately half of the mice in the study groups. It now is customary to express longevity by the index “maximum life-span” (MLS) rather than average life-span because various modifications of animal maintenance, in the attempt to optimize conditions, may inordinately affect the average. In the case of the study just discussed, either index revealed an increase in longevity in the RCI group of more than 50% (8). Similar studies of RCI in the case of yeast, Saccharomyces cerevisiae, have provided results of up to 75% extension of mean and maximum life-span (5). Extension of life expectancy can be achieved in rodents even when RCI is begun in midlife or beyond (9). For example, RCI was initiated when cohorts of two different strains of mice were one year of age. Caloric intake by the mice was gradually reduced over a two-month period resulting in an average loss of 25%–30% of body weight. Body survival of the mice was extended from approximately 33 to 37 weeks (~12%) in one strain and from 24 to 29 weeks (~20%) in the other. RCI not only extends longevity but appears also to preserve good health (10). Some of the physiological functions that are prolonged in more “youthful condition” include blood sugar levels, sensitivity to beneficial effects of insulin, amounts of body fat, capacity for DNA repair, lower incidence of tumors, and, as discussed in the following section, more vigorous immunity. It has been stated that: “about 80 to 90% of the ~300 age-sensitive changes examined in rodents fed a CR [caloric restriction] diet . . . exhibited a (delayed aging profile,”) (8). RCI-MEDIATED DELAY OF IMMUNOSENESCENCE Because it is widely believed that the major effect of aging of the immune system is on the T-cell population, the effects of RCI on T-cell aging have concerned several investigators. There is a substantial decline in numbers of lymphocytes present in the blood (lymphopenia) during the course of aging of certain strains of mice (11) but not in all strains. That decline in lymphocytes appeared to be selective; the number of granulocytes more than doubled over the same period. Similarly, the numbers of naive CD4+ and CD8+ T cells declined in the blood whereas the numbers of B lymphocytes increased significantly with advancing age (11). RCI imposed at the time of weaning did not affect the age-related decline in total T cells but did retard the decline in numbers of naive CD4+ and CD8+ T cells. Unlike the blood cell populations, the actual numbers of CD4+ and CD8+ cells in the spleen remained constant with advancing age in mice fed ad libitum. However, mice on the restricted diet

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experienced a decline in both the CD4 and CD8 subsets. That decline was correlated with an overall decline in weight and total cellularity of the spleen. Mice maintained on the unrestricted diet experienced no decline in those parameters; rather, there was an increase in spleen weight and cellularity as well as in total CD4 and CD8 T cells with advancing age. The most significant finding of the investigation (11) was the delay in the loss of naive CD4+ and CD8+ T cells associated with caloric restriction. The retention of naive T cells was paralleled by an apparently more-gradual loss of T-cell precursors (CD4+CD8+ doublepositive cells) present in the thymus. It should be noted that the size of the thymus declined as in the ad libitum-fed mice but remained more cellular. The preceding review of the work from Harrison’s laboratory (11) provides a comprehensive focal point around which to organize the data from most of the studies on the effects of RCI on aging of immunity. For example, there is general agreement that RCI promotes the retention of naive T cells during aging and diminishes the rate of naive-to-memory cell shift (12–14). RCI initiated at the time of weaning (15) of mice or begun at one year of age (9) retards the decline in T-cell mediated cytotoxicity (CTLs) by preserving both CTL precursors and the helper CD4+ cells (15). RCI begun at weaning of mice sustains the youthful ability of T cells to generate IL-2 and to proliferate in response to appropriate mitogens (12). RCI initiated in midadulthood reduces the output of IL-6 (a proinflammatory cytokine ) associated with aging (16). From the proceding discussion, it seems likely that the reduced intake of calories begun after weaning or later in midlife helps to protect the immune system from the ravages of aging. It must be cautioned, however, that moresystematic and broadened studies are required before a dogmatic assertion can be justified. For example, it is important to obtain clear insight as to the effects of RCI on the resistance to infections in the aging. There have been few attempts to test the hypothesis that RCI can reduce the frequency and severity of infections associated with aging. One informative analysis was concerned with susceptibility to, and severity of, influenza virus infections in mice (17). There is ample evidence that aging impairs the ability of both mice and humans to cope with influenza and that the underlying reason is a flawed immune system (18–20). It was, therefore, an important advance to demonstrate that RCI significantly retarded the age-associated increased susceptibility to influenza (17). Immunological resistance to influenza is primarily dependent on Class I MHC-restricted, CD8+ CTL (21,22) although a significant role for CD4+ T helper cells has been demonstrated (23). In any case, the increased resistance of aged mice to influenza provided by RCI is attributable to retarded aging of T cells. The preceding discussion of influenza infections in aged mice suggests a point that should be considered in evaluating the effects of CR on immunologi-

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Fig. 5-1. Age and stress increase the mortality associated with influenza infection. Three- and 22-month-old male C57BL/6 mice were infected intranasally with 24 HAU influenza A/PR8 virus. Restraint stress (RST) was initiated one day prior to infection and continued nightly throughout. Differences in survival were determined 12 days after infection when no additional virus-mediated mortality was expected. Numbers in parentheses = (number of survivors/total animals per group) *p ≤ .05 compared to three-month controls. ** p ≤ .05 compared to 22-month controls. (From ref. 24, with permission.)

cal competence of aging rodents or other homoiotherms. The point is that we will not know just how effective RCI can be until (a) quantitative studies, such as limiting dilution analyses of T-cell frequencies, have been completed, and (b) analyses of reserve immune potential in comparison to that of young adults has been performed. Perhaps the effects of RCI are to elevate immune potential to a level that can generate an improved response to an acute antigenic challenge but not to a level that replenishes age-exhausted reserve potential. To clarify the point, consider a study (24) of influenza in aged mice that were subjected to restraint stress (Fig. 5-1). Both the rate of death and the number of deaths increased in the group of stressed mice compared to aged mice not stressed. In contrast, there was no effect of stress on the severity of infection in young mice; i.e., the rate and number of deaths were the same whether or not the young mice were stressed. Those results suggest that stress reduced immune potential in both young and aged mice. However, the loss of potential

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was not detectable in young mice, which retained adequate capacity to cope with the infection; but the loss of potential in aged mice reduced the immune capacity in a portion of the animals below the level necessary to cope with the infection. It should be quite informative to evaluate the effects of RCI in a similarly designed study. HOW DOES RCI PROMOTE LIFE-SPAN EXTENSION? Numerous explanations have been offered to account for the effects of RCI on life-span. Among those now considered creditable are: delay in neuroendocrine and/or immunological regression; depression of metabolic rate and body temperature; increased macromolecular repair, especially DNA; reduction and reversal of oxidative stress and damage (see ref. 8). Those explanations are interrelated rather than mutually exclusive. The case for oxidative stress as a major causative factor associated with aging and its moderation by RCI has been masterfully presented (8) and we do not attempt to repeat it here. A brief discussion of the studies concerned with expression (activation and repression) of genes associated with aging is warranted, not because we yet know the genetic basis of aging but to emphasize the technical approach that is likely to yield key genetic information in the near future. The technique of “gene expression profiling” that utilizes DNA oligonucleotide arrays to determine expression or repression of genes has been employed by several groups of investigators to analyze the changes at the gene level with age and the effects of RCI on such changes. One of the first studies of differential gene expression associated with senescence was an analysis of replicative senescence as exhibited by cultures of human fibroblasts (25). That study was not intended to be exhaustive; rather, it was an analysis that included a number of genes previously suggested to be affected by aging. A total of 84 genes were successfully analyzed by sequencing polymerase chain reaction (PCR)-amplified gene tags. Of those, 27 were known genes and 37 were novel, unidentified genes. The authors’ enhanced differential display technique together with Northern blots confirmed that 12 known genes and 11 of the novel genes were expressed differentially in the young and senescent cells. Among the more interesting differences in the senescent compared to the young fibroblasts were: the elevated expression of manganese superoxide dismutase (Mn-SOD), insulin-like growth factor binding protein 5 (IGFBP-5) and IFNγ genes; and the much-reduced expression of the genes encoding calmodulin binding protein 80K-L and laminin A. A survey of differential gene expression in aged rat brain, heart, and liver, estimated to have assessed the expression of 10,000-plus genes, revealed that approximately 2% of the genes (perhaps 200) were affected by the aging process (26). That agrees remarkably well with the results of another survey

