Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology, characterized by the presence of inflammatory synovitis accompanied by destruction of joint cartilage and bone. Treatment with vasoactive intestinal peptide (VIP) prevents experimental arthritis in animal models by downregulation of both autoimmune and inflammatory components of the disease. The aim of this study was to characterize the protective effect of VIP on bone erosion in collagen-induced arthritis (CIA) in mice. We have studied the expression of different mediators implicated in bone homeostasis, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), receptor activator of nuclear factor-κB (RANK), receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), IL-1, IL-4, IL-6, IL-10, IL-11 and IL-17. Circulating cytokine levels were assessed by ELISA and the local expression of mediators were determined by RT-PCR in mRNA extracts from joints. VIP treatment resulted in decreased levels of circulating IL-6, IL-1β and TNFα, and increased levels of IL-4 and IL-10. CIA-mice treated with VIP presented a decrease in mRNA expression of IL-17, IL-11 in the joints. The ratio of RANKL to OPG decreased drastically in the joint after VIP treatment, which correlated with an increase in levels of circulating OPG in CIA mice treated with VIP. In addition, VIP treatment decreased the expression of mRNA for RANK, iNOS and COX-2. To investigate the molecular mechanisms involved, we tested the activity of NFκB and AP-1, two transcriptional factors closely related to joint erosion, by EMSA in synovial cells from CIA mice. VIP treatment in vivo was able to affect the transcriptional activity of both factors. Our data indicate that VIP is a viable candidate for the development of treatments for RA.

The extracellular matrix (ECM) is a complex and specialized three-dimensional macromolecular network, present in nearly all tissues, that also interacts with cell surface receptors on joint resident cells. Changes in the composition and physical properties of the ECM lead to the development of many diseases, including osteoarthritis (OA). OA is a chronic degenerative rheumatic disease characterized by a progressive loss of synovial joint function as a consequence of the degradation of articular cartilage, also associated with alterations in the synovial membrane and subchondral bone. During OA, ECM-degrading enzymes, including urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), cleave ECM components, such as fibronectin (Fn), generating fibronectin fragments (Fn-fs) with catabolic properties. In turn, Fn-fs promote activation of these proteinases, establishing a degradative and inflammatory feedback loop. Thus, the aim of this review is to update the contribution of ECM-degrading proteinases to the physiopathology of OA as well as their modulation by Fn-fs.

Objectives. Fibroblast-like synoviocytes (FLSs) are crucial players in the pathogenesis of synovitis in rheumatic diseases. Targeting FLS activation represents an approach to the development of therapeutic strategies. Our aim was to investigate whether the microenvironment of inflamed joints could modulate the expression of IL-22 and IL-22R1 on OA and RA FLSs. We also examined the effect of IL-22 on FLS activation as well as on their IL-17-related responses. Methods. IL-22 and IL-22R1 expression was studied by RT-PCR and immunoblotting. Proliferation was measured by an ELISA kit. IL-17 receptors, p19IL-23 and alarmins were analysed by RT-PCR. IL-17 receptor expression was evaluated by flow cytometry. MMP1 and IL-23 were measured by ELISA. S100A8/A9 expression was detected by immunofluorescence and ELISA. Signal transducer and activator of transcription 3 (STAT3) phosphorylation was quantified using a cell-based ELISA kit. Results. IL-22 and IL-22R1 were expressed constitutively in FLSs. We demonstrated that S100A8 and S100A9 were synthesized in FLSs. We reported that inflammatory mediators increased the expression of the IL-22/IL-22R1 axis, amplifying FLS activation. IL-22 enhanced FLS proliferation and up-regulated MMP1 and S100A8/A9 production. STAT3 phosphorylation was induced after IL-22 treatment and the stimulatory effect of IL-22 on S100A8/A9 was reduced after the activities of Janus kinase 2 (JAK2) and JAK3 were blocked. We showed an inhibitory action of IL-22 on IL-23 and IL-17RC expression in RA FLSs and on IL-17RA in OA FLSs. Conclusion. Therapies based on the pharmacological disruption signalling of IL-22 could be beneficial for the treatment of rheumatic diseases. The restricted expression of IL-22R1 to non-lymphoid cells could lead to a reduction of side effects mediated by immune responses.

