Regulation of TNF-Induced Osteoclast Differentiation.

Increased osteoclast (OC) differentiation and activity is the critical event that results in bone loss and joint destruction in common pathological bone conditions, such as osteoporosis and rheumatoid arthritis (RA). RANKL and its decoy receptor, osteoprotegerin (OPG), control OC differentiation and activity. However, there is a specific concern of a rebound effect of denosumab discontinuation in treating osteoporosis. TNFα can induce OC differentiation that is independent of the RANKL/RANK system. In this review, we discuss the factors that negatively and positively regulate TNFα induction of OC formation, and the mechanisms involved to inform the design of new anti-resorptive agents for the treatment of bone conditions with enhanced OC formation. Similar to, and being independent of, RANKL, TNFα recruits TNF receptor-associated factors (TRAFs) to sequentially activate transcriptional factors NF-κB p50 and p52, followed by c-Fos, and then NFATc1 to induce OC differentiation. However, induction of OC formation by TNFα alone is very limited, since it also induces many inhibitory proteins, such as TRAF3, p100, IRF8, and RBP-j. TNFα induction of OC differentiation is, however, versatile, and Interleukin-1 or TGFß1 can enhance TNFα-induced OC formation through a mechanism which is independent of RANKL, TRAF6, and/or NF-κB. However, TNFα polarized macrophages also produce anabolic factors, including insulin such as 6 peptide and Jagged1, to slow down bone loss in the pathological conditions. Thus, the development of novel approaches targeting TNFα signaling should focus on its downstream molecules that do not affect its anabolic effect.

Chondroprotective and anti-inflammatory role of melanocortin peptides in TNF-α activated human C-20/A4 chondrocytes.

BACKGROUND AND PURPOSE: Melanocortin MC(1) and MC(3 ) receptors, mediate the anti-inflammatory effects of melanocortin peptides. Targeting these receptors could therefore lead to development of novel anti-inflammatory therapeutic agents. We investigated the expression of MC(1) and MC(3) receptors on chondrocytes and the role of α-melanocyte-stimulating hormone (α-MSH) and the selective MC(3) receptor agonist, [DTRP(8) ]-γ-MSH, in modulating production of inflammatory cytokines, tissue-destructive proteins and induction of apoptotic pathway(s) in the human chondrocytic C-20/A4 cells. EXPERIMENTAL APPROACH: Effects of α-MSH, [DTRP(8) ]-γ-MSH alone or in the presence of the MC(3/4) receptor antagonist, SHU9119, on TNF-α induced release of pro-inflammatory cytokines, MMPs, apoptotic pathway(s) and cell death in C-20/A4 chondrocytes were investigated, along with their effect on the release of the anti-inflammatory cytokine IL-10. KEY RESULTS: C-20/A4 chondrocytes expressed functionally active MC(1,3) receptors. α-MSH and [DTRP(8) ]-γ-MSH treatment, for 30 min before TNF-α stimulation, provided a time-and-bell-shaped concentration-dependent decrease in pro-inflammatory cytokines (IL-1ß, IL-6 and IL-8) release and increased release of the chondroprotective and anti-inflammatory cytokine, IL-10, whilst decreasing expression of MMP1, MMP3, MMP13 genes.α-MSH and [DTRP(8) ]-γ-MSH treatment also inhibited TNF-α-induced caspase-3/7 activation and chondrocyte death. The effects of [DTRP(8) ]-γ-MSH, but not α-MSH, were abolished by the MC(3/4) receptor antagonist, SHU9119. CONCLUSION AND IMPLICATIONS: Activation of MC(1) /MC(3) receptors in C-20/A4 chondrocytes down-regulated production of pro-inflammatory cytokines and cartilage-destroying proteinases, inhibited initiation of apoptotic pathways and promoted release of chondroprotective and anti-inflammatory cytokines. Developing small molecule agonists to MC(1) /MC(3) receptors could be a viable approach for developing chondroprotective and anti-inflammatory therapies in rheumatoid and osteoarthritis.