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of changing gene expression in skeletal muscle of aged mice, which revealed a change in 113 of 6347 genes evaluated (1.8%) (27,28). Analysis of the genes affected, as determined in the latter study, showed the following: 58 (0.9%) of the genes evaluated showed increased expression in age muscle cells whereas 55 (0.9%) were depressed by more than twofold compared to young muscle; among the genes having increased expression, a significant proportion of them encode stress factors such as Hsp 27, Hsp 71, protease Do, and the gene for GADD 45, which is induced by damaged DNA. Genes that are enhanced in senescent fibroblasts such as IGFBP-5 were also found to be elevated in aged skeletal muscle. Among those genes showing repressed expression in aged muscle, the majority encode proteins integral to energy metabolism and biosynthesis. The studies on age-associated changes in gene expression of skeletal muscle were extended to include an evaluation of the consequences of RCI on gene expression (27,28). It was found that: “Of the four major gene classes that displayed consistent age-associated alterations (i.e., stress response, biosynthesis, protein metabolism and energy metabolism), 84% (26/31) were either completely or partially suppressed by CR [caloric restriction]” (28). In addition, RCI diminished the expression of several genes that encode DNA repair enzymes. It is apparent that the technique of “gene expression profiling” as discussed above is a powerful tool for analyses of aging at the gene level. It was estimated that the 6347 genes evaluated in the study of aged skeletal muscle (27,28) represent some 5–20% of the mouse genome. Additional, recent studies of brain and other postmitotic tissues have revealed that there are likely to be organspecific gene expression patterns associated with aging. Furthermore, it has been demonstrated that aging of replicating tissues produces a very different pattern of gene effects compared to postmitotic tissues (29). It will be challenging but rewarding to adapt the expression profiling technique to the analysis of aging of the cells of the immune system. DIETARY RESTRICTION VS MALNUTRITION The degree of RCI imposed on the aging experimental animals varied widely in the studies reviewed above, from roughly 75% to 35% of the ad libitum control groups. It seems that there is no agreed-upon, standard degree of RCI that produces the maximum extension of longevity rather than the life-shortening effects of malnutrition. An acceptable limit of RCI at present appears to be a diet that provides for body weight gain of weanling rodents, although the body weight may level off at less than half that of ad libitum-fed controls. In humans this would be clinically unacceptable, at present. The extent of lifespan prolongation is proportional to the degree of RCI, a finding that has

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prompted the comment that “food consumption above an optimal level (that is, the amount below which CR [caloric restriction] has a life-threatening effect on the animal) progressively shortens longevity” (8). Thus, both too little and too much food intake are life threatening. Finding a level of caloric intake that is “ideal,” i.e., a level that promotes both maximum life-span and robust health, may be impossible, especially for humans. It must be kept in mind that studies on laboratory animal longevity are performed under conditions that exclude infections, stress, and other environmental insults and that provide balanced nutrition even though calorically restricted. Moreover, it is clear that a given degree of RCI affects life-span and other parameters to different extents in different strains of inbred rodents. Therefore, there probably will be no prescribable amount of dietary restriction for all individuals of an outbred population such as humans. Similarly, finding a level of food consumption that promotes maximum lifespan may not be ideal for promoting optimum immune competence and ability to cope with infections. To date, sophisticated studies on immune competence as related to RCI are largely lacking. The data that are available, such as those from studies on influenza infections in mice (17) and RCI-mediated retention of naive T cells (11,12), are quite encouraging but more studies are needed, especially of pathogenic infections. Elevated or repressed gene expression, which seems to reflect aging and appears to be inhibited by RCI, might, in some cases, be a reflection of improper nutrition. How might an effect of improper nutrition be manifested at the gene level? There is considerable literature concerned with that topic (30–32), which we do not attempt to review here. Instead, we describe one potent example, viz., the interrelationship between fatty acids, the PPAR, and the substance leptin. It has been proposed that the life-span-lengthening effect of RCI is primarily a reflection of the reduction in fat mass, especially visceral fat, an effect mediated in part by the cytokine/hormone, leptin (33). That concept has been disputed (34) and evidence has been cited (35) opposing the proposed role of leptin. New evidence has appeared that does not resolve the issue but seems to emphasize the importance of the issue. Leptin is a small protein produced especially by adipocytes. Levels of leptin act through leptin receptors in the hypothalamus to signal the presence of sufficient energy stores. The response is a reduction in appetite and an increase in energy expenditure, thus preventing serious obesity. A genetic deficiency in mice, either of leptin or its receptor, results in severe obesity. In a recent study (36), the leptin gene was overexpressed in a group of young rats and a group of aged rats. In the young rats, plasma-free fatty acids and triacylglycerol declined markedly. Leptin mRNA nearly disappeared from adipocytes and there was a substantial increase in fatty-acid-oxidizing

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enzymes. Furthermore, the transcription factor that regulates expression of those enzymes, PPARα, was significantly elevated. The results of overexpressing the leptin gene in aged rats were entirely different, in fact, the opposite of the results in the young rats. Although the level of expression of the leptin gene was higher in the aged than in the young rats, leptin appeared to be without effect. Analysis of signaling elements in the hypothalamus of untreated young and aged rats revealed that the factor SOCS-3 (suppressor of cytokine signaling-3) was about threefold higher in the aged animals. In the gene-transfected rats, SOCS-3 was about threefold higher in the aged rats’ adipocytes. It was suggested, therefore, that elevated SOCS-3 which has been proposed as an inhibitor for leptin (37,38), was responsible for the failure of aged rats to respond to leptin. Another possibility is the presence of inordinately high levels in aged animals of the circulating leptin-binding protein (39); that possibilitiy has not been explored, to our knowledge. The circulating levels of leptin can change rapidly during feeding or fasting and the level of leptin is not merely an indicator of fat mass (40). Leptin-deficient and leptin receptor-deficient animals are immunologically defective and susceptible to infection (reviewed in ref. 41). The levels of leptin decline sharply during starvation and the deficiency of leptin appears, in part, to account for the immune deficiency associated with severe malnutrition. Defective immunity associated with starvation is ameliorated by administering leptin. A question worth repeating here is: how to distinguish between acceptable RCI and malnutrition? With regard to that question,consideration should be given to utilizing levels of free and bound leptin (i.e., bound to leptin-binding protein and soluble leptin receptor) as an index of food restriction. Perhaps the rate of decline of leptin following the initiation of a dietary restriction regimen could be used to discriminate between life-extending and life-shortening RCI regimens. It should be mentioned that it has not been determined directly (as far as we know) that leptin levels are influenced by RCI. In humans, the level of serum albumin has often been used as an indication of nutritional status (e.g., ref. 42). In studies of protein-calorie malnourished, aged humans (79–80 years of age), the study population was divided into three groups according to serum albumin concentration: (a) albumin >35 g/L; (b) albumin 30–35 g/L; and (c) albumin <30 g/L. Immunological parameters were assessed in subjects of those three groups. There was a direct correlation between the degree of malnutrition and the loss of immunological integrity, which included number of peripheral blood T cells, response of T cells to mitogen, and several tests of cell-mediated immunity. Furthermore, relatively low serum albumin levels and T-cell expression of the IL-2 receptor (CD25) were strongly correlated with susceptibility to pulmonary infection (42). It will be interesting and possibly of clinical importance to learn how leptin concentration and serum

