Alpha-MSH

-MSH James M. Lipton1,* and Anna Catania2 1

Zengen Inc., 21800 Oxnard Street, Suite 980, Woodland Hills, CA 91367, USA

2

Third Division of Internal Medicine, Ospedale Maggiore di Milano IRCCS, Via F. Sforza 35, 20122 Milano, Italy * corresponding author tel: 818-887-8688, fax: 818-884-5988, e-mail:[email protected] DOI: 10.1006/rwcy.2000.13002.

SUMMARY -Melanocyte-stimulating hormone (-MSH), a melanotropin or melanocortin (ACTH- or -MSH-like) peptide, is known for its neuroimmunomodulatory properties. This molecule serves as an endogenous mediator in the brain, pituitary, circulation, peripheral tissues, and/or between host cells that modulates the production and actions of proinflammatory agents. Exogenous -MSH suppresses inflammatory responses in animal models. Furthermore, the peptide is increased in clinical inflammatory disease in humans, presumably as a natural countermeasure to inflammation. The tridecapeptide -MSH and its C-terminal tripeptide amino acid sequence KPV (MSH 11±13) have parallel anti-inflammatory effects in animal models and in in vitro tests on host cells. There is recent evidence that these peptides also have antimicrobial properties that can benefit the host.

BACKGROUND

Discovery The history of -MSH began with an initial observation of the influence of the pituitary gland in control of skin color in amphibia (Eberle, 1988). There were many attempts to identify the amino acid sequence of isolates of highly purified skin-darkening agents. Harris and Lerner (1957) determined the amino acid sequence of porcine -MSH, a tridecapeptide with blocked N-terminus and amidated C-terminus. Subsequent studies revealed that the -MSH

Cytokine Reference

sequence is part of ACTH; isolation and structural analysis of -MSH from a number of mammals and fishes showed a high sequence conservation of the molecule during evolution. The importance of the peptide in the control of inflammation, fever, and microbial invasion was not known for many years after the amino acid sequence was described.

Alternative names -MSH was originally named for its capacity to darken skin color in amphibia. The peptide was subsequently called melanotropin. According to the IUPAC-IUB convention, the full name of the hormone that covers melanin dispersion is melanocytestimulating hormone, abbreviated MSH, and the trivial name is melanotropin. The prefix  denotes the basic tridecapeptide derived from ACTH (Eberle, 1988). The term opiomelanocortins is generally used for any and all of the peptides derived from the proopiomelanocortin precursor. Melanocortin relates only to ACTH/-MSH-derived peptides.

Structure -MSH is derived from the precursor proopiomelanocortin (POMC) (Figure 1). The genetic code for MSH resides in the third exon of the POMC gene that also contains the genetic information for -LPH, ACTH, and related peptides. Like other prohormones, cleavage sites of POMC occur between pairs of basic amino acid residues (-Lys-Lys-, -ArgLys-, -Arg-Arg-, -Lys-Arg-) that are recognized by

Copyright#2000 Academic Press

1384 James M. Lipton and Anna Catania specific endopeptidases. Two of these enzymes, PC1 and PC2, are involved in processing POMC peptides. Their cleavage specificities are distinct: PC1 cleaves POMC into corticotropin and -lipotropin; PC2 cleaves POMC into -endorphin and -MSH (Benjannet et al., 1991). -MSH (1±13) is cleaved from the ACTH amino acid sequence and therefore shares the peptide backbone of ACTH (1±13). However, -MSH lacks the amino acid sequence required to stimulate adrenal cells. The -MSH peptide is normally acetylated at the N-terminal and amidated at the C-terminal; the predominant form in the pituitary is diacetylated. Although the -MSH 6±9 amino acid sequence (His Phe Arg Trp) is considered the core sequence underlying the melanotropic activity of the peptide, it is clear that the 11±13 amino acid sequence (Lys Pro Val) is especially important in host response functions (Lipton and Catania, 1997).

Main activities and pathophysiological roles Details of the activities of -MSH peptides are provided in sections below. In brief, the peptides modulate fever and inflammation and defend against microbial invasion. It is notable that in in vivo tests in which the peptides are administered alone in dosage adequate to influence host responses, there is no discernible influence on normal physiology. Extremely high doses are required to lower normal temperature; it has not been possible to reach lethal concentrations except by injecting massive amounts into the cerebral ventricles. The latter concentrations are unlikely to be reached even after systemic administration of any quantity. From the evidence currently in hand, it appears that the peptides exert salutary control over host responses, preventing them from reaching dangerous levels, and they directly kill invading microorganisms. That their influences are part of an ancient control system is supported by the rises in circulating peptides during host challenge and in disease. The proposed role of these peptides in pathophysiology is that of defender against excessive self-responses and against direct invasion.

GENE AND GENE REGULATION

Accession numbers Accession numbers for the human POMC gene, exon three: J00292, J00293, V0569

Chromosome location In humans, the chromosome site for the POMC gene, the precursor of -MSH, is 2 P (Zabel et al., 1983).

Regulatory sites and corresponding transcription factors -MSH is derived from the third exon of POMC DNA. A single gene encodes POMC in humans; this gene is unusual in that it possesses three promoter regions that control transcription: P1, P2, and P3. P2 controls transcription in the normal pituitary gland; P3 is weakly active in a variety of peripheral tissues; P1 is the predominant promoter in some cancers (Kraus et al., 1993).

PROTEIN

Sequence See Figure 1.

Description of protein As indicated above, -MSH is a tridecapeptide. It is normally N-acetylated and amidated at the C-terminus.

Important homologies -MSH has the same amino acid sequence as the domain of ACTH from which it is derived. -MSH shares the Met Glu His Phe Arg Trp Gly sequence with -MSH.

Figure 1 -MSH derivation and amino acid sequence.

-MSH 1385

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Knowledge of -MSH distribution in tissues has changed substantially with development of modern localization techniques. It is now clear that low concentrations of -MSH are, or can be, expressed ubiquitously. However, there are distinct regional differences in that certain cells express the peptide constitutively (Table 1). It is remarkable that particularly high concentrations of -MSH are found in the brain and in barrier organs such as skin and gut. -MSH was originally isolated from the pars intermedia of the pituitary gland. Small amounts of the peptide are also synthesized by the corticotrophs of the pars distalis, especially in young animals (Usategui et al., 1976). Immunocytochemical analysis of normal adult pituitaries for -MSH-containing cells showed that, although there is great interindividual variation in the number of -MSH cells in both the pars intermedia and pars distalis, the cells are usually more concentrated in the latter site (Coates et al., 1986). The number of melanotrophs in the human pituitary decreases with age; pituitaries of anencephalics do not contain -MSH cells (Visser and Swabb, 1979). -MSH occurs in widespread regions of the brain in a distribution that is relatively consistent across species (Eberle, 1988). Immunoreactive cell bodies are concentrated in three regions: first, the arcuate or infundibular nucleus of the hypothalamus, from which fibers project throughout the hypothalamus and into the preoptic region, thalamus, midbrain (periaqueductal gray), amygdala, lateral septum, and associated telencephalic structures; second, the nucleus tractus solitarius of the brainstem, from which both locally distributed and descending fibers arise; and third, the dorsolateral hypothalamus/zona incerta, with projections that extend into the dorsal hippocampus and cerebral cortex. Whereas cells in the first two regions contain POMC mRNA and POMC derivatives such as -endorphin (Bloch et al., 1979;

Table 1 Some cell types that secrete -MSH Melanotrophs of pars intermedia and pars distalis

