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Alzheimer's disease and dementia are among the most significant current healthcare challenges given the rapidly growing elderly population, and the almost total lack of effective therapeutic interventions. Alzheimer's disease pathology has long been considered in terms of accumulation of amyloid beta and hyperphosphorylated tau, but the importance of neuroinflammation in driving disease has taken greater precedence over the last 15-20 years. Inflammatory activation of the primary brain immune cells, the microglia, has been implicated in Alzheimer's pathogenesis through genetic, pre-clinical, imaging and post-mortem human studies, and strategies to regulate microglial activity may hold great promise for disease modification. Neuroinflammation is necessary for defence of the brain against pathogen invasion or damage but is normally self-limiting due to the engagement of endogenous pro-resolving circuitry that terminates inflammatory activity, a process that appears to fail in Alzheimer's disease. Here we discuss the potential for a major regulator and promoter of resolution, the receptor FPR2, to restrain pro-inflammatory microglial activity, and propose that it may serve as a valuable target for therapeutic investigation in Alzheimer's disease. [Abstract copyright: This article is protected by copyright. All rights reserved.]

Neuroinflammation driven primarily by microglia directly contributes to neuronal death in many neurodegenerative diseases. Classical anti-inflammatory approaches aim to suppress pro-inflammatory mediator production, but exploitation of inflammatory resolution, the active endogenous process whereby inflammation is terminated, may also be of benefit. A key driver of peripheral inflammatory resolution, formyl peptide receptors 2/3 (Fpr2/3), is expressed by microglia, but its therapeutic potential in neurodegeneration remains unclear. Here, we sought to provide a proof-of-principle that Fpr2/3 targeting could reverse inflammatory microglial activation induced by the potent bacterial inflammogen lipopolysaccharide (LPS). Stimulation of murine BV2 microglia with LPS triggered release of nitric oxide, TNFα and IL-10, upregulated surface expression of CD38 and CD40 and suppressed CD206, all of which were reduced by subsequent treatment with the Fpr2/3 ligand C43. Cellular exposure to LPS also stimulated NADPH oxidase and mitochondrial derived reactive oxygen species production, effects reversed by C43 treatment. Mechanistic studies showed C43 to act through p38 MAPK phosphorylation and reduction of LPS-induced NFκB nuclear translocation through prevention of IκBα degradation. Here, we provide proof-of-concept data indicating exploitation of the pro-resolving receptor Fpr2/3 as a promising therapeutic strategy in neuroinflammatory conditions.

Background and PurposeMicroglial inflammatory activity is thought to be a major contributor to the pathology of neurodegenerative conditions such as Alzheimer’s disease (AD), and strategies to restrain their behaviour are under active investigation. Classically, anti-inflammatory approaches aim to suppress pro-inflammatory mediator production, but exploitation of inflammatory resolution, the endogenous process whereby an inflammatory reaction is terminated, has not been fully investigated as a therapeutic approach in AD. In this study, we sought to provide proof-of-principal that the major pro-resolving actor, formyl peptide receptor 2, Fpr2, could be targeted to reverse microglial activation induced by the AD-associated pro-inflammatory stimulus, oligomeric β-amyloid (oAβ).Experimental ApproachThe immortalised murine microglial cell line BV2 was employed as a model system to investigate the pro-resolving effects of the Fpr2 ligand QC1 upon oAβ-induced inflammatory, oxidative and metabolic behaviour. Cytotoxic behaviour of BV2 cells was assessed through use of co-cultures with retinoic acid-differentiated human SH-SY5Y cells.Key ResultsStimulation of BV2 cells with oAβ at 100nM did not induce classical inflammatory marker production but did stimulate production of reactive oxygen species (ROS), an effect that could be reversed by subsequent treatment with the Fpr2 ligand QC1. Further investigation revealed that oAβ-induced ROS production was associated with NADPH oxidase activation and a shift in BV2 cell metabolic phenotype, activating the pentose phosphate pathway and NADPH production, changes that were again reversed by QC1 treatment. Microglial oAβ-stimulated ROS production was sufficient to induce apoptosis of bystander SH-SY5Y cells, an effect that could be prevented by QC1 treatment.Conclusion and ImplicationsIn this study, we provide proof-of-concept data that indicate exploitation of the pro-resolving receptor Fpr2 can reverse damaging oAβ-induced microglial activation. Future strategies aiming to restrain neuroinflammation in conditions such as AD should examine pro-resolving actors as a mechanism to harness the brain’s endogenous healing pathways and limit neuroinflammatory damage.

