Melanocortin Regulation of Inflammation

The melanocortin system consists of the melanocortins, two endogenous antagonists, the agouti-signaling protein (agouti) and agouti-related peptide (AgRP), and five receptors. The melanocortins, including adrenocorticotropic hormone (ACTH), and α-, β-, and γ-melanocyte-stimulating hormones (α-, β-, γ-MSHs), are derived from post-translational processing of a common precursor pro-opiomelanocortin (POMC). In addition to the well-documented adrenal responses stimulated by ACTH and pigmentary effects induced by α-MSH, other physiological functions, including energy homeostasis, sexual activity, exocrine secretion, as well as anti-inflammatory and immunomodulatory actions, exerted by these versatile neuropeptides upon the host, have also been reported extensively ( 1 – 3 ). The first clinical experiment applying ACTH in the treatment of patients with severe rheumatoid arthritis, rheumatic fever, and certain other conditions, was performed by Hench et al. ( 4 , 5 ), the Nobel Laureates in Physiology or Medicine in 1950 for their milestone discoveries on ACTH and the adrenal hormone cortisol ( 6 ). At that time, ACTH-induced improvement in clinical features in rheumatoid arthritis patients was thought to be via stimulation of hypothalamus-pituitary-adrenal gland axis and production of glucocorticoids. Our understanding of melanocortin physiology and pharmacology has greatly improved with the recognition and cloning of melanocortin receptors (MCRs). Indeed, it was not until 2002 that local activation of melanocortin-3 receptor (MC3R) by ACTH, independent of glucocorticoid, was demonstrated to be also responsible for ACTH efficacy in gouty arthritis ( 7 ). This important study suggested that selective MC3R agonists might be utilized as novel anti-inflammatory agents for clinical management of chronic inflammatory conditions ( 7 ). In this review, we will briefly introduce the melanocortin system, including melanocortin peptides, MCRs, and intracellular signaling pathways. Then, the anti-inflammatory effects of melanocortins in vitro and in vivo , the underlying molecular mechanisms, as well as potential therapeutic use of melanocortins, will be elaborated.

The versatile actions of melanocortin peptides are mediated by five MCRs, MC1R to MC5R, which are named according to the sequence of their cloning. As members of Family A G protein-coupled receptors, MCRs share the common hallmark structure consisting of seven α-helical transmembrane domains, an extracellular N terminus and intracellular C tail, connected by three alternating extracellular and three intracellular loops. Sequence comparison of human MCRs reveals high homologies, from 38% identity between MC2R and MC4R to 60% identity between MC4R and MC5R ( 27 ). As shown in Table 2 , the MCR exerts diverse expression patterns, which might contribute to their multifaceted physiological functions. As challenging and popular drug targets, the MCRs have triggered intensive efforts to develop more potent and selective melanocortin agonists and antagonists. Table 1 includes some of the most commonly-used peptide and small molecule ligands for the MCRs. Table 2 The distribution of the MCRs. MCR mRNA expression Techniques Species a References MC1R Present in skin and melanocytes; Absent in thyroid and testis Northern blotting Human (Cloudman S91 or WM264-1 melanoma cells) ( 60 , 61 ) Present in periaqueductal gray matter; Absent in amygdala, septal area, thalamus, midbrain, and hypothalamus In situ hybridization Rat ( 62 ) Present in macrophages, monocytes, lymphocytes, neutrophils, and astrocytes RT-PCR b , S1 nuclease assays, and degenerate PCR Murine (RAW264.7), human (THP-1 or A172 cells, neutrophils, or peripheral blood mononuclear cells) ( 63 – 67 ) MC2R Present in zona fasciculata and zona glomerulosa of adrenal gland; Absent in medulla and capsule of adrenal gland, pituitary, liver, lung, thyroid, and kidney In situ hybridization and Northern blotting Rhesus macaque ( 61 ) Present in chondrocytes and osteoblasts RT-PCR