The Role Of Tfh Cells In The Pathogenesis Of Primary Sjogren’s Syndrome

Objective:Follicular helper T cells (Tfh cells) were recently founded one kind ofCD4+T helper lymphocyte subsets, whose occurrence, differentiation andfunction were independent of Th1, Th2, Th17, Treg cells. The majorphenotype of the Tfh cells whose main function is to help B cells to produceantibodies is CXCR5, the Tfh cells’ chemokine receptor. The Tfh cells thatexpress CXCR5can migrate in response to CXCL13(human B lymphocytechemotactic factor1, BLC-1, which is generated by the follicular dendriticcells) and relocate to follicles, promote B cell proliferation, differentiation,immunoglobulin class switching, so involved in the immune response. Thisco-localization of CD4T cells with B cells is critical for T-B interactions.Besides, both BCL-6, the master regulator of Tfh differentiation and IL-21,the main effector of Tfh cells also play important role in the differentiation ofB cells. Sjogren’s syndrome is generally considered as the B lymphocyteproliferative diseases. Pathogenic role of Tfh cells in Sjogren’s syndrome isoccasionally reported abroad, but there is no domestic research in this area.The purpose of this project is to detect CXCR5, IL-21levels in the labialgland of the patients with Sjogren’s syndrome using immunohistochemistry, todetect apoptosis in the labial gland of the patients with Sjogren’s syndromeusing the TUNEL, to explore the expression, localization and biologicalfunctions of Tfh cells in the labial gland of Sjogren’s syndrome patients.Materials and Methods:We chose15cases of Sjogren’s syndrome patients as disease group in therheumatism of the Second Hospital of Hebei Medical University duringMarch2011to December2012, including4cases with pulmonary fibrosis,2cases with renal tubular acidosis,2cases with blood system damage (immune thrombocytopenic decrease),7cases with no organ system damage. Beforelabial salivary gland biopsy, all the patients were untreated. We chose11casespatients with mucous cyst, lower lip trauma or other non-autoimmune diseasein oral surgery as a control group. All the biopsies were placed into4%Paraformaldehyde for24hours. Standard paraffin preparations were prepared,sectioned at a thickness of5μm, and stained with hematoxylin and eosin.Detect the expression of CXCR5, IL-21by Microscope usingImmunohistochemical technology. Observe the differences of CXCR5, IL-21between the two groups on the expression, positioning, and its relationshipwith the infiltration of lymphocytes, plasma cells, GC formation. Detect theapoptosis situation of labial gland using TUNEL assay, count the number ofpositive cells under the microscope. In tissue sections, the pale yellow tobrownish yellow nucleus of labial gland acinar and ductal epithelial cell is thepositive signs. Five rating scale were divided according the mean number ofpositive cells by counting five high-power field:0score, Positive cells <1%;1score, Positive cells1%-25%;2score, Positive cells26%-50%;3score,Positive cells51%-75%;4score, Positive cells＞75%. Positive signal isdivided into three categories according to the degree of staining: weak stainingintensity (+): light yellow or individual cells are yellow to brownish-yellow,denoted1; strong staining intensity (+++): yellow to brown staining, denoted3; Moderate-intensity staining: between weakly positive and strongly positive,denoted2. Last comprehensive score equal to the score that staining intensityscore×the percentage score of positive cells. Comprehensive score≥1ispositive expression,<1compared with negative. Statistical analyses wereperformed by using SPSS13.0（SPSS Company, Chicago，Illinois，USA）The data were expressed as individual values withx s. Differences in themean CXCR5/IL-21/apoptosis levels of various groups were analyzed byusing analysis of variance. Differences between the two groups were analyzedby using T test (two-tailed). Pearson correlation coefficient was used to assessthe correlation between the levels of CXCR5/IL-21and laboratory data (whilethe ranked data used spearman’s correlation coefficient). Values of P <0.05 were considered significant.Results:1Under microscope, we can see that the control group had acinar withunform size, conduit arranged in neat rows, no expansion, there was no or asmall amount of inflammatory cell infiltration in stromal; in disease group,acinar had different sizes, glandular atrophy partly, especially around thecatheter, there had one or more focal lymphocytic infiltration in stromal.2The differences of CXCR5expression level between disease andcontrol groups were statistically significant (P=0.000), and we could detectthe expression of CXCR5in duct cells and lymphocytes around the duct,acinar cells were not detected positive cells, the expression of CXCR5wasrelated to the pathological grade (P=0.000, r=0.816), prompted that theCXCR5expression was related to the number of lymphocyte focus.3The differences of IL-21expression level between disease and controlgroups were statistically significant (P=0.008), and we could detect theexpression of IL-21in duct cells and lymphocytes around the duct, acinar cellswere not detected positive cells, the expression of IL-21was related to thepathological grade (P=0.001, r=0.628), prompted that the IL-21expressionwas related to the number of lymphocyte focus.4The differences of apoptosis score by TUNEL between disease andcontrol groups were statistically significant (P=0.000), there are a largenumber of apoptotic cells both among acinar cells and ductal cells in the tissuesections of disease group.5According to different organ damage, the disease group was dividedinto4groups: blood system damage, interstitial lung fibrosis, renal tubularacidosis, and no organ damage. The CXCR5expression of them were(5.00±1.41),(3.50±1.00),(5.00±1.41) and (4.29±1.80) points, respectively, thedifference among the4groups was not significant, P=0.663; The IL-21expression of them were (6.00±0.00),(2.5±1.91),(6.00±4.24) and (2.86±2.19)points, respectively. The difference among the4groups was not significant,too, P=0.311. 6The correlation analysis showed that the CXCR5expression level wascorrelated with the IL-21expression level, with a correlation coefficient0.666,P=0.000; the expression of IL-21was correlated with the last comprehensivescore of apoptosis detected by TUNEL, with a correlation coefficient0.599,P=0.001; the CXCR5expression was correlated with the last comprehensivescore of apoptosis detected by TUNEL, with a correlation coefficient0.513,P=0.007. Respectively inspect the correlation of IgG, IgA, IgM level withCXCR5expression level, results were (P=0.389, P=0.626, P=0.359) had nosignificance; Respectively inspect the correlation of IgG, IgA, IgM level withIL-21expression level, results were (P=0.788、P=0.377、P=0.250), had nosignificance, too.Conclusions:1No matter the expression of CXCR5, IL-21, or the comprehensivescore of apoptotic cells in the labial glands of disease group was higher thancontrol group, tip: Tfh cells may be involved in the pathogenesis of primarySjogren’s syndrome.2Both the expression of CXCR5and IL-21were positively correlatedwith the number of apoptotic cells in disease group, this result pointed at thatIL-21,CXCR5may promote apoptosis, which prompted that Tfh cells may beassociated with apoptosis.