Targeting the melanocortin receptor system for anti-stroke therapy.

The melanocortin receptors are a subfamily of G-protein-coupled, rhodopsin-like receptors that are rapidly being acknowledged as an extremely promising target for pharmacological intervention in a variety of different inflammatory pathologies, including stroke. Stroke continues to be a leading cause of death worldwide, with risk factors including smoking, diabetes, hypertension and obesity. The pathophysiology of stroke is highly complex: reintroduction of blood flow to the infarcted brain region is paramount in limiting ischaemic damage caused by stroke, yet a concomitant inflammatory response can compound tissue damage. The possibilities of pro-resolving treatments that target this inflammatory response have only recently begun to be explored. This review discusses the endogenous roles of the melanocortin system in reducing characterized aspects of inflammation, and how these, together with potent neuroprotective actions, suggest its potential as a therapeutic target in stroke.

Anti-inflammatory and antiosteoclastogenesis properties of endogenous melanocortin receptor type 3 in experimental arthritis.

The development of biological therapies has improved management of rheumatoid arthritis. However, costs and unresponsiveness to therapy in a sizeable proportion of patients limit their use, making it imperative to identify new targets for drug development programs. Here we investigated the melanocortin-receptor type 3 (MC(3)) pathway. Gene-deficient mice were subjected to a model of serum-transfer-induced arthritis and joints analyzed for gene expression (cytokines, MCs) and morphology. Pharmacological analyses were also conducted in this model. Osteoclastogenesis was studied from bone marrow cells. Mc(3)(-/-) mice displayed an exacerbated inflammatory arthritis, associated with prominent bone erosion and higher articular expression of Rankl. Osteoclastogenesis studied from Mc(3)(-/-) bone marrow cells revealed a higher degree of responsiveness to Rankl, linked to prolonged NF-κB activation compared to wild types. Up-regulation of a discrete set of inflammatory genes, including Il-1ß, Il-6, and Nos2, was measured in Mc(3)(-/-) mice, and a marked up-regulation of joint Mc(3) accompanied arthritis resolution in wild-type mice. Administration of an MC(3) agonist, D[Trp8]-γ-MSH, attenuated disease incidence and severity in wild-type but not Mc(3)(-/-) mice. Overall, these findings identify MC(3)-mediated signaling as a beneficial pathway in experimental arthritis; hence this receptor is a novel target for the development of therapeutics for arthritis.

Saliva: physiology and diagnostic potential in health and disease.

Saliva has been described as the mirror of the body. In a world of soaring healthcare costs and an environment where rapid diagnosis may be critical to a positive patient outcome, saliva is emerging as a viable alternative to blood sampling. In this review, we discuss the composition and various physiological roles of saliva in the oral cavity, including soft tissue protection, antimicrobial activities, and oral tissue repair. We then explore saliva as a diagnostic marker of local oral disease and focus particularly on oral cancers. The cancer theme continues when we focus on systemic disease diagnosis from salivary biomarkers. Communicable disease is the focus of the next section where we review the literature relating to the direct and indirect detection of pathogenic infections from human saliva. Finally, we discuss hormones involved in appetite regulation and whether saliva is a viable alternative to blood in order to monitor hormones that are involved in satiety.

A role for MC3R in modulating lung inflammation.