b Human (primary articular chondrocytes or osteoblasts) ( 68 , 69 ) MC3R Present in brain and placenta; Absent in adrenal gland, pancreas, stomach, kidney, spleen, liver, and lung Northern blotting Rat or canine ( 70 , 71 ) Present in cortex, thalamus, hypothalamus, hippocampus, amygdala, and septum In situ hybridization Rat or mice ( 70 , 71 ) Present in monocytes and macrophages RT-PCR b and Southern blotting Mouse (peritoneal macrophages) or human (THP-1 cells) ( 64 , 72 ) MC4R Present in brain; Absent in heart, ovary, testis, spleen, kidney, lung, stomach, colon, pancreas, jejunum, cortex, cerebellum, salivary gland, liver, adrenal, Northern blotting Canine or rat ( 73 , 74 ) Present in thalamus, hypothalamus, dentate gyrus, cortex, amygdala, and CA1 and CA2 regions of hippocampus; Absent in CA3 and CA4 regions of hippocampus. In situ hybridization Mouse ( 73 ) Present in thalamus, hypothalamus, brainstem, spinal cord, olfactory cortex, hippocampal formation, amygdala, septal region, corpus striatum, bed nucleus of the stria terminalis, midbrain, pons, and medulla oblongata. In situ hybridization Rat ( 74 , 75 ) MC5R Present in skeletal muscle, lung, spleen, brain, Absent in melanoma cells, adrenal tissues, and placenta Northern blotting Rat, mouse or human (A375 melanoma cells) ( 76 ) Present in skin, skeletal muscle, thymus, bone marrow, adrenal gland, testis, ovary, and uterus; Absent in lung, kidney, esophagus, stomach, duodenum, jejunum, ileum, colon, liver, pancreas, prostate, seminal vesicle, spinal cord, dorsal root ganglion, sciatic nerve and soleus muscle RNase protection assays Mouse ( 19 , 77 ) B lymphocytes RT-PCR b Mouse (Ba/F3 lymphocytes) ( 78 ) Splenic lymphocytes Radioligand binding assay Rat (mononuclear lymphocytes) ( 79 ) T lymphocytes Immunohistochemistry Mouse (CD25+CD4+ regulatory T cells) ( 80 ) a indicates the species from which the tissues or cell types are collected; b reverse transcription-PCR . The MC1R is the classical MSH receptor expressed in skin and hair follicles that regulates pigmentation. However, MC1R, with the highest affinity for α-MSH, is expressed in virtually all cell types responsive to the anti-inflammatory action of melanocortins. It was also shown that normal human monocytes and mouse brain contain a low number of MC1R binding sites which is upregulated by various stimuli such as lipopolysaccharide (LPS) or combinations of cytokines, and traumatic brain injury, respectively ( 81 , 82 ). In addition, by measuring ionized calcium binding adaptor molecule-1 staining (Iba-1) in microglial cells, a known marker for cerebral inflammation, Schaible et al. showed that α-MSH ( 11 – 13 ), probably by targeting the MC1R, significantly attenuates cerebral inflammation, as evidenced by reduced activation of Iba-1 positive cells in the ipsilateral hemisphere ( 82 ). Two synthetic MC1R agonists, BMS-470539 and AP1189, were also reported to exhibit anti-inflammatory activities in various inflammation models ( 33 – 35 ). The MC2R, the classical ACTH receptor, is expressed in the adrenal cortex that regulates adrenal steroidogenesis and cell proliferation (

ACTH Effects in vitro The fact that ACTH is superior to corticosteroids in treating certain inflammatory diseases has raised a possibility that ACTH might have some therapeutic effects other than stimulating the levels of endogenous cortisol. In normal human keratinocytes, ACTH 1−39 was shown to suppress NFκB activation stimulated by tumor necrosis factor-α (TNF-α), likely via enhancing nuclear translocation of the NFκB inhibitor IκBα to the nucleus ( 103 ). Further immunofluorescent labeling and western blot studies detected MC1R and MC2R in normal human keratinocytes, which might mediate the peptide-induced suppression on activation of NFκB ( 103 ). In addition, the ability of ACTH to directly modulate local CNS inflammation, a factor in, and perhaps initiates, many CNS diseases, has been reported in several in vitro studies. Using rat brain cultures containing oligodendroglia, astrocytes, and microglia preincubated