Study On The Role Of FLS Regulated By IL-34 In The Mechanism Of RA

Background: Rheumatoid arthritis(RA) belongs to chronic progressive autoimmune disease, mainly characterized by synovial hyperplasia, cartilage and bone destruction, but the definite pathogenesis is still unclear. Now it has been considered that the pathogenesis is related to cytokine network disorder, hyper-proliferation and invasiveness of synovial tissue and imbalance of T cell subsets. Interleukins 34(IL-34) was discovered in 2008 with the function of sustaining the survival, development and activation of macrophages and dendritic cells, mainly through and colony stimulating factor 1 receptor(CSF-1R). In the serum and synovial joints of patients with RA, the content of IL-34 was higher than the normal control and was positively correlated with RF and anti-CCP antibody. Fibroblast-like synoviocytes(FLS) play an important role in the onset of rheumatoid arthritis, CSF-1R was highly expressed on their surface, IL-34 might regulate the function of FLS through its receptors, which might be involved in the pathogenesis and process of disease.Objective: This study was to explore the role of IL-34 on the pathogenesis and progress of RA, and research on the pathogenesis mechanism of RA FLS regulated by IL-34.Methods: The serum were collected and isolated from RA patients, the levels of IL-34 in the serum from RA patients and healthy controls were detected by enzyme-linked immunosorbent assay(ELISA) and the relationship between the volume of IL-34 and the clinical index also were analysed. FLS from RA patients were cultured, when developed into 4~6 generations using flow cytometry to appraise FLS’ purity and the expression level of IL-34 receptor(CSF-1R) on the surface of FLS, added recombinant IL-34 in the cultured FLS, detected the levels of cytokines in the supernatant fluid by protein chip technology. Increase the strains of FLS, the results of protein chip were validated by reverse transcription-polymerase chain reaction(RT-PCR) and ELISA, IL-6, cox-2 mRNA expression and IL-6, PGE2 secretion by FLS were detected in the presence or absence of IL-34 or with the treatment of IL-34 receptor antagonist. Total RNA was extracted from IL-34-stimulated FLS, the level of caspase8 mRNA was measured by RT-PCR. To observe the effects of IL-34 on FLS apoptosis, cell proliferation was determined by MTT method after IL-34 stimulation, extracted total protein from FLS, the phosphorylation of Erk 1/2, JNK- 1/2/3, P38, the NF-kappa B were measured by Western blot, the level of IL-6, cox-2 mRNA were tested by RT-PCR and the secretion of IL-6, PGE2 of FLS were determined by ELISA after treatment with corresponding signaling pathway inhibitors and IL-34. Separated PBMC of the healthy control in vitro, and isolated CD4+T cells by magnetic bead, the percentage and purity of CD4+T cells were by obtained by flow cytometry, the purity of CD4+T cells must be attained above 95%, put the isolated CD4+T cells and RA FLS in a direct contact system or in a transwell coculture system, treatment with CD3/CD28 or CD3/CD28/IL-34, the percentage Th17 cells was measured by flow cytometry. IL-6 levels in the co-culture supernatant were detected by ELISA method, the coculture system of RA FLS and peripheral blood CD4+T cells from healthy control were treated with IL-6 receptor antagonist, the percentage of Th17 cells was tested by flow cytometry.Results: The levels of serum IL-34 of RA patients were significantly increased compared to healthy subjects. Meanwhile, the levels of IL-34 were positively correlated relationship with ESR, CRP, RF, anti CCP antibody. CSF-1R was highly expressed on the surface of RA FLS, recombinant IL-34 can significantly promote the RA FLS to secrete a variety of mediaters in vitro, especially IL-6, PGE2, ENA-8, IL-8, GRO and MCP-1, IL-34 could also promote FLS’ proliferation, inhibited its apoptosis, CSF-1R antagonist inhibited FLS cells to secrete IL-6 and PGE2, it showed that intracellular p-ERK 1/2, p-P38, p-JNK-1/2/3 and p-NF-kappa B significantly increased with IL-34 stimulation in the RA FLS, the total ERK 1/2, P38, JNK-1/2/3 and NF-kappa B keep unchanged. IL-6 secretion by IL-34-stimulated FLS was significantly reduced after treatment with P38, JNK-1/2/3 and NF-kappa B inhibitors. Similarly the expression of PGE2 was also obvious distured by P38 and NF-kappa B inhibitors. In the PBMC, CSF-1R could only be detected on the surface of monocytes, but not on surface of T, B lymphocytes, neutriphils whether they were activated or not. The percentage of Th17 had no difference between the RA FLS and healthy CD4+T cells directed contact coculture system and transwell coculture system when were added with anti-CD3/CD28 antibody or anti-CD3/CD28 antibody/IL-34, anti-CD3/CD28 antibody/IL-34 could significantly promote the generation of Th17 and secretion of IL-6 in the co-culture system, the amount of IL-6 secreted by IL-34-stimulated FLS was significantly lower than which in the coculture system, IL-6 receptor antagonist could obviously reduce the differentiation of Th17 in the co-culture system.Conclusion: The levels of IL-34 in serum from RA patient were closely related with the onset and progress of RA, IL-34 could facilitate the proliferation of FLS and prevent their apoptosis as well, simultaneously IL-34 could promote the secretion of various cytokines, chemokines and soluble proteins. It also promoted the secretion of IL-6 in FLS through the JNK/P38/NF-κB signaling pathway, PGE2 secretion was related to the P38/NF-κB signaling pathway as well. At last, IL-34 might up-regulate the generation of Th17 through overexperssion of IL-6 by FLS in RA.