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albumin concentration compare as indicators of malnutrition or as guides to choosing RCI consistent with extending life-span but avoiding malnutrition. From a global perspective, malnutrition is a major problem causing severe and frequent infections among persons of all ages. There is a clear relationship between malnutrition and parasitic diseases in humans (43,44). In order to explore the relationship between malnutrition, depressed innate immunity, and leishmaniasis, a mouse model of malnutrition was developed (45). Groups of mice were provided with diets representing mild, moderate, and severe protein undernutrition beginning at the time they were weaned. The diets representing mild, moderate, and severe protein undernutrition provided, respectively, 12%, 12%, and 18% fewer calories than the control diet. Over the course of 28 days, the gain in body weight in the mildly undernourished group was about half that in the control group. There was no gain or loss in body weight after weaning in the moderately undernourished group. The severely undernourished group lost body weight steadily over the 28 days of observation. The progression and severity of L. donovani infection were worse in all three of the undernourished groups and correlated with the loss of innate immune competence caused by malnutrition. The effects of protein malnutrition on immune competence are devastating whereas RCI, within limits, seems to enhance immunity. Not surprisingly, susceptibiity to infections and/or severity of infections rises sharply with protein malnutrition. In contrast, RCI within limits seems to enhance resistance to infection. Therefore, as judged by the criteria of immune competence and resistance to infection, RCI and protein undernutrition exert very different physiological effects on organisms. Mild malnutrition, which is not uncommon among the elderly (46,47), will not enhance life expectancy and will increase the chance of acquiring an infectious disease. REFERENCES 1. Clark GH. Thales to Dewey, 2nd ed. The Jefferson, MD: Trinity Foundation, 1989. 2. McCay CM, Crowell MF, Maynard LA. The effect of retarded growth upon the length of the lifespan and upon the ultimate body size. J Nutr 1935;10:63–79. 3. Weindruch R, Walford RL. The Retardation of Aging and Disease by Dietary Restriction. Springfield, IL: Thomas, 1998. 4. Larsen PL, Clarke CF. Extension of life-span in Caenorhabditis elegans by a diet lacking coenzyme Q. Science 2002;295:120–123. 5. Jiang JC, Jaruga E, Repnevskaya MV, Jazwinski SM. An intervention resembling caloric restriction prolongs life span and retards aging in yeast. FASEB J 2000;14: 2135–2137. 6. Sohal RS, Orr WC. In: Esser K, Martin GM, eds. Molecular Aspects of Aging New York: Wiley, 1995:109–127. 7. Roth GS, Lane MA, Ingram DK, et al. Biomarkers of caloric restriction may predict longevity in humans. Science 2002;297:811.

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8. Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science 1996;273:59–63. 9. Weindruch R, Gottesman SRS, Walford RL. Modification of age- related immune decline in mice dietarily restricted from or after midadulthood. Proc Natl Acad Sci USA 1982;79:898–902. 10. Yu BP, Masoro ET, Murata I, et al. Life span study of SPF Fischer 344 male rats fed ad libitum or restricted diets: longevity, growth, lean body mass and disease. J Gerontol 1982;37:130–141. 11. Chen J, Astle CM, Harrison DE. Delayed immune aging in diet-restricted B6 CBAT6 F1 mice is associated with preservation of naïve T cells. J Gerontol Biol Sci 1998;53A:B330–B337. 12. Venkatraman JT, Attwood VG, Turturro A, et al. Maintenance of virgin T cells and immune functions by food restriction during aging in long-lived B6D2F1 female mice. Aging: Immunol Infect Dis 1994;5:13–25. 13. Fernandes G, Venkatraman JT, Turturro A, et al. Effect of food restriction on life span and immune functions in long-lived Fischer-344 X Brown Norway F1 rats. J Clin Immunol 1997;17:85–95. 14. Miller RA. The aging immune system: Primer and prospectus. Science 1996;273: 70–74. 15. Miller RA, Harrison DE. Delayed reduction in T cell precursor frequencies accompanies diet-induced lifespan extension. J Immunol 1985;134:1426–1429. 16. Volk MJ, Pugh TD, Kim MJ, et al. Dietary restriction from middle age attenuates age-associated lymphoma development and interleukin 6 dysregulation in C57BL/ 6 mice. Cancer Res 1944;54:3054–3061. 17. Effros RB, Walford RL, Weindruch R, Mitcheltree C. Influences of dietary restriction on immunity to influenza in aged mice. J Gerontol Biol Sci 1991;46: B142–B147. 18. Bender BS, Johnson MP, Small PA Jr. Influenza in senescent mice: Impaired cytotoxic T-lymphocyte activity is correlated with prolonged infection. Immunology 1991;72:514–519. 19. Bender BS, Small PA Jr. Heterotypic immune mice lose protection against influenza virus infection with senescence. J Infect Dis 1993;168:873–880. 20. Powers DC. Influenza A virus-specific cytotoxic T lymphocyte activity declines with advancing age. J Am Geriatr Soc 1993;41:1–5. 21. Yap KL, Ada GL, McKenzie IFC. Transfer of specific cytotoxic T lymphocytes protects mice inoculated with influenza virus. Nature 1978;273:238–240. 22. Bender BS, Croghan T, Zhang L, Small PA Jr. Transgenic mice lacking class I major histocompaatibility complex-restricted T cells have delayed viral clearance and increased mortality after influenza virus challenge. J Exp Med 1992;175: 1143–1151. 23. Taylor SF, Cottey RJ, Zander DS, Bender BS. Influenza infection of β 2microglobulin-deficient (β2 m–/–) mice reveals a loss of CD4+ T cell functions with aging. J Immunol 1997;159:3453–3459. 24. Padgett DA, MacCallum RC, Sheridan JF. Stress exacerbates age-related decrements in the immune response to an experimental influenza viral infection. J Gerontol Biol Sci 1998;53A:B347–B353.

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25. Linskens MHK, Feng J, Andrews WH, et al. Cataloging altered gene expression in young and senescent cells using enhanced differential display. Nucleic Acids Res 1995;23:3244–3251. 26. Goyns MH, Charlton MA, Dunford JE, et al. Differential display analysis of gene expression indicates that age-related changes are restricted to a small cohort of genes. Mech Ageing Dev 1998;101:73–90. 27. Lee C-K, Klopp RG, Weindruch R, Prolla TA. Gene expression profile of aging and its retardation by caloric restriction. Science 1999;285:1390–1393. 28. Weindruch R, Kayo T, Lee C-K, Prolla TA. Microarray profiling of gene expression in aging and its alteration by caloric restriction in mice. J Nutr 2001;131: 9185–9235. 29. Ly DH, Lockhart DJ, Lerner RA, Schultz PG. Mitotic misregulation and human aging. Science 2000;287:2486–2492. 30. Clarke SD, Abraham S. Gene expression: Nutrient control of pre- and posttranscriptional events. FASEB J 1992;6:3146–3152. 31. Berdanier CD. Nutrient-gene interactions: Today and tomorrow. FASEB J 1994; 8:1. 32. Kim M-J C, Berdanier CD. Nutrient-gene interactions determine mitochondrial function: Effect of dietary fat. FASEB J 1998;12:2243–2248. 33. Barzilai N, Gupta G. Revisiting the role of fat mass in the life extension induced by caloric restriction. J Gerontol Biol Sci 1999;54A:B89–B96. 34. Masoro ET. Commentary on “Revisiting the Role of Fat Mass in the Life Extension Induced by Caloric Restriction.” J Gerontol Biol Sci 1999;54A:B97. 35. Harrison DE, Archer JR, Astle CM. Effects of food restriction on aging: separation of food intake and adiposity. Proc Natl Acad Sci USA 1984;81:1835–1838. 36. Wang Z-W, Pan W-T, Lee Y, et al. The role of leptin resistance in the lipid abnormalities of aging. FASEB J 2001;15:108–114. 37. Bjorback C, Elmquist JK, Frantz JD, et al. Identification of SOCS-3 as a potential mediator of central leptin resistance. Mol Cell 1998;1:619–625. 38. Bjorback C, El-Haschimi K, Frantz JD, Flier JS. The role of SOCS-3 in leptin signaling and leptin resistance. J Biol Chem 1999;274:30,059–30,065. 39. Patel N, Brinkman-Van der Linden EC, Altmann SW, et al. OB-BP1/Siglec-6, a leptin-and sialic acid-binding protein of the immunoglobulin superfamily. J Biol Chem 1999;274:22,729–22,738. 40. Ahima RS, Prabakaran D, Mantzoros C, et al. Role of leptin in the neuroendocrine response to fasting. Nature 1996;382:250–252. 41. Faggioni R, Feingold KR, Grunfeld C. Leptin regulation of the immune response and the immunodeficiency of malnutrition. FASEB J 2001;15:2565–2571. 42. Lesourd B. Protein undernutrition as the major cause of decreased immune function in the elderly: Clinical and functional implications. Nutr Rev 1995;53: S86–S94. 43. Badaro R, Jones TC, Lorenco R, et al. A prospective study of visceral leishmaniasis in an endemic area of Brazil. J Infect Dis 1986;154:639–649. 44. Harrison LH, Naidu TG, Drew JS, et al. Reciprocal relationships between undernutrition and the parasitic disease visceral leishmaniasis. Rev Infect Dis 1986;8:447–453.