Eberle, 1988

Corticotrophs in humans

Eberle, 1988

Human melanoma

Loir et al., 1997, 1998

Placental cells

Kreiger, 1982

Larsson, 1980; Civelli et al., 1982; Gee et al., 1993), cells in the dorsolateral hypothalamus do not stain for other POMC peptides (Guy et al., 1980). Although evidence from studies of human tissue is limited, -MSH is most concentrated in the pituitary (nanomolar range), whereas it occurs in lower but substantial concentrations in the hypothalamus, locus coeruleus, and the substantia innominata (DeÂsy and Pelletier, 1978; O'Donohue et al., 1979; Arai et al., 1986). The highest concentration of immunoreactive -MSH within the brain is in the hypothalamus (Arai et al., 1986), where the peptide exists mainly in the desacetylated form (Parker et al., 1981); substantial concentrations also occur in the substantia innominata (Candy et al., 1985). The peptide is likewise found in the cerebrospinal fluid (CSF) of monkeys and humans (Rudman et al., 1973). Human CSF also contains an anti--MSH protein (20±40 kDa) that binds/neutralizes the peptide (Rudman et al., 1974; Scimonelli and Eberle, 1987). It may be, because -MSH receptors are estimated to be 35±45 kDa proteins (Scimonelli and Eberle, 1987; Mountjoy et al., 1992), that this antagonist is simply an excess of unattached receptors. This would be conceptually similar to the circumstance of soluble cytokine receptors, such as the soluble IL-1 and TNF receptors, that are believed to modulate the actions of the cytokines by binding them. In considering the role of -MSH in modulation of host responses, it is appropriate to recognize that the peptide is located, as stated above, within barriers to invasion and challenge, such as the skin and gut. -MSH occurs in the skins of both albino and pigmented rats and in human epidermis (Thody et al., 1983); -MSH in rat skin is not of pituitary origin because it is also present in hypophysectomized animals (Thody et al., 1983). Immunoreactive -MSH is found in the mucosal barrier of the gastrointestinal tract, especially concentrated in the duodenum but also found in substantial concentrations in the ileum, jejunum, and colon in intact and hypophysectomized rats (Fox and Kraicer, 1981). The nonhypophysial origin of the peptide within these barriers between the host and the external environment is not proof of a suppressive host response influence of the local peptide; however, with evidence of the anticytokine actions of -MSH described below, the presence of the peptide in these barriers is consistent with such an influence. -MSH also occurs in other peripheral tissues such as the placenta (Clark et al., 1978), testes, and ovaries (Bardin et al., 1987), and the adrenal medulla (Evans et al., 1983). Although in these sites the molecule might participate in entirely disparate functions, it is not unreasonable to ask if its role in these peripheral

1386 James M. Lipton and Anna Catania tissues is also to modulate actions of cytokines produced in the course of normal metabolism and during host challenge and/or to counter microbial invasion. Of particular importance, -MSH is produced by multiple cell types in response to host challenge (Table 2). Of great importance to understanding the role of -MSH in host responses is the variation in its concentration in the circulation in disease and in Table 2 Production of -MSH by cells activated in the host response Human monocyte/ macrophages

Rajora et al., 1996

Murine monocyte/ macrophages

Star et al., 1995

Lymphocytes

Smith and Blalock, 1995

Keratinocytes

Chakraboty and Pawelek, 1993

Murine microglia

Delgado et al., 1998

acute challenge to the host. An increased plasma MSH concentration would indicate that the molecule becomes available to influence host reactions taking place in widespread regions of the body. Such changes in plasma -MSH concentration do occur; they are described in a following section.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators -MSH is induced by proinflammatory agents (Table 3) and, in turn, the peptide inhibits the production of proinflammatory mediators (Table 5). It is likely that the latter influence occurs via prevention of activation of nuclear factor B (NFB), as described in the section on In vitro findings. Inhibition of this transcription factor by -MSH results from preservation of IB.

Table 3 Examples of -MSH inducers

RECEPTOR UTILIZATION

LPS

Catania et al., 1998a

Antigen

Taylor and Streilein, 1996

LPS + IFN

Delgado et al., 1998

PMA

Lipton et al., 1999

TNF

Rajora et al., 1996

HIV envelope glycoprotein gp 120

Catania et al., 1998b

The receptors that underlie the effects of -MSH and related melanocortins have been identified and cloned. Five G protein-linked receptors (MC-1R through MC-5R) are currently recognized. When transfected into carrier cells, these receptors increase intracellular cAMP upon stimulation with melanocortin molecules. The receptors are widely distributed in peripheral tissues and in the brain (Tatro, 1996). All of the receptors react to -MSH and one subtype,

Table 4 Melanocortin receptor subtypes and loci in host cells and brain Receptor subtype

Loci

References

MC-1R

Human monocyte/macrophages

Rajora et al., 1996

Rodent monocyte/macrophages

Star et al., 1995

Human neutrophils

Catania et al., 1996

Periaqueductal gray

Xia et al., 1995

Whole murine brain

Rajora et al., 1997a

MC-2R

Adrenal cortex

Mountjoy et al., 1992; Cammas et al., 1995

MC-3R

Human monocyte/macrophages

Taherzadeh et al., 1999

Hypothalamus, septum, thalamus, other

Gantz et al., 1993; Roselli-Rehfuss et al., 1993

MC-4R

Limbic structures, hypothalamus, hindbrain, spinal cord

Mountjoy et al., 1992; Gantz et al., 1993

MC-5R

Human monocyte/macrophages

Chhajlani and Wikberg, 1992; Taherzadeh et al., 1999

Human and mouse brains

Chhajlani and Wikberg, 1992; Gantz et al., 1994

-MSH 1387 MC-2R, is believed to be the ACTH receptor. As indicated in Table 4, there are differences in ligand specificity of the receptor subtypes. All subtypes have been demonstrated in human cells and several have been isolated from other species. These receptors are the smallest G protein-linked receptors yet described and, like the anti-inflammatory effects of the peptides, appear to be highly conserved across species. That melanocortin peptides characteristically increase intracellular cAMP is consistent with evidence that increases in cAMP in host cells are associated with anti-inflammatory influences. There is evidence that the IP3 pathway is also activated in cells transfected with MC-3R plasmid vector (Konda et al., 1994).

IN VITRO ACTIVITIES

In vitro findings The primary effects of -MSH in vitro are inhibition of production and action of proinflammatory cytokines (Table 5) and preservation of I and Table 5 -MSH inhibits production of proinflammatory mediators IL-1

Catania et al., 1998a, 1998b

IL-2

Taylor and Streilein, 1996

IL-6

Catania et al., 1998a, 1998b

IL-8

Lipton et al., 1999

TNF

Rajora et al., 1997a

IFN

Lipton and Catania, 1997

Neopterin

Rajora et al., 1996

Nitric oxide

Star et al., 1995

therefore inhibition of NF activation (Table 6). The -MSH peptides also inhibit PMN migration (Catania et al., 1995) and they induce antiinflammatory IL-10 (Bhardwaj et al., 1996). -MSH peptides likewise inhibit HIV-1 in chronically infected U1 cells and in actively infected monocyte/macrophages (Barcellini et al., 1998). Antimicrobial Activity of -MSH Peptides -MSH and shorter amino acid sequences derived from it have recently been found to have antimicrobial influences against two major representative pathogens: Staphylococcus aureus and Candida albicans. -MSH (1±13) and its C-terminal tripeptide (11±13, KPV) significantly inhibited S. aureus colony formation and reversed the enhancing effect of urokinase on colony formation. Antimicrobial effects occurred over a broad range of concentrations, including the physiological (picomolar) range. Lower concentrations of -MSH peptides likewise reduced viability and germ tube formation of the yeast C. albicans. The most effective peptides were those bearing the C-terminal tripeptide of the -MSH sequence, i.e. -MSH (1±13), (6±13), and (11±13). The -MSH sequence (4±10), important for melanotropic effects, was also effective but significantly less potent. ACTH (1±39), the precursor of -MSH, and an intermediate sequence of it, ACTH (18±39), which does not include the -MSH amino acid sequence, had no significant candidacidal effects. These antimicrobial influences of -MSH peptides are probably mediated by their induction of cAMP. Indeed, this messenger was significantly augmented in peptidetreated yeast and the adenylyl cyclase inducer forskolin significantly reduced C. albicans colony formation. Reduced killing of pathogens is a detrimental consequence of therapy with common anti-inflammatory drugs and -MSH has potent anti-inflammatory