In this study we set out to ascertain whether melanocortin peptides could be potential therapeutic agents in allergic and non-allergic models of lung inflammation by identifying the receptor(s) involved using a molecular, genetic and pharmacological approach. Western blot analyses revealed expression of the melanocortin receptor (MCR) type 1 and 3 on alveolar macrophages from wild-type mice. Alveolar macrophage incubation, with the selective MC3R agonist [D-TRP(8)]-gamma-MSH and pan-agonist alpha-MSH but not the selective MC1R agonist MS05, led to an increase in cAMP in wild-type macrophages. This increase occurred also in macrophages taken from recessive yellow (e/e; bearing a mutant and inactive MC1R) mice but not from MC3R-null mice. In an allergic model of inflammation, the pan-agonist alpha-MSH and selective MC3R agonist [D-TRP(8)]-gamma-MSH displayed significant attenuation of both eosinophil and lymphocyte accumulation but not IL-5 levels in wild-type and recessive yellow e/e mice. However in MC3R-null mice, alpha-MSH failed to cause a significant inhibition in these parameters, highlighting a preferential role for MC3R in mediating the anti-inflammatory effects of melanocortins in this model. Utilising a non-allergic model of LPS-induced lung neutrophilia, the pan-agonist alpha-MSH and selective MC3R agonist [D-TRP(8)]-gamma-MSH displayed significant attenuation of neutrophil accumulation and inhibition of TNF-alpha release. Thus, this study highlights that melanocortin peptides inhibit leukocyte accumulation in a model of allergic and non-allergic inflammation and this protective effect is associated with activation of the MC3R. The inhibition of leukocyte accumulation is via inhibition of TNF-alpha in the non-allergic model of inflammation but not IL-5 in the allergic model. These data have highlighted the potential for selective MC3R agonists as novel anti-inflammatory therapeutics in lung inflammation.

Inflamed phenotype of the mesenteric microcirculation of melanocortin type 3 receptor-null mice after ischemia-reperfusion.

The existence of anti-inflammatory circuits centered on melanocortin receptors (MCRs) has been supported by the inhibitory properties displayed by melanocortin peptides in models of inflammation and tissue injury. Here we addressed the pathophysiological effect that one MCR, MCR type 3 (MC3R), might have on vascular inflammation. After occlusion (35 min) and reopening of the superior mesenteric artery, MC3R-null mice displayed a higher degree of plasma extravasation (45 min postreperfusion) and cell adhesion and emigration (90 min postreperfusion). These cellular alterations were complemented by higher expression of mesenteric tissue CCL2 and CXCL1 (mRNA and protein) and myeloperoxydase, as compared with wild-type animals. MC1R and MC3R mRNA and protein were both expressed in the inflamed mesenteric tissue; however, no changes in vascular responses were observed in a mouse colony bearing an inactive MC1R. Pharmacological treatment of animals with a selective MC3R agonist ([D-Trp(8)]-gamma-melanocyte-stimulating hormone; 10 microg i.v.) produced marked attenuation of cell adhesion, emigration, and chemokine generation; such effects were absent in MC3R-null mice. These new data reveal the existence of a tonic inhibitory signal provided by MC3R in the mesenteric microcirculation of the mouse, acting to down-regulate cell trafficking and local mediator generation.

[D-Trp8]-gamma-melanocyte-stimulating hormone exhibits anti-inflammatory efficacy in mice bearing a nonfunctional MC1R (recessive yellow e/e mouse).

Two melanocortin receptors (MC1 and MC3R) have been identified as main transducers of the anti-inflammatory effects of natural and synthetic melanocortins. In this study, we have taken advantage of the recent description of the selective MC3R agonist [d-Trp(8)]-gamma-melanocyte-stimulating hormone (MSH) and of the recessive yellow (e/e) mouse, bearing a nonfunctional MC1R, thereby incrementing our knowledge on this topic. Culturing peritoneal macrophages of recessive yellow (e/e) mice with [d-Trp(8)]-gamma-MSH led to accumulation of cAMP, indicating MC3R receptor functionality: this effect was blocked by a neutralizing antibody against MC3R. Likewise, release of the chemokine KC by urate crystals was attenuated by [d-Trp(8)]-gamma-MSH, and this effect was prevented by synthetic [Ac-Nle(4)-c[Asp(5)-2'-Nal(7),Lys(10)]alpha-MSH(4-10)-NH(2) (SHU9119)] and natural [agouti-related protein (AGRP)] MC3R antagonists but not by the MC4R antagonist Ac-Cys-Nle-Arg-His-d-2-Nal-Arg-Trp-Cys-NH(2) (HS024). Systemic treatment of mice with [d-Trp(8)]-gamma-MSH inhibited KC release and polymorphonuclear cell accumulation elicited by urate crystals in the murine peritoneal cavity. SHU9119 and AGRP prevented the inhibitory actions of [d-Trp(8)]-gamma-MSH, whereas HS024 was inactive. We also demonstrate here that [d-Trp(8)]-gamma-MSH displays a dual mechanism of action by inducing the anti-inflammatory protein heme-oxygenase 1 (HO-1). Treatment with the HO-1 inhibitor zinc protoporphyrin IX exacerbated the inflammatory response elicited by urate crystals and abrogated the anti-inflammatory effects of [d-Trp(8)]-gamma-MSH. In conclusion, these data support the development of the selective MC3R agonist [d-Trp(8)]-gamma-MSH for the treatment of inflammatory pathologies, based on a dual mechanism of cytokine/chemokine inhibition and induction of the anti-inflammatory protein HO-1.