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45. Anstead GM, Chandrasekar B, Zhao W, et al. Malnutrition alters the innate immune response and increases early visceralization following Leishmania donovani infection. Infect Immun 2001;69:4709–4718. 46. Chandra RK. Nutritional regulation of immunity and risk of infection in old age. Immunology 1989;67:141–147. 47. Sullivan DH, Sun S, Walls RC. Protein-energy undernutrition among elderly hospitalized patients: A prospective study. JAMA 1999;281:2013–2019.

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Epilogue VV: What have you learned from science. EC: Only one thing: that one ought to wash one’s hands before touching nature. —Erwin Chargaff, Heraclitean Fire

In view of the considerable time and effort we have devoted to the preparation of this monograph, and the hope that it is both comprehensive and comprehensible, we now expose our ideas as to where research efforts should be concentrated. There follows a listing and brief discussion of four research programs that, in our view, are both feasible and likely to be most rewarding. The listing is not in any order of priority. First is a comprehensive study of the effects of aging on cellular membranes. It has been apparent for years that changes in membranes occur with age as well as with variations in diet and availability of fatty acids. It is now clear that signaling platforms (rafts) are of major importance in the functions of T cells, B cells, MPs, and neutrophils, and that changes with age in membrane composition, especially phospholipids and cholesterol, alter the capabilities for intracellular signaling (both qualiltative and quantitative). It is likely that oxidative stress is a major factor in causing the changes in membranes. Senescence of mitochondria and disruption of the electron transport and proton pumping chain is probably a major contributor to oxidative damage. In some types of cells such as MPs and neutrophils, aging influences the disposal of the products of the oxidative burst. In all cells in which NO and O2 are momentarily present in uncombined form, it is likely that they may interact within the hydrophobic regions of membrane lipid bilayers. It has been demonstrated that in those areas of cellular membranes the reaction between NO and O2 is 300 times faster than in aqueous media (1). Furthermore, it was concluded that about 90% of the interaction between NO and O2 occurs within membranes even though only 3% of the volume of cells is occupied by membranes. The principal product of From: Aging, Immunity, and Infection By J. F. Albright and J. W. Albright © Humana Press Inc., Totowa, NJ

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that interaction between NO and O2 is the highly destructive peroxynitrite (ONOO–) radical. Not only are mitochondria likely to be a major source of reactive oxygen species (ROS) in aged cells, they also are a principal target of ROS. For example, damage to the production of NADPH, which is required within mitochondria for regenerating reduced glutathione (GSH) can lead to intramitochondrial redox imbalance (2). As a consequence, ROS levels rise and produce severe mitochondrial DNA damage, lipid peroxidation, and marked reduction in adenosine 5'-triphosphate (ATP) output. One of the findings that makes the oxidative theory of aging so compelling is the effect of RCI on senescencemediated mitochondrial dysfunction (3). The skeletal muscle mitochondria of aging RCI rats were significantly less likely to leak protons than in the case of similarly aged rats fed ad libitum. That was also true of cardiac muscle mitochondria, and evidence was adduced that RCI reduces proton leakage from respiratory enzyme complex I of the electron transport chain (4). A second research program that is likely to be particularly rewarding concerns the age-associated moderation of the immunological repertoire and the apparent narrowing and focusing of the immune responses. That is suggested, in part, by the emergence with age of clones of CD8+ T cells, of B cells (especially CD5+), and to some extent of CD4+ T cells (see Chap. 4). A major aspect of this program should deal with the molecular patterns of the microorganisms for which those clones are specific and the manner in which the immunogenic fragments are presented to responsive cells. In this regard, an outstanding, recent contribution revealed that in elderly subjects who are seropositive for the CMV a substantial portion of the peripheral blood CD8+ cells are in clones of CMV-specific CTL (5). In some individuals, up to 25% of the total CD8+ population belongs to one or a few large clones. Thus, it appears that CMV is able to drive emergence of specific clones in aging subjects. Are most of the emergent CD8 T-cell clones specific for viruses? Are those viruses of the latent/cryptic variety? Or, perhaps, the emergence of clones of cells relates to opportunistic fungal infections. What is the possible importance of the molecular patterns of commensal gut microorganisms in driving the emergence of those clones? There is evidence that at least some of the MPs of elderly subjects are in an activated state (see refs. 6 and 7). Whether or not most or all of them are activated is uncertain. The behavior of those activated MPs needs to be determined (e.g., location, ability to recirculate, spectrum and amounts of cytokines, chemokines, and soluble receptors they produce). Whether or not those activated MPs can and do function as APCs, and the identity of the immunogenic fragments they proffer to lymphocytes, would seem to be important items for research. In that regard, MPs are responsible for clearing dying cells and debris,

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which they do with remarkable efficiency. That responsibility may increase with age. The possibility that the oligoclonality of T cells and B cells with advancing age is a reflection of presentation of immunogenic fragments of modified self deserves considerably more attention than it has received to date. A third research program that has received little attention, probably because it has not been well formulated, concerns the possible relationship between aging of the immune system and aging of the GI system. Three of the more obvious ways in which the two systems might influence each other are: (a) the effect of RCI on the intestinal flora and a role for the latter in contributing nutritionally to RCI-enhanced longevity; (b) effects on the immune system of age-associated translocation of microorganisms and substances derived from commensal organisms of the GI system; and (c) age-related changes of gutassociated lymphoid tissue (e.g., mesenteric node, Peyer’s patches) and of B1 cells in particular (8). Part of the following discussion is quite speculative as there is meager evidence to either support or refute the notions presented. The intestinal microflora comprises some 1014 bacterial cells and more than 400 bacterial species (9). In the normal human adult, the microflora in the upper small intestine is not numerically large, some 103–104 organisms per mL contents. The number of organisms in that segment of the intestine is regulated by the relatively acidic conditions (gastric acid) and the movement of the microvilli. The density of microorganisms increases dramatically in the lower ileum (106–108 per mL contents) and especially in the colon (1011–1012 organisms per mL). In the distal ileum Gram-negative organisms begin to outnumber the Gram-positive and there is a substantial number of anaerobes. In the colon, anaerobes outnumber aerobes by 1000 to 1. The colon is a region of high oxidative stress owing in part to its high activity of xanthine oxidase and the large number of catalase-negative bacteria (10,11). It has been described as a “free radical time bomb” (11). Both the structure and functions of the GI tract are influenced by the bacterial content. For example, in germ-free animals the intestinal wall is thinner and less cellular, the villi are smaller, the crypts shallower, and the total mucosal area significantly less (see ref. 9, and refs. therein). In addition, there are fewer lymphocytes and MPs, and the Peyer’s patches are small and with few GCs. After exposure to enteric bacteria, the GI tract of germ-free animals rapidly acquires a more conventional appearance. At the other extreme is the condition of bacterial overgrowth. Several conditions may contribute to overgrowth including less-acid conditions (such as increased gastric pH associated with aging), reduced villous sweeping of the contents along the lumen, reduced peristaltic activity, and intestinal malabsorption and diet. A critical question is whether or not those conditions, some or all of which may occur in elderly, can result in translocation of bacteria

