James M. Lipton

Targeting Melanocortin Receptors as a Novel Strategy to Control Inflammation

Adrenocorticotropic hormone and α-, β-, and γ-melanocyte-stimulating hormones, collectively called melanocortin peptides, exert multiple effects upon the host. These effects range from modulation of fever and inflammation to control of food intake, autonomic functions, and exocrine secretions. Recognition and cloning of five melanocortin receptors (MCRs) has greatly improved understanding of peptide-target cell interactions. Preclinical investigations indicate that activation of certain MCR subtypes, primarily MC1R and MC3R, could be a novel strategy to control inflammatory disorders. As a consequence of reduced translocation of the nuclear factor κB to the nucleus, MCR activation causes a collective reduction of the major molecules involved in the inflammatory process. Therefore, anti-inflammatory influences are broad and are not restricted to a specific mediator. Short half-life and lack of selectivity could be an obstacle to the use of the natural melanocortins. However, design and synthesis of new MCR ligands with selective chemical properties are already in progress. This review examines how marshaling MCR could control inflammation.

The neuropeptide α-MSH has specific receptors on neutrophils and reduces chemotaxis in vitro

Abstract The proopiomelanocortin-derived peptide α-melanocyte stimulating hormone (α-MSH) has potent anti-inflammatory effects in all animal models of inflammation against which it has been tested. Understanding of the mechanism by which this occurs is incomplete, although there is recent evidence for α-MSH receptors in murine and human macrophages and for modulation of production of proinflammatory cytokines and related mediators by α-MSH. Because of the prominence of neutrophils in early stages of inflammatory reactions where α-MSH is effective, we examined human neutrophils for evidence of mRNA for α-MSH receptors and for inhibition of neutrophil chemotaxis. There was accumulation of mRNA for melanocortin receptor 1 (MC1) in RT PCR product from neutrophils stimulated with interferon and LPS. In subsequent studies α-MSH inhibited migration of neutrophils from most normal volunteers when the cells were placed in FMLP or IL-8 gradients. The inhibition by α-MSH could be traced to alterations in cAMP in neutrophils. The presence of α-MSH receptor message in neutrophils is consistent with the established anti-inflammatory effects of the peptide. Direct inhibition of neutrophil chemotaxis likely contributes to the anti-inflammatory activity of α-MSH.

Melanocortin peptides inhibit production of proinflammatory cytokines and nitric oxide by activated microglia

Inflammatory processes contribute to neurodegenerative disease, stroke, encephalitis, and other central nervous system (CNS) disorders. Activated microglia are a source of cytokines and other inflammatory agents within the CNS and it is therefore important to control glial function in order to preserve neural cells. Melanocortin peptides are pro‐opiomelanocortin‐derived amino acid sequences that include α‐melanocyte‐stimulating hormone (α‐MSH) and adrenocorticotropic hormone (ACTH). These peptides have potent and broad anti‐inflammatory effects. We tested effects of α‐MSH (1‐13), α‐MSH (11‐13), and ACTH (1‐24) on production of tumor necrosis factor α (TNF‐α), interleukin‐6 (IL‐6), and nitric oxide (NO) in a cultured murine microglial cell line (N9) stimulated with lipopolysaccharide (LPS) plus interferon γ (IFN‐γ). Melanocortin peptides inhibited production of these cytokines and NO in a concentration‐related fashion, probably by increasing intracellular cAMP. When stimulated with LPS + IFN‐γ, microglia increased release of α‐MSH. Production of TNF‐α, IL‐6, and NO was greater in activated microglia after immunoneutralization of endogenous α‐MSH. The results suggest that α‐MSH is an autocrine factor in microglia. Because melanocortin peptides inhibit production of proinflammatory mediators by activated microglia they might be useful in treatment of inflammatory/degenerative brain disorders. J. Leukoc. Biol. 63: 740–745; 1998.

Alpha-melanocyte-stimulating hormone reduces endotoxin-induced liver inflammation.