Microglial inflammatory activity is thought to be a major contributor to the pathology of neurodegenerative conditions such as Alzheimer’s disease (AD), and strategies to restrain their behaviour are under active investigation. Classically, anti-inflammatory approaches are aimed at suppressing proinflammatory mediator production, but exploitation of inflammatory resolution, the endogenous process whereby an inflammatory reaction is terminated, has not been fully investigated as a therapeutic approach in AD. In this study, we sought to provide proof-of-principle that the major proresolving actor, formyl peptide receptor 2, Fpr2, could be targeted to reverse microglial activation induced by the AD-associated proinflammatory stimulus, oligomeric β-amyloid (oAβ). The immortalised murine microglial cell line BV2 was employed as a model system to investigate the proresolving effects of the Fpr2 ligand QC1 upon oAβ-induced inflammatory, oxidative, and metabolic behaviour. Cytotoxic behaviour of BV2 cells was assessed through the use of cocultures with retinoic acid-differentiated human SH-SY5Y cells. Stimulation of BV2 cells with oAβ at 100 nM did not induce classical inflammatory marker production but did stimulate production of reactive oxygen species (ROS), an effect that could be reversed by subsequent treatment with the Fpr2 ligand QC1. Further investigation revealed that oAβ-induced ROS production was associated with NADPH oxidase activation and a shift in BV2 cell metabolic phenotype, activating the pentose phosphate pathway and NADPH production, changes that were again reversed by QC1 treatment. Microglial oAβ-stimulated ROS production was sufficient to induce apoptosis of bystander SH-SY5Y cells, an effect that could be prevented by QC1 treatment. In this study, we provide proof-of-concept data that indicate exploitation of the proresolving receptor Fpr2 can reverse damaging oAβ-induced microglial activation. Future strategies that are aimed at restraining neuroinflammation in conditions such as AD should examine proresolving actors as a mechanism to harness the brain’s endogenous healing pathways and limit neuroinflammatory damage.

Human melanocortin MC1 and MC3 receptors expressed on C-20/A4 chondrocytes exhibit chondroprotective and anti-inflammatory effects when activated by melanocortin peptides. Nearly 9 million people in the UK suffer from osteoarthritis, and bacterial infections play a role in its development. Here, we evaluate the effect of a panel of melanocortin peptides with different selectivity for human melanocortin MC1 (α-MSH, BMS-470539 dihydrochloride) and MC3 ([DTrp8]-γ-MSH, PG-990) receptors and C-terminal peptide α-MSH11-13(KPV), on inhibiting LPS-induced chondrocyte death, pro-inflammatory mediators and induction of anti-inflammatory proteins. C-20/A4 chondrocytes were treated with a panel of melanocortin peptides prophylactically and therapeutically in presence of LPS (0.1 μg/ml). The chondroprotective properties of these peptides determined by cell viability assay, RT-PCR, ELISA for detection of changes in inflammatory markers (IL-6, IL-8 and MMP-1, -3 and -13) and western blotting for expression of the anti-inflammatory protein heme-oxygenase-1. C-20/A4 expressed human melanocortin MC1 and MC3 receptors and melanocortin peptides elevated cAMP. LPS stimulation caused a reduction in C-20/A4 viability, attenuated by the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride, and MC3 receptor agonists PG-990 and [DTrp8]-γ-MSH. Prophylactic and therapeutic regimes of [DTrp8]-γ-MSH significantly inhibited LPS-induced modulation of cartilage-damaging IL-6, IL-8, MMPs −1,-3 and −13 mediators both prophylactically and therapeutically, whilst human melanocortin MC1 and MC3 receptor agonists promoted an increase in HO-1 production. In the presence of LPS, activation of human melanocortin MC1 and MC3 receptors provided potent chondroprotection, upregulation of anti-inflammatory proteins and downregulation of inflammatory and proteolytic mediators involved in cartilage degradation, suggesting a new avenue for osteoarthritis treatment.

Objective— Neutrophil recruitment is a key process in the pathogenesis of stroke, and may provide a valuable therapeutic target. Targeting the melanocortin (MC) receptors has previously shown to inhibit leukocyte recruitment in peripheral inflammation, however, it is not known whether treatments are effective in the unique cerebral microvascular environment. Here, we provide novel research highlighting the effects of the MC peptides on cerebral neutrophil recruitment, demonstrating important yet discrete roles for both MC 1 and MC 3 . Approach and Results— Using intravital microscopy, in 2 distinct murine models of cerebral ischemia-reperfusion (I/R) injury, we have investigated MC control for neutrophil recruitment. After global I/R, pharmacological treatments suppressed pathological neutrophil recruitment. MC 1 selective treatment rapidly inhibited neutrophil recruitment while a nonselective MC agonist provided protection even when coadministered with an MC 3/4 antagonist, suggesting the importance of early MC 1 signaling. However, by 2-hour reperfusion, MC 1 -mediated effects were reduced, and MC 3 anti-inflammatory circuits predominated. Mice bearing a nonfunctional MC 1 displayed a transient exacerbation of neutrophil recruitment after global I/R, which diminished by 2 hours. However importantly, enhanced inflammatory responses in both MC 1 mutant and MC 3 −/− mice resulted in increased infarct size and poor functional outcome after focal I/R. Furthermore, we used an in vitro model of leukocyte recruitment to demonstrate these anti-inflammatory actions are also effective in human cells. Conclusions— These studies reveal for the first time MC control for neutrophil recruitment in the unique pathophysiological context of cerebral I/R, while also demonstrating the potential therapeutic value of targeting multiple MCs in developing effective therapeutics.