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(microorganisms) or microbial components from the gut into the bloodstream or lymph. If that can and does happen in the elderly there may be several consequences including: (a) availability of antigens to drive selective clonal expansion of cells such as B (and B1) cells; (b) sustained low-level activation of MPs, neutrophils, and certain other cells; and (c) chronic sub-clinical stress. The beneficial effects of RCI might be explained in part by its ameliorating effect on the intestinal flora. It should be noted that dietary components significantly affect intestinal flora both qualitatively and quantitatively. Diets lacking in protein or vitamins (e.g., folate) or deficient in fiber can result in bacterial overgrowth and translocation (12–14). It has been asserted that: “The multiorgan system that composes the gastrointestinal tract has a large reserve capacity, and thus there is little change in gastrointestinal function because of aging in the absence of disease “ (15). There seems to be no reason to quarrel with that statement except to note that the “large reserve capacity” may not have been adequately tested in the elderly. Trauma and stress have been demonstrated to result in bacterial translocation in young subjects; less of those insults that have little or no effect in the young may produce substantial translocation in the elderly. In any case, as discussed in (15), inadequate gastric acid secretion (atrophic gastritis) occurs typically in about one-third of the elderly in the United States. Gastric motility is significantly reduced and gastric emptying may take up to twice as long in the elderly compared to young adults. As a consequence, bacterial overgrowth in the upper GI tract occurs commonly in the elderly. Although such overgrowth alone may not result in bacterial translocation into the bloodstream, the added effect of stress, trauma, and other disorders may overcome the barriers to intestinal bacterial transmigration. It has been noted (16) that whereas translocation “was initially viewed with skepticism by many clinicians, bacterial translocation is now generally considered responsible for a large proportion of complicating infections in hospitalized patients, with immunosuppressed patients, trauma patients, postsurgical patients, and transplant recipients considered at highest risk.” Escape of bacteria from the gut under conditions of stress, trauma, physical disorders such as poor blood flow in the gut, endotoxemia, immunosuppression, malnutrition, and so forth, often occurs by way of Peyer’s patches and mesenteric lymph node and thence to the thoracic duct and the blood stream. Under those abnormal conditions, it may be possible to detect live bacteria in the mesenteric node, the thoracic duct, liver, spleen, peritoneal cavity, and elsewhere. Bacteremia may develop and become life-threatening. Commensal bacteria, normally considered harmless and welcome for the protection they provide by competing with pathogenic organisms, may become morbid. The pathogenic potential of commensal microorganisms is kept in check by the GALT prima-

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rily as a function of polymeric IgA (17). A recent report (18) of an outstanding investigation now reveals that the IgA responsible for controlling the commensal microflora is specific antibody directed against cell wall antigens and proteins of the microorganisms. Furthermore, the IgA against commensal microorganisms is produced by cells of the B1 variety in a T-cell-independent manner. What is more, those B cells are not dependent on being nestled in a GC or lymphoid follicle in order to produce the anti-commensal IgA. It appears, subject to final, rigorous proof, that those B1 cells are activated directly by specific molecular patterns of the commensal microorganisms. There is much more to this exciting story, and, no doubt, more to come soon. Here, we are particualrly interested in the fact that commensal microorganisms elicit responses by B1 cells. Both B1 and B2 cells exist in the lamina propria, Peyer’s patches, and mesenteric lymph node. The B1 cells produce the IgA with which the commensal microorganisms are coated whereas conventional B2 cells produce the highly specific IgA antibodies that eliminate pathogenic bacteria that enter the gut (18,19). The reasons why commensal bacteria escape elimination by antibodies generated by B2 cells are not known but probably center on: the unusual types of cells that compose the mucosal immune response (abundance of T cells expressing γδ TCR and curious T cells bearing αα TCR); the selective action of TRLs that discriminate between commensals and pathogens; and mechanisms that inhibit the production of critical cytokines such as that employed by nonpathogenic Salmonella, which block the dissociation of transcription factor NFκB from its inhibitor, IκB (see ref. 20 and refs. cited therein). There is strong evidence that the B1 cells migrate out of the peritoneal cavity into the mesenteric lymph node where they encounter antigens of the commensal microorganisms (8). The B1 cells proliferate and differentiate in the node and the mature plasma cells enter the lamina propria of the intestine where they secrete IgA into the lumen. There are several observations that hint that aged subjects experience chronic, low-level, subclinical inflammation (6,7), for example, the activated (“semi-activated”) condition of MPs and neutrophils and the 10-fold greater sensitivity of the aged to endotoxins. Those and other observations can be coupled with the likelihood that commensal microorganisms bearing endotoxins or other toxins translocate in small numbers from the gut of the elderly. Therefore, there is the possibilitiy that chronic, subclinical inflammation and stress may contribute to immunosuppression and other disorders in the elderly including life shortening. We are reluctant to elevate that notion to the level of a hypothesis; rather, we suggest that it could prove rewarding to conduct studies along the following lines. First, conduct a study of the numbers and status of B1 cells in the peritoneal and pleural cavities of aged rodents and their abil-

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ity in the aged to migrate into the mesenteric lymph node and differentiate in that microenvironment. Second, do an analysis, both qualitative and quantitative, of the IgA present in the intestine of aged subjects along with an assessment of the proportion of commensal bacteria coated with IgA. It should be interesting to determine whether or not commensal bacteria lacking the coat of IgA can translocate out of the intestine. Third, it seems possible that the healthand longevity-enhancing effects of RCI might, in part, reflect extended, efficient control of commensal microorganisms by retarding the age-associated decline in B1 cell production of IgA. The final research program that seems to be of relatively high priority concerns the failure of GC development that is common in aged rodents and humans. This was reviewed in Chapter 4 where it was seen that prominent reasons for that age-related defect include defective FDCs, lack of essential chemokines and, possibly, defective involvement of factors such as LT α, a product of Th1 cells, which is required for normal GC formation (21) and may be deficient in the aged. As noted in Chapter 4, LTα and TNF act on stromal cells to stimulate production of chemokines that facilitate homing of B and T cells to appropriate regions in the spleen and lymph nodes (22,23). In actuality, the studies that have identified TNF, LTα, LTβ, and TNFR1 as being involved in the generation of chemokines required for secondary lymphoid tissue organization have been performed with appropriate gene-targeted (“knockout”) mice or by use of specific neutralizing antibodies. Another member of the TNF family, osteoprotegerin ligand (OPGL), has been identified as being involved in lymphoid tissue organization (24). OPGL was found to be identical to TRANCE (TNF-related activation-induced cytokine), also known as RANKL (receptor activator of NFκB ligand). Thus, OPGL is a ligand for RANK. OPGL is expressed on T cells and RANK on DCs as well as T cells. What might occur to the organization of those tissues in the prolonged absence of adequate concentrations of the essential chemokines, or the age-associated derangement of receptors for the chemokines or the TNFR1, is not known. We suggest that this is a study that should be done. There is evidence already that chemokine production is severely depressed in aged rodents. For example, we have found that NK cells of aged mice are almost completely lacking the ability to generate a battery of chemokines that are readily produced by NK cells from young mice (unpublished). Finally, there is the possibility that functional secondary lymphoid organs might be induced de novo in aged individuals. This possibility is suggested by the recent studies of ectopic lymphoid neogenesis at sites of inflammation (25,26). Initial studies have been done on creation of ectopic lymphoid tissue as a means of immunotherapy of cancer (27). The approach to generating ectopic lymphoid tissue in the elderly involves, first, determining which