Alpha-Melanocyte-stimulating hormone (MSH) is a potent anti-inflammatory agent in many models of inflammation, suggesting that it inhibits a critical step common to different forms of inflammation. We showed previously that alpha-MSH inhibits nitric oxide (NO) production in cultured macro-phages. To determine how alpha-MSH acts in vivo, we induced acute hepatic inflammation by administering endotoxin (LPS) to mice pretreated with Corynebacterium parvum, alpha-MSH prevented liver inflammation even when given 30 min after LPS administration. To determine the mechanisms of action of alpha-MSH, we tested its influence on NO, infiltrating inflammatory cells, cytokines, and chemokines. Alpha-MSH inhibited systemic NO production, hepatic neutrophil infiltration, and increased hepatic mRNA abundance for TNF alpha, and the neutrophil and monocyte chemokines (KC/IL-8 and MCP-1). We conclude that alpha-MSH prevents LPS-induced hepatic inflammation by inhibiting production of chemoattractant chemokines which then modulate infiltration of inflammatory cells. Thus, alpha-MSH has an effect very early in the inflammatory cascade.

Sodium valproate inhibits production of TNF-α and IL-6 and activation of NF-κB

Sodium valproate (VPA) is frequently used to treat epilepsy and convulsive disorders. Several reports have indicated that anti-epileptic drugs (AED) affect the immune system, but the mechanism has not been clear. We examined whether the commonly used AEDs, diazepam (DZP), carbamazepine (CBZ), phenobarbital (PB), phenytoin (PHT), and VPA, can inhibit activation of the nuclear transcription factor kappa B (NF-kappaB), in human monocytic leukemia cells (THP-1) and in human glioma cells (A-172). NF-kappaB is essential to the expression of the kappa light chain of immunoglobulin and proinflammatory cytokines. Electrophoretic mobility shift assays (EMSA) of nuclear extracts demonstrated that VPA inhibits NF-kappaB activation induced by lipopolysaccharide (LPS), but the other AEDs do not. Western blot analysis revealed that this inhibition is not linked to preservation of expression of IkappaBalpha protein. Chloramphenicol acetyltransferase (CAT) assay indicated that NF-kappaB-dependent reporter gene expression is suppressed in glioma cells pretreated with VPA. VPA significantly inhibited LPS-induced production of TNF-alpha and IL-6 by THP-1 cells, whereas other AEDs did not. The findings are consistent with the idea that VPA suppresses TNF-alpha and IL-6 production via inhibition of NF-kappaB activation. Our results suggest that VPA can modulate immune responses in vitro. These findings raise the possibility that such modulation might occur with clinical use of VPA.

α-MSH Modulates Local and Circulating Tumor Necrosis Factor-α in Experimental Brain Inflammation

Tumor necrosis factor (TNF-α) underlies pathological processes and functional disturbances in acute and chronic neurological disease and injury. The neuroimmunomodulatory peptide α-MSH modulates actions and production of proinflammatory cytokines including TNF-α, but there is no prior evidence that it alters TNF-α induced within the brain. To test for this potential influence of the peptide, TNF-α was induced centrally by local injection of bacterial lipopolysaccharide (LPS). α-MSH given once i.c.v. with LPS challenge, twice daily intraperitoneally (i.p.) for 5 d between central LPS injections, or both i.p. and centrally, inhibited production of TNF-α within brain tissue. Inhibition of TNF-α protein formation by α-MSH was confirmed by inhibition of TNF-α mRNA. Plasma TNF-α concentration was elevated markedly after central LPS, indicative of an augmented peripheral host response induced by the CNS signal. The increase was inhibited by α-MSH treatments, in relation to inhibition of central TNF-α. Presence within normal mouse brain of mRNA for the α-MSH receptor MC-1 suggests that the inhibitory effects of α-MSH on brain and plasma TNF-α might be mediated by this receptor subtype. The inhibitory effect of α-MSH on brain TNF-α did not depend on circulating factors because the effect also occurred in brain tissuein vitro. This indicates that α-MSH can act directly on brain cells to inhibit their production of TNF-α. If central TNF-α contributes to pathology in CNS disease and injury, and promotes inflammation in the periphery, agents that act on brain α-MSH receptors should decrease the pathological TNF-α reaction and promote tissue survival.