with cytotoxic agents, ACTH 1−39 was shown to protect mature oligodendroglia and oligodendroglia progenitor cell (OPC) from death induced by staurosporine, AMPA, NMDA, kainate, quinolinic acid, or reactive oxygen species (ROS) ( 104 , 105 ). In their studies, preincubation of the cytotoxic agents caused 50–75% death of mature oligodendroglia but little or no death of astrocytes or microglia ( 104 ). Since oligodendroglia and OPC uniquely provide metabolic support to neurons/axons, the protection of oligodendroglia and OPC from several excitotoxic and inflammation-related insults is of great importance in keeping the integrity of CNS ( 104 ). Using rat glial cultures, the same group demonstrated that ACTH 1−39 induces proliferation of OPC and accelerates differentiation of platelet-derived growth factor receptor-α (a phenotypic marker for OPC) positive OPC to a later stage characterized by greater expansion of oligodendroglia myelin-like sheets compared to untreated cells ( 105 ). Adenylyl cyclase was suggested to be involved in ACTH-mediated regulation of OPC proliferation, differentiation, and anti-inflammatory actions based on the findings that (1) OPC have delayed maturation and accelerated proliferation when adenylyl cyclase is activated in vitro ( 106 , 107 ), (2) pituitary adenylyl cyclase-activating polypeptide-deficient mice have earlier onset of myelination, less time for axonal development, and synapse formation, as well as less neuronal plasticity ( 108 ), and (3) cAMP-inducing agents prevent oligodendroglial excitotoxicity and protect mature oligodendroglia from excitotoxic agents ( 109 ). Furthermore, the same group showed that ACTH also protects rat forebrain neurons, the most vulnerable cells in CNS, from apoptotic, excitotoxic, and inflammation-related damage in a similar manner as mature oligodendroglia and OPC ( 110 ). Since excitotoxic damage to neurons is an important cause to several experimental and clinical CNS diseases, it is reasonable to explain the therapeutic benefits of ACTH in several inflammatory animal models of CNS disorders ( 110 ). However, the contribution from direct effects on oligodendroglia, OPC, or neurons within the brain and whether such beneficial actions can be observed in vivo in human patients remain to be investigated.

It is well-known that ACTH-mediated anti-inflammatory effects can be achieved through glucocorticoid-dependent and -independent manners. Regarding glucocorticoid-dependent action, ACTH is unique among melanocortins since it is the only peptide that activates MC2R. Generally, stimuli such as stress or inflammation trigger the synthesis and release of corticotropin-releasing hormone from the paraventricular nucleus of the hypothalamus, which further stimulates the release of ACTH from the pituitary. ACTH then circulates to the adrenal gland, where it activates MC2R located on the adrenal cortex and induces rapid synthesis of cortisol. Cortisol then activates glucocorticoid receptor and triggers downstream anti-inflammatory actions through genomic and non-genomic pathways, eventually resulting in decreased production of cytokines, chemokines, and inducible NO synthase, increased anti-inflammatory mediators, and phagocytosis of apoptotic neutrophils [reviewed in ( 6 )]. It is also reported that glucocorticoids produced in the skin can also modulate local inflammation, although the underlying mechanism is not clear ( 115 ). It is not until 2002, 50 years after the approval of ACTH for the treatment of many inflammatory conditions, that Getting et al. revealed the retention of anti-inflammatory effects of ACTH in adrenalectomized rats with knee gout, suggesting the existence of hypothalamus-pituitary-adrenal- and glucocorticoid-independent anti-inflammation mechanism ( 7 ). Until now, extensive in vitro and in vivo studies, as discussed above, have undoubtedly supported the notion that ACTH can exert its anti-inflammatory effects directly through activation of certain MCRs expressed in peripheral immune cells and hypothalamic neural cells.

