The ability of melanocortin peptides to reduce macrophage release of pro-inflammatory mediators and increase anti-inflammatory mediators is a well-understood phenomenon with a number of groups contributing to this field. For example Capsoni et al. (2009) stimulated peripheral blood derived monocytes in vitro with monosodium urate crystals in the presence of αMSH (pan agonist) or (CKPV)₂ (a biologically potent Lys-Pro-Val peptide linked by Cys–Cys residues) and found significant reductions in IL-1β, IL-8, and TNFα release into supernatants (Capsoni et al., 2009). We treated the RAW264.7 mouse macrophage cell line with ACTH_1–39 (pan agonist) and MTII, amore stable αMSH derivative with higher affinity for MC₃ and MC₄ promoted an increase in cAMP accumulation and release of anti-inflammatory cytokine IL-10, the later effect being abrogated when cells were treated with H-89 (protein kinase A [PKA] inhibitor) suggesting signaling down-stream of MC₃ and not MC₄ (as RAW264.7 did not express MC₄) was through the cAMP–PKA pathway (Lam et al., 2006). In similar experiments both ACTH and MTII elicited in a cAMP–PKA dependent manner cytoprotective and anti-inflammatory heme oxygenase-1 (HO-1) but not heme oxygenase-2, heat shock protein 70 and 90 in RAW267.4 cells (Lam et al., 2005). Mandrika et al. (2001) showed dual signaling mechanism by which αMSH inhibited nitric oxide production by RAW267.4 macrophage. One pathway dependent of MC₁/cAMP activation and the other causing inhibition of NFκB translocation in a cAMP-independent manner. Other specialized cells of the macrophage lineage such as microglia treated with αMSH or ACTH substantially reduced TNFα, IL-6, and nitric oxide increases caused by LPS + IFNΓ activation, suggesting that MC peptides exert their anti-inflammatory actions on peripheral as well as central cells of the phagocytic lineage. There is evidence of active MC receptors because the same study reported that αMSH could induce cAMP accumulation in both resting and activated microglia (Delgado et al., 1998).

Rat macrophage isolated from gouty knee joints also accumulated intracellular cAMP upon treatment with melanocortin peptide; ACTH (Getting et al., 2002). More recently functionality of MC receptors by cAMP readout on murine alveolar macrophage was confirmed after incubation with αMSH, [D-Trp⁸]ΓMSH (MC₃/MC₅ agonist), and MSO5 (MC₁ agonist; Getting et al., 2008; Joseph et al., 2010). Together these studies suggest that the cAMP pathway represents a common underlying mechanism for melanocortin to deliver anti-inflammatory effects. Manna and Aggarwal stimulated human monocytes with various inflammatory agents including TNFα, LPS, ceramide, and okadaic acid to obtain NFκB activation, which was diminished in cells treated with αMSH (Manna and Aggarwal, 1998). The agonistic effect appeared to be cAMP-dependent such that inhibitors of adenylate cyclase and PKA reversed the inhibitory effect of αMSH. Furthermore αMSH inhibition of degradation of the NFκB inhibitory subunit IκBα and nuclear translocation of p65 subunit were also noted (Manna and Aggarwal, 1998).

Collectively these data appear to show that the anti-inflammatory actions of αMSH are dependent on MC₁, MC₃, and/or MC₅ receptor activation and triggers inhibition of NFκB in a cAMP-dependent and independent manner through molecular links which are yet to be deciphered.

As discussed so far, melanocortins are able to dampen macrophage release of pro-inflammatory mediators in inflammatory settings. These phenomena were exploited further by Capsoni et al. (2009) who showed that melanocortins could influence the monocytes ability to recruit and prime neutrophils. Chemotaxis of neutrophils treated with αMSH or (CKPV)₂ was examined using a boyden chamber assay and supernatants collected from monosodium urate crystal-stimulated monocytes as the source of chemoattractants. Chemotaxis was significantly reduced in neutrophils treated with agonist compared to controls. Furthermore reduced chemiluminescence (as a marker of reactive oxygen intermediates) production by pre-treated primed neutrophils was observed, in line with other studies showing melanocortin peptides can inhibit chemotaxis and generation of reactive oxygen species (Catania et al., 1996; Getting et al., 1999a; Capsoni et al., 2009).