Antimicrobial effects of α-MSH peptides

The presence of the ancient antiinflammatory peptide α‐melanocyte‐stimulating hormone [α‐MSH (1–13), SYSMEHFRWGKPV] in barrier organs such as gut and skin suggests a role in the nonspecific (innate) host defense. α‐MSH and and its carboxy‐terminal tripeptide (11–13, KPV) were determined to have antimicrobial influences against two major and representative pathogens: Staphylococcus aureus and Candida albicans. α‐MSH peptides 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. Small concentrations of α‐MSH peptides likewise reduced viability and germ tube formation of the yeast C. albicans. Antimicrobial influences of α‐MSH peptides could be mediated by their capacity to increase cellular cAMP. Indeed, this messenger was significantly augmented in peptide‐treated yeast and the potent adenylyl cyclase inhibitor dideoxyadenosine (ddAdo) partly reversed the killing activity of α‐MSH peptides. Reduced killing of pathogens is a detrimental consequence of therapy with anti‐inflammatory drugs. Because α‐MSH has potent anti‐inflammatory effects we determined influences of α‐MSH on C. albicans and S. aureus killing by human neutrophils. α‐MSH peptides did not reduce killing but rather enhanced it, likely as a consequence of the direct antimicrobial activity. α‐MSH peptides that combine antipyretic, antiinflammatory, and antimicrobial effects could be useful in treatment of disorders in which infection and inflammation coexist. J. Leukoc. Biol. 67:233–239; 2000.

α-MSH Modulates Experimental Inflammatory Bowel Disease

Abstract Rajora, N., G. Boccoli, A. Catania and J. M. Lipton. α -MSH modulates experimental inflammatory bowel disease. Peptides 18(3), 381–385, 1997.—The mechanisms underlying inflammatory bowel disease (IBD) remain obscure but the importance of inflammatory processes is clear and most pharmacological therapies inhibit inflammation. The search for more effective agents with low toxicity continues. To test the possibility that the antiinflammatory/anticytokine peptide α -MSH can be used to control IBD, the peptide was administered to a murine colitis model. The peptide treatment had marked salutary effects: it reduced the appearance of fecal blood by over 80%, inhibited weight loss, and prevented disintegration of the general condition of the animals. Mice given α -MSH showed markedly lower production of TNF α by tissues of the lower colon stimulated with concanavalin A; the inhibitory effect of α -MSH on production of inflammatory nitric oxide by lower bowel tissue was even greater. The combined results indicate that α -MSH modulates experimental IBD, perhaps by inhibiting production within the gut of the local proinflammatory agents TNF α and nitric oxide, or by inhibiting inflammatory processes closely linked to these mediators.

α-MSH and its receptors in regulation of tumor necrosis factor-α production by human monocyte/macrophages

The hypothesis that macrophages contain an autocrine circuit based on melanocortin [ACTH and α-melanocyte-stimulating hormone (α-MSH)] peptides has major implications for neuroimmunomodulation research and inflammation therapy. To test this hypothesis, cells of the THP-1 human monocyte/macrophage line were stimulated with lipopolysaccharide (LPS) in the presence and absence of α-MSH. The inflammatory cytokine tumor necrosis factor (TNF)-α was inhibited in relation to α-MSH concentration. Similar inhibitory effects on TNF-α were observed with ACTH peptides that contain the α-MSH amino acid sequence and act on melanocortin receptors. Nuclease protection assays indicated that expression of the human melanocortin-1 receptor subtype (hMC-1R) occurs in THP-1 cells; Southern blots of RT-PCR product revealed that additional subtypes, hMC-3R and hMC-5R, also occur. Incubation of resting macrophages with antibody to hMC-1R increased TNF-α concentration; the antibody also markedly reduced the inhibitory influence of α-MSH on TNF-α in macrophages treated with LPS. These results in cells known to produce α-MSH at rest and to increase secretion of the peptide when challenged are consistent with an endogenous regulatory circuit based on melanocortin peptides and their receptors. Targeting of this neuroimmunomodulatory circuit in inflammatory diseases in which myelomonocytic cells are prominent should be beneficial.

The Neuropeptide Alpha-Melanocyte-Stimulating Hormone Inhibits Experimental Arthritis in Rats

alpha-Melanocyte-stimulating hormone (alpha-MSH) modulates inflammatory processes in models of acute inflammation and in models of sepsis/septic shock/adult respiratory-distress syndrome (ARDS). Because this neuropeptide inhibits actions of cytokines and other mediators of imflammation that are also believed to underlie aspects of chronic inflammation, tests were performed to compare the effects of repeated administration of the peptide with those of prednisolone and saline on the development of adjuvant arthritis in rats. alpha-MSH (50 micrograms), injected i.p. twice daily, markedly inhibited the clinical and histological signs of experimental arthritis and moderated the weight loss observed in control animals. Prednisolone (100 mg/kg), given twice per day, prevented development of arthritis but caused marked and progressive weight loss. The results confirm the potent anti-inflammatory influence of alpha-MSH, in this case in a model of chronic inflammation that has immune components.