BACKGROUND AND PURPOSE: Melanocortin MC1 and MC3 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 MC1 and MC3 receptors on chondrocytes and the role of α-melanocyte-stimulating hormone (α-MSH) and the selective MC3 receptor agonist, [DTRP8]-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, [DTRP8]-γ-MSH alone or in the presence of the MC3/4 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 MC1,3 receptors. α-MSH and [DTRP8]-γ-MSH treatment, for 30 min before TNF-α stimulation, provided a time-and-bell-shaped concentration-dependent decrease in pro-inflammatory cytokines (IL-1β, 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 [DTRP8]-γ-MSH treatment also inhibited TNF-α-induced caspase-3/7 activation and chondrocyte death. The effects of [DTRP8]-γ-MSH, but not α-MSH, were abolished by the MC3/4 receptor antagonist, SHU9119. CONCLUSION AND IMPLICATIONS: Activation of MC1/MC3 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 MC1/MC3 receptors could be a viable approach for developing chondroprotective and anti-inflammatory therapies in rheumatoid and osteoarthritis.

Myostatin is a powerful negative regulator of skeletal muscle mass in mammalian species. It plays a key role in skeletal muscle homeostasis and has now been well described since its discovery. Myostatin is capable of inducing muscle atrophy via its inhibition of myoblast proliferation, increasing ubiquitin-proteasomal activity and downregulating activity of the IGF-Akt pathway. These well-recognized effects are seen in multiple atrophy causing situations, including injury, diseases such as cachexia, disuse and space flight, demonstrating the importance of the myostatin signalling mechanism. Based on this central role, significant work has been pursued to inhibit myostatin's actions in vivo. Importantly, several new studies have uncovered roles for myostatin distinct from skeletal muscle size. Myostatin has been suggested to play a role in cardiomyocyte homeostasis, glucose metabolism and adipocyte proliferation, all of which are examined in detail below. Based on these effects, myostatin inhibition has potential to be widely utilized in many Western diseases such as chronic obstructive pulmonary disease, type II diabetes and obesity. However, if myostatin inhibitors are to successfully translate from bench-top to bedside in the near future, awareness must be raised on these non-traditional effects of myostatin away from skeletal muscle. Indeed, further research into these novel areas is required.

Objective— To determine whether the inhibitory action of the antiallergic cromone “mast cell stabilizing” drugs on polymorphonuclear leukocyte (PMN) trafficking is mediated through an annexin-A1 (Anx-A1) dependent mechanism. Methods and Results— Intravital microscopy was used to monitor the actions of cromones in the inflamed microcirculation. Reperfusion injury provoked a dramatic increase in adherent and emigrated leukocytes in the mesenteric vascular bed, associated with augmented tissue levels of myeloperoxidase. Nedocromil, 2 to 20 mg/kg, significantly ( P −/− mice. Short pretreatment of human PMNs with nedocromil, 10 nmol/L, inhibited cell adhesion ( P Conclusion— We propose a novel mechanism to explain the antiinflammatory actions of cromones on PMN trafficking, an effect that has long puzzled investigators.

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-1β, 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.

Background and purpose: Over three decades of research evaluating the biology of melanocortin (MC) hormones and synthetic peptides, activation of the MC type 1 (MC1) receptor has been identified as a viable target for the development of novel anti-inflammatory therapeutic agents. Here, we have tested a recently described selective agonist of MC1 receptors, BMS-470539, on leucocyte/post-capillary venule interactions in murine microvascular beds. Experimental approach: Intravital microscopy of two murine microcirculations were utilized, applying two distinct modes of promoting inflammation. The specificity of the effects of BMS-470539 was assessed using mice bearing mutant inactive MC1 receptors (the recessive yellow e/e colony). Key results: BMS-470539, given before an ischaemia–reperfusion protocol, inhibited cell adhesion and emigration with no effect on cell rolling, as assessed 90 min into the reperfusion phase. These properties were paralleled by inhibition of tissue expression of both CXCL1 and CCL2. Confocal investigations of inflamed post-capillary venules revealed immunostaining for MC1 receptors on adherent and emigrated leucocytes. Congruently, the anti-inflammatory properties of BMS-470539 were lost in mesenteries of mice bearing the inactive mutant MC1 receptors. Therapeutic administration of BMS-470539 stopped cell emigration, but did not affect cell adhesion in the cremasteric microcirculation inflamed by superfusion with platelet-activating factor. Conclusions and implications: Activation of MC1 receptors inhibited leucocyte adhesion and emigration. Development of new chemical entities directed at MC1 receptors could be a viable approach in the development of novel anti-inflammatory therapeutic agents with potential application to post-ischaemic conditions.

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-Trp8]-γ-melanocyte-stimulating hormone; 10 μg 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.—Leoni, G., Patel, H. B., Sampaio, A. L. F., Gavins, F. N. E., Murray, J. F., Grieco, P., Getting, S. J., Perretti, M. Inflamed phenotype of the mesenteric microcirculation of melanocortin type 3 receptor-null mice after ischemia-reperfusion.

