Effect of calcitonin gene-related peptide,neuropeptide Y,substance P,and vasoactive intestinal peptide on interleukin-1beta,interleukin-6 and tumor necrosis factor-alpha production by peripheral whole blood cells from rheumatoid arthritis and osteoarthritis patients

In the present study, we have investigated the in vitro effect of calcitonin-related peptide (CGRP), neuropeptide Y (NPY), substance P (SP) and vasoactive intestinal peptide (VIP) at concentrations of 10(-8), 10(-9) and 10(-10) M on the production of different proinflammatory cytokines or chemokines such as IL-1beta, IL-6 and TNFalpha by peripheral whole blood cells from patients with rheumatoid arthritis, as well as from osteoarthritis patients studied as a control group without immunoinflammatory background. We have found that CGRP, NPY, SP and VIP stimulated significantly the production of those cytokines and chemokines in rheumatoid arthritis patients. In general, the stimulation was higher at the 10(-9) M concentration, with SP and VIP, and in rheumatoid arthritis patients compared to osteoarthritis ones. Neuropeptides did not significantly modify the LPS-induced cytokine production by whole blood cells. The results indicate that physiological concentrations of the neuropeptides studied can modulate the inflammatory and immunological response, stimulating significantly the production of inflammatory cytokines by human whole blood cells in rheumatoid arthritis patients, as well as, in a minor way, in osteoarthritis patients.     

Immunological evaluation of rheumatoid arthritis patients treated with itolizumab

Rheumatoid arthritis is an autoimmune disease characterized by joint inflammation that affects approximately 1% of the general population. Itolizumab, a monoclonal antibody specific for the human CD6 molecule mainly expressed on T lymphocytes, has been shown to inhibit proliferation of T cells and proinflammatory cytokine production in psoriasis patients. We have now assessed the immunological effect of itolizumab in combination with methotrexate in rheumatoid arthritis by analyzing clinical samples taken from 30 patients enrolled in a clinical trial. T and B cell subpopulations were measured at different time points of the study. Plasma cytokine levels and anti-idiotypic antibody response to itolizumab were also evaluated. The combined treatment of itolizumab and methotrexate led to a reduction in the frequency of T cell subpopulations, and plasma levels of proinflammatory cytokines showed a significant decrease up to at least 12 weeks after treatment ended. No anti-idiotypic antibody response was detected. These results support the relevance of the CD6 molecule as a therapeutic target for the treatment of this disease.     

The microRNA miR-23b suppresses IL-17-associated autoimmune inflammation by targeting TAB2, TAB3 and IKK-α

Inflammatory cytokines such as interleukin-17 (IL-17) promote inflammatory autoimmune diseases. Although several microRNAs (miRNAs) have been shown to regulate autoimmune pathogenesis by affecting lymphocyte development and function, the role of miRNAs in resident cells present in inflammatory lesions remains unclear. Here we show that miR-23b is downregulated in inflammatory lesions of humans with lupus or rheumatoid arthritis, as well as in the mouse models of lupus, rheumatoid arthritis or multiple sclerosis. IL-17 downregulates miR-23b expression in human fibroblast-like synoviocytes, mouse primary kidney cells and astrocytes and is essential for the downregulation of miR-23b during autoimmune pathogenesis. In turn, miR-23b suppresses IL-17-, tumor necrosis factor α (TNF-α)- or IL-1β-induced NF-κB activation and inflammatory cytokine expression by targeting TGF-β-activated kinase 1/MAP3K7 binding protein 2 (TAB2), TAB3 and inhibitor of nuclear factor κ-B kinase subunit α (IKK-α) and, consequently, represses autoimmune inflammation. Thus, IL-17 contributes to autoimmune pathogenesis by suppressing miR-23b expression in radio-resident cells and promoting proinflammatory cytokine expression.     

Adenoviral transfer of cyclin-dependent kinase inhibitor genes suppresses collagen-induced arthritis in mice

In rheumatoid synovial tissues, synovial fibroblasts are activated by proinflammatory cytokines and proliferate to develop hyperplastic pannus tissues, which irreversibly damage the affected joints. We recently reported that the cyclin-dependent kinase inhibitors p16(INK4a) and p21(Cip1) are not expressed in vivo in rheumatoid synovial fibroblasts, but are readily inducible in vitro. This observation was followed by the successful treatment of rat adjuvant arthritis by local p16(INK4a) gene transfer, showing that the inhibition of the cell cycle of the synovial cells ameliorates the arthritis. In this study, we show that another animal model of rheumatoid arthritis, murine collagen-induced arthritis, can be effectively treated by local gene transfer of p21(Cip1) as well as that of p16(INK4a). The anti-arthritic effects were observed even when the treatment was conducted after the arthritis had developed. Furthermore, the effects included suppression of the expression of proinflammatory cytokines such as IL-1ss, IL-6, and TNF-alpha. Our results demonstrate that the ectopic expression of cyclin-dependent kinase inhibitors not only prevents synovial overgrowth but also ameliorates the proinflammatory milieu in the affected joints. The induction of p21(Cip1) in rheumatoid synovial tissues by pharmacological agents may also be an effective strategy to treat rheumatoid arthritis.     

