Variation Of Helper T Cell 17/ Regulatory T Cells In The Peripheral Blood And Their Related Cytokines Of Patients With Rheumatoid Arthritis And The Clinical Significance

Background Rheumatoid arthritis(RA) is the most commen autoimmunity diseas that lead to disability.Recent study displays that imbalance of T helper cells 17(Th17) and regulatory T cells (Treg) plays an important role in the pathogenesis and development of RA.The two cell subsets all belong to CD4+T cell.Treg can secrete inhibitive cytokines transforming growth factor-β(TGF-β) and so on,then express immunoregulation.Th17 dependents on interleukin(IL)-23,secretes inhibitive inflammatory cytokines IL-17,IL-6 and so on,then express inflammatory response.But the specific variation tendency and clinical significance of Th17/Treg and the related cytokines IL-17,TGF-β,IL-6 and IL-23 have not been clear.Objective To detect the positive expression level of Th17 and Treg in the peripheral blood and the content of related cytokines(IL-17,IL-6,IL-23,transforming growth factor-β(TGF-β)) in the serum and knee-joint synovia of RA patients, compare the disparities with healthy people, analyze the relationship with duration length,disease activity, bone erosion, drug treatment,ect,in order to discuss the function and cilnical significance of Thl7 and Treg cells in RA.Methods 57 RA patients and 32 healthy volunteers(gender and age matching) were collected in the Rheumatology and Immune Department of the First Affiliated Hospital of Anhui Medical University during December in the year of 2009 to May in the year of 2009. Meanwhile RA patients were divided into groups according to duration length,disease activity, bone erosion and drug treatment. Flow cytometry(FCM) was used to analyze the level of interleukin(IL)-17 positive CD3 positive CD8 negative T(Th17) cells and Foxp3 positive CD25 positive CD3 positive CD8 negative T(Treg) cells from the peripheral blood of experimental subjects,enzyme linked immunosorbent assay (ELISA) was used to examine the levels of related cytokine IL-17,IL-6,IL-23 and TGF-βfrom the serum of experimental subjects and the knee-joint synovia of RA patients. Compared the level of IL-17,IL-6,IL-23 and TGF-βin the knee-joint synovia of RA patients with their serum.Compared the positive expression level of Th17 and Treg,the ratio of Thl7/Treg,the level of IL-17,IL-6,IL-23 and TGF-βin RA patients with normal controls,analyzing the relationship with the clinical data.Results1. Th17 and Treg Expression and Related Cytokines Level Comparison:Compared with normal controls(n=32),both the positive expression level of IL-17+CD3+CD8-T cells(Th17) and the ratio of Thl7/Treg in the peripheral blood of RA patients(n=57) increased significantly (4.09%±1.85% vs 2.32%±1.42%,P=0.000;1.14±0.61 vs 0.34±0.17,P=0.000), while the positive expression level of Foxp3+CD25+CD3+CD8-T cells (Treg) was markedly lower (3.92%±1.56% vs 7.04%±2.20%,P=0.000); The level of IL-6,IL-17 and IL-23 in the serum of RA patients were significantly elevated(P<0.05),but the level of TGF-βchanged insignificantly(P>0.05).2. Th17/Treg and Related Cytokines Relevance:Th17 expression percentage and Th17/Treg ratio were positive correlated with serum IL-17,IL-6 and IL-23 level in RA peripheral blood(P<0.05),but TGF-β(P>0.05);Treg expression percentage hasn't obvious correlated with serum IL-17,IL-6,IL-23 and TGF-βlevel (P>0.05);the level of IL-17,IL-6 and IL-23 in the serum of RA peripheral blood was positive correlated,but TGF-β. 3. Joint Fluid and Serum Related Cytokines Comparison:The level of IL-17 in the knee-joint synovia of RA patients(n=8) increased compared with that in their serum, but IL-6,IL-23 and TGF-β.4. Th17/Treg and Disease Progression:Compared with early RA patients (disease progressio≤2years,n=11),the level of Th17,Treg and the ratio of Thl7/Treg in chronic RA patients (disease progressio>2 years,n=46) didn't markedly changed(P>0.05), the