The Study On The Correlation Of IL-17 And Chemokine Receptor CCR4 And CCR6 With Rheumatoid Arthritis

IntroductionRheumatoid arthritis(RA) is a chronic inflammatory autoimmune disease characterized by joint involvement.It be known that RA is caused by many factors,T cells play a key role in the pathogenesis and progress of RA,recently we found that RA was closely related to immune dysfunction,and a variety of complex factors involved in the joint damage and the process of systemic disorder of patients with RA. Inflammatory cytokines such as tumor necrosis factorα(TNF-α) and interleukin-1(IL-1) are involved in the pathogenesis of RA,and the beneficial effects of antagonists to these cytokines have been reported,which support the view that cytokine plays a role in the development of RA.Recently,IL-17 is secreted by the novel T helper subset,which is different from Th1 and Th2 cells,and can produces interleukin(IL)-17,IL-6 and TNF-α,but not IFN-γ,and IL-4,this cell is called Thl7 cell.While IL-17 is expressed by T cells,interleukin -17 reptor(IL-17R) is expressed in virtually subtotal cells and tissues.Functionally,IL-17 has been classified as a proinflammatory mediator responsible for inducing inflammatory effectors in target cells.IL-17 plays a key role in the regulation of immune and inflammatory response,in the homeostasis of several tissues,and the progression of autoimmune diseases.IL-17 may be an important activator of T cell-driven inflammation,as IL-17 shares properties with TNF-αand IL-1 cytokines,may thus contribute to the pathogenesis of RA.What role of the IL-17 play in the pathogenesis of RA is still not very clear currently.Latest research shows that there is Memory/effect Th17 cell in the subgroups of the CCR6⁺ in normal human peripheral blood and inflammation tissue of patients.CCR6⁺ CCR4⁺ T cells can produce a large number of IL-17 specifically,but not IFN-γ,at the same time,these cells also express transcriptional factor,which plays a critical role in the development of the Th17 is the orphan unclear receptor RORrt.At present,it has to be studied further that wether CCR4 and CCR6 can be characteristic signs of Th17 cells.This experiment will study the level difference of IFN-γ,IL-10,IL-17 of Chinese patients with active R_A patients,no active RA patients,systemic lupus erythematosus(SLE) patients and healthy human serum and also study the expression differences of CCR4,CCR6 on surface of blood CD4⁺ T cells.In order to clear the relevance of IL-17,CCR4,CCR6 and RA disease as well as the IL-17 and CCR4,CCR6.Discuss the role of Th17 cells in the pathogenesis of RA,in search of cell factor target to provide a scientific basis for clinical treatment of RA.Material1.ObjectSamples of peripheral blood from 65 patients diagnosed as RA(20 men and 45 women,age 18-60 years,mean 41±13 years) based on the 1987 criteria of the American College of Rheumatology,precluding severity cerebral vessels,liver,kidney disease and other autoimmune disorder,35 active R.A,30 no active RA,according to standards of judgement of active stage,satisfied 3 or more than 3 of 5 index,contain:â‘ 3 or morethan 3 arthrocele;â‘¡morning stiffness＞45min/per day;â‘¢ESR≧ 28 mm/h;â‘£3 or more than 3 tendemess of joints;⑤average power of grip with both hands,male≦192mmHg,female≦146mmHg;35 patients diagnosed as SLE(10 men and 25 women,age 18-60 years,mean 39±13 years) based on the 1997 criteria of the American College of Rheumatology,precluding severity cerebral vessels,liver,kidney disease and other autoimmune disorder;50 healthy controls(17 men and 33 women, age 18-60 years,mean 38±12 years) who are serum determination of liver enzyme and renal function is normal,hepatitis surface antigen and antibody to hepatitis C is negative,no disease of immune system,were included in the study.SLE patients and healthy controls were matched for age and gender with RA patients. 2.Equipment and reagentsMost of the equipment and reagents were obtained from BECTON DICKINSON company,including flowcytometer(FACS-Aria);10ml EDTA venipuncture vials;the mAbs:anti-CD3(PerCP conjugated),anti-CD4(FITC conjugated),anti-CCR6(PE conjugated),anti-CCR4(PE-Cy7 conjugated).Human IFN-γ,IL-10,IL-17 ELISA kit were obtained from R&D company.And other reagents were prepared with oneself: erythrocytolysin(KHCO₃:1.0g,NH₄CL:8.25g,EDTA·Na₂::0.037g,Water:1000ml). Methods1.Detection of IFN-γ,IL-10,IL-17 levels in serumSerum IFN-γ,IL-10,IL-17 levels were determined by enzyme linked immunosordent assay(ELISA)(R&D company).Read absorbance of each well on a spectrophotometr using 450nm.Draw standard curve according to the standard preparation of kit.Calculation IFN-γ,IL-10,IL-17 levels.2.Detection of CCR4 and CCR6 in whole blood surfaceFor each RA patient,SLE patient and healthy control,we used the following Ab combinations:PerCP,FITC,PE,PE-Cy7.100ul whole blood was