The Expression And Clinical Significance Of Soluble Form Of Costimulatory Molecule PD-1in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune arthritis characterized byprogressive joint destruction. The main clinical manifestations of RA is chronic,symmetry, multi-synovial arthritis and extra-articular lesions. The occurrence of RA isabout0.4%in China. Although the etiology of RA remains a mystery, current studysuggested that RA is a T cell-mediated autoimmune diseases and excessive activation ofT lymphocytes play an important role in the immunopathological process.According to the two-signal model, naive T cells require two necessary signals tobecome fully activated. The first signal, which gives specificity to the immune response,is provided by the interaction of antigen-major histocompatibility complex (Ag-MHC)complex with T cell receptor (TCR). The second signal is delivered by manycostimulatory molecules on the antigen-presenting cells (APCs) to promote T cellactivation, proliferation and cytokine secretion. The most intensively characterizedcostimulatory molecules are those of the B7/CD28family. The PD-1（Programmeddeath-1）, which belongs to CD28family, is encoded by PDCD1gene. PD-1with itstwo ligands, PD-L1and PD-L2, triggers inhibitory signals to inhibit T and B cellactivation and production of cytokines. In addition, PD-1expression plays an importantrole in the occurrence and development of a series of diseases. Studies showed thatPD-1receptor was a promising target in the intervention of human malignant cancers,autoimmune disease and viral diseases.This study is to explore the function of soluble PD-1(sPD-1) in the immunologicalpathogenesis of RA by detection of soluble PD-1expression in peripheral blood of RApatients, osteoarthritis patients and healthy controls, and by the analysis of correlationbetween sPD-1expression and clinical stage, clinical symptoms and laboratoryparameters; Furthermore, PDCD1genotyping and the mRNA level of PD-1Δex3, IFN-γ,IL-4, IL-10, IL-17A and IL-21cytokine in peripheral blood of RA patients wereanalyzed to determine the molecular mechanism of abnormal expression of soluble PD-1in RA patients.PARTⅠ The Expression and Function of Soluble PD-1inPatients of Rheumatoid ArthritisObjective Explore the expression and clinical significance of sPD-1in RA patients.Observe the influence of sPD-1in PD-1/PD-L1pathway. Methods The expression ofsPD-1in the serum of RA patients, OA patients and healthy controls was detected byELISA and the correlation between sPD-1and clinical data was analyzed. At the sametime peripheral blood mononuclear cells (PBMC) was isolated and mRNA level offlPD-1, PD-1Δex3was detected by RealTime RT-PCR. The proliferation of CD4+T cellsaffected by sPD-1was also determined in vitro. Results The expression of sPD-1was38.39±5.45ng/ml in RA, significantly higher than in HC (20.56±1.29ng/ml, P=0.0003)group and OA group (21.88±2.35ng/ml, P=0.0493). RA patients in acute episode hadhigher expression of sPD-1than patients in relief episode (P=0.0027). The expressionof sPD-1is positive correlated to RF and the counts of joints involved (r=0.2668，P=0.0411；r=0.4365，P=0.0003). The expression of membranous PD-1(mPD-1) inCD4+T cells was significantly higher in RA group than in HC group (P <0.001) and OAgroup (P <0.001). The mRNA expression of PD-1Δex3and flPD-1was significantlyhigher in RA group than in HC (P=0.0346, P=0.0057) and OA (P=0.0401, P=0.0457)group. PD-L1inhibited the proliferation of CD4+T cells, and this inhibition could bereversed by sPD-1(P=0.0035). Conclusion The expression of sPD-1was significantlyup-regulated in RA, and positive correlated to the clinical stage, clinical symptoms andprognosis. Soluble PD-1may block the PD-1/PD-L1signaling pathway, and played animportant role in the immunopathological process of RA.PARTⅡ The Molecular Mechanism of AbnormalExpression of Soluble PD-1in Rheumatoid Arthritis Patients1. Expression of cytokines in peripheral blood of RA patients and thecorrelation with PD-1Δex3Objective Explore the expression of IFN-γ、IL-4、IL-10、IL-17A and IL-21cytokines in peripheral blood of RA patients. Analyze the correlation between PD-1Δex3and the imbalanced cytokines in RA patients. Methods The expression ofIFN-γ、IL-4、IL-10in the serum was detected by ELISA. Peripheral Blood MononuclearCells (PBMC) was isolated from peripheral blood of subjects and IFN-γ、IL-4、IL-10、IL-17A、IL-21mRNA level was detected by Real Time Rt-PCR. Analyze the correlationbetween PD-1Δex3and the imbalanced cytokines in RA patients. Results Theexpression of IFN-γ、IL-4、IL-10were up-regulated in the different activation phases ofT cells in vitro. Real Time PCR demonstrated elevated mRNA expression levels of fourcytokines in RA group than in HC and OA group, including IFN-γ(P=0.0421, P=0.1637), IL-4(P=0.0359, P=0.0499), IL-17A(P=0.0420, P=0.0428), IL-21(P=0.0425, P=0.2471). Further more, the expression of IFN-γ、IL-4、IL-17A were positivecorrelated to the expression of PD-1Δex3(r=0.4633，P=0.0026；r=0.4109，P=0.0069and r=0.4065，P=0.0092). Conclusion The abnormal activation of T cell subsets andtheir related inflammatory cytokines may affect the expression of sPD-1.2. PDCD1gene polymorphism is associated with the expression of sPD-1inrheumatoid arthritisObjective Screening the single nucleiotide polymorphisms(SNPs) in PDCD1exon3coding regions and the intron/exon boundaries, analyze the association of candidateSNPs with RA. Study the expression of sPD-1in different genetic background of RApatients. Methods Polymerase chain reaction amplification and direct sequencing wereused to screen PDCD1exon3coding regions and intron/exon boundaries forpolymorphisms. The association analysis of single SNP with RA is dertermined bylogistic regression. We also studied the expression of sPD-1in different genotyps of RApatients. Results We finaly found3SNPs in the target sequence, SNP2and SNP3werenewly discovered SNP sites. The three SNPs were predicated to locate within the targetsequence of splicing factor SRp40, SC35and SF2/ASF by bioinformatics software. Thefrequency of G allele of SNP1was significantly greater in RA patients than in healthycontrols (P=0.021), statistically significant differences were found for genotypes ofSNP1(P=0.037) and the log-additive model were accepted as the best inheritancemodel. The expression of sPD-1in G/G and A/G RA patients were significantly higherthan in A/A patients (P=0.0237and0.0488). Conclusion Previous studied therelationship between SNP1genotype and sPD-1expression in RA patients. Our resultssuggested that sPD-1was involved in the immune tolerance of RA. The abnormally high expression of sPD-1in the pathological state may be affected by the geneticvariants which might change the alternative splicing of PD-1pre-mRNA, and thespecific molecular mechanisms require further study.