In this study we have characterized the anti-inflammatory profile of a selective melanocortin type 3 receptor (MC3-R) ligand [D-Trp8]-gamma-MSH, validating in vitro results with analyses in mice deficient for this receptor subtype. In wild-type (WT) macrophages, [D-Trp8]-gamma-MSH activated MC3-R (as tested by accumulation of cyclic AMP) and inhibited (approximately 50%) the release of interleukin (IL)-1 and the chemokine KC (CXCL1), but was ineffective in cells taken from MC3-R null mice. In vivo, administration of 3-30 microg [D-Trp8]-gamma-MSH significantly inhibited leukocyte influx and cytokine production in a model of crystal-induced peritonitis, and these effects were absent in MC3-R null mice or blocked by coadministration of an MC3-R antagonist. Finally, in a model of gouty arthritis, direct injection of urate crystals into the rat joint provoked a marked inflammatory reaction that was significantly inhibited (approximately 70%) by systemic or local administration of [D-Trp8]-gamma-MSH. In conclusion, using an integrated transgenic and pharmacological approach, we provide strong proof of concept for the development of selective MC3-R agonists as novel anti-inflammatory therapeutics.

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.

Adrenocorticotrophic hormone (ACTH(1-39)) and the melanocortins (alpha, beta and gamma-melanocyte-stimulating hormone [MSH]) are derived from a larger precursor molecule known as the pro-opiomelanocortin (POMC) protein. They exert their numerous biological effects by activating 7 transmembrane G-protein coupled receptors (GPCR), leading to adenylyl cyclase activation and subsequent cAMP accumulation within the target cell. To date, 5 melanocortin receptors (MCR) have been identified and termed MC1R to MC5R, they have been shown to have a wide and varied distribution throughout the body, being found in the central nervous system (CNS), periphery and immune cells. Melanocortins have a multitude of actions including: (i) modulating disease pathologies including arthritis, asthma, obesity; (ii) affecting functions, for example erectile dysfunction, skin tanning; and (iii) organ systems, for example cardiovascular system. Recently a mechanistic approach has been identified with alpha-MSH preventing NF-kappaB activation via the preservation and expression of IkappaBalphaprotein. This leads to a reduction of pro-inflammatory mediators including cytokines and inhibition of adhesion molecule expression, with subsequent reduction in leukocyte emigration. Development of selective ligands with an appropriate pharmacokinetic profile will enable a pharmacological evaluation of the potential beneficial effects of the melanocortins. In this review I have discussed the potential mechanistic action for the melanocortins and some of the disease pathologies shown to be modulated. This review proposes targeting the MCR with the ultimate aim of controlling many of the diseases that we face today.

Cardiovascular disease is a major cause of mortality within the western world affecting 2.7 million British people. This review highlights the beneficial effects of naturally occurring hormones and their peptides, in myocardial ischaemic-injury (MI) models, a disease pathology in which cytokines and neutrophils play a causal role. Here we discuss two distinct classes of endogenous peptides: the steroid inducible annexin 1 and the melanocortin peptides. Annexin 1 and the melanocortins counteract the most important part of the host inflammatory response, namely, the process of leukocyte extravasation, as well as release of proinflammatory mediators. Their biological effects are mediated via the seven transmembrane G-protein-coupled receptors, the fMLP receptor family (or FPR), and the melanocortin receptors, respectively. Pharmacological analysis has demonstrated that the first 24 amino acids of the N-terminus (termed Ac2-26) are the most active region. Both exogenous annexin 1 and its peptides demonstrate cardioprotectiveness and continuing work is required to understand this annexin 1/FPR relationship fully. The melanocortin peptides are derived from a precursor molecule called the POMC protein. These peptides display potent anti-inflammatory effects in human and animal models of disease. In MI, the MC3R has been demonstrated to play an important role in mediating the protective effects of these peptides. The potential anti-inflammatory role for endogenous peptides in cardiac disease is in its infancy. The inhibition of cell migration and release of cytokines and other soluble mediators appears to play an important role in affording protection in ischaemic injury and thus may lead to potential therapeutic targets. ©2006 with author. Published by TheScientificWorld, Ltd. Research Advisory Board, St. Bartholomew’s and the Royal London School of Medicine. (Grant Code RAB04/PJ/4); British Heart Foundation (Grant Code FS/03/100/16326).

