The Expression And Significance Of ICOS And Bcl-6 Protein In The Peripheral Blood And Labial Gland Tissue Of Patients With Primary Sjogren’s Syndrome

Sjogren’s syndrome(SS) is one of the diffuse connective tissue diseases which is characterized by dry mouth and dry eyes due to the decreasing level of tear and saliva, even multiple organs dysfunction is involved. The pathological feature of SS is high infiltration of lymphocytes. SS is divided into two types:primary SS and secondary SS. The basic pathogenesis of Primary sjogren’s syndrome(PSS) is immune dysfunction, in which exocrine gland duct epithelial cells may play a key role in antigen presenting. T and B cells proliferate after cell recognition then B cells differentiate into plasma cells, producing a large number of immune globulins and autoantibodies, which lead to excessive abnormal humoral immune responses, causing inflammation and tissue damage.In this process there is a certain type of CD4 + T cell subsets, which are called follicular helper T cell(Tfh cell) and could facilitate B cells in immune response. CXCR5 chemokine receptors are the main surface markers. They highly express specific ICOS, Bcl- 6, CD40, PD-1 and IL-21, exerting its biological function and playing an indispensable role.Objective: Currently, the pathogenic role of Tfh cells in SS is still not very comprehensive in domestic and international studies. In the study, we detect the percentage of CD4+CXCR5+T cells in peripheral blood of SS patients by flow cytometry(FCM), CXCR5 m RNA and ICOS m RNA in peripheral blood by real-time PCR(RT-PCR), and ICOS and Bcl-6 protein expression in labial gland tissue by immunohistochemical method(IHC) and try to explore the quantitative and functional change of Tfh cells in the pathogenesis of sjogren’s syndrome.Method: 1 Thirty PSS patients from the Department of Immunology and Rheumatology of the Second Hospital of Hebei Medical University were chose as PSS group from January 2014 to January 2015, among which 2 cases were male and 28 cases were female with the average age of(50.47±9.96) years(ranged from 40 to 65 years). 20 subjects had no systemic damage, 2 patients were complicated with pulmonary fibrosis, 1 case with pulmonary hypertension, 5 cases with immune thrombocytopenia and 2 patients with renal tubular acidosis. All the patients did not receive any medication including hormone or immune inhibitors before enrolled in the trial. 1.1 Flow Cytometry(FCM): Thirty PSS patients were used as PSS group and 20 cases of healthy volunteers as healthy control. 1 ml peripheral venous blood with heparin anticoagulation was drawn from each subject with an empty stomach, and the percentage of CD4+CXCR5+T cells of two groups was detected by flow cytometry. Difference between the two groups and the correlation of clinical indicators. 1.2 RT-PCR: Thirty PSS patients were used as PSS group and 20 cases of healthy volunteers as healthy control. 5 ml peripheral venous blood with EDTA anticoagulation was drawn from each subject with an empty stomach, and the CXCR5 m RNA, ICOS m RNA expression quantity changes of two groups was detected by RT-PCR.Difference between the two groups and the correlation of clinical indicators. Samples through the primer design, total RNA synthesis c DNA, reverse transcription, RT-PCR reaction system, the establishment of real-time amplification curves for each sample and the sample amplification product dissolve curve, relative quantitative analysis results calculated for each sample. 1.3 Immunohistochemistry(IHC):Samples of labial gland from 30 PSS patients were treated with 4% paraformaldehyde for fixation, paraffin embedding, HE staining and immunohistochemical staining. Samples from 20 cases of patients with non-autoimmune diseases such as trauma, submucosal cyst and etc were collected as control. ICOS and Bcl-6 protein expression was detected and difference between the two groups and correlations with lymphocyte infiltration as well as formation of the germinal center were analyzed. 2 clinical indicators:Data of immunoglobulins(Ig A, Ig G, Ig M), blood sedimentation(ESR), anti SSA/SSB antibody were recorded and analyzed for the correlation with other indices. 