level of IL-6 and IL-23 in the serum increased(P<0.05), but TGF-βand IL-17(P>0.05).5. Th17/Treg and Disease Activity: Compared with low active stage(disease activity score in 28 joints(DAS28)≤5.1,n=15) ,the level of Th17 and the ratio of Thl7/Treg in RA patients at high active stage(DAS28>5.1,n=42) increased significantly(P<0.05),but the level of Treg and IL-17/IL-6/TGF-β/IL-23 didn't markedly changed(P>0.05);The level of Th17 and the ratio of Thl7/Treg was positive correlated significantly with disease activity indexes (include number of tender joints of 28 counted (TJC),pain visual analog scale(VAS) of patients,DAS28,etc)(P<0.05), the level of IL-6 was positive correlated significantly with pain VAS of patients(P<0.05),the level of TGF-βwas negative correlated significantly with erythrocyte sedimentation rate(ESR) (P<0.05),but Treg, IL-17 and IL-23(P>0.05).6. Th17/Treg and Bone Destruction: Compared with RA patients in the inseverely osteoclasia stage (hands X-ray stageⅠandⅡ,n=34),the level of Th17 cells ,the ratio of Thl7/Treg and the level of IL-23 of RA patients in the severely osteoclasia stage (hands X-ray stageⅢandⅣ,n=23) increased significantly(P<0.05),but Treg,IL-17,IL-6 and TGF-β(P>0.05); factors of logistic regression analysis discovers some risk factors of bone erosion,such as the level of IL-23 and disease duration(P<0.05). 7. Drug Therapy for Th17/Treg Influence:Analysis of variance shows that,compared uncured RA patients (incipience,use glucocorticoid(GC) and non-steroidal anti-inflammatory drugs(NSAIDs) less than one month and use disease modifying anti-rheumatic drugs(DMARDs) less than six month,n=33) with NC group(n=32), the level of Th17,IL-17,IL-6,IL-23 and the ratio of Thl7/Treg increased significantly, the level of Treg and TGF-βdecreased significantly(p<0.05); Compared unnormal cured RA patients (use GC and NSAIDs more than one month and use DMARDs less than six month.n=13) and normal cured RA patients (use DMARDs more than six month,n=11) with uncured RA patients,the level of Th17 and the ratio of Thl7/Treg decreased significantly(P<0.05),but Treg(P>0.05); Compared normal cured RA patients with unnormal cured RA patients, the ratio of Thl7/Treg decreased significantly(P<0.05),but the level of Th17 and Treg(P>0.05); comparison of incured,unnormal cured and normal cured RA patients,the level of IL-17,IL-6,IL-23 and TGF-βdidn't markedly changed(P>0.05). Conclusion1. Th17 cells increased significantly,Treg cells decreased,and Th17/Treg percentage increased significantly in RA patients; Compared early period RA with the later period RA, Th17 and Treg hasn't changed;the imbalance of T lymphocyte subsets Th17/Treg may be the important reasons of onset of RA, and with the Th17 dominant.2. The level of Th17 related cytokines IL-17,IL-6 and IL-23 also increased significantly, and the three cytokines strong positive correlated;but the level of Treg related cytokines TGF-βhasn't changed; Compared early period RA,the level of serum IL-6 and IL-23 in the later period RA increased significantly;the level of IL-17 in joint fluid increased significantly than that in the serum of RA patients; IL-6 and IL-23 promoted disease progress, IL-17 gathered to joint cavity, participate in RA pathological process3. The level of Th17 and the ratio of Thl7/Treg increased significantly in RA patients at active stage,and positive correlated with disease activity indexes,such as TJC, pain VAS of patients,DAS28,etc; the level of serum IL-6 positive correlated with pain VAS of patient, the level of serum TGF-βnegative correlated with ESR;the level of Th17,IL-17 and the ratio of Thl7/Treg increased significantly in RA patients at inseverely osteoclasia stage;high level of Th17 and Th17/Treg maybe through the whole disease process, induce disease activity and osteoclasia.4. Through anti-rheumatic drugs can reduce Th17 cells and Th17 / Treg ratio,adjust Th17/Treg lymphocyte subsets balance.