Cross-talk between IL-1 and IL-6 signaling pathways in rheumatoid arthritis synovial fibroblasts.

The balance between pro- and anti-inflammatory cytokines plays an important role in determining the severity of inflammation in rheumatoid arthritis (RA). Antagonism between opposing cytokines at the level of signal transduction plays an important role in many other systems. We have begun to explore the possible contribution of signal transduction cross-talk to cytokine balance in RA by examining the effects of IL-1, a proinflammatory cytokine, on the signaling and action of IL-6, a pleiotropic cytokine that has both pro- and anti-inflammatory actions, in RA synovial fibroblasts. Pretreatment with IL-1 suppressed Janus kinase-STAT signaling by IL-6, modified patterns of gene activation, and blocked IL-6 induction of tissue inhibitor of metalloproteases 1 expression. These results suggest that proinflammatory cytokines may contribute to pathogenesis by modulating or blocking signal transduction by pleiotropic or anti-inflammatory cytokines. The mechanism of inhibition did not require de novo gene activation and did not depend upon tyrosine phosphatase activity, but, instead, was dependent on the p38 stress kinase. These results identify a molecular basis for IL-1 and IL-6 cross-talk in RA synoviocytes and suggest that, in addition to levels of cytokine expression, modulation of signal transduction also plays a role in regulating cytokine balance in RA.     

Cytokines in rheumatoid arthritis: is it all TNF-alpha?

Anti-TNF-alpha therapy has shown clear efficacy in the treatment of rheumatoid arthritis (RA). Since some patients do not respond and the treatment is suspensive, combination therapy may be of interest. Other cytokines produced by monocytes such as IL-1, IL-12, IL-18 are also involved. The secretion of these cytokines is regulated by subsets of T-lymphocytes. Among these, IL-17 producing Th1 cells appear to contribute directly to the destructive process. Furthermore, this T-cell contribution enhances the action of monocyte derived proinflammatory cytokines. Using models of human RA synovium inflammation and bone resorption, ex vivo results suggest that combination therapy may be of interest. Acting on more than one cytokine may increase the percentage of responding RA patients as well as the degree of individual patient response.     

The role of human T-lymphocyte-monocyte contact in inflammation and tissue destruction

Contact-mediated signaling of monocytes by human stimulated T lymphocytes (TL) is a potent proinflammatory mechanism that triggers massive upregulation of the proinflammatory cytokines IL-1 and tumor necrosis factor-alpha. These two cytokines play an important part in chronic destructive diseases, including rheumatoid arthritis. To date this cell-cell contact appears to be a major endogenous mechanism to display such an activity in monocyte-macrophages. Since TL and monocyte-macrophages play a pivotal part in the pathogenesis of chronic inflammatory diseases, we investigated the possible ligands and counter-ligands involved in this cell-cell interaction. We also characterized an inhibitory molecule interfering in this process, apolipoprotein A-I. This review aims to summarize the state of the art and importance of contact-mediated monocyte activation by stimulated TL in cytokine production in rheumatoid arthritis and mechanisms that might control it.     

Prolactin and growth hormone induce differential cytokine and chemokine profile in murine peritoneal macrophages in vitro: involvement of p-38 MAP kinase, STAT3 and NF-kappaB

The role of immune-neuroendocrine interactions in the autoimmune diseases is well recognized. Autoimmune rheumatoid diseases in their active phase have been characterized by high levels of prolactin (PRL) as well as proinflammatory cytokines which suggest a co-relationship between them. In the present study, we have investigated the profile of cytokines secreted by macrophages on treatment with PRL and growth hormone (GH) in vitro. Significantly enhanced production of cytokines IL-1beta, IL-12p40 and IFN-gamma was observed on treatment of macrophages with PRL or GH. However, higher doses of PRL (1000 ng/ml) induced the production of anti-inflammatory cytokine IL-10, with significant abrogation in production of proinflammatory cytokines. It is further observed that PRL and GH induced the production of chemokines MIP-1alpha and RANTES. PRL but not GH selectively induced significantly enhanced production of MCP-1 and IP-10. It is further shown that p38 MAP kinase, STAT3 and NF-kappaB could play a differential regulatory role in PRL or GH induced production of cytokines by macrophages.     