The issue of which melanocortin receptor (MC-R) is responsible for the anti-inflammatory effects of melanocortin peptides is still a matter of debate. Here we have addressed this aspect using a dual pharmacological and genetic approach, taking advantage of the recent characterization of more selective agonists/antagonists at MC1 and MC3-R as well as of the existence of a naturally defective MC1-R mouse strain, the recessive yellow (e/e) mouse. RT-PCR and ultrastructural analyses showed the presence of MC3-R mRNA and protein in peritoneal macrophages (Mφ) collected from recessive yellow (e/e) mice and wild-type mice. This receptor was functional as Mφ incubation (30 min) with melanocortin peptides led to accumulation of cAMP, an effect abrogated by the MC3/4-R antagonist SHU9119, but not by the selective MC4-R antagonist HS024. In vitro Mφ activation, determined as release of the CXC chemokine KC and IL-1β, was inhibited by the more selective MC3-R agonist γ2-melanocyte stimulating hormone but not by the selective MC1-R agonist MS05. Systemic treatment of mice with a panel of melanocortin peptides inhibited IL-1β release and PMN accumulation elicited by urate crystals in the murine peritoneal cavity. MS05 failed to inhibit any of the inflammatory parameters either in wild-type or recessive yellow (e/e) mice. SHU9119 prevented the inhibitory actions of γ2-melanocyte stimulating hormone both in vitro and in vivo while HS024 was inactive in vivo. In conclusion, agonism at MC3-R expressed on peritoneal Mφ leads to inhibition of experimental nonimmune peritonitis in both wild-type and recessive yellow (e/e) mice.

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.

Introduction: Osteoarthritis (OA) is a degenerative joint disease partially mediated by the catabolic cytokine TNF-α, leading to progressive and permanent degeneration of cartilage. Silymarin, an extract from the ripe seeds of Silybum marianum , possesses anti-inflammatory properties that could help resolve inflammation in chondrocytes. This study aims to assess the chondroprotection and anti-inflammatory effects of silymarin on TNF-α induced cell death, pro-inflammatory cytokine and matrix-metalloproteinase release. Methods: Human C-20/A4 chondrocytic cells were treated with silymarin (2–20 μM) alone or in the presence of dexamethasone (10 μM) for 30 min prior to TNF-α (60 pg/ml) stimulation for 6 h. Cell viability was determined by MTT assat. Cell free supernatants were collected to detect IL-6, IL-8 and MMP-1 release by ELISA. RT-PCR was used to detect IL-6, IL-8 and MMP-1 gene expression. Data are expressed as Mean ± SD of n = 4 determinations in triplicate. * P P

Skeletal muscle loss correlates with mortality across chronic pathological disorders. Chronic O2 reduction induces atrophy in healthy mountaineers and hypoxemic COPD patients. Hypoxic rats and hypo...

Melanocortin receptors (MCS, MC1–MC5) are GPCRs, activated with different affinities by the melanocortin peptides (?-, ?-, ?-MSH and ACTH). They are widely distributed throughout the body displaying a multitude of actions however their role in reproductive physiology is unclear. Previously, we have shown a reduction of pituitary hormone content and abnormalities in testes morphology in male MC3 null mice. The aim of this study was to characterise MCS expression in the male mouse reproductive axis and to investigate the effects of MCS agonists/antagonists on in vitro testicular testosterone production (TP). MCS expression patterns were determined in mRNAs from hypothalami, pituitaries and testes of Balb/c and C57BL/6 male mice by two-step reverse transcriptase (RT) - quantitative (q) PCR and analysed using qBaseplus. To examine the functions of MCS in gonads, testes of Balb/c and C57BL/6 mice were hemi-sected and incubated with the potent MC3 and, to a lesser extent, MC4 agonist ([D-TRP8]-?2-MSH; 0.3–10 ?g/ml) in the presence or absence of hCG (50 mIU/ml) and/or a MC3/MC4 antagonist and MC1/MC5 agonist (SHU9119; 10 ?g/ml) for 5 h at 35 °C in air. There were no significant differences in the pattern of MCS expression between C57BL/6 and Balb/c. The most abundant MC in the hypothalamus and pituitary appears to be MC3 whilst in the testis it was MC2. TP in C57BL/6 mice is 50% less than in Balb/c: confirming published data that the former mice are hypoandrogenic. Activation of MC3 and MC4 using [D-TRP8]-?2-MSH had no or inhibitory effects on basal TP in Balb/c and C57BL/6, respectively. The same ligand decreased TP in both strains in the presence of hCG. In contrast, SHU9119±hCG increased TP threefold compared to basal levels independently of ligand dose in Balb/c. This study suggests that activation of specific MCS can alter TP however further work is required.

The effects of the natural and synthetic ligands for the melanocortin receptor type 3 (MC3-R) have been evaluated in a murine model of experimental gout. Systemic treatment of mice with γ2-melanocyte-stimulating hormone (γ2-MSH) and the synthetic agonist MTII inhibited accumulation of KC, interleukin-1 beta (IL-1β), and PMN elicited by urate crystals in the peritoneal cavity. In vitro, macrophage (Mø) activation, determined as release of KC and IL-1β, was inhibited by γ2-MSH and MTII. The mixed MC3/4-R antagonist SHU9119 prevented the inhibitory actions of γ2-MSH and MTII in vitro and in vivo, whereas the selective MC4-R antagonist HS024 was without effect. Western blotting also showed the presence of MC3-R protein on murine peritoneal Mø. Furthermore, agonism at the MC3-R evoked accumulation of cAMP within the Mø, which was inhibited by SHU9119. Thus, naturally occurring melanocortins, as well as the synthetic long-acting compound MTII, activate MC3-R on peritoneal Mø to inhibit the experimental inflammatory response.