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chemokines, cytokines, and receptors are deficient and, second, the order and amounts in which to supply those substances and, third, the appropriate sites. Studies of that type are under way in young animals (28). If it were possible to introduce at those sites dendritic cells bearing key immunogenic peptides of the pathogens that afflict the elderly, many of the infectious diseases of the elderly might be prevented. REFERENCES 1. Liu X, Miller MJS, Joshi MS, et al. Accelerated reaction of nitric oxide with O2 within the hydrophobic interior of biological membranes. Proc Natl Acad Sci USA 1998;95: 2175–2179. 2. Jo SH, Son MK, Koh HJ, et al. Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase (IDPm). J Biol Chem 2001;276:16,168–16,176. 3. Lal SB, Ramsey JJ, Monemdjou S, et al. Effects of caloric restriction on skeletal muscle mitochondrial proton leak in aging rats. J Gerontol. Biol Sci Med Sci 2001;56:B116–B122. 4. Gredilla R, Sanz A, Lopez-Torres M, Barja G. Caloric restriction decreases mitochondrial free radical generation at complex I and lowers oxidative damage to mitochondrial DNA in the rat heart. FASEB J 2001;15:1589–1591. 5. Khan N, Shariff N, Cobbold M, et al. Cytomegalovirus seropositivity drives the CD8 T cell repertoire toward greater clonality in healthy elderly individuals. J. Immunol. 2002;169:1984–1992. 6. Catania A, Airaghi L, Motta P, et al. Cytokine antagonists in aged subjects and their relation with cellular immunity. J Gerontol Biol Sci 1997;52A:B93–B97. 7. Albright JW, Albright JF. Soluble receptors and other substances that regulate proinflammatory cytokines in young and aging humans. J Gerontol Biol Sci 2000; 55A:B20-B25. 8. Fagarasan S, Honjo T. T-independent immune response: New aspects of B cell biology. Science 2000;290:89–92. 9. Simon GL, Gorbach SL. The human intestinal microflora. Dig Dis Sci 1986;31 (S9):147S–162S. 10. Bulger EM, Helton WS. Nutrient antioxidants in gastrointestinal diseases. Gastroenteral Clin North Amer 1998;27:403–419. 11. McCord JM. Radical explanations for old observations. Gastroenterology 1987;92:2026–2034. 12. Alverdy JC, Aoys E, Moss GS. Total parenteral nutrition promotes bacterial translocation from the gut. Surgery 1988;104:185–190. 13. Hosoda S. The gastrointestinal tract and nutrition in the aging process: An overview. Nutr Rev 1992;50:372–373. 14. Gorbach SL, Goldin BR. Nutrition and the gastrointestinal microflora. Nutr Rev 1992;50:378–381. 15. Saltzman JR, Russell RM. The aging gut: Nutritional issues. Gastroenterology Clin North Amer 1998;27:309–324. 16 Wells CL. Colonization and translocation of intestinal bacterial flora. Transplant Proc 1996;28:2653–2656.

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17. Berg RD. Bacterial translocation from the gastrointestinal tract. Adv. Exp. Med. Biol. 1999;473:11–30. 18. Macpherson AJ, Gatt D, Sainsbury E, et al. A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria. Science 2000;288:2222–2226. 19. Kroese FG, de Waard R, Bos NA. B-1 cells and their reactivity with the murine intestinal microflora. Sem Immunol 1996;8:11–18. 20. Xavier RJ, Podolsky DK. How to get along—friendly microbes in a hostile world. Science 2000;289:1483–1484. 21. Matsumoto M, Mariathason S, Nahm MH, et al. Role of lymphotoxin and the type I TNF receptor in the formation of germinal centers. Science 1996;271:1289–1291. 22. Ngo VN, Korner H, Gunn MD, et al. Lymphotoxin α/β and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen. J Exp Med 1999;189:403–412. 23. Cyster JG. Chemokines and cell migration in secondary lymphoid organs. Science 1999;286:2098–2102. 24. Kong Y-Y, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999; 397:315–323. 25. Ludewig B, Odermatt B, Landmann S, et al. Dendritic cells induce autoimmune diabetes and maintain disease via de novo formation of local lymphoid tissue. J Exp Med 1998;188:1493–1501. 26. Hjelmstrom P. Lymphoid neogenesis: De novo formation of lymphoid tissue in chronic inflammation through expression of homing chemokines. J Leukocyte Biol 2001;69:331–339. 27. Schrama D, thor Straten P, Fischer WH, et al. Targeting of lymphotoxin-α to the tumor elicits an efficient immune response associated with induction of peripheral lymphoid-like tissue. Immunity 2001;14:111–119. 28. Luther SA, Bidgol A, Hargreaves DC, et al. Differing activities of homeostatic chemokines CCL 19, CCL 21, and CXCL 12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis. J Immunol 2002;169:424–33.

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Index A

Antigens T cells, 141–142 APC, 66 secondary lymphoid organs, 186 T cell interaction, 171 T helper cells, 162–166 ARNT, 89 Arylhydrocarbon receptor nuclear translocator (ARNT), 89

Acquired immunodeficiency syndrome (AIDS), 43 cell membrane receptors, 44t Activated T cell(s) nuclear factor. See Nuclear factor of activated T cells (NFAT) Adaptive/acquired immunity, 135–197 Adenovirus, 45, 45t cell membrane receptors, 44t AFC export, 184 within GC, 189–191 Affinity maturation within GC, 189–191 Aging, 1–15 demographics, 1–4 future health, 3–4 infectious diseases, 5–7, 6f mortality, 8 neutrophils, 103–105 theory, 12 worldwide, 2 AIDS, 43 cell membrane receptors, 44t AmpC enzymes, 40 Anatomical-functional relationships age-associated changes, 23–24 Antibiotic resistance bacterial variation, 39–42 Antibody forming cell (AFC) export, 184 within GC, 189–191 Antigen-processing cells (APC), 66 secondary lymphoid organs, 186 T cell interaction, 171 T helper cells, 162–166 f: figure t: table

B Bacteremia, 30–31 Bacteria antibiotic resistance, 39–42 attachment, 29–30 bladder, 24 host cells invasion, 34–35 infections, 20–24 age-associated susceptibility, 24–28 mucosal surfaces, 28–42 lectins-glycoconjugates, 33t type II secretion mechanism, 33–34 Bacteroides, 23t B cells, 183–184 aging, 191–194 clonal expansion within GC, 189–191 diversification, 183–184 follicular microenvironment GC, 187 transpiring, 187 Beta-lactam, 39 Beta-lactamases, 39 Bifidobacteria, 23t Biliary stent blockage biofilm, 37t

233

234 Biliary tract infection biofilm, 37t Biofilms, 36–38, 37t Bladder bacteria, 24 B lymphocytes. See B cells B memory cells, 190 Body composition, 12 Bone marrow B cells, 193f Bordetella pertussis, 29 lectins, 33t Borrelia burgdorferi lectins, 33t C Calcineurin nuclear factor of activated T cells, 165, 166, 179–181, 179f Candida albicans lectins, 33t Caries biofilm, 37t Catheters Hickman biofilm, 37t urinary cystitis, 37t CD3+ cells, 140–141 TCR signal transduction, 178–179 CD4+ cells, 140–141, 151–153, 215 functionally distinct subsets, 154–161 interleukin-4, 165–166 memory, 148 CD8+ cells, 151–152, 215 memory, 149, 150 functionally distinct subsets, 154–161 Cellular redox genes, 87t Central memory T cells (TCM), 147f Chemokine circuit GC, 188

Index Chlamydia bacterial type II secretion mechanism, 34 host cell invasion, 34 Chlamydia pneumoniae, 21t Ciprofloxacin, 40 Clonal expansion of B cells within GC, 189–191 Clostridia, 23t Clostridium difficile lectins, 33t CMV, 45t, 46 Contact lens biofilm, 37t Coronaviruses, 21t, 45t COX-2, 90–94 CXCL 13, 188 Cyclic AMP, 93 Cystitis urinary catheter biofilm, 37t Cytokine(s) natural killer cells, 110–112 produced by neutrophils, 99t signaling suppressor of, 220 Cytomegalovirus (CMV), 45t, 46 D DC. See Dendritic cells (DC) Death leading causes, 5t Dendritic cells (DC), 162–166, 164f, 181 dermal, 163 follicular, 186 interdigitating, 186 interstitial, 163 Langerhan’s cell-derived, 163 myeloid, 163 plasma cytoid, 163 Dental caries biofilm, 37t Dermal dendritic cells (DC), 163 Developed nations population, 3f

Index Dietary restriction vs malnutrition, 218–220 Drug efflux pumps, 40 E Effector memory T cells (TEM), 147f Elderly. See also Aging infectious diseases, 20t United States projections, 4 Endocarditis native valve biofilm, 37t Enterobacteria, 23t Enterococci, 21t vancomycin, 41 Enteropathogenic Escherichia coli (EPEC), 32 Epstein-Barr virus cell membrane receptors, 44t Erythrocytes, 32 ESBL, 40 Escherichia coli, 21, 21t, 29 bacterial type II secretion mechanism, 34 host cell invasion, 34 lectins, 33t Eubacteria, 23t Extended spectrum beta-lactamases (ESBL), 40 F FDC, 186 Flora normal gastrointestinal, 23t Follicular dendritic cells (FDC), 186 Free radicals, 12 Fungal infections, 50–51 Fungi, 23t Future health, 3–4 G Gastrointestinal tract infections, 22 normal flora, 23t