Table 6 -MSH inhibits NFB activation and IB degradation Cells/tissue

Peptide influence

Reference

U-937 cells (human lymphoma)

Inhibited NFB activation/degradation of IB

Manna and Aggarwal, 1998

Human glioma in vitro/ murine brain in vivo

Inhibited NFB activation/degradation of IB

Ichiyama et al., 1999a

Murine brain in vivo, systemic peptide

Inhibited NFB activation/degradation of IB

Ichiyama et al., 1999b

Peripheral NF / CNS peptide in mouse

Inhibiton of peripheral activation of NFB by systemic peptide

Ichiyama et al., 2000a, 2000b

Human glioma

Autocrine regulation of NFB activation

Ichiyama et al., 1999a

1388 James M. Lipton and Anna Catania effects. Therefore, it was important to determine whether -MSH inhibits C. albicans killing by human neutrophils. -MSH peptides did not reduce killing but rather enhanced it, likely as a consequence of its direct antimicrobial activity. The addition of antimicrobial activity to the established anti-inflammatory and antipyretic effects suggest that the peptides might be useful in treatment of disorders in which both inflammation and infection occur.

Regulatory molecules: Inhibitors and enhancers Research on enhancement and inhibition of in vitro activities of -MSH has been limited. In general, LPS and proinflammatory cytokines induce production and release of the peptide. Any agents that modulate their stimuli reduce production of the peptide.

Bioassays used -MSH peptides characteristically increase cAMP within those cells that bear melanocortin receptors. Thus, such increases in normal or receptor plasmid transfected cells can be used to screen for receptor ligands, establish antagonists, etc.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles -MSH peptides have clear effects on pigmentation in amphibians; the precise role of dermal -MSH in human skin pigmentation, if any, remains unclear. The ACTH peptide occurs in higher concentrations in the skin, it acts on similar receptors, and it appears to be more potent than -MSH in stimulating melanogenesis. Thody and Graham (1998) suggest that -MSH should not be viewed solely as a pigmentary peptide since it has many different actions and its primary role in the skin may be to maintain homeostasis. The widespread distribution of -MSH and its receptors make it difficult to link this agent to a specific function or functions. It is unlikely that a peptide with such a lengthy history has a role in a single function such as regulation of coat color or body weight (Hagen et al., 1999). However, the

potency of the peptide in control of inflammation and microbial invasion, the most ancient host responses, suggest that the peptide is pivotal in these functions.

Species differences To date there are no established differences across species in host responses to -MSH ligands. The antipyretic and anti-inflammatory effects of the peptides have been demonstrated in mice, rats, rabbits, cats, dogs, and squirrel monkeys. The consistency is perhaps not surprising given the consistency across species of the amino acid sequences in -MSH peptides.

Knockout mouse phenotypes There are no -MSH knockout animals to date. This is in part because the peptide is derived from POMC and no animals have been produced in which genes for PC1/PC2 are specifically disrupted. There are mice deficient in two melanocortin receptor subtypes: MC-1R and MC-4R. The phenotype for MC-1R mice is yellow coat and adult obesity (recessive yellow mouse). Recent evidence indicates that -MSH peptides have anti-inflammatory effects in the MC-1R knockout animals (Lipton et al., 1999), indicating that this receptor subtype is not essential for this function. As with other POMC-derived peptides, it appears that related receptor subtypes can and do participate when one subtype is deleted. Inactivation of MC-4R results in maturity-onset obesity associated with hyperphagia, hyperinsulinema, and hyperglycemia (Huszar et al., 1997). Whether MC-4R knockout animals have deficient anti-inflammatory response to -MSH peptides has not been established.

Transgenic overexpression At this time no transgenic animals overexpressing -MSH have been produced. However, in preparation for development of such animals, a plasmid vector encoding -MSH and containing enhancers and promoters driving transcriptions of IL-6 and -MSH genes was inserted into human glioma cells (Ichiyama et al., 2000a). NFB activation induced by LPS was inhibited in the transfected cells. Western analysis indicated that this inhibition was linked to preservation of IB2 protein. Chloramphenicol acetyltransferase assay indicated that NFB-dependent reporter gene was suppressed in glioma cells with -MSH vector. Fluorescence staining confirmed that

-MSH 1389 these glioma cells express -MSH receptors. The combined evidence of -MSH receptors, -MSH production, and -MSH production-associated inhibition of the inflammatory NFB pathway suggests an autocrine circuit based on the peptide in these transfected cells. These results predict that animals that overexpress -MSH will have reduced inflammatory reactions. Agouti protein antagonizes the effects of -MSH and other melanocortins. The molecule appears to compete with -MSH for the melanocortin receptor MC-1R. It is believed that there is a novel signaling mechanism whereby -MSH and Agouti protein function as independent ligands, with each inhibiting the binding of the other and transducing opposing signals through a single receptor. It may be that such a relation with Agouti protein also occurs with other melanocortin receptor subtypes. That Agouti protein hypersecreting mice have augmented inflammatory responses was indicated by the reactions of the animals to systemic endotoxin (Lipton et al., 1999). These mice had greater increases in serum IL-6 following systemic injections of LPS. This increase may occur by virtue of inhibition of endogenous -MSH at the level of the melanocortin receptor.

Pharmacological effects To date, -MSH peptides have been shown to modulate inflammation in over 20 animal models (Lipton and Catania, 1997). These peptides suppress all in vivo forms of inflammation commonly recognized (Table 7).

Table 7

These anti-inflammatory effects are believed to result from three actions of the peptides: 1. Direct action on host cells 2. Direct action on host cells within the brain 3. Actions on melanocortin receptors in the brain Direct Action on Host Cells Although both neutrophils and macrophages are important in many types of inflammation, there is a tendency to associate neutrophils with acute or developing inflammation and macrophages with longerterm or chronic inflammatory reactions. Evidence described in the in vitro section indicates that -MSH peptides inhibit neutrophil migration in vitro (Catania et al., 1996) This inhibition also occurs in vivo (Chiao et al., 1997). Also described in the in vitro section are the effects of -MSH on NFB activation, or inflammatory cytokine production, and related host responses. Despite the latter in vitro data, there is no systemic evidence of -MSH peptide influence or macrophage counts in in vivo models of chronic inflammation. Direct Action on Host Cells within the Brain There is substantial evidence, described in the in vitro section, of direct inhibitory actions of -MSH peptides on inflammatory actions in nonneuronal cells within the brain, both astrocytes and microglia. The latter cells share fuctions of inflammatory host cells in the periphery. There is also evidence that the peptides modulate CNS inflammation within the living brain. Such an influence may be very important in view of the recent evidence that local inflammatory processes, such as the induction of TNF, contribute to

Commonly recognized forms of inflammation

Forms of inflammation

Representative references

Acute inflammation

Hiltz and Lipton, 1990 (dermal inflammation); Chiao et al., 1997 (renal inflammation)

Delayed hypersensitivity

Hiltz and Lipton, 1990 (dermal inflammation); Grabbe et al., 1996

Chronic inflammation

Ceriani et al., 1994 (arthritis model); Rajora et al., 1997b (inflammatory bowel disease model)

Systemic inflammation

Catania et al., 2000

CNS inflammation

Huh et al., 1997 (experimental stroke); Ichiyama et al., 1999a, 1999b (generalized CNS inflammation)

