Dissection of the Anti-Inflammatory Effect of the Core and C- Terminal (KPV) -Melanocyte-Stimulating Hormone Peptides

In this study, we analyzed the anti-inflammatory effects of -melanocyte stimulating hormone (MSH)11–13 (KPV) in comparison with other MSH peptides in a model of crystal-induced peritonitis. Systemic treatment of mice with KPV, -MSH, the core melanocortin peptide His-Phe-Arg-Trp, and the melanocontin receptor 3/4 agonist Ac-Nle-c[Asp,D-Phe,Lys]NH2 ACTH4-10 (MTII) but not the selective MC1-R agonist H-Ser-Ser-Ile-Ile-SerHis-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 (MS05) resulted in a significant reduction in accumulation of polymorphonuclear leukocyte in the peritoneal cavity. The antimigratory effect of KPV was not blocked by the MC3/4-R antagonist Ac-Nle-c[Asp,DNal,Lys]NH2 ACTH4-10 (SHU9119). In vitro, macrophage activation, determined as release of KC and interleukin (IL)-1 was inhibited by -MSH and MTII but not by KPV. Furthermore, macrophage activation by MTII led to an increase in cAMP accumulation, which was attenuated by SHU9119, whereas KPV failed to increase cAMP. The anti-inflammatory properties of KPV were also evident in IL-1 -induced peritonitis inflammation and in mice with a nonfunctional MC1-R (recessive yellow e/e mice). In conclusion, these data highlight that the Cterminal MSH peptide KPV exhibits an anti-inflammatory effect that is clearly different from that of the core MSH peptides. KPV is unlikely to mediate its effects through melanocortin receptors but is more likely to act through inhibition of IL-1 functions. The pro-opiomelanocortin gene product undergoes posttranslational processing to form the endogenous ligands of the melanocortin receptors [ -, -, -melanocyte stimulating hormone (MSH)] and adrenocorticotropin (ACTH), which all contain the common amino acid motif His-Phe-Arg-Trp (HFRW) tetrapeptide (Wikberg et al., 2000; Getting, 2002). Five melanocortin receptors (MC-Rs) have been cloned and are positively coupled to adenylate cyclase, thus receptor activation leads to increases in intracellular cAMP (Wikberg et al., 2000; Getting, 2002). These endogenous peptides are endowed with anti-inflammatory properties, including inhibition of tumor necrosis factor, interleukin (IL)-1, and the CXC chemokine KC release (Getting, 2002) as well as adhesion molecule expression (Kalden et al., 1999). This is possibly due to their ability to inhibit nuclear transcription factorB activation (Manna and Aggarwal, 1998; Kalden et al., 1999) and protection of I B degradation (Ichiyama et al., 1999), thus affecting the humoral and cellular phases of inflammation (Hiltz and Lipton, 1989; Lipton and Catania, 1998). These anti-inflammatory properties have been highlighted in several experimental models of acute and chronic inflammation (for a recent review, see Getting, 2002). At present, there is a lot of confusion within the field of whether a single MC-R mediates the anti-inflammatory effects of melanocortin peptides. One of the receptors, MC1-R, has long been regarded as the receptor responsible for the anti-inflammatory effects of -MSH and related peptides (Wikberg et al., 2000), whereas more recently we have proposed a central role for MC3-R (Getting et al., 1999, 2001). The MC1-R mRNA, but not protein, expression has been found in an array of cells, including monocytes, B-lymphocytes, NK cells, a subset of cytoxic T cells (Neumann Andersen et al., 2001), dendritic cells (Becher et al., 1999) as well as mast cells (Adachi et al., 1999). The expression of MC3-R mRNA and protein has been detected in rodent peritoneal and knee joint macrophages (MØ). Importantly, the receptor is functional because its activation leads to cAMP accumulation. In a series of inflammatory models, the relatively selective agonists have been shown to down-regulate the host inflammatory response, and this inhibition was abrogated in the presence of MC3-R, but not MC4-R antagonists (Getting et al., 1999, 2001, 2002). This work was supported by the Arthritis Research Campaign UK (Grant G0571). M.P. is a Senior Fellow of the Association pour la Recherche sur le Cancer, UK. H.S. was supported by the Swedish Research Council (VR, medicin) and Melacure Therapeutics AB. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.051623. ABBREVIATIONS: MSH, melanocyte stimulating hormone; ACTH, adrenocorticotropin; MC-R, melanocortin receptor; IL, interleukin; MØ, macrophage; MSU, monosodium urate; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; PMN, polymorphonuclear leukocyte. 0022-3565/03/3062-631–637$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 306, No. 2 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 51623/1082430 JPET 306:631–637, 2003 Printed in U.S.A. 631 at A PE T Jornals on July 8, 2017 jpet.asjournals.org D ow nladed from Different fragments of the anti-inflammatory melanocortin peptide -MSH have been investigated for their efficacy, including the core region of -MSH4–10 (MEHFRWG) and the C-terminal peptide -MSH11–13 (KPV). -MSH4–10, identical to ACTH4–10, has been shown to inhibit prostaglandin E1 generation and edema formation in rat skin (Gecse et al., 1980) and PMN migration and IL-1 and KC release in a model of crystal-induced inflammation (Getting et al., 1999). KPV ( -MSH11–13) has been reported to reduce experimental pyresis while the core region was inactive (Richards and Lipton, 1984). KPV can also inhibit carrageenan-induced edema formation in the mouse (Hiltz and Lipton, 1990). A similar observation was noted by the same group in a model of picryl chloride in the mouse (Hiltz and Lipton, 1989) and endogenous pyrogen injected into the mouse paw (Hiltz et al., 1992). A potential mechanism of action for KPV, in analogy to that reported for -MSH, is its ability to inhibit nuclear factorB activation (Mandrika et al., 2001), potentially leading to inhibition of proinflammatory cytokine synthesis. There is a great interest from pharmaceutical companies to exploit the potent anti-inflammatory effects of the melanocortins. However, there has been much confusion regarding the mechanism behind the effects of the different MSH peptides and receptors. In this study, we systematically investigated the anti-inflammatory effects of core and C-terminal MSH peptides to understand the molecular mechanisms underlying the efficacy of these peptides. We have used an integrated approach taking advantage of recent selective MC-R antagonists, as well as a strain of mice (recessive yellow e/e; Robbins et al., 1993) without a functional MC1-R. The effects of other melanocortin peptides were also studied for comparative purposes. Materials and Methods