Role Of IL-21 In The Induction Of T Cell Activation And Proinflammatory Cytokine Secretion In Rheumatoid Arthritis

BACKGROUDRheumatoid arthritis (RA) is a familiar chronic, systemic and inflammatoryautoimmune disease, in which main clinical features are chronic and symmetricpoly-synovium joints inflammation and extra-articular lesions. RA patients will loseall labor force at the late stage, characterized by the joints deformity. It increased theheavy burden for society and patient's family.An inflamed synovium is central to the pathophysiology of RA, which is also keyfeature in the induction of joint destruction. The pathogenesis of RA is still elusive.Recent years, increasing evidences have proved that RA is closely associated withabnormal immunological response in the joints. Cellular immune dysregulation playsan important role in the pathogenesis of RA. There are large number of CD4~+ T cellinfiltrates in synovial tissues, these cells are activated and produce high levels ofproinflammatory cytokines, such as TNF-α, IL-1, IL-2, IL-6, IL-10, IL-12, IL-15,IL-18 and IFN-γ,. Importantly, TNF-αand IL-1 are major proinflammatory cytokinesin the induction of joint damage.Interleukin-21 (IL-21) is a recently discovered cytokine and is a novel classⅠcytokine. IL-21 was identified as a four-helix-bundle cytokine that is mosthomologous to IL-15and also has significant homology to IL-2 and IL-4. IL-21R hadthe highest homology to IL-2Rβand IL-4Ruand IL-9R. IL-21 is secreted by CD4~+T-helper cells and selectively modifies both humoral and cell-mediated immuneresponses through an interaction with various cell types including T, B, NK cells anddendritic cells. IL-21R is expressed in T, NK, B and dendritic cells and plays an important role in regulating these cell functional responses. Like IL-2, IL-21 exhibitsa broad capacity to regulate T cell functions. IL-21 is also critical for normal humoralimmunity, and synergizes with anti-CD40 mAb to stimulate B cell activation, clonalexpansion and maturation.It is not known the exact role of IL-21 and it's receptor in the pathogenesis ofhuman diseases. Some data have shown that IL-21 is involved in the diseasedevelopment of Crohn's disease and systemic scleroderma, suggesting that IL-21 mayalso play a role in the pathogenesis of RA.OBJECTIVETo investigate the role of IL-21 in the pathogenesis of RA, we analyzed theIL-21R expression in peripheral blood and synovial fluid mononuclear cells, as wellas its in situ expression in synovium and IL-21 induced peripheral blood T cell (PB-T)and synovial fluid T cell (SF-T) activation, proliferation and TNF-αand IFN-γ,secretion. This could help us determine the correlation of RA with IL-21, and find anew and effective approach to treat RA.METHODS14 cases of RA, 12 cases of osteoarthritis (OA) and 8 cases of acute injuriedpatients' knee joint synovial tissue were obtained. They were immediately embeddedin OCT compound, frozen in liquid nitrogen, and stored at—80℃forimmunohistochemistry. Heparinized peripheral blood samples were obtained from 38patients with RA and 24 patients with OA. In addition, blood samples from 26 healthyvolunteers were also taken for comparison. Synovial fluid (SF) was also taken from12 patients with RA and 10 patients with OA. In addition, SF from 7 patients who hadno any visible and microscopic signs of inflammation after arthroscopic examinationwas used as controls. The diagnosis of RA and OA patients was described accordingto the advised criterion of American College Rheumatology (ACR). None receivedany immunosuppressants and target cytokine therapy. The frequency of IL-21 R+ cellsin PBMC and SFMC was determined on a flow cytometry. PB- and SF-CD4~+ T cellswere purified and stimulated with coated anti-CD3 mAb and IL-21R expression wasdetermined at various time intervals of 8, 12, 24 and 48 h of culture. After stimulated with coated anti-CD3 and IL-21, the role of IL-21 inducing PB-T and SF-T cellactivation, proliferation