TLR9 recognizes unmethylated CpG-rich, pathogen-derived DNA sequences and represents the component of the innate immune system that heavily influences adaptive immunity and may contribute to the immunological disturbances in rheumatoid arthritis (RA). Accumulating data indicate that BM of RA patients participates in the pathogenesis of this disease as a site of proinflammatory cytokines overproduction and lymphocytes activation. Here, we investigated the functionality of TLR9 and its role in the modulation of RA BM B-cell functions. We report that BM B cells isolated from RA patients express TLR9 at the mRNA and protein levels acquired at the stage of preB/immature B-cell maturation. Stimulation of BM CD20 1 B cells by CpG-containing oligodeoxynucleotide-enhanced expression of activation markers (CD86 and CD54) triggered IL-6 and TNF-a secretion and cell proliferation. Significantly higher levels of eubacterial DNA encoding 16S-rRNA were found in BM samples from RA than osteoarthritis patients. Moreover, RA BM B cells exerted higher expression of CD86 than their osteoarthritis counterparts, suggesting their in situ activation via TLR9. Thus, our data indicate that TLR9 may participate in direct activation and proliferation of B cells in BM, and therefore could play a role in the pathogenesis of RA.Key words: B cells . Inflammation . Rheumatoid arthritis . TLR9 Supporting Information available online Introduction TLR9, a member of the family of pathogen recognition receptors, recognizes unmethylated CpG-rich, pathogen-derived DNA sequences [1]. Stimulation of TLR9 by these ligands trigger signaling pathway common for all TLR that results in NF-kB transcription factor activation and production of proinflammatory cytokines, including TNF-a, 3], whose overexpression plays an important role in the development and perpetuation of rheumatoid arthritis (RA) [4]. Furthermore, via activation of antigen presenting cells, TLR9 exerts potent effect on the development of acquired immunity [5]. It was shown that unmethylated DNA sequences containing CpG motifs enhance capacity of antigen presentation, induce cell proliferation, immunoglobulin class switch recombination and production of inflammatory mediators by human peripheral blood (PB) and mouse B lymphocytes [5][6][7][8][9]. Interestingly, TLR9-transduced signaling pathway contributes to the activation of autoreactive, rheumatoidfactor-producing B cells [10]. Moreover, experimental data suggest that TLR9 and its ligands participate in the pathogenesis of RA [11,12]. Intra-articularly injected bacterial DNA containing CpG motifs induces transient arthritis [11] and aggravates collagen-induced [14][15][16]. Furthermore, BM may serve as a secondary lymphoid organ, where an effective inflammatory response with strong antigen presentation, exceeding that observed in lymph nodes, can develop resulting in functional effector and memory T-cell generation [17,18]. Interestingly, the development of pathological processes in RA such as synovitis, joint cartilage and bone destructio...

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Background IL-21 is secreted by activated T cells and modulates immune cell functions with both proinflammatory and anti-inflammatory effects. IL-21 receptor (IL-21R), homologous to IL-2Rβ and IL-4Rα, associates with the gamma common chain upon ligand binding. It was recently described that IL-21R is overexpressed in the inflamed synovial membrane and on leucocytes of rheumatoid arthritis patients. Objective Previously we have shown that blockade of the IL-21 pathway with soluble IL-21R-Fc resulted in a reduction of clinical signs of arthritis in rodent models. To understand potential mechanisms of IL-21 regulation in arthritis, we analyzed serum immunoglobulin levels, and cytokine expression in the paws, serum, and collagen-restimulated splenocytes, in response to IL-21 pathway blockade. Methods Arthritis was induced in DBA/1 male mice with bovine type II collagen. Animals were treated with either soluble mIL-21R-Fc, which neutralizes murine IL-21 bioactivity, with TNFRII-Fc or with control IgG. Spleens from each group of treated mice were cultured in vitro with collagen and assayed for cytokine secretion. Cytokines and anticollagen-specific IgG