1. The role played by endogenous lipocortin 1 in the anti-migratory action exerted by dexamethasone (Dex) on monocyte recruitment in an in vivo model of acute inflammation was investigated by use of several neutralizing polyclonal antibodies raised against lipocortin 1 or a lipocortin 1-derived N-terminus peptide (peptide Ac2-26). The efficacy of peptide Ac2-26 in inhibiting monocyte and polymorphonuclear leucocyte (PMN) recruitment was also tested. 2. Intraperitoneal (i.p.) injection of zymosan A (1 mg) produced a time-dependent cell accumulation into mouse peritoneal cavities which followed a typical profile of acute inflammation: PMN influx was maximal at 4 h post-zymosan (between 15 and 20 x 10(6) cells per mouse), and this was followed by an accumulation of monocytes which peaked at the 24 h time-point (between 10 and 15 x 10(6) cells per mouse). 3. Dex administration to mice reduced zymosan-induced 4 h PMN infiltration and 24 h monocyte accumulation with similar efficacy: approximately 50% of inhibition of recruitment of both cell types was achieved at the dose of 30 micrograms per mouse (approximately 1 mg kg-1, subcutaneously (s.c.)). Maximal inhibitions of 64% and 67% on PMN and monocyte recruitment, respectively, were measured after a dose of 100 micrograms per mouse (approximately 3 mg kg-1, s.c.). 4. Dex (30 micrograms s.c.) inhibited monocyte (53%) and PMN (69%) accumulation in response to zymosan application in mice which had been treated with a non-immune sheep serum (50 microliters s.c.). In contrast, the steroid was no longer active in reducing cell accumulation in mice which had been passively immunized against full length human recombinant lipocortin 1 (serum LCS3), or against lipocortin 1 N-terminus peptide. 5. Treatment of mice with vinblastine (1 mg kg-1, intravenously (i.v.)) produced a remarkable leucopenia as assessed 24 h after administration. This was accompanied by a 60% reduction in 4 h-PMN influx, and by a 27% reduction in 24 h-monocyte accumulation, measured after zymosan administration. The inhibitory effect of Dex on monocyte recruitment was not significantly modified in vinblastine-treated mice, with 36% and 57% of inhibition calculated at the dose of 30 micrograms Dex, and 70% and 60% of inhibition at 100 micrograms Dex, in vehicle- and vinblastine-treated mice, respectively. 6. Treatment of mice with peptide Ac2-26 dose-dependently attenuated PMN influx at 4 h post-zymosan with a significant effect at 100 micrograms per mouse (45% of inhibition, n-9, P < 0.05) and a maximal effect of 61% inhibition at the highest dose tested of 200 micrograms s.c. (n = 14, P < 0.05). No effect of peptide Ac2-26 (200 micrograms s.c.) was seen on zymosan-induced 24 h monocyte recruitment. In contrast, administration of 200 micrograms peptide Ac2-26 every 6 h was effective in reducing the number of monocytes harvested from the inflamed peritoneal cavities at 24 h post-zymosan: 9.40 +/- 0.58 x 10(6) monocytes per mouse (n = 13) and 5.74 +/- 0.34 monocytes per mouse (n = 14) in vehicle- and peptide Ac2-26-treated mice, respectively (P < 0.05). 7. Finally, peptide Ac2-26 produced a concentration-dependent inhibition of the rate of phagocytosis of mouse resident peritoneal macrophages as measured by flow cytometry, with a maximal reduction of 34% at the highest concentration tested of 100 micrograms ml-1 (n = 8 experiments performed in duplicate; P < 0.05). 8. In conclusion, this study suggests that in vivo monocyte recruitment during acute inflammation is, at least in part, under the negative modulatory control of endogenous lipocortin 1 (as seen after administration of Dex by using the specific antisera) and exogenous lipocortin 1 mimetics (as observed with peptide Ac2-26. In addition to the neutrophil, we can now propose that the monocyte also can be a target for the in vivo anti-inflammatory action of lipocortin 1.

The effect of altering hippocampal nitric oxide (NO) levels on basal and N-methyl-D-aspartate receptor-evoked release of GABA has been studied in freely moving rats. N-Methyl-D-aspartate (NMDA) increased extracellular GABA in a concentration-dependent manner. The nitric oxide synthase inhibitor L-nitro-arginine-methyl ester (L-NAME; 100 microM) increased basal GABA release, and also enhanced release of GABA evoked by NMDA (100 microM) compared with the same concentration of NMDA infused alone. 200 microM L-NAME increased basal dialysate GABA, but to a lesser extent than the 100 microM concentration of the drug, and the NMDA-induced release of GABA was decreased. 1.0 mM L-NAME significantly decreased basal extracellular GABA, while abolishing the NMDA-evoked release of the amino acid. The actions of L-NAME were not mimicked by its much less active isomer D-nitro-arginine-methyl ester. The NO donor S-nitroso-N-acetylpenicillamine decreased dialysate GABA at a 500 microM concentration but increased the extracellular level of the transmitter when infused at 1.0 and 2.0 mM concentrations. These data suggest that NO may mediate both excitatory and inhibitory functions in vivo.

