The Mechanisms Of BLyS/APRIL And Their Receptors On Participating In T Cell Responses In Immune-mediated Arthritis And The Therapeutic Effects Of TACI-Ig Fusion Protein

B lymphocyte stimulator (BLyS) is one of tumor necrosis factor superfamily members, which derived from monocytes/macrophages, dentritic cells, neutrophils, and malignant B cells. A proliferation-inducing ligand (APRIL) is a close homolog of BLyS. BLyS/APRIL regulate the size and function of B cell pool, promote immunoglobulin (Ig) class switching and recombination as the vital cytokine via binding to transmembrane activator and CAML interactor (TACI), B cell maturation antigen (BCMA), B cell-activating factor receptor (BAFF-R) respectively. Recently, people found that activated T cells also produced BLyS and APRIL, and expressed BAFF-R and TACI. BLyS/APRIL played important roles in differentiation and activation of T helper lymphocyte (Th), but there was no report about effect of BLyS/APRIL in T cell mediated inflammatory immune responses.TACI-Ig contains the BLyS/APRIL-binding extracellular portion of the TACI molecule fused to the Fc portion of human IgG and can be used to neutralize BLyS and APRIL and prevent them from binding to their receptors. To explore the effect of BLyS/APRIL in Th mediated inflammatory immune response, we chose the rat adjuvant-induced arthritis (AA) model which a model involves primarily T cells autoimmune reaction to joints in this study to observe effects of TACI-Ig on A A rats, and to examine how BLyS/APRIL plays a therapeutic role by affecting the T cells.Objective:In this study, we examined the effects of BLyS and APRIL and receptors (BCMA, TACI, BAFF-R) in T cells mediated inflammatory immune responses, also examined the correlationships between the BLyS level and arthritis extent, investigated the therapeutic effects of TACI-Ig on rat AA, and explored how BLyS/APRIL affected the T cells.Methods:The model of rat AA was induced by a single intradermal injection of0.1ml of CFA (complete Freund’s adjuvant) into the right hind metatarsal footpad of rat. Animals were divided into eight groups randomly, including TACI-Ig groups (0.7,2.1and6.3mg/kg, sc, d16～d34), rhTNFR:Fc group (2.8mg/kg, sc, d16～d34), MTX (0.5mg/kg, ig, dl6～d34), IgG-Fc group (6.3mg/kg, sc, d16～d34), normal group and model group.Secondary foot claw degree of swelling was examined by volume method. Rats were inspected daily for signs of arthritis by arthritis index and body weight. AA rat joints were observed by X-ray. We used hematoxylin and eosin (HE) stain method for histopathological evaluation of joints and spleens. Serum IL-1β, TNF-α, PGE2, BLyS, APRIL, IL-17, IL-2, IL-10, TGF-β1, IFN-γ, IgG1, IgG2a, IgM, IgA and synovium BLyS and APRIL levels were detected by ELISA. Expression of BLyS, CD3, CD20and CD68in spleen, Expression of BLyS, APRIL, CD21, CD138and PNAd in synovium tissue were detected by immunohistochemistry.(CD3+CD4+),(CD4+CD25+),(CD4+CD44+) and (CD4+CD62L+) markers on CD4+T cells in peripheral blood, spleen, thymus were detected by flow cytometry.Results:1. Establishment and evaluation of rats with adjuvant-induced arthritisFCA induced rat A A, the secondary inflammatory reaction occurred as swollen joints characteristic by polyarthritis. X-ray showed that articular soft tissue swelled significantly, no bone erosion and joint space change occurred. Histopathological showed that hydrops in articular cavity, inflammatory exudation, synoviocytes proliferation, pannus formation, microvascular hyperplasia, infiltration with inflammatory cells, and synoviocytes violated cartilage surface. Immune responses resulted in increased cellularity and size in the spleen follicles, periarterial lymphatic sheath and marginal zone, and appearance of prominent germinal center in white pulp.2. Imbalance of T cell subset and cellular immune are important characteristicCompared with normal group, the concentrations of