1390 James M. Lipton and Anna Catania Alzheimer's disease, multiple sclerosis, stroke, and other CNS disorders. Actions on Melanocortin Receptors in the Brain Actions on melanocortin receptors in the brain that activate descending anti-inflammatory neural pathways and thereby modulate peripheral inflammation have a very powerful effect which has been documented in several animal models (Table 8). The major theory of the mechanism of the peripheral anti-inflammatory action of central MSH peptides is derived from evidence of descending neural pain modulation pathways and concepts of neurogenic inflammation. In brief, inflammation is normally promoted by neural influences. In the case of an injury at a localized site in the skin of the foot, for example, neural signals travel in fine fibers to the dorsal roots of the spinal cord. Signals may ascend to the brain to signal pain, but in the case of inflammation, there is a descending train of signals back down the terminals that previously transduced sensory (afferent) information. These now efferent fibers release inflammatory agents in the vicinity of the injury or invasion. These agents, including calcitonin gene-related peptide and substance P, increase vascular permeability and induce histamine release, thereby promoting inflammation in concert with local neutrophils and other host cells. This neurogenic inflammation can be inhibited by injections into the brain ventricles of -MSH peptides. -MSH peptide receptors within the brain (perhaps MC-1R, 3R, 4R, and 5R) drive descending anti-inflammatory signals that are believed to inhibit release of the chemical mediators released from peripheral nerve fibers. The main proof of this is that severing the spinal cord

blocks the anti-inflammatory effects of centrally administered -MSH peptides. 2-Adrenergic blockers given systemically have a similar effect, indicating that a 2-receptor is part of the inhibiting circuit. It appears then that normally both central and peripheral -MSH receptors participate in modulation of inflammation. Mice with inflammation in the hind paw and spinal transection do not show an anti-inflammatory effect of central -MSH. However, these same animals with transactions show reduced inflammation when -MSH is given systemically. This result indicates that there are at least two pools of -MSH receptors that participate in control of inflammation: one pool within the brain, another in the periphery.

Interactions with cytokine network The primary conclusions regarding the influence of -MSH on the cytokine network in vivo is that the peptides modulate actions of proinflammatory cytokines and/or of production of the cytokines. Central administration of -MSH antiserum increased plasma ACTH and corticosterone responses induced by central injection of IL-1 in rats (Papadopoulos and Wardlaw, 1999). Recent evidence (Huang et al., 1999) indicates that systemic injection of LPS suppresses food intake and that -MSH suppresses this cytokine-mediated effect even further, resulting in even lower intake. Nanogram doses of -MSH given centrally block increased nociception induced by central IL-6 (Oka et al., 1995). -MSH given i.p. to mice inhibited PGE production by hippocampal tissue in vivo (Weidenfeld et al., 1995). Central -MSH also blocks the immunosuppressive effect of central IL-1 (Weiss et al., 1994).

Table 8 Inhibition of peripheral inflammation by central -MSH peptides Agents/reactions

Central peptide

Reference

Picryl chloride

-MSH 1±13

Lipton et al., 1991

Picryl chloride

NDP--MSH 1±13

Dulaney et al., 1992

IL-1

-MSH 1±13/D-Val subs

Watanabe et al., 1993

IL-1 , IL-8, LTB4, PAF

-MSH 1±13; -MSH 11±13 (IL-1 )

Ceriani et al., 1994

IL-1 / 2-adrenergic blockade or spinal transection

-MSH 1±13

Macaluso et al., 1994

Circulating TNF

-MSH 1±13

Rajora et al., 1997a

Circulating/lung, liver TNF/NO; lung MPO

-MSH 1±13; antiserum

Delgado et al., 2000

Endotoxemia

-MSH 1±13

Lipton et al., 1999

-MSH 1391 IL-1 -induced inflammation was inhibited by -MSH peptide and fragments given systemically or centrally (Watanabe et al., 1993). Migration of neutrophils into air pouches injected with IL-1 was inhibited by coadministration of -MSH (Perretti et al., 1993). Both -MSH (1±13) and 11±13 inhibited anorexia caused by central IL-1 (Uehara et al., 1992). -MSH (1±13) and D-val (11±13) inhibited acute inflammation in mice caused by IL-1 , IL-6, and TNF (Hiltz et al., 1992). In rabbits, -MSH inhibited fever caused by IL-6 and TNF (Martin et al., 1991). Ten milligrams of -MSH given i.c.v. inhibited IL-1-induced elevations in plasma ACTH and corticosterone and decreases in NK cell activity (Weiss et al., 1991). Central IL-1 in rats rapidly suppressed cellular immune responses in peripheral lymphocytes; central -MSH blocked these effects (Sundar et al., 1989). IL-1 induced fever, increased production of serum amyloid P, augmentation of circulating neutrophils were all inhibited by -MSH; the peptide also prevented IL-1 induction of corticosterone and depression of contact sensitivity (Daynes et al., 1987).

Endogenous inhibitors and enhancers The posttranslational processing of POMC, which yields the mature hormones, includes a number of steps: glycosylation, phosphorylation, tissue-specific proteolytic cleavage, amidation, and acetylation. Some of these posttranslational modifications can be regulated by neuronal factors (Lamacz et al., 1991) (Table 1). For instance, dopamine inhibits acetylation of -MSH and thus reduces secretion of the biologically active form in the frog (Lamacz et al., 1991). Administration of the dopamine antagonist haloperidol to rats resulted in a 4- to 6-fold (time- and dosedependent) increase in the concentration of pars intermedia POMC mRNA (Chen et al., 1983). In contrast, ergocryptine, a dopamine antagonist, decreased POMC mRNA in the rat pars intermedia 2- to 3-fold (Chen et al., 1983). Stress-induced activation of -MSH secretion may be due, in part, to decreased activity of tuberohypophysial dopamine neurons in the intermediate lobe (Lookingland et al., 1991). Endogenous dopamine inhibits -MSH release from the rat hypothalamus via D2-dopamine receptors (Tiligada and Wilson, 1989). There appears to be a feedback circuit between -MSH and dopamine; central -MSH selectively activates tuberoinfundibular dopaminergic neurons, thereby inhibiting its own release (Lindley et al., 1990). Although the evidence is incomplete, several other neurotransmitters likewise influence -MSH

secretion. Endogenous GABA tonically inhibits release of -MSH from rat hypothalamic slices (Mabley et al., 1991). Activation of 5-hydroxytryptamine (5-HT) receptors stimulates -MSH release and apomorphine blocks this stimulatory effect, suggesting that there is a direct antagonism between dopaminergic and serotoninergic regulation of MSH release (Carr et al., 1991). Stimulation of  opioid receptors inhibits the activity of intermediate lobe tuberohypophysial dopamine neurons and increases secretion of -MSH from the melanotrophs (Manzanares et al., 1990). -Adrenoreceptor activation causes release of -MSH in response to certain stresses, such as intermittent foot shock (Berkenbosch et al., 1984). Histamine has a stimulatory effect on MSH secretion, probably through release of epinephrine (Knigge and Warberg, 1991). Corticotropinreleasing factor (CRF), which plays a major role in induction of ACTH secretion, does not seem important in control of -MSH release. PFU 83, a rat monoclonal antibody to CRF, did not affect resting or ether-induced -MSH secretion (Van Oers et al., 1989). Also, CRF given in doses that caused a marked increase in plasma ACTH in rabbits did not promote -MSH release (Catania et al., 1991).

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects To evaluate potential use of plasma -MSH concentrations as estimates of disease, the first step was to determine normal values in healthy subjects (Catania et al., 1998a). Plasma concentration of MSH (Eurodiagnostica RIA kit, MalmoÈ, Sweden) in 234 normal blood donors was 21.30  0.63 pg/mL (mean  SE; range 1.5±75.2). There was no difference between mean concentrations of the peptide in males and females (P > 0.05). To determine whether plasma concentrations of -MSH fluctuate over time, the peptide was measured every 15 minutes over a 180minute period in five normal subjects. No major variations were observed (repeated measures ANOVA on ranks: P=0.7). To learn whether concentrations of -MSH are altered with aging, -MSH in plasma of 125 normal elderly subjects (79.63  5.8 years, range 66±95) was measured. Mean plasma -MSH in aged subjects was lower than in young controls (15.87  0.8 pg/mL, P< 0.001).