α-Melanocyte stimulating hormone (α-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.

Small-diameter sensory neurones found in the rat dorsal root ganglia (DRG) include cells sensitive to glial cell line-derived neurotrophic factor (GDNF), which express the inhibitory peptide somatostatin (SOM). Here we addressed the functional relationship between GDNF and sensory neurone-derived SOM. Topical application of GDNF through the rat isolated dorsal horn of the spinal cord promoted activity-induced release of SOM from central terminals of sensory neurones. Once released by sensory neurones, SOM is known to act, at least in part, by opposing the action of Substance P (SP) in neurogenic inflammation. Therefore, we evaluated GDNF ability to modulate two well-documented effects of peripherally and centrally administered SP. Local application of GDNF in the mouse air pouch reduced SP-induced leukocyte migration. This effect of GDNF was mimicked by the SOM analog octreotide (OCT) and required intact SOM neuronal pools. Intrathecal injection of GDNF activated rat lumbar dorsal horn neurones and inhibited intrathecal SP-induced thermal hypersensitivity. This effect of GDNF was reversed by the SOM antagonist c-SOM and mimicked by OCT. In conclusion we propose GDNF regulation of neuronal SOM release as a novel mechanism that, if explored, may lead to new therapeutic strategies based on local release of somatostatin.

In cartilage research bovine articular cartilage is used as an alternative to human tissue. However, animal material is subject to availability and primary cultures undergo senescence, limiting their use. Here we report the immortalization of primary bovine chondrocytes, which could be used as a surrogate for freshly isolated chondrocytes. Chondrocytes were isolated from cartilage explants and immortalized using 1.0 µg/ml benzo[alpha]pyrene. For 3-dimensional culture, chondrocytes were resuspended in 0.5% low-melt agarose at high density (HD) and cultured for 24 h prior to determining changes in expression profile and morphology. A13/BACii chondrocytes acquired a ‘flat’ irregular morphology and a foetal-like cell volume (1,509.59 ± 182.04 µm3). The human cell line C-20/A4 showed a statistically similar volume and length to A13/BACii. Two-dimensional-cultured A13/BACii expressed elevated levels of type I collagen (col1), reduced levels of type II collagen (col2) compared to freshly isolated chondrocytes and an overall col2 to col1 expression ratio (col2:col1) of 0.11 ± 0.01. Upon 3-dimensional encapsulation, there was a significant rise in col2 expression in both A13/BACii and C-20/A4, suggesting a capacity for redifferentiation in both cell lines with a return of col2:col1 values of A13/BACii to values previously observed in primary chondrocytes. A13/BACii chondrocytes expressed aggrecan, matrix metalloproteinase (MMP)-3, MMP-9 and MMP-13, further supporting indications of the differentiated phenotype. Here we report the creation of a novel chondrocytic cell line and demonstrate its strong potential for redifferentiation upon HD 3-dimensional encapsulation, providing an alternative to conventional dedifferentiated cell lines and primary culture.

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.

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 (Mφ) 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 Mφ. HO-1 induction in peritoneal Mφ 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 Mφ, and proposes that this may be of functional relevance in determining MC-R-dependent control of the host inflammatory response.

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.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.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.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.

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 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 37-kDa protein annexin 1 (Anx-1; lipocortin 1) has been implicated in the regulation of phagocytosis, cell signaling, and proliferation and is postulated to be a mediator of glucocorticoid action in inflammation and in the control of anterior pituitary hormone release. Here, we report that mice lacking the Anx-1 gene exhibit a complex phenotype that includes an altered expression of other annexins as well as of COX-2 and cPLA2. In carrageenin- or zymosan-induced inflammation, Anx-1-/- mice exhibit an exaggerated response to the stimuli characterized by an increase in leukocyte emigration and IL-1beta generation and a partial or complete resistance to the antiinflammatory effects of glucocorticoids. Anx-1-/- polymorphonuclear leucocytes exhibited increased spontaneous migratory behavior in vivo whereas in vitro, leukocytes from Anx-1-/- mice had reduced cell surface CD 11b (MAC-1) but enhanced CD62L (L-selectin) expression and Anx-1-/- macrophages exhibited anomalies in phagocytosis. There are also gender differences in activated leukocyte behavior in the Anx-1-/- mice that are not seen in the wild-type animals, suggesting an interaction between sex hormones and inflammation in Anx-1-/- animals.

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.