levels were also measured in the serum by ELISA. Cytokine mRNA levels in the paws were evaluated by quantitative PCR analysis. Results Treatment of mice with IL-21R-Fc or TNFRII-Fc reduced clinical and histological signs of collagen-induced arthritis. IL-6 mRNA in paws and serum IL-6 levels were decreased after IL-21R-Fc treatment. IFNγ mRNA was increased in paws of IL-21R-Fctreated mice. Collagen-specific spleen cell responses from IL-21R-Fc-treated mice exhibited increased IFNγ and IL-2, and reduced IL-6 and IL-17 levels. Serum levels of total IgG 1 were also reduced in response to IL-21R-Fc treatment. Conclusion These data demonstrate a role for IL-21 in the modulation of collagen-specific T-cell responses and the pathology of arthritis, supporting a rationale for blockade of the IL-21 pathway in rheumatoid arthritis. Background TNFα is a key cytokine in rheumatoid arthritis (RA) physiopathology. We recently demonstrated that a new cationic liposome formulation allowed intravenous delivery of a small interfering RNA (siRNA) targeting TNFα and efficiently restoring the immunological balance in an experiment model of RA. Since 30% of patients do not respond to anti-TNF biotherapies, however, there is a need to develop alternative therapeutic approaches. Objective Strong association of other proinflammatory cytokines with the pathogenesis of RA prompted us to investigate which cytokine other than TNFα could be targeted for therapeutic benefit using RNA interference. Methods Two siRNA sequences were designed for IL-1β, IL-6 and IL-18 proinflammatory cytokines, and their efficacy and specificity were validated in vitro on J774.1 mouse macrophage cells, measuring both mRNA and protein levels following a lipopolysaccharide challenge. For in vivo administration, siRNAs were formulated as lipoplexes with the RPR209120/DOPE liposome and a carrier DNA, and were injected intrav...

Chaperonins have classically been thought of as intracellular molecules involved in the correct folding of proteins. Their expression is upregulated during times of stress such as heat (hence their common nomenclature as heat shock proteins [HSP]), anoxia, hypoglycaemia and reactive oxygen species [1]. These are conditions found in infected tissues or in tissues with chronic inflammation such as the rheumatoid synovium. In their intracellular location they protect the cell from apoptotic death due to stress. Increasingly chaperonins have been recognised to subserve extracellular functions for which they have received the name 'chaperokines' since they bind to specific receptors on the cell surface and activate cells of the innate immune system to secrete inflammatory cytokines, chemokines and small molecular weight mediators such as prostaglandins [2]. Indeed, an early event in inflammation is cell stress/necrosis leading to the release of HSP60 and HSP70 that binds via a CD14-mediated mechanism to Toll-like receptors 2 and 4 [2] as part of the 'danger' signal [3]. The secretion of tumour necrosis factor alpha, IL-1, IL-12 and other chemokines prepares the environment for a TH1 adaptive immune response. It is now recognised that some chaperonins, such as BiP and HSP27, may activate the innate immune system to secrete anti-inflammatory cytokines, such as IL-10 [4,5] that may skew the adaptive immune response to TH2. Recent work by our group has shown that BiP can not only prevent but also treat ongoing collagen-induced arthritis in DBA/1 mice [6], suggesting that chaperonins may down modulate ongoing TH1 responses. Thus, it may be possible to suppress rheumatoid inflammation by administration of appropriate chaperonins such as BiP. Finally, chaperonins may be important system regulators determining the outcome between TH1 and Th2 immune responses. References 1. Pockley AG: Heat shock proteins as regulators of the immune response. Lancet 2003, 362:469-476. 2. Asea A: Chaperokine-induced signal transduction pathways. Exerc Immunol Rev 2003, 9:25-33. 3. Matzinger P: The danger model: a renewed sense of self. Science 2002, 296:301-305. 4. De AK, Kodys KM, Yeh BS, Miller-Graziano C: Exaggerated human monocyte IL-10 concomitant to minimal TNF-alpha induction by heatshock protein 27 (Hsp27) suggests Hsp27 is primarily an antiinflammatory stimulus.