The melanocortin peptide HP228 displays protective effects in acute models of inflammation and organ damage.

The clinically efficacious melanocortin peptide HP228 has here been investigated for its anti-inflammatory efficacy. In this study we have investigated the efficacy of HP228 in murine acute models of inflammation and myocardial ischaemia. Systemic treatment of mice with HP228 inhibited neutrophil accumulation in zymosan; urate crystal and carrageenan induced inflammatory models. In the urate model this was due to inhibition of pro-inflammatory chemokines and cytokines, whilst different mechanisms exist for zymosan peritonitis and carrageenan-induced air-pouch inflammation. HP228 was next evaluated in a model of myocardial ischaemia, another condition where cytokines and neutrophils are thought to play a causal role. HP228 caused a 50% reduction in myocardial damage following reperfusion. HP228 therefore inhibits the most important facet of the host inflammatory response namely leukocyte migration. These data show for the first time that the clinically efficacious peptide HP228 displays protective effects in models of inflammation and organ damage.

Melanocortin receptor signaling in RAW264.7 macrophage cell line.

Melanocortin peptides modulate cytokine release and adhesion molecule expression. Here we have investigated the early cell-signaling pathway responsible for the induction of interleukin-10 (IL-10) in RAW264.7 cells. Cell incubation with ACTH(1-39) or MTII (melanotan II) did not alter ERK1/2 and JNK phosphorylation, while p38 phosphorylation and intracellular cAMP accumulation occurred within minutes. ACTH(1-39) and MTII provoked a time-dependent accumulation of IL-10 that was abrogated by the PKA inhibitor H-89 and only partially blocked by the p38 MAPK inhibitor SB203580. Thus, in RAW264.7 cells, IL-10 induction by the melanocortins is via the PKA pathway, and this mechanism could contribute to their anti-inflammatory profile.

In vitro and in vivo induction of heme oxygenase 1 in mouse macrophages following melanocortin receptor activation.

RAW264.7 cell incubation with adrenocorticotrophin (ACTH) led to a time-dependent (4-24 h) and concentration-related (1-100 ng/ml) induction of heme oxygenase (HO)-1, and this was a specific effect, because the pattern of expression of other cellular proteins (HO-2, heat shock proteins 70 and 90) was not modified by ACTH. Combined RT-PCR and Western blot analyses revealed expression of the melanocortin receptor (MC-R) types 1 and 3, but not 4, in these cells. However, use of more selective agonists (including melanotan (MTII)) indicated a predominant role for MC3-R in the induction of HO-1 expression and activity. Relevantly, ACTH and MTII incubation with primary peritoneal macrophages (Mphi) also induced HO-1 expression. The potential link between MC3-R dependent cAMP formation and HO-1 induction was ascertained by the following: 1) ACTH and MTII produced a concentration-dependent accumulation of cAMP in RAW264.7 cells, and 2) whereas a selective inhibitor of cAMP-dependent protein kinase A abrogated ACTH- and MTII-induced HO-1 expression, a soluble cAMP derivative promoted HO-1 induction both in RAW264.7 cells and primary Mphi. HO-1 induction in peritoneal Mphi was also detected following in vivo administration of MTII, and appeared to be functionally related to the antimigratory effect of this melanocortin, as determined with a specific inhibitor (zinc protoporphyrin IX). In conclusion, this study highlights a biochemical link between MC-R activation and HO-1 induction in the Mphi, and proposes that this may be of functional relevance in determining MC-R-dependent control of the host inflammatory response.