To decipher what role MC₃ has on leukocyte interaction with the inflamed mesentery, experiments were performed using pharmacological agents and mice nullified for MC₃ where the superior mesenteric artery was occluded for 35 min followed by reopening to allow for blood reperfusion. Treatment with [D-Trp⁸]ΓMSH, a MC₃/MC₅ agonist did not alter cell rolling but decreased cell adhesion and emigration compared to vehicle control an effect not observed in Mc3r−/− mice. These data were mirrored by augmented KC and MCP-1 responses in mesenteric tissues of Mc3r−/− mice suggesting MC₃ is able to regulate leukocyte interaction with postcapillary venules and regulate levels of pro-inflammatory molecules in ischemia reperfusion injured tissues (Leoni et al., 2008). Moreover, whilst agonizing more than one MC receptor, the vasculoprotective properties of [D-Trp⁸]ΓMSH are solely transduced by MC₃.

Of interest, the modulation of chemokines occurred in the tissue and not with respect to plasma levels, moreover, levels of the cytokines IL-1β and TNFα were not affected by pharmacological treatment with [D-Trp⁸]ΓMSH or absence of the MC₃ gene. The “selective” effect on tissue chemokine levels may be secondary to inhibition of activation of resident cells, such as tissue macrophages and connective tissue type mast cells (Kubes and Granger, 1996; Tailor et al., 1999).

In similar experimental settings, the effects of a small molecule MC₁ agonist, compound BMS-470539 (Kang et al., 2006) inhibited cell adhesion, and emigration, an effect that was lacking in yellow^e/e mice. Interestingly, unlike Mc3r−/− mice, the yellow^e/e mice (expressing non-functional MC₁) displayed a comparable leukocyte adhesion and emigration response to wild types. Collectively these two studies identify a complex scenario whereby although both MC₁ and MC₃ are expressed in the inflamed mesentery, MC₁ may not be endogenously activated in ischemia reperfusion injury whereas MC₃ is both exogenously and endogenously activated in this setting (Leoni et al., 2010). Future studies addressing the hypothetical generation of selective agonists post-ischemia reperfusion might help deciphering this apparent discrepancy.

Until a decade ago, the mainstream school of thought was that the anti-inflammatory actions of melanocortin agonists were solely through MC₁ activation (Lipton and Catania, 1997; Luger et al., 2003). We used two mouse colonies, one bearing a non-functional MC₁ (yellow^e/e mice) and the other knocked out for MC₃ (Mc3r−/− mice), to conclude that MC₃ can be engaged by agonists with anti-inflammatory properties. Cultured primary peritoneal macrophage from yellow^e/e mice were treated with [D-Trp⁸]ΓMSH displaying a dose-dependent increase in cAMP accumulation, an effect reversed in the presence of an antibody to MC₃. The release of the chemokine KC was abrogated in the presence of [D-Trp⁸]ΓMSH with the antagonist agouti related protein (AGRP) abolishing the inhibitory effect.

In another study mice pre-treated with ACTH were injected with MSU crystals to induce peritonitis. A reduced accumulation of PMN was observed in the peritoneal cavity of mice treated with ACTH compared to vehicle control, data that was mirrored by decreased levels of KC also within the cavity. Interestingly co-administration of SHU9119 (an MC₃/MC₄ antagonist) with ACTH inhibited the agonists effects suggesting ACTH was acting through MC₃ and further confirmed by the detection of MC₃ but not MC₄ at the mRNA transcript level on peritoneal macrophage (Getting et al., 1999b). At a latter date, Getting et al. (2003) in the same peritonitis model using more selective MC₃ agonists and yellow^e/e mice confirmed that indeed agonism at MC₃ inhibited pro-inflammatory cytokines (IL-1) and chemokines (KC) and accumulation of neutrophils in the inflamed peritoneal cavity (Getting et al., 2003). Collectively these data suggest MC₃ more than MC₁ (at least in mouse) to be pivotal in bringing about the anti-inflammatory effects observed following treatment with these agonists (Getting et al., 1999b, 2003, 2006).