1392 James M. Lipton and Anna Catania -MSH was measured in 106 newborns at delivery and during the first week of postnatal life. -MSH concentration was greater in premature than in fullterm neonates. Plasma -MSH was more elevated in complicated than in uncomplicated delivery and the peptide significantly decreased 12 hours after birth in term newborns (Mauri et al., 1993).

Role in experiments of nature and disease states Alterations in circulating -MSH have been observed in a number of diseases (Table 9). HIV Infection In research on HIV-infected patients, plasma concentrations of -MSH in patients of different CDC groups were compared. Circulating -MSH was elevated in plasma of HIV-infected patients of the CDC groups III and IV (Catania and Lipton, 1993; Catania et al., 1994). Further research on relations between MSH and disease progression in HIV-infected patients showed that greater concentrations of -MSH are associated with reduced disease progression. The association between elevated -MSH and reduced AIDS-related events or death was even more pronounced in patients with baseline CD4+ T cells < 200/L (Airaghi et al., 1999). This observation was not surprising because production of proinflammatory

cytokines that promote HIV replication in infected cells were all inhibited by -MSH. Indeed, IL-1, IL-6, and TNF were reduced by -MSH in whole blood of HIV-positive patients (Catania et al., 1998b). Further, recent research in HIV-infected cells shows that -MSH and its C-terminal tripeptide Lys-ProVal inhibit HIV-1 replication in chronically infected promonocytic U1 cells and in acutely infected human myocytes (Barcellini et al., 1998). Sepsis Syndrome Although in normal human subjects injected with endotoxin there was an increase in plasma -MSH (Catania et al., 1995), during early phases of naturally occurring sepsis syndrome concentrations of the peptide were reduced (Catania et al., 2000). Plasma -MSH returned to normal values in patients who recovered and remained low in those who died. Reduction of -MSH is likely to have detrimental consequences in patients with sepsis syndrome and to contribute to severity of systemic inflammation. Consistent with this idea, a negative correlation was found between concentrations of -MSH and TNF in plasma of septic patients. Although it is possible that reduced -MSH and elevated TNF coexisted in patients with more severe disease, it may well be that reduction in endogenous -MSH promoted TNF production. Addition of -MSH to LPS-stimulated whole blood samples of septic patients inhibited production of TNF and IL-1 in a concentrationdependent manner.

Table 9 Plasma concentrations of -MSH in human diseases Condition

Comments

Reference

HIV infection

Increased in plasma of CDC III and IV patients

Catania et al., 1993, 1994

HIV infection

Reduced disease progression in patients with elevated plasma -MSH concentrations

Airaghi et al., 1999

Rheumatoid arthritis

Increased in synovial fluid of patients with greater inflammation; normal in plasma

Catania et al., 1994

Acute myocardial infarction (AMI)

Increased in plasma during AMI in patients receiving thrombolytic agents

Airaghi et al., 1995

Multiple sclerosis

Increased in patients with greater disability score

Catania et al., unpublished results

Sepsis syndrome

Reduced in plasma during critical phase of sepsis syndrome/septic shock

Catania et al., 2000

Hemodialysis

Increased in plasma

Catania et al., unpublished results

Parkinson's disease

Increased in CSF

Rainero et al., 1988b

Alzheimer's disease

Reduced in brain tissue

Arai et al., 1986; Rainero et al., 1988a

-MSH 1393 Rheumatoid Arthritis -MSH was found in the synovial fluid of arthritis patients and its concentration was greater in the forms marked by greater inflammation. The greatest concentration of the peptide occurred in synovial fluid of patients with systemic juvenile arthritis. MSH was also detectable in the synovium of patients with osteoarthtritis, but its concentration was much lower than in patients with either rheumatoid or juvenile arthritis (Catania et al., 1994). Plasma concentrations of peptide were not increased in any group. This observation suggests local production of -MSH within the inflamed joint. Myocardial Infarction -MSH increases in the circulation of patients with acute myocardial infarction receiving thrombolytic therapy (Airaghi et al., 1995). Thrombolytic therapy and reinstitution of flow to the ischemic zone is an effective means of controlling ischemic tissue injury. However, because inflammation caused by ischemia and reperfusion is an important component of myocardial infarction, management of patients with acute myocardial infarction should include reduction of inflammation as well. The results suggest that MSH, which has potent anti-inflammatory effects, might be useful in this condition. Multiple Sclerosis In preliminary studies -MSH was measured in plasma and spinal fluid of patients with definite multiple sclerosis of the remitting-relapsing or secondary progressive type. The data show that patients with greater disability score (EDSS  4) have increased circulating -MSH relative to patients with low disability scores who have concentrations of the peptide similar to normal subjects (Catania et al., 1998a). These data suggest that changes of circulating -MSH occur in multiple sclerosis much as in other inflammatory disorders in humans. That is, in the presence of greater severity of inflammation, there is an increase in -MSH as a compensatory reaction. Hemodialysis Patients with renal insufficiency treated with chronic hemodialysis show signs of systemic inflammation. Therefore, -MSH might be released into the circulation to counteract effects of proinflammatory mediators in hemodialysis patients. -MSH was indeed increased in plasma of patients on hemodialysis and its concentration was greater than in subjects

with severe renal insufficiency not treated with hemodialysis (Catania et al., unpublished results). Parkinson's Disease Mean CSF -MSH concentration was 2-fold greater in parkinsonian patients as compared with control subjects (Rainero et al., 1988b). These results suggest a functional relation between dopaminergic and melanotropinergic systems in the human brain. Alzheimer's Disease The concentration of -MSH was low in Alzheimer's brains. This result may be consistent with a CNS inflammatory reaction in this disease (Arai et al., 1986) that is not compensated for by the low peptide concentrations. Melanoma Research in melanoma patients showed increased -MSH levels in melanoma patients' plasma and high immunoreactive -MSH content in melanoma metastases. Further, -MSH receptor expression in melanoma cells was increased by various effectors able to stimulate melanogenesis (Ghanem et al., 1986).

References Airaghi, L., Lettino, M., Manfredi, M. G., Lipton, J. M., and Catania, A. (1995). Endogenous cytokine antagonists during myocardial ischemia and thrombolytic therapy. Am. Heart J. 130, 204±211. Airaghi, L., Capra, R., Pravettoni, G., Maggiolo, F., Suter, F., Lipton, J. M., and Catania, A. (1999). Elevated concentrations of plasma -MSH are associated with reduced disease progression in HIV-infected patients. J. Lab. Clin. Med. 133, 309±315. Arai, H., Morji, T., Kosaka, K., and Iisuka, R. (1986). Extrahypophyseal distribution of -melanocyte stimulating hormone (-MSH)-like immunoreactivity in postmortem brains from normal subjects and Alzheimer type dementia patients. Brain Res. Bull. 377, 305±310. Barcellini, W., La Maestra, L., Clerici, G., Lipton, J. M., and Catania, A. (1998). Inhibitory influences of -MSH peptides on HIV-1 expression in monocyte cells. 12th World AIDS Conference, Geneva Bardin, C. W., Chen, C. L. C., and Morris, P. L. (1987). Proopiomelanocortin-derived peptides in testis, ovary, and tissues of reproduction. Rec. Prog. Horm. Res. 43, 1±28. Benjannet, S., Rondeau, N., Day, R., Chretien, M., and Sedah, N. G. (1991). PC1 and PC2 are protein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc. Natl Acad. Sci. USA 88, 3564±3568. Berkenbosch, F., Vermes, I., and Tilders, F. J. H. (1984). The -adrenoreceptor-blocking drug propranolol prevents secretion of immunoreactive -endorphin and -melanocyte-stimulating