Inhibitory effects of TSG-6 Link module on leukocyte-endothelial cell interactions in vitro and in vivo.

OBJECTIVE: The authors investigated whether the anti-inflammatory protein tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) and its Link module (Link_TSG6) could affect the complex multistep process of leukocyte/endothelial cell (EC) interaction. METHODS: Mouse mesenteries were inflamed with interleukin (IL)-1beta and the extent of leukocyte rolling, adhesion, and emigration was determined after 2 h. Link_TSG6 and a single-point mutant (termed K13E) were given intraperitoneally together with the cytokine. Human neutrophil chemotaxis and transmigration were determined in vitro in response to IL-8 and/or TNF-alpha. TSG-6, Link_TSG6, and K13E were added to the leukocytes or the EC monolayers. RESULTS: Co-injection of Link_TSG6 with IL-1beta selectively inhibited cell flux, adhesion, and emigration as analyzed in mesenteric postcapillary venules. The fewer cells that rolled in the animals treated with Link_TSG6 displayed a velocity similar to that measured in vehicle-treated mice. In vitro, Link_TSG6 did not affect neutrophil chemotaxis or EC activation but did inhibit neutrophil transmigration across EC monolayers. The latter effect was shared by full-length TSG-6 and observed equally in response to IL-8 or TNF-alpha. CONCLUSIONS: These data restrict the site of action for at least some of the anti-inflammatory effects ascribed to TSG-6/Link_TSG6 to the microenvironment of the extravasating leukocyte.

MC-3 receptor and the inflammatory mechanisms activated in acute myocardial infarct.

Investigation of the mechanisms activated by endogenous inhibitory pathways can lead to identification of novel targets for cardiovascular inflammatory pathologies. Here we exploited the potential protective role that melanocortin receptor type 3 (MC3-R) activation might play in a myocardial ischemia-reperfusion injury model. In resting conditions, mouse and rat heart extracts expressed MC3-R mRNA and protein, without changes following ischemia-reperfusion. At the cellular level heart macrophages, but not fibroblasts or cardiomyocytes, expressed this receptor, as demonstrated by immunogold labeling. In vivo, administration of the melanocortin agonist MTII (10 microg per mouse equivalent to 9.3 nmol) 30 min prior to ischemia (25 min) attenuated mouse heart 2 h reperfusion injury by approximately 40%, an effect prevented by the mixed MC3/4-R antagonist SHU9119 but not by the selective MC4-R antagonist HS204. Similar results were obtained when the compound was given at the beginning of the reperfusion period. Importantly, delayed myocardial damage as measured 24 h post-reperfusion was equally protected by administration of 10 microg MTII. The focus on MC3-R was also substantiated by analysis of the recessive yellow (e/e) mouse, bearing a mutated (inactive) MC1-R, in which MTII was fully protective. Myocardial protection was associated with reduced markers of systemic and local inflammation, including cytokine contents (interleukin-1 and KC) and myeloperoxidase activity. In conclusion, this study has highlighted a previously unrecognized protective role for MC3-R activation on acute and delayed heart reperfusion injury. These data may open new avenues for therapeutic intervention against heart and possibly other organ ischemia-reperfusion injury.

Modulation of inflammation and response to dexamethasone by Annexin 1 in antigen-induced arthritis.