More recently MC₃ has been implicated in the regulation of macrophage precursor differentiation to osteoclasts. Inflammatory arthritis was induced by injection of an arthritogenic serum into wild type and Mc3r−/−, observing a higher prevalence and severity of disease observed in the latter genotype (Patel et al., 2010a). In a series of real time PCR analyses of extracts from the mouse ankle joint, a discrete set of inflammatory genes (13 out of 96) were upregulated in Mc3r−/− including IL-1β, IL-6, NOS2, CCR4, CXCR3, CCL2 as compared to wild types. It is worthy to note here that major macrophage secretory products include IL-1β and IL-6 (Gordon, 2003).

Upon microscopic analyses of the ankle joints, a significantly higher number of joints were affected by bone erosion, as indicated by histological scores and number of TRAP-positive osteoclasts within the Mc3r−/− joint. This observation was corroborated by the higher levels of RANKL (a key driver of osteoclast formation) mRNA in Mc3r−/− ankle joints (Lacey et al., 1998). Although the exact mechanism was not characterized, some conclusion could be reached by the study of osteoclast formation in vitro. Bone marrow-derived macrophage from wild type and Mc3r−/− mice were differentiated to osteoclasts in the presence of M-CSF and RANKL. A defect was noted such that a higher number of osteoclasts were generated from macrophage absent of the MC₃ gene compared to MC₃ sufficient cells. The Mc3r−/− osteoclasts displayed an increased “eating” ability such that when cultured on calcium phosphate coated wells significantly more resorption pits could be observed and quantified. Interestingly Mc3r−/− cells had increased and sustained RANKL-mediated NFκB signaling compared to wild types; this finding could provide mechanistic support to the increased CCL2 synthesis observed by these cells (Patel et al., 2010a). Together with the study by Cornish et al. (2003), our work implicates a role for melanocortins and their melanocortin receptors in the regulation of macrophage differentiation to specialized cells, example being here the osteoclast. It is plausible that MCs may modulate macrophage differentiation in other specialized cells such as the microglia or Kupffer cells, with further implications for their therapeutic potential.

Resolution of inflammation is an important process required to reset tissue/cells to a state of normalization after insult/injury. During this process a number of endogenous pathways are activated in order to regain homeostatic balance after inflammation. An exciting enhancement to the field of melanocortin biology in inflammation has been the recent finding that AP214, a modified αMSH analog, possesses prophagocytic and pro-resolving effects (Montero-Melendez et al., 2011), in line with the profile reported for resolvins and lipoxins (Schwab et al., 2007).

AP214 inhibits neutrophil recruitment in the zymosan peritonitis model, an effect that could be due, at least in part, to a modulation of macrophage phagocytic abilities of the particles. In vitro, AP214-treated biogel-elicited macrophages were incubated with zymosan particles to determine the percentage of phagocytic cells and number of ingested particles, observing an increment in both parameters. Such an effect of AP214 was also observed with respect to efferocytosis since this MSH analog augmented phagocytosis of human apoptotic neutrophils by mouse macrophages, an effect that was absent when Mc3r−/− macrophages were used. In in vivo settings, injection of apoptotic neutrophils into murine peritoneal cavities pre-treated with AP214 led to an increase in macrophage ingestion of neutrophils compared to vehicle control. This recent study has uncovered a new angle in which melanocortins and their receptors can affect the inflammatory reaction providing strong evidence for genuine pro-resolving activities centered on tight regulation of macrophage functions.