1394 James M. Lipton and Anna Catania hormone in response to certain stress stimuli. Endocrinology 115, 1051±1059. Bhardwaj, R. J., Schwarz, A., and Becher, E. (1996). Proopiomelanocortin-derived peptides induce IL-10 production in human monocytes. J. Immunol. 156, 473±478. Bloch, B., Bugnon, C., Fellmann, D., Lenys, D., and Gouget, A. (1979). Neurons of the rat hypothalamus reactive with antisera against endorphins, ACTH, MSH and -LPH. Cell Tissue Res. 204, 1±15. Cammas, F. M., Kapas, S., Barker, S., and Clark, A. J. L. (1995). Cloning, characterization and expression of a functional mouse ACTH receptor. Biochem. Biophy. Res. Commun. 212, 912±918. Candy, J. M., Perry, R. H., Thompson, J. E., Johnson, M., and Oakley, A. E. (1985). Neuropeptide localization in the substantia innominata and adjacent regions of the human brain. J. Anat. 140, 309±327. Carr, J. A., Saland, L. C., Samora, A., Benavidez, S., and Krobert, K. (1991). In vivo effects of serotonergic agents on -melanocyte-stimulating hormone secretion. Neuro endocrinology 54, 616±622. Catania, A., and Lipton, J. M. (1993). -MSH peptides in host responses: from basic evidence to human research. Ann. NY Acad. Sci. 680, 412±423. Catania, A., Martin, L. W., and Lipton, J. M. (1991). Dexamethasone facilitates -MSH release in the rabbit. Brain Res. Bull. 26, 727±730. Catania, A., Airaghi, L., Manfredi, M. G., Vivirito, M. C., Milazzo, F., Lipton, J. M., and Zanussi, C. (1993). Proopiomelanocortin-derived peptides and cytokines: relations in patients with acquired immunodeficiency syndrome. Clin. Immunol. Immunopathol. 66, 73±79. Catania, A., Gerloni, V., Procaccia, S., Airaghi, L., Manfredi, M. G., Lomater, C., Grossi, L., and Lipton, J. M. (1994). The anticytokine neuropeptide alpha-melanocyte-stimulating hormone in synovial fluid of patients with rheumatic diseases: comparisons with other anticytokine molecules. NeuroImmunoModulation 1, 321±328. Catania, A., Suffredini, A. F., and Lipton, J. M. (1995). Endotoxin causes -MSH release in normal human subjects. NeuroImmunoModulation 2, 258±262. Catania, R., Rajora, N., Capsoni, F., Minonzio, F., Star, R. A., and Lipton, J. M. (1996). The neuropeptide -MSH has specific receptors on neutrophils and reduces chemotaxis in vitro. Peptides 17, 675±679. Catania, A., Airaghi, L., Garofalo, L., Cutuli, M., and Lipton, J. M. (1998a). The neuropeptide -MSH in AIDS and other conditions in humans. Ann. NY Acad. Sci. 840, 848±856. Catania, A., Garofalo, L., Cutuli, M., Gringeri, A., Santagostino, E., and Lipton, J. M. (1998b). Melanocortin peptides inhibit production of proinflammatory cytokines in whole blood samples of patients infected with HIV. Peptides 19, 1099±1104. Catania, A., Cutuli, M., Garofalo, L., Airaghi, L., Valenza, F., Gattinoni, L., and Lipton, J. M. (2000). Production of proinflammatory cytokines in septic patients: modulation by  melanocyte stimulating hormone. Crit. Care Med. (in press). Ceriani, G., Diaz, J., Murphree, S., Catania, A., and Lipton, J. M. (1994). The neuropeptide alpha-melanocyte-stimulating hormone inhibits experimental arthritis in rats. NeuroImmunoModulation 1, 28±32. Chakraboty, A., and Pawelek, J. (1993). MSH receptors in immortalized human epidermal keratinocytes: a potential mechanism for coordinate regulation of the epidermal-melanin unit. J. Cell Physiol. 157, 344±350.

Chen, C. L., Dionne, F. T., and Roberts, J. L. (1983). Regulation of pro-opiomelanocortin mRNA levels in rat pituitary by dopaminergic compounds. Proc. Natl Acad. Sci. USA 80, 2211±2215. Chhajlani, V., and Wikberg, J. E. S. (1992). Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA. FEBS Lett. 309, 417±420. Chiao, H., Kohda, Y., McLeroy, P., Craig, L., Housini, I., and Star, R. A. (1997). Alpha-melanocyte-stimulating hormone protects against renal injury after ischemia in mice and rats. J. Clin. Invest. 99, 1165±1172. Civelli, O., Birnberg, N., and Herbert, E. (1982). Detection and quantitation of pro-opiomelanocortin mRNA in pituitary and brain tissue from different species. J. Biol. Chem. 257, 6783± 6787. Clark, D., Thody, A. J., Shuster, S., and Bowers, H. (1978). Immunoreactive -MSH in human plasma in pregnancy. Nature 273, 163±164. Coates, P. J., Doniach, I., Hale, A. C., and Rees, L. H. (1986). The distribution of immunoreactive -melanocyte-stimulating hormone cells in the adult human pituitary gland. J. Endocrinol. 11, 335±342. Daynes, R. A., Robertson, B. A., Cho, B. H., Burnham, D. K., and Newton, R. (1987). Alpha-melanocyte-stimulating hormone exhibits target cell selectivity in its capacity to affect interleukin 1-inducible responses in vivo and in vitro. J. Immunol. 139, 103±109. Delgado, R., Carlin, A., Airaghi, L., Demitri, M. T., Meda, L., Galimberti, D., Baron, P. L., Lipton , J. M., and Catania, A. (1998). Melanocortin peptides inhibit production of proinflammatory cytokines and nitric oxide by activated microglia. J. Leukoc. Biol. 63, 740±745. Delgado, R., Demitri, M. T., Carlin, A., Meazza, C., Villa, P., Ghezzi, P., Lipton, J. M., and Catania, A. (2000). Inhibition of systemic inflammation by central action of the neuropeptide -MSH. NeuroImmunoModulation (in press). DeÂsy, L., and Pelletier, G. (1978). Immunohistochemical localization of -melanocyte stimulating hormone (-MSH) in the human hypothalamus. Brain Res. 154, 377±381. Dulaney, R., Macaluso, A., Woerner, J., Hiltz, M., Catania, A., and Lipton, J. M. (1992). Changes in peripheral inflammation induced by CNS actions of an -MSH analog and of endogenous pyrogen. Prog. Neuroendocrinimmunol. 5, 179±186. Eberle, A. N. (1988). In ``The Melanotropins'' (ed S. Karger), Basel. Evans, C. J., Erdelyi, E., Weber, E., and Barchas, J. D. (1983). Identification of proopiomelanocortin-derived peptides in the human adrenal medulla. Science 221, pp. 957±960. Fox, J. A. E. T., and Kraicer, J. (1981). Immunoreactive -melanocyte stimulating hormone, its distribution in the gastrointestinal tract of intact and hypophysectomized rats. Life Sci. 28, 2127±2132. Gantz, I., Miwa, H., Konda, Y., Shimoto, Y., Tashiro, T., Watson, S. J., Del Valle, J., and Yamada, T. (1993). Molecular cloning, expression and gene localization of a fourth melanocortin receptor. J. Biol. Chem. 268, 15174±15179. Gantz, I., Shimoto, Y., Konda, Y., Miwa, H., Dickinson, C. J., and Yamada, T. (1994). Molecular cloning, expression and characterization of a fifth melanocortin receptor. Biochem. Biophy. Res. Commun. 200, 1214±1220. Gee, C. E., Chen, C. C., Roberts, J. L., Thompson, R., and Watson, S. J. (1993). Identification of pro-opiomelanocortin neurons in rat hypothalamus by the in situ cDNA-mRNA hybridisation. Nature 306, 374±376. Ghanem, G., LieÂnard, D., Hanson, P., Lejeune, F., and FruÈling, J. (1986). Increased serum -melanocyte stimulating hormone