Interleukin-17: the missing link between T-cell accumulation and effector cell actions in rheumatoid arthritis?

The prominence of T cells and monocyte/macrophages in rheumatoid synovium suggests T cells may localize and amplify the effector functions of monocyte/macrophages in rheumatoid disease. However, while T cells are abundant in rheumatoid joints, classic T-cell derived cytokines are scarce, especially when compared to the levels of monokines IL-1 beta and TNF-alpha. For this reason, it has been speculated that monocyte/macrophages may act independently of T cells in rheumatoid disease and that the role of T cells may be more or less irrelevant to core disease mechanisms. The question of T-cell influence requires re-evaluation in light of the characterization of IL-17, a T-cell derived cytokine that is abundant in rheumatoid synovium and synovial fluid. IL-17 has a number of pro-inflammatory effects, both directly and through amplification of the effects of IL-1 beta and TNF-alpha. IL-17 is able to induce expression of pro-inflammatory cytokines and stimulate release of eicosanoids by monocytes and synoviocytes. Furthermore, IL-17 has been implicated in the pathogenesis of inflammatory bone and joint damage through induction of matrix metalloproteinases and osteoclasts, as well as inhibition of proteoglycan synthesis. In animal models of arthritis, intra-articular injection of IL-17 results in joint inflammation and damage. The recognition of IL-17 as a pro-inflammatory T cell derived cytokine, and its abundance within rheumatoid joints, provides the strongest candidate mechanism to date through which T cells can capture and localize macrophage effector functions in rheumatoid arthritis. As such, IL-17 warrants consideration for its potential as a therapeutic target in rheumatoid arthritis.     

Regulation of cytokines, cytokine inhibitors, and acute-phase proteins following anti-TNF-alpha therapy in rheumatoid arthritis.

Treatment with a chimeric mAb to TNF-alpha has been shown to suppress inflammation and improve patient well-being in rheumatoid arthritis (RA), but the mechanisms of action of such treatment have not been fully explored. Here we show that in vivo administration of anti-TNF-alpha Ab, using a longitudinal analysis, results in the rapid down-regulation of a spectrum of cytokines, cytokine inhibitors, and acute-phase proteins. Marked diurnal variation in the serum levels of some of these were detected. These results were consistent with the concept of a cytokine-dependent cytokine cascade, and the degree of clinical benefit noted after anti-TNF-alpha therapy is probably due to the reduction in many proinflammatory mediators apart from TNF-alpha, such as IL-6, which reached normal levels within 24 h. Serum levels of cytokine inhibitors such as soluble p75 and p55 TNFR were reduced as was IL-1 receptor antagonist. Reductions in acute-phase proteins occurred after serum IL-6 fell and included serum amyloid A, haptoglobin, and fibrinogen. The latter reduction could be of importance, as it is a risk factor for atherosclerosis, which is augmented in RA patients.     

Inhibitor of Apoptosis Proteins (IAPs) and Their Antagonists Regulate Spontaneous and Tumor Necrosis Factor (TNF)-induced Proinflammatory Cytokine and Chemokine Production

Inhibitor of apoptosis proteins (IAPs) play a major role in determining whether cells undergo apoptosis in response to TNF as well as other stimuli. However, TNF is also highly proinflammatory through its ability to trigger the secretion of multiple inflammatory cytokines and chemokines, which is arguably the most important role of TNF in vivo. Indeed, deregulated production of TNF-induced cytokines is a major driver of inflammation in several autoimmune conditions such as rheumatoid arthritis. Here, we show that IAPs are required for the production of multiple TNF-induced proinflammatory mediators. Ablation or antagonism of IAPs potently suppressed TNF- or RIPK1-induced proinflammatory cytokine and chemokine production. Surprisingly, IAP antagonism also led to spontaneous production of chemokines, particularly RANTES, in vitro and in vivo. Thus, IAPs play a major role in influencing the production of multiple inflammatory mediators, arguing that these proteins are important regulators of inflammation in addition to apoptosis. Furthermore, small molecule IAP antagonists can modulate spontaneous as well as TNF-induced inflammatory responses, which may have implications for use of these agents in therapeutic settings.     