α -MSH Effects in vitro The molecular mechanisms of α-MSH-mediated anti-inflammatory functions have been studied extensively in vitro (summarized in Table 3 ). α-MSH-mediated inhibition of NF-κB activation, a well-known master regulator of inflammation that controls the expression of many cytokines, cytokine receptors, chemokines, growth factors, and adhesion molecules, was first discovered by Manna and Aggarwal, who demonstrated that α-MSH completely abolishes TNF-α-induced NF-κB phosphorylation in a dose- and time-dependent manner ( 124 ). It also blocks LPS-, okadaic acid-, and ceramide-mediated activation of NF-κB in human monocytic, epithelial, glioma, and lymphoid cells ( 124 ). Subsequently, similar effects exerted by α-MSH were reported in a variety of cell types including human microvascular endothelial cells ( 125 ), human melanocytes and melanoma cells ( 126 ), human glioma cells ( 127 , 128 ), human pulmonary epithelial cells ( 129 ), human keratinocytes ( 103 ), mast cells ( 130 ), Schwann cells ( 131 ), human macrophages and neutrophils ( 132 ), human dermal fibroblast cells ( 133 ), and rat small intestine cells ( 152 ). Suppression of NF-κB translocation is achieved through generation of cAMP, activation of protein kinase A, and protection of IκBα from phosphorylation ( 124 ). Recently, α-MSH was reported to attenuate TNF-α-induced matrix metalloproteinase-13 expression by regulating activation of p38 and NF-κB in HTB-94 cells (a human chondrosarcoma cell line expressing MC1R), indicating that α-MSH might be used to prevent matrix metalloproteinase-13-mediated collagen degradation ( 153 ). Table 3 Molecular mechanisms of α-MSH-mediated anti-inflammatory functions. Actions of α-MSH Effectors Cell types investigated References Inhibition of transcription factor NF-κB Human monocytic U937, epithelial HeLa, glioma H4, lymphoid Jurkat cells, mast cells, Schwann cells, microvascular endothelial cells, dermal fibroblast cells, melanocytes and melanoma cells, keratinocytes, macrophages/neutrophils, and rat small intestine cells ( 124 – 133 ) Suppression of proinflammatory cytokines TNF-α Human A-172 and THP-1 cells, murine fibroblast L292 cells, and microglial N9 cells ( 63 , 64 , 134 , 135 ) IL-1, IL-6, and IL8 Rat PBMCs, murine peritoneal macrophages, murine microglial N9 cells, HaCaT cells ( 7 , 134 , 136 , 137 ) Groα HaCaT cells ( 138 ) KC Murine peritoneal macrophages ( 139 ) Suppression of non-cytokine proinflammatory mediators NO RAW 264.7 cells, THP-1 cells, human melanoma FM55 cells, murine microglial N9 cells, rat PBMCs and astrocytes ( 65 , 134 , 135 , 140 – 143 ) PGE 2 Human melanoma FM55 and HaCaT cells, and rat astrocytes ( 142 , 144 ) ROS Rat peritoneal neutrophils ( 145 ) Suppression of adhesion molecules ICAM-1 Human normal and malignant cells, dermal papilla cells and fibroblasts, and murine mast cells ( 130 , 133 , 146 , 147 ) CD40 and CD86 Human monocytes and peripheral blood-derived dendritic cells ( 81 , 148 ) VCAM-1 HMEC-1 and HDMECs ( 125 , 149 ) E-selectin HMEC-1 and HDMECs ( 125 , 149 ) Induction of cytokine suppression IL-10 Human monocytes ( 150 , 151 ) Extensive studies have demonstrated that α-MSH exerts the anti-inflammatory actions by suppressing the expression of proinflammatory cytokines such as TNF-α, interferon-γ (IFN-γ), IL-1, IL-6, IL-8, and KC. Lipton and colleagues showed that α-MSH, presumably by acting through MC1R, inhibits bacterial endotoxin-induced TNF-α production in human glioma cells ( 63 ). Similar inhibitory effects on TNF-α production exerted by α-MSH were observed in human monocyte/macrophages ( 64 ), melanocyte and melanoma cells ( 126 ), and human keratinocytes ( 103 ). Taylor et al. demonstrated that constitutive picomolar α-MSH detected in normal aqueous humor of human, rabbits, and mice is able to suppress the production of antigen-stimulated IFN-γ ( 154 , 155 ). In addition, after preincubation of human peripheral blood mononuclear cells (PBMCs) with mitogen and different concentrations of α-MSH, Luger et al. showed that, in human PBMCs, the mitogen-induced transcription and biological activity of IFN-γ are significantly blocked by α-MSH in a dose-dependent manner, as evidenced by changes in IFN-γ mRNA expression and the major histocompatibility complex class I antigen expression, respectively ( 156 ). The production of other proinflammatory cytokines, including IL-1, IL-6, IL-8, Groα, and KC, when co-incubated with or without proinflammatory stimuli, are also inhibited by α-MSH ( 7 , 136 – 138 ). The ability of α-MSH in suppressing TNF-α-, IFN-γ-, or LPS-induced expression of intercellular adhesion molecule-1 (ICAM-1) have been reported in human normal and malignant cells, human dermal papilla cells and fibroblasts, and murine mast cells ( 130 , 133 , 146 , 147 ). The inhibition of LPS-induced vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression were also reported in human microvascular endothelial cells (HMEC-1) and human d

With the aid of numerous cell culture systems, a variety of mechanisms as discussed above, including inhibition of nuclear transcription factor NF-κB activation, suppression of proinflammatory cytokine production and adhesion molecule expression, restraint of proinflammatory non-cytokine regulators, induction of cytokine suppressors, as well as modulation of lymphocyte function and proliferation, have been demonstrated to contribute to α-MSH-mediated anti-inflammatory effects, which are further validated in animal models of experimentally induced fever, rheumatoid arthritis, ARDS, inflammatory skin and bowel diseases, and brain inflammation ( 160 ). Similar to ACTH, it is thought that the anti-inflammatory effects of α-MSH might be mediated through activation of specific MCRs expressed in peripheral immune cells and hypothalamic neurons. For instance, MC3R was shown to be required for ACTH- and α-MSH-mediated anti-inflammatory effects in rheumatoid arthritis ( 7 ). The α-MSH-induced inflammation-modulatory signaling pathways in the CNS are thought to be mediated by neural MC4R ( 176 ). MC1R and MC5R might be involved in α-MSH-mediated modulation of proinflammatory cytokines and collagens in human articular chondrocytes ( 68 ). Furthermore, protective effects of α-MSH in autoimmune uveoretinitis require a functional MC5R ( 177 ).

