IL-31 Levels In Peripheral Blood And Synovial Fluid Of RA Patients And The Relationship Between IL-31 Levels And Joint Inflammation

OBJECTIVE:This study was based on detecting the expression of Interlekin-31(IL-31)in peripheral blood and joint synovial fluid of rheumatoid arthritis(RA)and examining the expressions of inflammatory cells and immunoglobulin in rheumatoid arthritis and osteoarthritis(OA),to analyze the relationship between IL-31 and rheumatoid arthritis.Methods:1.This study included 96 patients with rheumatoid arthritis,50 patients with osteoarthritis,and55 healthy controls.At the same time,clinical data of RA patients were collected,including gender,age,course of disease,swollen joint count(SJC),tenderness joint count(TJC),and health assessment questionnaire(HAQ);DAS-28(Disease Activity Score-28,DAS-28)was used to reflect the disease activity of patients with rheumatoid arthritis;at the same time,rheumatoid factor(RF),erythrocyte sedimentation rate(ESR),and Anti-cyclic citrullinated peptide antibody(ACPA),C-reactive protein(CRP),serum immunoglobulin levels Ig(Immunoglobulin)A,IgG,IgM and other laboratory test results were collected.2.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of IL-31 in serum(n=96)and joint synovial fluid(n=11)of patients with rheumatoid arthritis.The rate turbidimetry method was used to quantitatively detect the levels of IgG,IgM,and IgA in synovial fluid and analyze the correlation between serum IL-31 level and serum RF and ACPA,and correlation between peripheral blood IL-31 level and synovial fluid IgG,IgM,IgA.3.Joint synovial fluid(n=11)centrifugation,smear of cell sediment,swine Giemsa staining,observe inflammatory cells with microscope and count.4.Flow cytometry analysis was used to observe the proportion of CD4~+and CD4~+IL-4~+T cells,CD4~+IFN-?~+T cells in peripheral blood mononuclear cells from rheumatoid arthritis patients(n=18)and healthy controls(n=10),and IL-31 expression in CD4~+IL-4~+T,CD4~+IFN-?~+T cells.5.HE staining was used to observe the infiltration of synovial tissue inflammatory cells,and immunohistochemical analysis was performed to analyse the infiltration of CD4~+T,CD19~+B,IFN-?~+T,and IL-4~+T cells in synovial tissue of rheumatoid arthritis patients(n=8).Results:1.Compared with OA group[84.66(1.10,330.80)pg/mL]and healthy control group[67.83(0,553.30)pg/mL],serum IL-31 expression level in RA group[457.30(169.60,1007)pg/mL]significantly increased(p<0.0001,p<0.0001);IL-31 levels of joint synovial fluid in RA group[1002(226.30,5663)pg/mL]were significantly higher than those in OA group[0(0,171.30)pg/mL](p<0.0001).And in RA patients with a high DAS-28 score,the level of IL-31[2144(1689,3566)pg/mL]was significantly higher than moderate[783.20(575.10,974.80)pg/mL],low DAS-28score[344.70(289.40,425.70)pg/mL]and patients in remission[81.40(0,171)pg/mL](p=0.0287,p<0.0001,p<0.0001).2.By analyzing the correlation between IL-31 and rheumatoid arthritis related indexes,we found that serum ACPA levels were positively correlated with IL-31 in patients with RA(r=0.641,p<0.0001),synovial IgG was positively correlated with serum IL-31(r=1.000,p=0.0004),and synovial IgG was correlated with synovial fluid IL-31(r=1.000,p=0.0004).There was no correlation between RF,CRP,ESR and IL-31 levels,and there was no correlation between IgA,IgM and IL-31levels(p>0.05).3.Compared with OA patients,the synovial fluid inflammatory cells infiltration in RA patients increased.The white blood cells count was[4.77(3.91,6.39)×10~6/mL]vs[2.12(1.69,7.23)×10~6/mL](p=0.031),the neutrophil cells count was[2.46(1.98,3.45)×10~6/mL]vs[1.52(1.23,5.83)×10~6/mL](p=0.0025)and the plasma cells count was[1.01(0.88,1.39)×10~6/mL]vs[0.18(0.05,0.31)×10~6/mL](p=0.0001),lymphocyte cells count was[1.22(0.87,1.88)×10~6/mL]vs[0.38(0.16,0.88)×10~6/mL](p=0.003).The infiltration of neutrophil,plasma,and lymphocytes cells increased,and the differences were statistically significant.4.Compared with the healthy control group,the proportion of CD4~+IL-31~+T cells[1.35(1.10,2.13)%vs 0.28(0.07,0.49)%],CD4~+IFN-?~+T cells[19.09(14.95,20.76)%vs 9.79(1.35,13.32)%]and CD4~+IL-4~+T[3.10(1.70,4.62)%vs 1.23(1.03,1.58)%](p<0.0001,p=0.0008,p=0.0096)in RA patients all increased.The proportion of IFN-?~+IL-31~+T cells in RA patients[0.61(0.39,1.21)%]is significantly higher than that of healthy controls[0.14(0.04,0.23)%](p<0.0001);The ratio of IL-4~+IL-31~+T cells in RA[0.17(0.10,0.81)%]was significantly higher than that of healthy controls[0.06(0.05,0.13)%](p=0.0155).5.Compared with OA patients,HE staining of joint synovial tissue in RA patients showed increased lymphocyte infiltration,and immunohistochemical analysis showed CD4~+T[0.59(0.43,0.67)]vs[0.38(0.16,0.44)],CD19~+B cells[0.45(0.36,0.51)vs 0.02(0.01,0.35)]significantly increased(p<0.05),of which Th1[0.18(0.16,0.22)vs 0.11(0.08,0.13)],Th2 cells[0.09(0.08,0.13)vs 0.06(0.05,0.08)]infiltration increased,and Th1 cell infiltration increased mainly(p<0.01).Conclusions:1.IL-31 is elevated in peripheral blood and synovial fluid of patients with rheumatoid arthritis,and is positively correlated with ACPA and IgG,indicating that IL-31 may play an important role in the progression of rheumatoid arthritis.2.IL-31 may be closely related to the disease activity of rheumatoid arthritis,and may be a valuable biomarker that reflects the severity of rheumatoid arthritis.3.Th1 cells in peripheral blood of rheumatoid arthritis increase,and IL-31 in peripheral blood may be mainly secreted by Th1 cells.