235 GC, 184–190 actions within, 189–191 B-cell follicular microenvironment, 187 Gene product alpha growth-related, 99 Genes, 13 cellular redox, 87t expression profiling, 217 transcription, 85 Geriatric infectious diseases, 20t Germinal centers (GC), 184–190 actions within, 189–191 B-cell follicular microenvironment, 187 Gerontogenes, 13 virtual, 13 Glycosylphosphatidylinositol (GPI), 169 Gompertz hazard function, 8 GPI, 169 Growth-related gene product alpha, 99 Growth-related oncogene, 99 H Haemophilus influenzae host cell invasion, 34 lectins, 33t respiratory infection, 21t Health future, 3–4 Helicobacter pylori lectins, 33t Herpes simplex, 45t cell membrane receptors, 44t Herpes simplex virus 1 (HSV-1), 46 Herpes simplex virus 2 (HSV-2), 46 Herpesviridae, 46 Hickman catheters biofilm, 37t HIF, 89 HIF-1 beta, 89 HIV, 43 cell membrane receptors, 44t

236 Hospitalization rate, 6 Host cells bacterial invasion, 34–35 HSV-1, 46 HSV-2, 46 Human immunodeficiency virus (HIV), 43 cell membrane receptors, 44t Humoral immune response Th2 cells, 154–155 Hydrogen peroxide, 75–76 Hypoxia-inducible factor (HIF), 89 Hypoxia-inducible factor 1 beta (HIF-1 beta), 89 I ICU pneumonia biofilm, 37t IDC, 186 IL-2, 151 aging, 158t production, 177 IL-3 aging, 158t IL-4 aging, 158t CD4+ T cells, 165–166 Th1/2 differentiation, 162–163 IL-5 aging, 158t IL-10 aging, 158t production, 177 Th1/2 differentiation, 162–163 IL-12, 78–79 aging, 158t IL-15, 151 Immune response humoral Th2 cells, 154–155 Immune system, 213–231 Immunity adaptive/acquired, 135–197 innate pattern recognizing receptors, 62–72

Index Immunosenescence reduced calorie intake, 214–217 Infectious disease(s) aging, 5–7, 6f bacterial, 20–24 age-associated susceptibility, 24–28 mucosal surfaces, 28–42 biliary tract biofilm, 37t fungal, 50–51 gastrointestinal tract, 22 geriatric, 20t morbidity, 6 nosocomial biofilm, 37t resistance, 19–53 natural/innate, 61–117 respiratory tract, 20–21 Haemophilus influenzae, 21t pathogens, 21t urinary tract, 20–21 pathogens, 21t viral, 42–47 Influenza, 5, 21t, 45t, 215 cell membrane receptors, 44t mortality, 216f Innate immunity pattern recognizing receptors, 62–72 Innate infection resistance senescence, 61–117 Interdigitating dendritic cells (IDC), 186 Interferon gamma, 91 aging, 158t Interleukin-2 (IL-2), 151 aging, 158t production, 177 Interleukin-3 (IL-3) aging, 158t Interleukin-4 (IL-4) aging, 158t CD4+ T cells, 165–166 Th1/2 differentiation, 162–163

Index Interleukin-5 (IL-5) aging, 158t Interleukin-10 (IL-10) aging, 158t production, 177 Th1/2 differentiation, 162–163 Interleukin-12 (IL-12), 78–79 aging, 158t Interleukin-15 (IL-15), 151 Interstitial dendritic cells (DC), 163 Isotype switching within GC, 189–191 J–K Japan life expectancy, 2 Killer-cell immunoglobulin-like receptor, 107 Killer cells lymphokine-activated, 105–115 kir, 107 KIR (killer-cell immunoglobulin-like receptor), 107 Klebsiella, 21, 21t Klebsiella pneumoniae lectins, 33t L Lactam, 39 Lactamases, 39 Lactobacilli, 23t Langerhan’s cell-derived dendritic cells (DC), 163 Lectins-glycoconjugates bacterial, 33t Legionella pneumophilia, 21t, 29 Leishmania mexicana, 29 Leptin, 219 Life expectancy increase, 2t Japan, 2 limits, 7–11 mean, 9 Life-span extension reduced calorie intake, 217–218

237 Lipid rafts, 169 Listeria monocytogenes, 26–28 host cell invasion, 34 Longevity, 213–231 rectangularization, 11 Lymph node stained section, 185f Lymphoid organs peripheral B-lymphocytes, 193f secondary APC, 186 Lymphokine-activated killer cells, 105–115 M Macrophages, 72–79, 83–84 age-related changes, 81–96 endocrinological influences, 94–96 reactive oxygen intermediates, 82–83 Malnutrition vs dietary restriction, 218–220 Mannose-binding protein, 62–63 Mannose receptor, 63–65 fragments, 64t MARCO, 70 Maximum life-span (MLS), 214 MCV, 43 Memory T cells, 148, 149, 150 aging, 151–152 effector, 147f elaboration and maintenance, 145–151 functionally distinct subsets, 154–161 Microorganisms ingestion and intracytoplasmic fate, 73–74 MLS, 214 Molluscum contagiosum virus (MCV), 43 Monocytes, 72–79 Morbidity infectious diseases, 6

238 Mortality aging, 8 Mycobacterium host cell invasion, 34 lectins, 33t Mycobacterium tuberculosis, 24–26 biofilms, 36 Mycoplasma galliseptum lectins, 33t Myeloid dendritic cells (DC), 163 Myeloperoxidase, 77f N Nalidixic acid, 40 Native valve endocarditis biofilm, 37t Natural/innate infection resistance senescence, 61–117 Natural killer (NK) cells, 105–115, 181 age-related impairment, 112–115 cytokines, 110–112 receptors, 106–110 Natural killer T (NK-T) cells, 109–110 cytokines, 110–112 Neutrophils, 96–105 aging, 103–105 antimicrobial functions, 96–100 cytokines produced by, 99t mobilization, 100–102 NFAT. See Nuclear factor of activated T cells (NFAT) Nitrogen, 75–76 Nitrogen oxide, 76, 83–84 NK cells, 105–115, 181 age-related impairment, 112–115 cytokines, 110–112 receptors, 106–110 NK-T cells, 109–110 cytokines, 110–112 Nosocomial infection biofilm, 37t Nuclear factor of activated T cells (NFAT), 165–166 calcineurin, 165, 166, 179–181, 179f translocation, 179f

Index transcription factors Th1 differentiation, 166 translocation, 179 Nutrition, 12, 213–231 O Older population. See also Aging infectious diseases, 20t United States projections, 4 Oldest humans worldwide, 2t Onchocerca ochengi, 48 Oncogene growth-related, 99 Orthopedic devices biofilm, 37t Osteomyelitis biofilm, 37t Oxidative damage, 12 P PALS, 184–185 Parasitemia course, 51f Pattern recognizing receptors (PRR) innate immunity, 62–72 PC export within GC, 189–191 Periarteriolar lymphoid sheath (PALS), 184–185 Periodontitis biofilm, 37t Peripheral lymphoid organs B-lymphocytes, 193f Peripheral T cells functions and diversity, 145–172 Peroxisome proliferator-activated receptors (PPAR), 71–72 PGE, 90–94 PGE2, 90 Phagocytic cells, 72–79, 96–105 microbial evasion, 80–81 professional, 72–79 Phagolysosome, 74