and proinflammatory cytokine secretion was measured byELISA, flow cytometry and a beta scintillation counter.RESULTS1. IL-21R was highly expressed in synovial tissues of RA patients. IL-21R~+ cellswere significantly increased in the synovial intimal lining and sublining layers in RApatients compared with those in controls. The number of IL-21R~+ cells among totalcell number was significantly higher in inflamed synovial tissue of RA patients (12.3±2.4％) compared with that from OA patients (2.1±0.6％) and healthy controls (1.2±0.3％) (P＜0.001). No difference was observed between OA patients and healthycontrols.2. After confirming in situ expression of IL-21R in synovial tissues of RApatients, we wanted to address the frequencies of IL-21R~+ cells in PBMC and SFMCusing a flow cytometry. CD4~+, CD8~+ T cells, B cells and NK cells in peripheral bloodand synovial fluid of RA patients expressed significantly higher levels of IL-21Rcompared with those in healthy controls (P＜0.05).3. IL-21R was rapidly decreased in healthy PB- and SF-CD4~+ T cells 8 h afteranti-CD3 in vitro stimulation, with a relatively low level of IL-21R expressionthereafter. However, the levels of IL-21R in RA CD4~+ T cells were found to bedecreased slowly after anti-CD3 stimulation, but still significantly higher comparedwith controls at 8, 12, 24 and 48 h of culture (P＜0.05). These results indicate thatIL-21R expression is mainly expressed in freshly isolated mononuclear cells fromperipheral blood and synovial fluid and disappear slowly after in vitro stimulation,suggesting that IL-21R may be involved in proinflammatory immune response insynovial membrane.4. Very low to absent levels of CD69 expression were detected on freshlyisolated PB-T cells from both RA and controls. After stimulation with coatedanti-CD3, CD69 expression was observed to be increased in both groups, but it wassignificantly higher in RA PB-T cells as compared to healthy controls (P＜0.05 at 8 hof culture). Interestingly, IL-21 further upregulated CD69 expression in PB-T cells, and the levels of CD69 were markedly higher in RA PB-T cells compared withcontrols (P＜0.05 at 8 and 12 h of culture). Moreover, CD69 expression was observedto be markedly increased in freshly isolated SF-T cells than in controls (P＜0.01).Interestingly, CD69 was upregulated in SF-T cells from both groups after IL-21stimulation, reached at the peak level 8 h, and decreased thereafter. However, itslevels were also significantly higher in RA SF-T cells than in healthy controls at 8, 12,24 and 48 h of culture (P＜0.05). Therefore, these data indicate that IL-21 stronglyinduces RAT cell activation.5. Since IL-21 could upregulate RA T cell activation, we further determined theproliferation-inducing effect of IL-2I on these T cells. PB- and SF-T cells from RAsynovial patients, when stimulated with coated anti-CD3 and IL-21, had asignificantly higher proliferative index than those from healthy controls. These resultsindicated that IL-21 is a strong inducer of RA T cells, suggesting that PB- and SF-Tcells are activated during inflammatory injury and become more responsive to IL-21.6. We explored whether IL-21 could contribute to proinflammatory cytokineproduction in the pathogenesis of RA. To this end, PB- or SF-T cells from RA patientswere stimulated with anti-CD3 and IL-21. T cell-derived cytokines (e.g., IFN-γ,,TNF-α, IL-2, IL-10) were measured by ELISA in culture supernatants. T ceils fromRA patients secreted significantly higher levels of TNF-αand IFN-γ,as. compared withthose from healthy controls (P＜0.05). These data demonstrated that IL-21 stronglytriggers RA T cell activation and induces proinflammatory cytokine secretion and maybe directly associated with the development of RA.CONCLUSION1. IL-21R is highly expressed in synovial tissue of RA patients.2. IL-21 may be an important proinflammatory cytokine in the pathogenesis.3. Targeted therapy directed blocking IL-21 may be helpful in the treatment ofRA.