A knowledgebase resource for interleukin-17 family mediated signaling

Interleukin-17 (IL-17) belongs to a relatively new family of cytokines that has garnered attention as the signature cytokine of Th17 cells. This cytokine family consists of 6 ligands, which bind to 5 receptor subtypes and induce downstream signaling. Although the receptors are ubiquitously expressed, cellular responses to ligands vary across tissues. The cytokine family is associated with various autoimmune disorders including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, asthma and psoriasis in addition to being implicated in the pathogenesis of cancer. In addition, this family plays a role in host defense against bacterial and fungal infections. The signaling mechanisms of the IL-17 family of proinflammatory cytokines are not well explored. In this study, we present a resource of literature-annotated reactions induced by IL-17. The reactions are catalogued under 5 categories, namely; molecular association, catalysis, transport, activation/inhibition and gene regulation. A total of 93 molecules and 122 reactions have been annotated. The IL-17 pathway is freely available through NetPath, a resource of signal transduction pathways previously developed by our group.     

The role of T-cell interleukin-17 in conducting destructive arthritis: lessons from animal models

Interleukin-17 (IL-17) is a T cell cytokine spontaneously produced by cultures of rheumatoid arthritis (RA) synovial membranes. High levels have been detected in the synovial fluid of patients with RA. The trigger for IL-17 is not fully identified; however, IL-23 promotes the production of IL-17 and a strong correlation between IL-15 and IL-17 levels in synovial fluid has been observed. IL-17 is a potent inducer of various cytokines such as tumor necrosis factor (TNF)-alpha, IL-1, and receptor activator of NF-kappaB ligand (RANKL). Additive or even synergistic effects with IL-1 and TNF-alpha in inducing cytokine expression and joint damage have been shown in vitro and in vivo. This review describes the role of IL-17 in the pathogenesis of destructive arthritis with a major focus on studies in vivo in arthritis models. From these studies in vivo it can be concluded that IL-17 becomes significant when T cells are a major element of the arthritis process. Moreover, IL-17 has the capacity to induce joint destruction in an IL-1-independent manner and can bypass TNF-dependent arthritis. Anti-IL-17 cytokine therapy is of interest as an additional new anti-rheumatic strategy for RA, in particular in situations in which elevated IL-17 might attenuate the response to anti-TNF/anti-IL-1 therapy.     

Suppressing IL-32 in monocytes impairs the induction of the proinflammatory cytokines TNFα and IL-1β

Targeting major proinflammatory cytokines such as IL-1β and TNFα is of great interest in patients with chronic inflammatory diseases, including rheumatoid arthritis, colitis, and psoriasis. The cytokine Interleukin (IL)-32 induces proinflammatory cytokines such as TNFα, IL-1β, IL-6, and chemokines. We previously used an IL-32 ligand-affinity column to purify proteinase 3, which is abundantly expressed in neutrophil and monocytic leukocytes but not in other cell types, and found that IL-32 is mainly produced by monocytic leukocytes. This evidence suggested that silencing endogenous IL-32 by short hairpin RNA (shRNA) in monocytic cells might reveal the precise function of endogenous IL-32. Indeed, lipopolysaccharide (LPS)- or phorbol myristate acetate (PMA)-induced proinflammatory cytokine production was significantly inhibited in shRNA/IL-32 stable clones as compared to control clones. Furthermore, macrophages in PMA-differentiated shRNA/IL-32 stable clones displayed remarkably impaired LPS- and IL-1β-induced proinflammatory cytokine production. These data suggest that IL-32 is not only involved in host defense against pathogens, but also might play a role in chronic inflammatory diseases. IL-32 production leads to major proinflammatory cytokine production during the initial immune response.     

Cytokines in the pathogenesis of rheumatoid arthritis

Cytokines regulate a broad range of inflammatory processes that are implicated in the pathogenesis of rheumatoid arthritis. In rheumatoid joints, it is well known that an imbalance between pro- and anti-inflammatory cytokine activities favours the induction of autoimmunity, chronic inflammation and thereby joint damage. However, it remains less clear how cytokines are organized within a hierarchical regulatory network, and therefore which cytokines may be the best targets for clinical intervention a priori. Here, we discuss the crucial effector function of cytokines in the immunological processes that are central to the pathogenesis of rheumatoid arthritis.     