-MSH 1395 (-MSH) in human malignant melanoma. Eur. Cancer Clin. Oncol. 22, 535±536. Grabbe, S., Bhardwaj, R. S., Mahnke, K., Simon, M. M., Schwarz, T., and Luger, T. A. (1996). -Melanocyte-stimulating hormone induces hapten-specific tolerance in mice. J. Immunol. 156, 473±478. Guy, J., Leclerc, R., Vaudry, H., and Pelletier, G. (1980). Identification of a second category of -melanocyte stimulating hormone (-MSH) neurons in the rat hypothalamus. Brain Res. Bull. 199, 135±146. Hagen, M. M., Rushing, P. A., Schwartz, M. W., Yagaloff, K. A., Burn, P., Woods, S. C., and Seeley, R. J. (1999). Role of the CNS melanocortin system in the response to overfeeding. J. Neurosci. 19, 2362±2367. Harris, J. I., and Lerner, A. B. (1957). Amino acid sequence of the -melanocyte-stimulating hormone Nature. 179, 1346±1347. Hiltz, M. E., and Lipton, J. M. (1990). -MSH peptides inhibit acute inflammation and contact hypersensitivity. Peptides 11, 979±982. Hiltz, M. E., Catania, A., and Lipton, J. M. (1992). Alpha-MSH peptides inhibit acute inflammation induced in mice by rIL-1 beta, rIL-6, rTNF-alpha and endogenous pyrogen but not that caused by LTB4, PAF and rII-8. Cytokine 4, 320±328. Huang, Q. H., Hruby, V. J., and Tatro, J. B. (1999). Role of central melanocortins in endotoxin-induced anorexia. Am. J. Physiol. 276, R864±R871. Huh, S.-K., Lipton, J. M., and Batjer, H. H. (1997). The protective effects of -melanocyte stimulating hormone on canine brainstem ischemia. Neurosurgery 40, 132±139. Huszar, D., Lynch, C. A., Fairchild-Huntress, V., Dunmore, J. H., Fang, Q., Berkemeier, L. R., Gu, W., Kesterson, R. A., Boston, B. A., Cone, R. D., Smith, F. J., Campfield, L. A., Burn, P., and Lee, F. (1997). Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88, 131±141. Ichiyama, T., Zhao, H., Catania, A., Furukawa, S., and Lipton, J. M. (1999a). -Melanocyte-stimulating hormone inhibits NF-IB activation and IBa degradation in human glioma cells and in experimental brain inflammation. Exp. Neurol. 157, 359±365. Ichiyama, T., Sakai, T., Catania, A., Barsh, G. S., Furukawa, S., and Lipton, J. M. (1999b). Systemically administered -melanocyte-stimulating peptides inhibit NF-B activation in experimental brain inflammation. Brain Res. 836, 31±37. Ichiyama, T., Campbell, I. L., Furukawa, S., Catania, A., and Lipton, J. M. (2000a). Autocrine -melanocyte-stimulating hormone inhibits NF-B activation in human glioma cells. J. Neurosci. Res. (in press). Ichiyama, T., Sakai, T., Catania, A., Barsh, G. S., Furukawa, S., and Lipton, J. M. (2000b). Inhibition of peripheral NF-B activation by central action of -melanocyte-stimulating hormone. J. Neuroimmunol. (in press). Knigge, U., and Warberg, J. (1991). The role of histamine in the neuroendocrine regulation of pituitary hormone secretion. Acta Endocrinol. 124, 609±619. Konda, Y., Gantz, I., Del Valle, J., Shimoto, Y., Miwa, H., and Yamada, T. (1994). Interaction of dual intracellular signaling pathways activated by the melanocortin-3 receptor. J. Biol. Chem. 269, 13162±13166. Kraus, J., Buchfelder, M., and Holt, V. (1993). Regulatory elements of the human proopiomelanocortin gene promoter. DNA Cell Biol. 12, 527±536. Kreiger, D. T. (1982). Placenta as a source of brain and pituitary hormones. Biol. Reprod. 26, 55±71. Lamacz, M., Tonon, M. C., Louiset, E., Cazin, L., and Vaudry, H. (1991). The intermediate lobe of the pituitary, model of

neuroendocrine communication. Arch. Intern. Physiol. Biochim. Biophys. 99, 205±219. Larsson, L. I. (1980). In ``Neural Peptides and Neuronal Communications'' (ed E. Costa and M. Trabucchi), Corticotrophin and -melanotropin in brain nerves: immunochemical evidence for axonal transport and processing, pp. 101±107. Raven Press, New York. Lindley, S. E., Lookinland, K. J., and Moore, K. E. (1990). Activation of tuberoinfundibular but not tuberohypophysial dopaminergic neurons following intracerebroventricular administration of alpha-melanocyte-stimulating hormone. Neuroendocrinology 51, 394±399. Lipton, J. M., and Catania, A. P. (1997). Antiinflammatory influence of the neuroimmunomodulator -MSH. Immunol. Today 18, 140±145. Lipton, J. M., Macaluso, A., Hiltz, M. E., and Catania, A. (1991). Central administration of the peptide -MSH inhibits inflammation in the skin. Peptides 12, 795±798. Lipton, J. M., Zhao, H., Ichiyama, T., Barsh, G. S., and Catania, A. (1999). Mechanisms of antiinflammatory actions of -MSH peptides. Ann. NY Acad. Sci. 885, 173±182. Loir, B., Bouchard, B., Morandini, R., Del Marmol, V., Deraemaecker, R., Garcia-Borron, J. C., and Ghanem, G. (1997). Immunoreactive alpha-melanotropin as an autocrine effector in human melanoma cells. Eur. J. Biochem. 244, 923±930. Loir, B., Sales, F., Deraemaecker, R., Morandini, R., GarciaBorron, J. C., and Ghanem, G. (1998). -Melanocortin immunoreactivity in human melanoma exudate is related to necrosis. Eur. J. Cancer. 34, 424±426. Lookingland, K. J., Gunnet, J. W., and Moore, K. E. (1991). Stress-induced secretion of -melanocyte-stimulating hormone is accompanied by a decrease in the activity of tuberohypophysial dopaminergic neurons. Neuroendocrinology 53, 91±96. Mabley, J., Wayman, C. P., and Wilson, J. F. (1991). Endogenous

-aminobutyric acid tonically inhibits release of -melanocytestimulating hormone from rat hypothalamic slices. Eur. J. Pharmacol. 209, 127±129. Macaluso, A., McCoy, D., Ceriani, G., Watanabe, T., Biltz, J., Catania, A., and Lipton, J. M. (1994). Antiinflammatory influences of -MSH molecules: central neurogenic and peripheral actions. J. Neurosci. 14, 2377±2382. Manna, S. K., and Aggarwal, B. B. (1998). Alpha-melanocytestimulating hormone inhibits the nuclear transcription factor NF-kappa B activation induced by various inflammatory agents. J. Immunol. 161, 2873±2880. Manzanares, J., Lookingland, K. J., and Moore, K. E. ( 1990). Kappa opioid-receptor-mediated regulation of -melanocyte-stimulating hormone secretion and tuberohypophy seal dopaminergic neuronal activity. Neuroendocrinology 52, 200±205. Martin, L. W., Catania, A., Hiltz, M. E., and Lipton, J. M. (1991). Neuropeptide alpha-MSH antagonizes IL-6- and TNF-induced fever. Peptides 12, 297±299. Mauri, A., Volpe, A., Martellotta, M. C., Barra, V., Piu, U., Angioni, G., Angioni, S., and Argiolas, A. (1993). Melanocyte-stimulating hormone during human perinatal life. J. Clin. Endocrinol. Metab. 77, 113±117. Mountjoy, K. G., Robbins, L. S., Mortrud, M. T., and Cone, R. D. (1992). The cloning of a family of genes that encode the melanocortin receptors. Science 257, 1248±1251. O'Donohue, T. L., Charlton, C. G., Helke, C. J., Miller, R. L., and Jacobowitz, D. M. (1979). In ``Peptides, Structure and Biological Function'' (ed E. Gross and J. Meienhofer), Identification of -melanocyte stimulating hormone