Index Plasma cell (PC) export within GC, 189–191 Plasma cytoid dendritic cells (DC), 163 Plasma membrane, 168 Pneumonia, 5 ICU biofilm, 37t Population demographic shift, 2–3 developed vs underdeveloped nations, 3f older United States, 4 Poxvirus, 43 PPAR, 71–72 Precursor T cells maturation, 156f Professional phagocytes, 72–79 Prostaglandin (PGE), 90–94 Prostaglandin E2 (PGE2), 90 Prostatitis biofilm, 37t Proteins molecular crosslinking, 13 postsynthetic modifications, 13 Proteus, 21, 21t Protozoan parasites, 47–50 PRR innate immunity, 62–72 Pseudomonas aeruginosa, 21, 21t bacterial type II secretion mechanism, 34 biofilms, 36 Q–R Quinolone-resistant bacteria, 39 Quorum sensing, 36–38 RCI, 213–217 immunosenescence, 214–217 life-span extension, 217–218 Reactive nitrogen intermediates (RNI), 75–76, 83–84 dysregulation, 85 Reactive oxygen, 75–76

239 Reactive oxygen intermediates (ROI), 75–76 dysregulation, 85 macrophages, 82–83 Reduced calorie intake (RCI), 213–217 immunosenescence, 214–217 life-span extension, 217–218 Respiratory syncytial, 21t Respiratory syncytial virus, 45t cell membrane receptors, 44t Respiratory tract infection, 20–21 Haemophilus influenzae, 21t pathogens, 21t Rhinoviruses, 21t, 45t RNI, 75–76, 83–84 dysregulation, 85 ROI, 75–76 dysregulation, 85 macrophages, 82–83 S Salmonella host cell invasion, 34, 35 lectins, 33t Salmonella typhimurium, 28 SEB, 176 Secondary lymphoid organs APC, 186 organization, 184–189 Senescence theories, 11–14 Septicemia, 5 Serratia marcescens lectins, 33t Shigella bacterial type II secretion mechanism, 34 host cell invasion, 34 Shigella flexneri lectins, 33t Signaling, 85 SOCS-3, 220 Spleen stained section, 185f

240 SR-A, 69–71 SR-B, 69–71 SR-C, 69 SR family, 69–71 Staphylococci, 23t vancomycin, 41 Staphylococcus aureus lectins, 33t Staphylococcus enterotoxin B (SEB), 176 Streptococci, 23t Streptococcus pneumoniae, 21t, 30–31 Streptococcus pyogenes lectins, 33t Supressor of cytokine signaling-3 (SOCS-3), 220 Survival curve rectangularization, 9, 10f Sutures biofilm, 37t T Taxol, 67 T cell(s). See also CD3+ cells; CD4+ cells; CD8+ cells activated nuclear factor. See Nuclear factor of activated T cells (NFAT) aging, 136–144 cognizable effects, 175–176 antigens, 141–142 APC interaction, 171 memory. See Memory T cells natural killer. See Natural killer T (NK-T) cells peripheral functions and diversity, 145–172 precursor maturation, 156f signaling immunological synapse, 167–172 T cell antigen receptor (TCR), 148, 169–172 CD3 complex

Index signal transduction, 178–179 signaling, 171 T cell receptor excision circles (TREC), 139 TCM, 147f TCR, 148, 169–172 CD3 complex signal transduction, 178–179 signaling, 171 T-dependent germinal cell formation aging, 191–194 TEM, 147f Th cells phenotypic characteristics, 154t Th1 cells differentiation interleukin-4, 162–163 NFAT transcription factors, 166 Th2 cells differentiation interleukin-4, 162–163 humoral immune response, 154–155 T helper 1 (Th1) cells differentiation interleukin-4, 162–163 NFAT transcription factors, 166 T helper 2 (Th2) cells differentiation interleukin-4, 162–163 humoral immune response, 154–155 T helper (Th) cells phenotypic characteristics, 154t Thymus age-associated dysfunction, 143–144 aging, 136–144 Tir (translocated intimin receptor), 32 Toll, 65–69 Toll-like receptors, 65–69 Toxoplasma gondii, 48, 50 Translocated intimin receptor, 32 Transposons, 41 TREC, 139

Index Trichinella spiralis, 48 Trypanosoma musculi, 49, 50 Tuberculosis, 24–26 Type IV pili, 36–37 U Underdeveloped nations population, 3f Uridine triphosphate (UTP), 93 Urinary catheter cystitis biofilm, 37t Urinary tract infection, 20–21 pathogens, 21t UTP, 93 V Vancomycin, 41 Vancomycin-resistant enterococci, 39 Varicella-zoster virus (VZV), 45t, 46 Vascular grafts biofilm, 37t

241 Vibrio cholerae bacterial type II secretion mechanism, 34 host cell invasion, 34 lectins, 33t Viral infections, 42–47 Virtual gerontogenes, 13 Viruses, 45t cell membrane receptors, 44t intracellular defense strategies, 48t VZV, 45t, 46 W–Y Worldwide aging, 2 Yersinia, 29 bacterial type II secretion mechanism, 34 host cell invasion, 34

INFECTIOUS DISEASE ™ VASSIL ST. GEORGIEV, Series Editor

Aging, Immunity, and Infection Joseph F. Albright and Julia W. Albright George Washington University School of Medicine, Washington, DC

With a growing world population of the aged in a state of immunological decline, there is an urgent need to develop new methods to delay or, better yet, prevent the loss of immune function. In Aging, Immunity, and Infection, the prominent immunogerontologists, Joseph and Julia Albright, critically review the major features and functions of the immune system that are most likely, or known, to be significantly altered by aging, and offer insightful analyses of the consequences for those aging subjects who must cope with infection. Topics of special interest include the demographics and theories of immunosenescence, the gradual breakdown of resistance to infection in the aged, and the effects of aging on selected mechanisms of both innate and adaptive immunity to infections. The Albrights also suggest how advances may be made in understanding the basic biology of immunosenescence, newer methods of treatment and prevention, and offer an evaluation of such provocative ideas as nutritional intervention and lifespan extension in immunosenescence. Chapter summaries—along with lists of key research areas and recent advances—provide a framework for greater insight into major aspects of the problem and its emerging solutions. Informative and forward-looking, Aging, Immunity, and Infection offers geriatricians, infectious disease specialists, and immunologists a state-of-the-art understanding of the deleterious effects of aging on the immune system, even as it provides a basis for research on how best to strengthen immunity in the elderly and reduce their susceptibility to infectious diseases. Features • Review of the effects of aging on innate and adaptive immunity to infectious diseases • Suggestions for advancing research in key areas of investigation

• Cutting-edge information on the pathogenesis of infections in geriatric patients • Critical discussion of lifespan extension and nutritional delay of immunosenescence

Contents 1 Human Aging: Present and Future. Demographics. Infectious Diseases of the Aging. Limits on Life Expectancy and Future Prospects. Theories of Senescence. Chapter Summary. References. 2 Aging and Altered Resistance to Infection. Relatively Common Bacterial Infections of Aging Humans. Selected Examples of Age-Associated Susceptibility to Bacterial Infections. Bacterial Interactions with Mucosal Surfaces. Antibiotic Resistance and Bacterial Variation. Viral Infections in Aging Humans. Protozoan Parasites in Aging Subjects. Fungal Infections in Aging Subjects. Chapter Summary. References. 3 Senescence of Natural/Innate Resistance to Infection. Pattern Recognizing Receptors of Innate Immunity. Phagocytic Cells: Monocytes/Macrophages. Microbial Evasion of

Phagocytic Destruction. Age-Related Changes in Macrophages. Phagocytic Cells: Neutrophils. Natural Killer/LymphokineActivated Killer Cells. Chapter Summary. References. 4 Aging of Adaptive/Acquired Immunity. Aging of the Thymus and Thymus-Derived (T) Cells. The Functions and Diversity of Peripheral T Cells. Summary: Known and Cognizable Effects of Aging T Cells. Differentiation, Functions, and Aging of B Cells. Chapter Summary. References. 5 Nutrition, Longevity, and Integrity of the Immune System. RCIMediated Delay of Immunosenescence. How Does RCI Promote Life-Span Extension? Dietary Restriction vs Malnutrition. References. Epilogue. Index.

90000

Infectious Disease™ AGING, IMMUNITY, AND INFECTION ISBN: 0-89603-644-8 E-ISBN: 1-59259-402-6 humanapress.com

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