Although β- and γ-MSHs share similar affinity for the immunomodulatory MC1R and MC3R as ACTH and α-MSH, the involvement of β- and γ-MSHs in the regulation of inflammation are less understood due to lack of experimental evidences. The central antipyretic actions of γ 2 -MSH during the inflammatory response was reported by Bock et al., who showed that intraseptal or intraventricular infusion of γ 2 -MSH inhibits LPS-induced fever in guinea pig ( 190 ). Recently, the anti-inflammatory activity of γ 2 -MSH were further confirmed by Getting and colleagues, who demonstrated that in an in vivo murine model of peritonitis and in vitro cultured macrophages, γ 2 -MSH causes a dose-dependent attenuation of polymorphonuclear leukocyte migration ( 37 ). This inhibition was shown to be associated with a reduction in KC and IL-1β levels ( 37 ). In addition, SHU9119 blocks the ability of γ 2 -MSH to inhibit neutrophil migration, KC, and IL-1β release, suggesting effects of γ 2 -MSH are mediated by the MC3R or MC4R or both ( 37 ). However, only the mRNA and protein of MC3R, but not MC4R, were confirmed by RT-PCR and western blotting in murine and rat peritoneal macrophages ( 37 ). The MC3R is functional since it responds to γ 2 -MSH stimulation in initiating production of intracellular cAMP and this effect is blocked in the presence of SHU9119 ( 37 ). They concluded that γ 2 -MSH can inhibit the experimental inflammatory response by selectively activating MC3R on peritoneal macrophages. Similar findings were observed in a recessive yellow mouse strain harboring a frameshift mutation in Mc1r gene that results in non-functional MC1R protein ( 191 ). Taking advantage of this mouse model and more selective melanocortin ligands, the same group showed that a functional MC1R is not necessary to elicit the anti-inflammatory actions of melanocortin peptides since (1) the recessive yellow mice does not display any signs of ongoing inflammatory response; (2) wild type and the recessive yellow mice showed no difference in MSU crystal-induced peritonitis as assessed by polymorphonuclear leukocyte migration and release of KC; (3) pre-administration of γ 2 -MSH reduces MSU crystal-induced polymorphonuclear leukocyte migration equally in wild-type and recessive yellow mice and this is associated with a reduction in IL-1β levels; (4) the selective MC1R agonist MS05 is inactive in inhibiting any of the inflammatory parameters in both wild type and recessive yellow mice ( 191 ). Using male Sprague–Dawley rats, Xia et al. showed that intravenous infusion of γ 2 -MSH reverses LPS-induced hypotension and tachycardia, attenuates systemic inflammatory responses to endotoxin, prevents LPS-induced IL-1β gene expression in the brain and peripheral tissues, and inhibits LPS-induced increases in plasma nitric oxide levels, indicating a novel approach for modulating systemic inflammation through pharmacological manipulation of γ 2 -MSH-mediated autonomic activity ( 192 ). The specialized anti-inflammatory functions of β- and γ-MSHs within the brain were also reported by Muceniece and colleagues. In an acute mouse neuroinflammation model, four tested melanocortins were shown to reduce LPS-induced increases in brain nitric oxide as measured by electron paramagnetic resonance, with an order of effectiveness: β-MSH ≥ γ 1 -MSH = γ 2 -MSH > α-MSH ( 193 ).

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. Conflict of Interest WW, D-YG, and Y-JL, Employees of Xiamen Huli Guoyu Clinic, Co., Ltd., Xiamen, China The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.