3 Statistical analysis: All the data were analyzed using statistical software SPSS 20.0. Measurement data is represented by x ±s. Comparison between two samples was performed by t-test. Correlations were analyzed by Spearman’s correlation analysis. Comparisons among three groups were performed by analysis of variance. The P values less than 0.05 were considered statistically significant. All the tests were fitted for two-tailed test and test level a is 0.05.Results:1 FCM: The percentage of CD4+CXCR5+T cells in PBMC from patients with PSS was significantly higher than that of healthy control group(16.84±6.38% vs 12.85±2.38%, P=0.001). There were no correlations between percentage of CD4+CXCR5+T cells in PBMC from patients with PSS and ESR, immunoglobulin Ig G, Ig A and Ig M(r=-0.096, P=0.614),(r=-0.280, P=0.133),(r=-0.276, P=0.139),(r=-0.287, P=0.123).There were no statistical differences between patients with positive SSA or/and SSB(Z=-0.915, P=0.360),(Z=-1.011, P=0.312).2 RT-PCR : Peripheral blood CXCR5 m RNA and ICOS m RNA expression in PSS group was significantly higher than healthy control(1.37±0.69 vs 0.57±0.12, P=0.000. 1.97±0.73 vs 1.54±0.58, P=0.036). There were no correlations between CXCR5 m RNA expression level and ESR, immunoglobulin Ig G, Ig A and Ig M(r=-0.065, P=0.733),(r=-0.051,P=0.788),(r=-0.177, P=0.350),(r=-0.259, P=0.167). There were no differences between patients with positive SSA/ SSB antibody and negative group(Z=0.000, P=1.000),(Z=-1.571, P=0.116).There were no correlations between ICOS m RNA expression level and ESR, immunoglobulin Ig G, Ig A and Ig M(r=0.024,P=0.898),(r=-0.166,P=0.382),(r=-0.266,P=0.156),(r=-0.394,P=0.031). There were no differences between patients with positive SSA/ SSB antibody and negative group(Z=-0.665,P=0.506),(Z=-1.442,P=0.149).3 IHC: The morphological characteristics of the labial gland in HE staining between PSS patients and healthy control are as follows: gland bubbles with uniform sizes and distribution, ducts with regular line and few or a small amount of inflammatory cells infiltration in interstitial tissue could be seen in control group, while gland bubbles with different sizes and distribution, atrophy gland especially around the ducts, and scattered or more than one focal lymphocytic infiltration were the typical pathological features of PSS groups in microscope. In immunohistochemical staining, ICOS protein expression level scores were 3.5(2, 6) and 1(0, 1) respectively in PSS and control group with statistical significance(Z=-5.54,P=0.000).ICOS protein expression was detected in ducts and peripheral lymphocytes as well, and occasionally in acinar cells of PSS group.ICOS protein expression significantly positively associated with the degree of peripheral lymphocyte infiltration(P=0.020), suggesting ICOS proteins associated with lymphocytic infiltration degree.Bcl-6 protein expression level scores were 6(2, 6) and 0(0, 1) respectively in PSS and control group with statistical significance(Z=-5.70,P=0.000).PSS group of duct cells in a tissue section and its peripheral lymphocytes were detected the Bcl-6 protein expression of acinar cells accidentally positive cells can be detected.Through statistical analysis of the Bcl-6 protein expression and catheter was significantly positively associated with the degree of peripheral lymphocyte infiltration(P=0.001), illustrate the Bcl-6 proteins related to the degree of lymphocytic infiltrates.4 Correlation analysis: There were positive correlations between the percentage of CD4+CXCR5 +T cells in PBMC and peripheral blood CXCR5 m RNA and ICOS m RNA expression level in patients with PSS(r=0.635,P=0.000. r=0.708, P=0.000). There were no correlations between the percentage of CD4+CXCR5 +T cells in PBMC expression ICOS protein, Bcl-6 protein in labial gland tissues(r=0.031, P=0.871),(r=0.006,P=0.976). There were positive correlations between PSS group of peripheral blood CXCR5 m RNA and ICOS m RNA expression level(r=0.665, P=0.000).PSS group of peripheral blood ICOS m RNA expression level and ICOS protein expression level in labial gland organization has no correlation(r=-0.044,P=0.817).PSS group of labial gland tissues ICOS protein and the Bcl-6 protein expression level was positively correlation(r=0.594,P=0.001).Conclusions:1 The percentage of CD4+CXCR5+T cells in PBMC from patients with PSS is significantly higher, suggesting Tfh cells probably favor the occurrence and development of PSS.2 Compared with the health control, there is positive correlation between peripheral blood CXCR5 and ICOS gene expression, Tfh cells may be by express ICOS genes play A role of biology3 Both the expression of ICOS and Bcl-6 were positively correlated with the number of lymphocyte infiltration in labial salivary gland of PSS patients, suggesting Tfh cells may play a key role in salivary gland tissue injury in PSS patients. But further study on the correlation with the peripheral blood Tfh cells is needed.