serum IFN-γ and IL-2(derived from Th1cells) and IL-17(derived from Th17cells) elevated significantly, whereas the concentrations of IL-10(derived from Th2cells) and TGF-β1(derived from Treg cells) reduced significantly in rats of AA. Compared with normal group, the expression of total (CD3+CD4+), activated (CD4+CD25+), memory (CD4+CD44+) and no-sensitizated CD4+T cell (CD4+CD62L+) significantly decreased in peripheral blood and spleen. These results suggested that imbalance of T cell subset and cellular immune are important characteristic.3. BLyS/APRIL and their receptors participated in the T cell response of AA ratsCompared with normal group, the concentrations of BLyS and APRIL elevated significantly in serum. We found markedly enhanced expression of BLyS in spleen of AA rats. To examine the correlation between the severity of the disease and the serum BLyS level, we measured coefficient of correlation between the secondary arthritis and histopathology and the BLyS level. Interestingly, the secondary arthritis (paw swelling and polyarthritis index) and spleen histopathology (cellularity of PALS, lymphoid follicles, marginal zone, red pulp, and the total number of GCs) significantly correlated with the production of serum BLyS. Immunohistochemistry and Western blot results showed that the expression of TACI and BCMA in synovium tissue increased and the expression of BAFF-R decreased. TACI-Ig up-regulated BAFF-R expression, down-regulated TACI and BCMA expression in synovium tissue, which further confirmed that the importance of BLyS/APRIL and their receptors in the T cell response of AA rats.4. BLyS and APRIL are important factors in the synovium inflammation of AA ratsCompared with normal group, the concentrations of BLyS and APRIL elevated significantly in synovium tissue of AA rats, respectively. Immunohistochemistry results showed that increased expression of BLyS in spleen of AA rats. Joints histopathology (synovial proliferation, cellular infiltration, pannus formation, and cartilage erosion) significantly correlated with the production of serum BLyS. TACI-Ig decreased CD21, CD138, PNAd expression in synovium tissue, which further confirmed that the BLyS and APRIL are important factors in the synovium inflammation of AA rats.5. TACI-Ig exerted its therapeutic effects through regulating the balance of T cell subsets and abnormal immune function of AA ratTACI-Ig (0.7,2.1,6.3mg/kg, sc, d16～d34) significantly decreased arthritis index and joint swollen degree. TACI-Ig (6.3mg/kg, sc, d16～d34) significantly improved soft tissue swelling. TACI-Ig (0.7,2.1,6.3mg/kg, sc, dl6～d34) alleviated joint and spleen histopathological manifestations. These results showed that TACI-Ig ameliorated arthritis symptom and alleviated histopathological manifestations of spleen and joint of the AA rats. TACI-Ig (0.7,2.1,6.3mg/kg, sc, d16～d34) decreased serum IgM, IL-1β, TNF-a, PGE2levels. TACI-Ig (0.7,2.1,6.3mg/kg, sc, dl6-d33) decreased BLyS and APRIL levels in blood and synovium tissue. TACI-Ig (0.7,2.1,6.3mg/kg, sc, d16～d34) inhibited hyperplasia of CD20(B cell), CD3(T cell), CD68(macrophage); TACI-Ig (0.7,2.1,6.3mg/kg, sc, d16～d34) modulated proportion of total (CD3+CD4+), activated (CD4+CD25+), memory (CD4+CD44+) and no-sensitizated CD4+T cell (CD4+CD62L+) in spleen and peripheral blood. These results suggested that therapeutic effects of TACI-Ig characteristic by modulation of AA rats T cell subsets and abnormal immune function.Conclusions:1. BLyS and APRIL play key roles in AA rats mediated by T cells. The secondary arthritis and histopathology significantly correlated with the production of serum BLyS;2. BLyS/APRIL and their receptors participated in the T cell response of AA rats;3. BLyS and APRIL are important factors in the synovium inflammation of AA rats;4. TACI-Ig exerted its therapeutic effects through regulating the balance of T cell subsets and abnormal immune function of AA rat.