Recent data suggest that IL-15 plays an important role in the pathogenesis of rheumatoid arthritis. In the present study, we hypothesized that elevated in the joints of rheumatoid arthritis, but not osteoarthritis, patients, IL-15 may exert its proinflammatory properties via the induction of IL-17, a cytokine known to stimulate synoviocytes to release several mediators of inflammation including IL-6, IL-8, GM-CSF and PGE2. To test this hypothesis, we first measured the levels of IL-17 and IL-15 using specific ELISA and found that synovial fluids of patients with rheumatoid arthritis, but not with osteoarthritis, contain high levels of these cytokines. A strong correlation between IL-15 and IL-17 levels in synovial fluids was observed. Among tested factors, LPS and TNF-α failed, IL-15 and IL-2 were equipotent, and PMA + ionomycin was far more efficient in the induction of IL-17 secretion by PBMCs isolated from healthy blood donors. Interestingly, synovial fluid cells, in contrast to PBMCs isolated from patients with rheumatoid arthritis, but not osteoarthritis, respond to PMA + ionomycin with much lower, comparable to IL-15-triggered IL-17 secretion. Moreover, PMA + ionomycin-triggered IL-17 secretion is completely or partially blocked in the presence of low doses of cyclosporin A or high doses of methylprednisolone, respectively. IL-15-triggered IL-17 secretion by PBMCs was completely inhibited by these drugs. Thus, our results suggest for the first time that IL-15 may represent a physiological trigger that via cyclosporin A and steroid sensitive pathways leads to the overproduction of IL-17 in the joints of rheumatoid arthritis patients.

Objective. To test the hypotheses that 1) proinflammatory cytokines affect osteoprotegerin (OPG) and soluble receptor activator of nuclear factor B ligand (sRANKL) production and therefore the OPG and sRANKL levels differ in rheumatoid arthritis (RA) patients in comparison with healthy individuals; and 2) anti-tumor necrosis factor ␣ (anti-TNF␣) therapy influences OPG and sRANKL levels.Methods. Sera were obtained from healthy individuals or RA patients receiving the combination of infliximab and methotrexate. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were isolated from RA patients. Fibroblast-like synoviocytes (FLS) were isolated from synovial tissue obtained at total knee replacement in RA patients. Supernatants from cells stimulated with cytokines were collected after culture in vitro. Concentrations of OPG and sRANKL were determined by enzymelinked immunosorbent assays.Results. A strong positive correlation between OPG concentration and age was observed in healthy individuals but not in RA patients. The OPG and sRANKL levels were higher in RA patients than in healthy controls. Cultured FLS spontaneously secreted much higher amounts of OPG than PBMCs or SFMCs. Proinflammatory cytokines enhanced OPG production. Anti-TNF␣ treatment resulted in the normalization of serum OPG and sRANKL levels in RA patients without influencing the OPG:sRANKL ratio.Conclusion. Although higher serum levels of OPG and sRANKL are present in RA patients than in healthy individuals, the ratio of OPG:sRANKL is similar. There is an age-dependent increase of OPG but not sRANKL levels in healthy subjects. Anti-TNF␣ treatment results in the normalization of elevated levels of OPG and sRANKL in RA patients.Bone remodeling and bone loss are controlled by a balance between tumor necrosis factor (TNF) superfamily molecules: osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF), receptor activator of nuclear factor B ligand (RANKL), receptor activator of nuclear factor B (RANK), and TNF-related apoptosisinducing ligand (TRAIL) (1-4). RANKL plays a key role in the regulation of osteoclastogenesis, osteoclast activation, dendritic cell survival, lymphocyte development, and lymph node organogenesis (5). Recent data Presented in part at

The hallmarks of rheumatoid arthritis (RA) are leukocytic infiltration of the synovium and expansiveness of fibroblast-like synoviocytes (FLS). The abnormal proliferation of FLS and their resistance to apoptosis is mediated, at least in part, by present in RA joints proinflammatory cytokines and growth factors. Because IL-15 exerts properties of antiapoptotic and growth factors, and is produced by RA FLS, we hypothesized that IL-15 participates in RA FLS activation. To test this hypothesis, we first examined whether RA FLS express chains required for high affinity functional IL-15R. Indeed, RA FLS express IL-15Rα at mRNA and protein levels. Moreover, we confirmed the presence of IL-2Rβ and common γ-chains. Interestingly, TNF-α or IL-1β triggered significant elevation of IL-15Rα chain at mRNA and protein levels. Next, we investigated the effects of exogenous or endogenous IL-15 on Bcl-2 and Bcl-xL expression, FLS proliferation, and apoptosis. Exogenous IL-15 enhanced RA FLS proliferation and increased the level of mRNA-encoding Bcl-xL. To test the role of endogenous IL-15 in the activation of RA FLS, an IL-15 mutant/Fcγ2a protein exerting properties of specific antagonist to the IL-15Rα chain was used. We found that blocking IL-15 biological activities using this protein substantially reduced endogenous expression of Bcl-2 and Bcl-xL, and RA FLS proliferation that was reflected by increased apoptosis. Thus, we have demonstrated that a distinctive phenotype of RA FLS, i.e., persistent activation, proliferation, and resistance to apoptosis, is related to the autocrine activation of IL-15Rs by FLS-derived IL-15.

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Tumor necrosis factor-alpha, acting through its receptors expressed on all cells of the body, is a key mediator of inflammation and immunity. However, its overproduction may also lead to pathologic changes. The latter situation occurs often in chronic inflammatory diseases such as rheumatoid arthritis. The concept suggesting tumor necrosis factor-alpha as a potential target emerged from experiments showing its key role in inducing many cytokines and mediators of inflammation. Several clinical trials targeting this cytokine in rheumatoid arthritis patients with a novel group of anti-tumor necrosis factor agents demonstrated reduced synovial inflammation and inhibition of bone and cartilage degradation. In addition to the therapeutic value of anti-tumor necrosis factor, analysis of laboratory changes not only proved the concept but provided new data, continuously expanding our understanding of the role of tumor necrosis factor-alpha in the pathogenesis of many diseases. These laboratory measures may also help the earlier identification of rheumatoid arthritis patients who have a less satisfactory response to this therapy.