Levels of gastrin-releasing peptide and substance P in synovial fluid and serum correlate with levels of cytokines in rheumatoid arthritis

It is well known that cytokines are highly involved in the disease process of rheumatoid arthritis (RA). Recently, targeting of neuropeptides has been suggested to have potential therapeutic effects in RA. The aim of this study was to investigate possible interrelations between five neuropeptides (bombesin/gastrin-releasing peptide (BN/GRP), substance P (SP), vasoactive intestinal peptide, calcitonin-gene-related peptide, and neuropeptide Y) and the three cytokines tumour necrosis factor (TNF)-α, IL-6, and monocyte chemoattractant protein-1 in synovial fluid of patients with RA. We also investigated possible interrelations between these neuropeptides and soluble TNF receptor 1 in serum from RA patients. Synovial fluid and sera were collected and assayed with ELISA or RIA. The most interesting findings were correlations between BN/GRP and SP and the cytokines. Thus, in synovial fluid, the concentrations of BN/GRP and SP grouped together with IL-6, and SP also grouped together with TNF-α and monocyte chemoattractant protein-1. BN/GRP and SP concentrations in synovial fluid also grouped together with the erythrocyte sedimentation rate. In the sera, BN/GRP concentrations and soluble TNF receptor 1 concentrations were correlated. These results are of interest because blocking of SP effects has long been discussed in relation to RA treatment and because BN/GRP is known to have trophic and growth-promoting effects and to play a role in inflammation and wound healing. Furthermore, the observations strengthen a suggestion that combination treatment with agents interfering with neuropeptides and cytokines would be efficacious in the treatment of RA. In conclusion, BN/GRP and SP are involved together with cytokines in the neuroimmunomodulation that occurs in the arthritic joint.     

Dipeptidyl peptidase IV activity and/or structure homologs: Contributing factors in the pathogenesis of rheumatoid arthritis?

Several of the proinflammatory peptides involved in rheumatoid arthritis pathogenesis, including peptides induced downstream of tumor necrosis factor-α as well as the monocyte/T cell-attracting chemokines RANTES and stromal cell-derived factor (SDF)-1α and the neuropeptides vasoactive intestinal peptide (VIP) and substance P, have their biological half-lives controlled by dipeptidyl peptidase IV (DPPIV). Proteolysis by DPPIV regulates not only the half-life but also receptor preference and downstream signaling. In this article, we examine the role of DPPIV homologs, including CD26, the canonical DPPIV, and their substrates in the pathogenesis of rheumatoid arthritis. The differing specific activities of the DPPIV family members and their differential inhibitor response provide new insights into therapeutic design.     

Bmx regulates LPS-induced IL-6 and VEGF production via mRNA stability in rheumatoid synovial fibroblasts

Discordant cytokine production is characteristic of chronic inflammatory conditions like rheumatoid arthritis (RA), and anti-cytokine therapeutics are becoming routinely used to treat RA in the clinic. Fibroblasts from rheumatoid synovium have been shown to contribute to cytokine production in inflamed joints; likewise these cells also produce cytokines in response to inflammatory mediators signalling through Toll like receptors (TLRs). Tyrosine kinase activity is essential to LPS-induced cytokine production, and we have previously implicated a role for the Tec kinase, Bmx, in inflammatory cytokine production. Here we show that Bmx kinase activity in RASF is increased following LPS stimulation and that Bmx is involved in the regulation of LPS-induced IL-6 and VEGF production via mRNA stabilisation. This is an important insight into the regulation of VEGF, which is involved in a wide range of different pathologies, and may lead to more effective design of novel anti-inflammatory/angiogenic therapeutics for conditions such as RA.     

Gene expression induced by interleukin-17 in fibroblast-like synoviocytes of patients with rheumatoid arthritis: upregulation of hyaluronan-binding protein TSG-6

Interleukin-17 (IL-17) has been characterized as a proinflammatory cytokine produced by CD4+ CD45RO+ memory T cells. Overproduction of IL-17 was detected in the synovium of patients with rheumatoid arthritis (RA) compared with patients with osteoarthritis. This study examines differentially expressed genes after the stimulation of fibroblast-like synoviocytes of RA patients by IL-17. Among these genes we identified the following: tumor necrosis factor-stimulated gene-6 (TSG-6), IL-6, IL-8, GRO-beta, and bone morphogenetic protein-6 with an expression 3.6-10.6-fold that in the unstimulated control. IL-17 augmented the expression of TSG-6, a hyaluronan-binding protein, in a time- and dose-dependent manner. IL-17 showed additive effects with IL-1beta and tumour necrosis factor-alpha on the expression of TSG-6, IL-6 and IL-8. The mitogen-activated protein kinase p38 seems to be necessary for the regulation of TSG-6 expression by IL-17, as shown by inhibition with SB203580. Our results support the hypothesis that IL-17 is important in the pathogenesis of RA, contributing to an unbalanced production of cytokines as well as participating in connective tissue remodeling.