BLyS-receptor Mediated Immune Responses In Rats With Adjuvant-induced Arthritis And The Effects Of A New Active Monomer CP-25

B lymphocyte stimulator (BLyS), a tumor neurosis factor ligand superfamily, binding to transmembrane activator or calcium-modulating cyclophylin ligand-interactor (TACI), B cell maturation antigen (BCMA) and B cell-activating factor receptor (BAFF-R), plays important roles in differentiation and activation of B cells. BLyS is expressed in a variety of cells types, such as monocytes, macrophages, dentritic cells and so on. Activated T cells also express BLyS and its receptors BAFF-R and TACI, regulate the function of T cells, promote the differentiation and activation of T cells, play an important role in T cell mediated autoimmune diseases.Total glucoside of paeony (TGP) is the first anti-inflammatory and immunoregulatory drug in treatment for RA. Phenylsulfinyl-paeoniflorin (paeoniflorin-6’-O-benzene sulfonate, CP-25) is esterification modified by Paeoniflorin (PAE), a monoterpene glucoside and the major components of TGP. It is highly lipid soluble, mainly absorbed from the small intestine, drug bioavailability is improved, effect of anti-inflammatory and immune regulation is obvious. It had obtained the country patent of invention (ZL 2012 1 0030616.4). How does CP-25 regulate immune response in rheumatoid arthritis (RA)? Whether does CP-25 plays its regulation function through the adjustment of T and FLS function and cell interaction? This is not clear.In this study, adjuvant-induced arthritis (AA) rats were used to explore the expression of BLyS and its receptors in immune organs and immune cells and to observe the effect of CP-25. The effect of CP-25 on the function of fibroblast-like synoviocyte (FLS) and regulating the interaction between FLS and CD4+ T cells was investigated, too.Objective:To observe the expression of BLyS and its receptors (BAFF-R、TACI、BCMA) in immune organs, immune cells and FLS and to investigate the effect of CP-25 on AA rats. The effect of CP-25 on the function of FLS and regulating the interaction between FLS and CD4+ T cells was investigated, too.Methods:The model of rat AA was induced by a single intradermal injection of 0.1 ml complete Freund’s adjuvant into the right hind metatarsal footpad of rat. Animals were divided into eight groups randomly, including normal group, model group, CP-25 groups (25,50 and 100 mg/kg), MTX (0.5mg/kg), TGP (50mg/kg) and PAE (50mg/kg).Rats were inspected daily for signs of arthritis by total score. We use hematoxylin and eosin (HE) stain method for histopathological evalution of spleen. The relative gene expression of BLyS and its receptors was assessed by quantitative real time polymerase chain reaction. (CD3+CD4+), (CD3+CD8+), (CD4+CD62L+), (CD4+CD44+), TACI and BAFF-R markers on CD4+T cells were detected by flow cytometry. Thymocytes and FLS proliferation was detected by Cell Counting kit-8. Concentrations of IL-1β, TNF-α, IL-6 were measured using enzyme-linked immunosorbent assay kits.Results:1. Expression of BLyS and its receptors (BAFF-R、TACI、BCMA) in immune organs, immune cells and FLSCompared with normal group, gene expression of BLyS, BAFF-R and BCMA elevated significantly in spleen tissue of AA rats, gene expression of TACI do not change. Gene expression of BLyS and BAFF-R elevated significantly in mesenteric lymph nodes tissue of AA rats, gene expression of TACI decreased, gene expression of BCMA do not change. Gene expression of BLyS, BAFF-R and BCMA elevated significantly in macrophages of AA rats, gene expression of TACI decreased. Gene expression of BLyS and BCMA elevated significantly in dentritic cells of AA rats, gene expression of TACI decreased, gene expression of BAFF-R do not change. Gene expression of BLyS, BAFF-R and BCMA elevated significantly in FLS of AA rats, gene expression of TACI do not change. Expression of BAFF-R elevated significantly in CD4+T cells of AA rats, expression of TACI decreased, expression of BCMA do not change.2. CP-25 inhibited the proliferation of BLyS-induced thymocytesCP-25(10-9,10-8,10-7,10-6,10-5 mol/L) significantly inhibited abnormal proliferation of BLyS-stimulated thymocytes from AA rats in a concentration-dependent manner.3. CP-25 inhibited the proliferation of BLyS-induced FLSCP-25 (10-7,10-6,10-5 mol/L) significantly inhibited the proliferation of BLyS-induced FLS from normal and AA rats in a concentration-dependent manner.4. BLyS-stimulated CD4+ T cells in enhancing FLS function and the effect of CP-25The proliferation and secretion of FLS subsequently facilitated in the presence of BLyS-stimulated CD4+ T cells. CP-25(10-8,10-7,10-6 mol/L) can inhibit the abnormal proliferation of FLS in a concentration-dependent manner in the co-cultures. CP-25 (10-8,10-7,10-6 mol/L) reduced the IL-1β level and CP-25 (10-7,10-6 mol/L) reduced the TNF-α and IL-6 levels remarkably in a concentration-dependent manner.5. Effects of CP-25 on total score and spleen histopathology in a rat AA modelCFA induced rat AA, the secondary inflammatory reaction occurred as swollen joints characteristic by polyarthritis on day 14 after immunization. CP-25 decreased total score and alleviated histopathological manifestations.6. CP-25 regulates the imbalance of T cell subsetCompared with normal group, the expression of CD3+CD4+ T helper cells significantly increased in AA rats, CP-25 (50, 100mg/kg) deceased proportion of CD3+CD4+ T helper cells. The expression of CD3+CD8+ cytotoxic T cells significantly decreased in AA rats, CP-25 (100mg/kg) increased proportion of CD3+CD8+ cytotoxic T cells. The expression of CD4+CD44+ memory CD4+T cells significantly increased in AA rats, CP-25 (50, 100mg/kg) decreased proportion of CD4+CD44+ memory CD4+T cells. The expression of no-sensitizated CD4+CD62L+ T cells does not change.7. Effects of CP-25 on levels of cytokines in AA FLSCompared with normal group, levels of IL-1β and TNF-a were significantly elevated in AA FLS. CP-25 (50, 100mg/kg) decreased IL-1β level and CP-25(100mg/kg) decreased TNF-α level.Conclusions:1. BLyS and its receptors participated in the T cell response of AA rats due to the abnormal expression of BLyS and its receptors in immune organs, immune cells and FLS in AA rats;2. CP-25 may exert its anti-inflammatory effects through suppressing the function of FLS and regulating the interaction between FLS and CD4+T cells.3. CP-25 decreased total score, alleviated histopathological manifestations, and modulated the imbalance of T cell subset, had a therapeutic effect on T cell mediated AA.