| OBJECTIVE Rheumatoid arthritis (RA) is a systemic autoimmune disease that primarilytargets the synovial membrane, cartilage, and bone. T cells and B cells play an importantrole in the pathogenesis of RA. The transmembrane activator and calcium modulator andcyclophilin ligand interactor-immunoglobulin (TACI-Ig), a recombinant fusion protein thatmodulates B and T cells activation by binding and neutralizing B lymphocyte stimulator(BLyS) and a proliferation-inducing ligand (APRIL), has been shown therapeutic effects onautoimmune disorders. The objective of this study was to investigate pathologicalmechanisms of BLyS/APRIL-receptors signaling and immunoregulatory efficacy ofTACI-Ig on mesenteric lymph node (MLN) of adjuvant-induced arthritis (AA) in rats.METHODS Our previous studies have shown that TACI-Ig had a therapeutic effect on adjuvant-induced arthritis (AA). In the present study, we investigate efficacy of TACI-Ig onmesenteric lymph node lymphocytes (MLNLs) of AA rats at the molecular level. SD ratswere immunized intradermally with0.1ml of Complete Freund’s adjuvant (CFA) into theright hind metatarsal rat footpad. Animals were divided into eight groups randomly,including TACI-Ig groups (0.7,2.1and6.3mg/kg), rhTNFR:Fc group (2.8mg/kg), MTXgroup (0.5mg/kg), IgG-Fc group (6.3mg/kg), normal group and model group. The levelsof BLyS, APRIL, interferon (IFN)-γ, interleukin (IL)-4, transforming growth factor beta(TGF)-β1, and IL-17were measured by enzyme-linked immunosorbent assay. Thelocalization and expression of TACI, B-cell maturation antigen (BCMA) and B cellactivating factor-receptor (BAFF-R) were investigated by immunohistochemistry andwestern blotting analysis in MLN. Proliferations of MLN lymphocytes activated byrecombinant human B lymphocyte stimulating factor (rhBLyS) were assayed by MTTmethod. Total (CD3+CD4+), activation (CD4+CD25+) and unsensitize (CD4+CD62L+)markers on CD4+T cell levels in MLN lymphocytes were analysed by flow cytometry invitro.RESULTS1Effects of TACI-Ig on histopathology of MLN in AA ratsOn day35, rats were sacrificed and subjected to histopathological examination. AA andIgG-Fc treated rats developed severe damage of MLN. This was characterized by markedhyperplasia of lymphatic follicle, increased size of paracortical areas, increased cellsnumbers of medullary cords and sinuses. In AA rats treated with TACI-Ig (0.7,2.1,6.3mg/kg), rhTNFR:Fc (2.8mg/kg) and MTX (0.5mg/kg) the histopathological scores of MLNdamage was significantly reduced.2Effects of TACI-Ig on the concentrations of IFN-γ, IL-4, TGF-β1and IL-17in MLNhomogenatesCompared with normal group, the concentrations of IFN-γ and IL-17elevated significantlyin MLN homogenates, whereas the concentrations of IL-4and TGF-β1reduced significantly of AA rats. Treatment with TACI-Ig (0.7,2.1,6.3mg/kg) significantlydecreased the concentrations of IFN-γ and IL-17and increased the concentrations of IL-4and TGF-β1. Moreover, the MLN from AA presented with a high ratio of Th1to Th2and alow ratio of Treg to Th17compared to the normal group, whereas the administration ofTACI-Ig (6.3mg/kg) significantly decreased the ratio of Th1and Th2and increased theratio of Treg and Th17. RhTNFR:Fc (2.8mg/kg) and MTX (0.5mg/kg) had the similarresults as TACI-Ig.3Effects of TACI-Ig on the levels of BLyS and APRIL in MLN homogenatesCompared with normal group, the levels of BLyS and APRIL elevated significantly inMLN of AA rats. Treatment with TACI-Ig (6.3mg/kg) significantly decreased the levels ofBLyS and APRIL. RhTNFR:Fc (2.8mg/kg) and MTX (0.5mg/kg) had the similar results asTACI-Ig.4Effects of TACI-Ig on TACI, BAFF-R and BCMA expression in MLN tissueImmunohistochemistry was used to examine the expression and localization of TACI,BAFF-R and BCMA in MLN. Tissue sections showed intense staining of medulla, andweaker staining of B cell follicles in superfacial cortex. Compared with normal group, wefound markedly enhanced expression of TACI and BCMA, and reduced expression ofBAFF-R in MLN of AA and IgG-Fc-treated rats. TACI-Ig at the concentration of2.1and6.3mg/kg significantly decreased TACI and BCMA expression, and increased BAFF-Rexpression in AA rats. RhTNFR:Fc (2.8mg/kg) had the similar results as TACI-Ig.5Effects of TACI-Ig on total protein expression of TACI, BAFF-R and BCMA inMLNIt was found that in the MLN lymphocytes expression of TACI and BCMA increasedsharply, while BAFF-R decreased in AA rats. Subcutaneous administration of TACI-Igcould down-regulate the total protein expression of TACI and BCMA, and up-regulate ofBAFF-R expression. RhTNFR:Fc (2.8mg/kg) had the similar results as TACI-Ig. MTX(0.5mg/kg) treatment could not significant alter above changes of receptors expression inAA rats. 6Effects of TACI-Ig on the rhBLyS-induced proliferation of MLN lymphocytes andT-lymphocytes subpopulations of AA rats in vitroRhBLyS (10-4μg/L) can lead to proliferative accentuation in MLNLs and TACI-Ig(10-5,10-4,10-3,10-2,10-1μg/L) significantly inhibit the rhBLyS stimulated proliferativereactions of lymphocytes. And TACI-Ig significantly decreased the enhanced total(CD3+CD4+T cell) and activated (CD4+CD25+T cell), enhanced the decreased unsensitized(CD4+CD62L+T cell) on CD4+T cell in MLN lymphocytes of AA rats.CONCLUSIONS1TACI-Ig decreased MLN histopathological scores, inhibited proliferative reactions ofMLN lymphocytes and alleviated histopathological manifestations.2TACI-Ig decreased the concentrations of BLyS, APRIL, IFN-γ and IL-17and increasedthe concentrations of IL-4and TGF-β1. And TACI-Ig decreased total (CD3+CD4+T cell)and activated (CD4+CD25+T cell), enhanced the unsensitized (CD4+CD62L+T cell) onCD4+T cell in MLN lymphocytes of AA rats. TACI-Ig might exert its anti-inflammatoryand immunoregulatory effects through inducing immune balance of Th1/Th2andTreg/Th17in peripheral MLN and regulating balance of T-lymphocytes subpopulations inMLN lymphocytes of AA rats.3TACI-Ig could down-regulate the total protein expression of TACI and BCMA, andup-regulate of BAFF-R expression. The mechanisms of TACI-Ig in MLN lymphocytes viaBLyS/APRIL-receptors-dependent signaling may play key roles in the pathogenesis ofautoimmune disorders. |
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