added and the 6ul of each Abs were added.After a 30-min incubation at room temperature,the erythrocytes were lysed using 3ml RBC lysing solution.The cells were washed twice with PBS and resuspended in 0.5ml PBS solution consist of 1%polyformaldehyde.There color flowcytometric analysis was performed and FACS CELLQUEST software was used to make analysis.3.Date and statistical analysisDetermine the group of lymphocytes by FSC and SSC.Using CD3⁺T lymphocytes and CD4⁺T lymphocytes as gates,analysis expressions of CCR4 and CCR6.SPSS13.0 software was used to make the statistical analysis.Comparisons of means of different groups were done using one-way ANOVA.Spearman rank correlations were used as a measure of correlation between IL-17 and CCR4,CCR6.P＜0.05 has statistical significance.Result1.Expression of IFN-γ,IL-10 and IL-17 levels in serum of RA patients,SLE patients and healthy controls(1) Expression of IFN-γlevels in serumThe serum levels of IFN-γwas significantly increased in patients with active RA and no active RA patients compared with healthy controls(P＜0.05);the serum levels of IFN-γwas significantly higher in patients with SLE than healthy controls(P＜0.05); there was no significant difference between active RA,no active RA patients and SLE patients(P＞0.05);there was also no difference between active RA and no active RA patients(P＞0.05).(2) Expression of IL-10 levels in serumThe levels of IL-10 was no significant difference between active RA,no active RA patients and healthy controls(P＞0.05);The serum levels of IL-10 was significantly higher in patients with SLE than healthy controls(P＜0.05);there was significant difference between active RA,no active RA patients and SLE patients(P＜0.05);there was also no difference between active RA and no active RA patients(P＞0.05).(3) Expression of IL-17 levels in serumThe serum levels of IL-17 was significantly increased in patients with active RA compared with healthy controls and SLE patients(P＜0.05);but there was no difference between no active RA patients and SLE patients,healthy controls(P＞0.05);the levels of IL-17 was no significant difference between SLE and healthy controls(P＞0.05); there was significant difference between active RA and no active RA patients(P＜0.05).2.The expression of CCR4 and CCR6 on peripheral blood(PB) CD4⁺T cells of RA,SLE and healthy controls(1) CCR4 expression on CD4⁺T cellsThe expression of CCR4 on CD4+T cells was significantly elevated in patients with active RA as compared with that of healthy controls(P＜0.01),but there was no differenttce between active RA patients and SLE patients(P＞0.05);the expression of CCR4 was significantly higher in patients with SLE than healthy controls(P＜0.05); there was no significant difference between no active RA and SLE patients,healthy controls(P＞0.05);there was difference between active RA and no active RA patients(P＜0.05).(2) CCR6 expression on CD4⁺T cellsThe expression of CCR6 on CD4⁺T cells was significantly elevated in patients with active RA patients as compared with that of SLE and healthy controls(P＜0.01); there was difference between no active RA patients and healthy controls(P＜0.05),but no difference between no active RA and SLE patients(P＞0.05);there was no significant difference between SLE and healthy controls(P＞0.05);there was difference between active RA and no active RA patients(P＜0.05).(3) CCR4⁺CCR6⁺T expression on CD4⁺T cellsThe expression of CCR4⁺CCR6⁺T on CD4⁺T cells was significantly elevated in patients with active RA patients as compared with that of SLE and healthy controls (P＜0.05);there was no difference between no active RA patients and SLE patients, healthy controls(P＞0.05);there was no significant difference between SLE and healthy controls(P＞0.05);there was difference between active RA and no active RA patients (P＜0.05).3.Correlations between IL-17 levels and CCR4 and CCR6 expression of RA patientsElevated serum IL-17 level have no correlation with the expression of CCR4 (P＞0.05),but was closely correlated with the expression of CCR6 and the level of CD4⁺CCR4⁺CCR6⁺T in active and no active RA patients(P＜0.05).Conclusion1.The serum levels of IFN-γwas significantly increased in patients with active RA and no active RA patients,but the levels of IL-10 was no significant change.It showed that it's key factor of Th1 cells enhance the response and may inhibit the establishment of a Th2 response,then disturb the balance between Th1/Th2,which lead to the occurrence of adult RA.2.The serum levels of IL-17 was significantly increased in patients with active RA compared with healthy controls and SLE patients.That indicated Th17 cells may be have correlation with the development of RA.3.The expression of CCR4 and CCR6 on CD4⁺T cells was significantly elevated in patients with active RA,There may be Close contact of the occurrence of RA,which is Th17 cells mediated...