OBJECTIVE: Annexin 1 (Anx-1) is a putative mediator of the antiinflammatory actions of glucocorticoids (GCs). This study investigated the role of Anx-1 in experimental arthritis and in GC-mediated inhibition of inflammation, using antigen-induced arthritis (AIA) in Anx-1 knockout (Anx-1(-/-)) mice. METHODS: Arthritis was induced by intraarticular injection of methylated BSA (mBSA) in mice preimmunized with mBSA. Disease was assessed after 7 days by histologic examination of the knee joints. Serum levels of anti-mBSA IgG were determined by enzyme-linked immunosorbent assay. Cytokine messenger RNA (mRNA) expression was detected by real-time polymerase chain reaction. RESULTS: A significant exacerbation of arthritis was observed in the Anx-1(-/-) mice compared with wild-type (WT) mice. This was associated with increased mRNA expression of synovial interleukin-1 beta, tumor necrosis factor alpha, interleukin-6, and macrophage migration inhibitory factor. Dexamethasone significantly reduced the histologic severity of synovitis and bone damage in the WT mice, but exerted no inhibitory effects in the Anx-1(-/-) mice, and also significantly reduced the serum levels of anti-mBSA IgG and the numbers of peripheral blood neutrophils and lymphocytes in WT mice, but had no such effect in Anx-1(-/-) mice. CONCLUSION: Anx-1 exerts endogenous antiinflammatory effects on AIA via the regulation of cytokine gene expression, and also mediates the antiinflammatory actions of dexamethasone in AIA.

Dissection of the anti-inflammatory effect of the core and C-terminal (KPV) alpha-melanocyte-stimulating hormone peptides.

In this study, we analyzed the anti-inflammatory effects of alpha-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, alpha-MSH, the core melanocortin peptide His-Phe-Arg-Trp, and the melanocontin receptor 3/4 agonist Ac-Nle4-c[Asp5,d-Phe7,Lys10]NH2 ACTH4-10 (MTII) but not the selective MC1-R agonist H-Ser-Ser-Ile-Ile-Ser-His-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-Nle4-c[Asp5,d-2Nal7,Lys10]NH2 ACTH4-10 (SHU9119). In vitro, macrophage activation, determined as release of KC and interleukin (IL)-1beta was inhibited by alpha-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-1beta-induced peritonitis inflammation and in mice with a nonfunctional MC1-R (recessive yellow e/e mice). In conclusion, these data highlight that the C-terminal 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-1beta functions.

The link module from human TSG-6 inhibits neutrophil migration in a hyaluronan- and inter-alpha -inhibitor-independent manner.

TSG-6 protein (the secreted product of the tumor necrosis factor-stimulated gene-6), a hyaluronan-binding protein comprised mainly of a Link and CUB module arranged in a contiguous fashion, has been shown previously to be a potent inhibitor of neutrophil migration in an in vivo model of acute inflammation (Wisniewski, H. G., Hua, J. C., Poppers, D. M., Naime, D., Vilcek, J., and Cronstein, B. N. (1996) J. Immunol. 156, 1609-1615). It was hypothesized that this activity of TSG-6 was likely to be mediated by its potentiation of inter-alpha-inhibitor anti-plasmin activity (causing a down-regulation of the protease network), which was reliant on these proteins forming a stable, probably covalent approximately 120-kDa complex. Here we have shown that the recombinant Link module from human TSG-6 (Link_TSG6; expressed in Escherichia coli) has an inhibitory effect on neutrophil influx into zymosan A-stimulated murine air pouches, equivalent to that of full-length protein (which we produced in a Drosophila expression system). The active dose of 1 microg of Link_TSG6 per mouse (administered intravenously) also resulted in a significant reduction in the concentrations of various inflammatory mediators (i.e. tumor necrosis factor-alpha, KC, and prostaglandin E(2)) in air pouch exudates. Link_TSG6, although unable to form a stable complex with inter-alpha-inhibitor (under conditions that promote maximum complex formation with the full-length protein), could potentiate its anti-plasmin activity. This demonstrates that formation of an approximately 120-kDa TSG-6.inter-alpha-inhibitor complex is not required for TSG-6 to enhance the serine protease inhibitory activity of inter-alpha-inhibitor. Six single-site Link_TSG6 mutants (with wild-type folds) were compared for their abilities to inhibit neutrophil migration in vivo, bind hyaluronan, and potentiate inter-alpha-inhibitor. These experiments indicate that all of the inhibitory activity of TSG-6 resides within the Link module domain, and that this anti-inflammatory property is not related to either its hyaluronan binding function or its potentiation of the anti-plasmin activity of inter-alpha-inhibitor.