An innovative avenue for anti-inflammatory therapy is inhibition of neutrophil extravasation by potentiating the action of endogenous anti-inflammatory mediators. The glucocorticoid-inducible protein annexin 1 and derived peptides are effective in inhibiting neutrophil extravasation. Here we tested the hypothesis that an interaction with the receptor for formylated peptide (FPR), so far reported only in vitro, could be the mechanism for this in vivo action. In a model of mouse peritonitis, FPR antagonists abrogated the anti-migratory effects of peptides Ac2-26 and Ac2-12, with a partial reduction in annexin 1 effects. A similar result was obtained in FPR (knock-out) KO mice. Binding of annexin 1 to circulating leukocytes was reduced (>50%) in FPR KO mice. In vitro, annexin binding to peritoneal macrophages was also markedly reduced in FPR KO mice. Finally, evidence of direct annexin 1 binding to murine FPR was obtained with HEK-293 cells transfected with the receptor. Overall, these results indicate a functional role for FPR in the anti-migratory effect of annexin 1 and derived peptides.

A cytotoxic cycle triggered by DNA single-strand breakage and poly (ADP-ribose) synthetase activation has been shown to contribute to the cellular injury during various forms of oxidant stress in vitro. The aim of this study was to investigate the role of poly (ADP-ribose) synthetase (PARS) in the process of neutrophil recruitment and in development of local and systemic inflammation. In pharmacological studies, PARS was inhibited by 3-aminobenzamide (10–20 mg/kg) in rats and mice. In other sets of studies, inflammatory responses in PARS−/− mice were compared with the responses in corresponding wild-type controls. Inhibition of PARS reduced neutrophil recruitment and reduced the extent of edema in zymosan- and carrageenan-triggered models of local inflammation. Moreover, inhibition of PARS prevented neutrophil recruitment, and reduced organ injury in rodent models of inflammation and multiple organ failure elicited by intraperitoneal injection of zymosan. Inhibition of PARS also reduced the extent of neutrophil emigration across murine mesenteric postcapillary venules. This reduction was due to an increased rate of adherent neutrophil detachment from the endothelium, promoting their reentry into the circulation. Taken together, our results demonstrate that PARS inhibition reduces local and systemic inflammation. Part of the antiinflammatory effects of PARS inhibition is due to reduced neutrophil recruitment, which may be related to maintained endothelial integrity.

Osteoarthritis (OA) is characterized by a loss of joint mobility and pain resulting from progressive destruction and loss of articular cartilage secondary to chondrocyte death and/ or senescence. Certain stimuli including nitric oxide (NO) and the pro-inflammatory cytokine tumor necrosis factor α (TNF-α have been implicated in this chondrocyte death and the subsequent accelerated damage to cartilage. In this study, we demonstrate that a corticotrophin releasing factor (CRF) family peptide, urocortin (Ucn), is produced by a human chondrocyte cell line, C-20/A4, and acts both as an endogenous survival signal and as a cytoprotective agent reducing the induction of apoptosis by NO but not TNF-α when added exogenously. Furthermore, treatment with the NO donor S-nitroso-N-acetyl-𝒟-ℒ-penicillamine upregulates chondrocyte Ucn expression, whereas treatment with TNF-α does not. The chondroprotective effects of Ucn are abolished by both specific ligand depletion (with an anti-Ucn antibody) and by CRF receptor blockade with the pan-CRFR antagonist α-helical CRH(9-41). CRFR expression was confirmed by reverse transcription-PCR with subsequent amplicon sequence analysis and demonstrates that C-20/A4 cells express both CRFR1 and CRFR2, specifically CRFR1α and CRFR2β. Protein expression of these receptors was confirmed by western blotting. The presence of both Ucn and its receptors in these cells, coupled with the induction of Ucn by NO, suggests the existence of an endogenous autocrine/paracrine chondroprotective mechanism against stimuli inducing chondrocyte apoptosis via the intrinsic/mitochondrial pathway.

The effect of altering hippocampal nitric oxide levels on basal and N-methyl-D-aspartate (NMDA) receptor-evoked release of glutamate and aspartate has been studied in freely moving rats. NMDA increased extracellular glutamate and aspartate in a concentration-dependent manner. The nitric oxide synthase inhibitor L-nitro-arginine-methyl ester (L-NAME; 100 microM) increased basal glutamate and aspartate release, and also enhanced release of these amino acids evoked by NMDA (100 microM) compared with the same concentration of NMDA infused alone. L-NAME at 200 microM increased basal dialysate glutamate, but not aspartate, to a lesser extent than the 100 microM concentration of the drug, and the NMDA-induced release of glutamate and aspartate was decreased. L-NAME at 1.0 mM did not significantly alter basal extracellular glutamate but significantly decreased dialysate aspartate, while abolishing the NMDA-evoked release of both amino acids. The actions of L-NAME were not mimicked by its much less active isomer D-nitro-arginine-methyl ester. The nitric oxide donor drug S-nitroso-N-penicillamine decreased dialysate glutamate and aspartate at a 500 microM concentration but increased the extracellular level of both amino acids when infused at 1.0 mM and 2.0 mM concentrations. These data suggest that nitric oxide may mediate both excitatory and inhibitory functions, according to the level of nitric oxide production in vivo.