Objectives. Vasoactive intestinal peptide (VIP) has demonstrated therapeutic effects in arthritis by inhibiting both innate and acquired immune responses. We investigated the potential effects of VIP in the regulation of Toll-like receptor (TLR) expression and function in synovial fibroblasts from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Methods. Cultured fibroblast-like synoviocytes (FLS) were obtained from patients with RA and OA. The effects of VIP on basal or TNF-α or lipopolysaccharide (LPS)-induced TLR2, TLR4 and MyD88 expression and its effects on TLR4-mediated CCL2 and CXCL8 chemokine production were studied by reverse transcription–polymerase chain reaction, western blotting and enzyme-linked immunosorbent assay. Results. TLR2, TLR4 and MyD88 mRNA expression was increased in RA FLS compared with OA FLS. The largest increase was observed for TLR4 and there was also overexpression at the protein level in RA FLS. TLR4 and MyD88 mRNA and proteins were induced by LPS and TNF-α in RA FLS. VIP down-regulated the induced but not the constitutive expression of TLR4 and MyD88 in RA FLS. VIP treatment decreased CCL2 and CXCL8 chemokine production in response to TLR4 activation with LPS in RA FLS. Conclusions. We demonstrate that VIP down-regulates LPS and TNF-α activation of TLR4 expression and the TLR4 functional response in terms of proinflammatory chemokine production. These studies suggest that the pleiotropic anti-inflammatory actions of VIP involve inhibitory effects on TLR4 expression and signalling.

Toll-like receptor 2 (TLR2) and -4 mediate signals from a great variety of bacterial gut products, giving the host a panel of microbe-recognizing receptors. Under homeostatic conditions, TLRs act as protective receptors of the intestinal epithelium. When homeostasis is disrupted in diseases such as inflammatory bowel disease, TLR2 and -4 are deregulated. Our study demonstrates, by using a trinitrobenzene sulfonic acid-induced colitis model of Crohn's disease, the constitutive expression and the up-regulation of TLR2 and -4 at messenger and protein levels in colon extracts, as well as in macrophages, dendritic cells, and lymphocytes from mesenteric lymphoid nodes. Vasoactive intestinal peptide (VIP) treatment induced a decrease of TLR2 and -4 expressions approaching ethanol control levels. Our results suggest that VIP modulation of TLR2 and -4 could be explained by two possible mechanisms. The first one would be the secondary reduction of TLR2 and -4 caused by the VIP-mediated decrease of inflammatory mediators such as interleukin-1β and interferon- γ, which synergize with bacterial products, contributing to the amplification of TLR presence in the intestine. The other possible mechanism would involve a VIP-mediated decrease of nuclear factor-κB, which would cause a direct down-regulation of TLR expression. In summary, the resultant physiological effect is the decrease of TLR2 and -4 expressions to homeostatic levels. Our study describes for the first time the role of a peptide present in the gut microenvironment as an effective modulator of the initial steps of acute inflammation, acting at local and systemic levels and leading to the restoration of the homeostasis lost after an established inflammatory/autoimmune disease.

Objective: Vasoactive intestinal peptide (VIP) has shown potent antiinflammatory effects in murine arthritis and ex vivo in human rheumatoid arthritis (RA) synovial cells. To investigate the potential endogenous participation of this system in the pathogenesis of RA, we analyzed the expression and regulation of VIP and its functional receptors in human fibroblast‐like synoviocytes (FLS) from patients with osteoarthritis (OA) and patients with RA.Methods: The expression of VIP was studied by reverse transcription–polymerase chain reaction (RT‐PCR), enzyme immunoassay, and immunofluorescence in cultured FLS, and by immunohistochemical analysis in synovial tissue. The expression and function of the potential VIP receptors in FLS were studied by RT‐PCR, determination of intracellular cAMP production, cell membrane adenylate cyclase (AC) activity, and interleukin‐6, CCL2, and CXCL8 production in response to VIP or specific agonists and antagonists.Results: VIP expression was detected in human FLS at the messenger RNA and protein levels, and it was significantly decreased in RA FLS compared with OA FLS. VIP receptor type 1 (VPAC₁) was the dominant AC‐coupled receptor in OA FLS, in contrast with RA FLS, in which VPAC₂ was dominant. Tumor necrosis factor α–treated OA FLS reproduced the VIP and VPAC receptor expression pattern of RA FLS. The antagonistic effects of VIP on FLS proinflammatory factor production were reproduced by VPAC₁‐ and VPAC₂‐specific agonists in OA FLS and RA FLS, respectively.Conclusion: VIP expression is down‐regulated in RA and in tumor necrosis factor α–treated FLS, suggesting that down‐regulation of this endogenous antiinflammatory factor may contribute to the pathogenesis of RA. In RA FLS, VPAC₂ mediates the antiinflammatory effects of VIP, suggesting that VPAC₂ agonists may be an alternative to VIP as antiinflammatory agents.