Endogenous lipid- and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor.

Aspirin (ASA) and dexamethasone (DEX) are widely used anti-inflammatory agents yet their mechanism(s) for blocking polymorphonuclear neutrophil (PMN) accumulation at sites of inflammation remains unclear. Here, we report that inhibition of PMN infiltration by ASA and DEX is a property shared by aspirin-triggered lipoxins (ATL) and the glucocorticoid-induced annexin 1 (ANXA1)-derived peptides that are both generated in vivo and act at the lipoxin A(4) receptor (ALXR/FPRL1) to halt PMN diapedesis. These structurally diverse ligands specifically interact directly with recombinant human ALXR demonstrated by specific radioligand binding and function as well as immunoprecipitation of PMN receptors. In addition, the combination of both ATL and ANXA1-derived peptides limited PMN infiltration and reduced production of inflammatory mediators (that is, prostaglandins and chemokines) in vivo. Together, these results indicate functional redundancies in endogenous lipid and peptide anti-inflammatory circuits that are spatially and temporally separate, where both ATL and specific ANXA1-derived peptides act in concert at ALXR to downregulate PMN recruitment to inflammatory loci.

Melanocortin peptides and their receptors: new targets for anti-inflammatory therapy.

alpha-Melanocyte stimulating hormone (alpha-MSH) and other melanocortin peptides are potent anti-inflammatory agents that exhibit efficacy in many animal models of acute and chronic inflammation. These peptides are produced both centrally and peripherally, and exert their biological actions via activation of membrane-bound receptors. To exploit the therapeutic potential of such anti-inflammatory peptides, it is essential that the receptor subtype mediating these effects is identified with certainty.

Activation of melanocortin type 3 receptor as a molecular mechanism for adrenocorticotropic hormone efficacy in gouty arthritis.

OBJECTIVE: To test the hypothesis that local activation of melanocortin receptor(s) by adrenocorticotropic hormone (ACTH) could be responsible, at least in part, for its efficacy in human gouty arthritis. METHODS: Monosodium urate monohydrate (MSU) crystals were administered into rat knee joints either alone or with ACTH or a selective melanocortin type 3 receptor (MC3-R) agonist. Neutrophil migration, arthritis score, increases in joint size, and cytokine levels were measured over time. MC3-R expression on rat knee joint macrophages was monitored by electron microscopy and intracellular accumulation of cyclic adenosine monophosphate. RESULTS: MSU crystals produced a knee joint inflammation that was time dependent and was characterized by cell influx and cytokine release that was sensitive to treatment with classic anti-arthritic drugs (indomethacin, colchicine, dexamethasone). Local, but not systemic, ACTH had an antiinflammatory effect in normal rats, a dose that did not alter circulating corticosterone (5 microg). This treatment was also effective in adrenalectomized rats. Rat knee joint macrophages expressed functional MC3-R. The MC3-R antagonist (SHU9119, 10 microg) blocked ACTH antiinflammatory actions, whereas antiinflammatory activity was retained with a selective MC3-R agonist (gamma(2)-melanocyte-stimulating hormone). CONCLUSION: This research provides evidence for a separate mechanism of action of ACTH in experimental gouty arthritis and points to a novel antiinflammatory target (selective agonists at MC3-R) for clinical management of human gouty arthritis and possibly other chronic inflammatory conditions.