1396 James M. Lipton and Anna Catania immunoreactivity in rat and human brain and cerebrospinal fluid, pp. 897±900. Pierce Chemical Company, Rockford, IL. Oka, T., Oka, K., Hosoi, M., and Hori, T. (1995). Intracerebroventricular injection of interleukin-6 induces thermal hyperalgesia in rats. Brain Res. 692, 123±128. Papadopoulos, A. D., and Wardlaw, S. L. (1999). Endogenous alpha-MSH modulates the hypothalamic±pituitary±adrenal response to the cytokine interleukin-1 beta. J. Neuroendocrinol. 11, 315±319. Parker, C. R., Barnea, A., Tilders, F. J. H., and Porter, J. C. (1981). Characterization of immunoreactive -melanocyte stimulating hormone in human brain tissue. Brain Res. Bull. 6, 275±280. Perretti, M., Appleton, I., Parente, L., and Flower, R. J. (1993). Pharmacology of interleukin-1-induced neutrophil migration. Agents Actions 38, C64±C65. Rainero, I., May, C., Kaye, J. A., Friedland, R. P., and Rapoport, S. I. (1988a). CSF -MSH in dementia of the Alzheimer type. Neurology 38, 1281±1284. Rainero, I., Kaye, J. A., May, C., Durso, R., Katz, D. I., Albert, M. L., Wolfe, N., Pinessi, L., Friedland, R. P., and Rapoport, S. I. (1988b). Alpha-melanocyte-stimulating hormonelike immunoreactivity is increased in cerebrospinal fluid of patients with Parkinson's disease. Arch. Neurol. 45, 1224±1227. Rajora, N., Ceriani, G., Catania, A., Star, R. A., Murphy, M. T., and Lipton, J. M. (1996). -MSH production, receptors, and influence on neopterin in a human monocyte/macrophage cell line. J. Leukoc. Biol. 59, 248±253. Rajora, N., Boccoli, G., Burns, D., Sharma, S., Catania, A. P., and Lipton, J. M. (1997a). -MSH modulates local and circulating TNF in experimental brain inflammation. J. Neurosci. 17, 2181±2186. Rajora, N., Boccoli, G., Catania, A., and Lipton, J. M. (1997b). -MSH modulates experimental inflammatory bowel disease. Peptides 18, 381±385. Roselli-Rehfuss, L., Mountjoy, K. G., Robbins, L. S., Mortrud, M. T., Low, M. L., Tatro, J. B., Entwistle, M. L., Simerly, R. B., and Cone, R. D. (1993). Identification of a receptor for gamma melanocortin and other proopiomelanocortin peptides in the hypothalamus and limbic system. Proc. Natl Acad. Sci. USA 90, 8856±8860. Rudman, D., Del Rio, A. E., Hollins, B. M., Houser, D. H., Keling, M. E., Sutin, J., Scott, J. W., Sears, R. A., and Rosenberg, M. Z. (1973). Melanotrophic-lipolytic peptides in various regions of bovine, simian and human brains and in simian and human cerebrospinal fluids. Encrinology 72, 32±379. Rudman, D., Del Rio, A. E., Chawla, R. K., Houser, D. H., and Sheen, S. (1974). An antimelanotrophic protein in human cerebrospinal fluid. Am. J. Physiol. 226, 693±697. Scimonelli, T., and Eberle, A. N. (1987). Photoaffinity labelling of melanoma cell MSH receptors. FEBS Lett. 226, 134±138. Smith, E. M., and Blalock, J. E. (1995). Human lymphocyte production of corticotropin and endorphin-like substances: association with leukocyte interferon. Proc. Natl Acad. Sci. USA 78, 7530±7534. Star, R. A., Rajora, N., Huang, J., Stock, R. C., Catania, A., and Lipton, J. M. (1995). Evidence of autocrine modulation of macrophage nitric oxide synthase by alpha-melanocyte-stimulating hormone. Proc. Natl Acad. Sci. USA 92, 8016±8020. Sundar, S. K., Becker, K. J., Cierpial, M. A., Carpenter, M. D., Rankin, L. A., Fleener, S. L., Ritchie, J. C., Simson, P. E., and

Weiss, J. M. (1989). Intracerebroventricular infusion of interleukin 1 rapidly decreases peripheral cellular immune responses. Proc. Natl Acad. Sci. USA 86, 6398±6402. Taherzadeh, S., Sharma, S., Chhajlani, V., Gantz, I., Rajora, N., Demitri, M. T., Kelly, L., Zhao, H., Catania, A., and Lipton, J. M. (1999). -MSH and its receptors in regulation of inflammatory tumor necrosis factor- (TNF) by human monocyte/macrophages. Am. J. Physiol. 276, R1289±1294. Tatro, J. B. (1996). Receptor biology of the melanacortins, a family of neuroimmunomodulatory peptides. NeuroImmunoModulation 3, 259±284. Taylor, A. W., and Streilein, J. W. (1996). Inhibition of antigenstimulated effector T cells by human cerebrospinal fluid. NeuroImmunoModulation 3, 112±118. Thody, A. J., and Graham, A. (1998). Does alpha-MSH have a role in regulating skin pigmentation in humans? Pigment Cell Res. 11, 265±274. Thody, A. J., Ridley, K., Penny, R. J., Chalmers, R., Fisher, C., and Shuster, S. (1983). MSH peptides are present in mammalian skin. Peptides 4, 813±816. Tiligada, E., and Wilson, J. F. (1989). D2- but not D1-dopamine receptors are involved in the inhibitory control of -melanocyte-stimulating hormone release from the rat hypothalamus. Exp. Brain Res. 74, 645±648. Uehara, Y., Shimizu, H., Sato, N., Tanaka, Y., Shimomura, Y., and Mori, M. (1992). Carboxyl-terminal tripeptide of alphamelanocyte-stimulating hormone antagonizes interleukin-1induced anorexia. Eur. J. Pharmacol. 220, 119±122. Usategui, R., Oliver, C., Vaudry, H., Lombardi, G., Rozemberg, I., and Muggia, A. (1976). Immunoreactive -MSH and ACTH levels in rat plasma and pituitary. Endocrinology 98, 189±196. Van Oers, J. W., Tilders, J. H., and Berkenbosch, F. (1989). Characterization and biological activity of a rat monoclonal antibody to rat/human corticotropin-releasing factor. Endocrinology 124, 1239±1246. Visser, M., and Swabb, D. F. (1979). Life span changes in the presence of -melanocyte-stimulating-hormone-containing cells in the human pituitary. J. Dev. Physiol. 1, 161±178. Watanabe, T., Hiltz, M. E., Catania, A., and Lipton, J. M. (1993). Inhibition of IL-1 - induced peripheral inflammation by peripheral and central administration of analogs of the neuropeptide -MSH . Brain Res. Bull. 32, 311±314. Weidenfeld, J., Crumeyrolle-Arias, M., and Haour, F. (1995). Effect of bacterial endotoxin and interleukin-1 on prostaglandin biosynthesis by the hippocampus of mouse brain: role of interleukin-1 receptors and glucocorticoids. Neuroendocrinology 62, 39±46. Weiss, J. M., Sundar, S. K., Cierpial, M. A., and Ritchie, J. C. (1991). Effects of interleukin-1 infused into brain are antagonized by alpha-MSH in a dose-dependent manner. Eur. J. Pharmacol. 192, 177±179. Weiss, J. M., Quan, N., and Sundar, S. K. (1994). Widespread activation and consequences of interleukin-1 in the brain. Ann. NY Acad. Sci. 741, 338±357. Xia, Y., Wikberg, J. E., and Chhajlani, V. (1995). Expression of melanocortin 1 receptor in periacqueductal gray matter. Neuroreport 6, 2193±2196. Zabel, B. U., Naylor, S. L., Sakaguchi, A. Y., Bell, G. I., and Shows, T. B. (1983). High-resolution chromosomal localization of human genes for amylase, proopiomelanocortin, somatostatin, and a DNA fragment (D3S1) by in situ hybridization. Proc. Natl Acad. Sci. USA 80, 6932±6936.