The Role And Mechanism Of CCL21/CCR7 On T Lymphocyte Migration And Infiltration In Primary Sj?gren's Syndrome

Objective:Primary Sj?gren's syndrome(pSS)is a chronic autoimmune disorder characterized by xerophthalmia and xerostomia with the autoantibodies production and hypergammaglobulinemia.The principal lesion in pSS is lymphocyte infiltration in exocrine glands.Up to now,the pathogenesis of pSS remains unclear,but worldwide investigators have reached a consensus that lymphocyte migration from blood circulatory system to tissue space plays an important role on it.In response to a series of chemokines,lymphocytes complete the trafficking process across endothelium and interstitium to target microenvironment via a multistep navigation mode.It's a complicated process that many sophisticated corpuscular interaction and lots of cytokines are involved.In recent years,the significance of chemokines and chemoreceptors in autoimmune disease has been recognized by degrees.CCL21 is also called secondary lymphoid tissue chemokine(SLC).It is a member of CC subfamily of chemokines.After combining with CCR7,it can assist many kinds of immunocytes including DC,T,B lymphocyte and NK cell to migrate selectively to target tissues and initiate the autoimmune reactions.At present,there is little investigation on CCL21/CCR7 in SS.For the purpose of exploring the role and the clinical significance of CCL21/CCR7 in the patients with SS,we detected the expression of CCR7 on the membrane of peripheral blood lymphocyes and the lymphocyte chemotaxis towards CCL21/CCR7.In addition,CCL21 can't educe its biological functions unless it combines with CCR7and subsequently activates the specified signal transduction pathways.JNK and p38MAPK acting as the important members of MAPK family,both can be activated by inflammatory factors and some G protein coupled receptors.They play important roles in many kinds of physiological and pathological processes including inflammation,stress,apoptosis,cell cycle,cell growth and cell movement.Nevertheless,there is no conclusion on which signal pathways in SS lymphocytes can be activated by CCL21/CCR7 complex and whether JNK and p38MAPK pathways mediate the lymphocyte chemotaxis towards CCL21/CCR7.Therefore,our study focusing on it tried to reveal the mechanism of exocrine glands damage in SS due to the lymphocyte migration and infiltration mediated by CCL21/CCR7.It will propose a new target for the treatment of SS.Methods:1.Detecting CCR7 level on lymphocytes by flow cytometry.We selected 32 patients with pSS,all of which met 2012 ACR classification for Sj?gren's syndrome.20 healthy dornors and 23 SLE patients were included as normal controls and disease controls,respectively.For the detection of CCR7 on the lymphocyte surface,venous blood from all subjects in the condition of empty stomach were incubated with FITC-labelled mouse antihuman CD4 mAb,PE-Cy5-labelled mouse antihuman CD8 mAb and PE-labelled mouse antihuman CCR7 mAb.The analysis was performed with flow cytometry.Meanwhile,the results of ESR,serum IgG,CRP and other clinical manifesttion in SS patients were noted.Meanwhile,ESSDAI and SSDDI were calculated and the relativities of them with CCR7 were analyzed.2.Chemotaxis assayPeripheral blood CD4~+T lymphocytes were isolated from all the patients with SS and normal controls.The cell trans-membrane test with 24-wells Transwell were conducted to assay the effect of CCL21/CCR7 and JNK,p38 MAPK pathways on the lymphocyte chemotaxis.3.Western blotThe protein levels of p-JNK and p-p38 MAPK in lymphocytes from different groups were detected by Western blot.4.Statistics analysisThe data was expressed with Mean±SD and manipulated with software Prism(version5.0;GraphPad Software,San Diego,CA).The diversity among multiple groups was compared by Kruskal-Wallis ANOVA with Dunn's post-test for multiple comparisons.The diversity between two groups was compared by Mann-Whitney U test,independent-sample t test or pair-sample t test.The correlation was analyzed by Pearson or Spearman correlation analysis.Results:1.The levels of CCR7 on lymphocytesThe levels of CCR7 on CD4+T and CD8+T cells from pSS patients were higher than that from normal controls(P<0.01).CD4+T cells from pSS expressed CCR7 more than CD8+T(P<0.01).However,in normal dornors,there was no difference on the levels of CCR7 expressed by the two kinds of cells(P>0.05).Furthermore,CCR7 was selectively and frequently expressed on CD8~+T cells in SLE patients(P<0.01).2.The correlation between CCR7 level and clinical indexesFrequency of CCR7~+CD4~+T cells in pSS patients with lymphadenopathy,splenomegaly,glandular swelling,or pulmonary involvement were significantly higher than those without these manifestations(p<0.05).Furthermore,CCR7 levels on CD4~+T cells from pSS patients were significantly correlated with ESR(r=0.183,p=0.015)and IgG(r=0.244,p=0.004),whereas CRP had no significant correlation with CCR7 levels(r=0.091,p=0.094).In pSS patients,ESSDAI was closely related to CCR7 level on CD4~+T cells(r=0.285,p=0.002),whereas SSDDI didn't correlated with it(r=0.102,p=0.075).3.The effect of CCL21 on lymphocyte chemotaxisCCL21 did not induce significant chemotactic migration of CD4~+and CD8~+T cells from normal subjects.However,CCL21 induced significant chemotactic migration of CD4~+and CD8~+T cells from patients with pSS.Furthermore,the CI of CD4~+T cells induced by CCL21was higher than that of CD8+T cells in pSS group.Moreover,The anti-CCR7mAb which blocked the pSS CD4~+T-cell chemotactic activity towards CCL21,could completely block the chemotaxis of the cells towards CCL21.The chemotaxis assays by Transwell confirmed that enhanced motility of CD4~+T cells from patients with pSS towards the chemokine CCL21 was the result of chemotaxis,but not chemokinesis.4.There was a close relationship between the CI of CD4~+T cells and the level of CCR7on them(r=0.550,p<0.001).In addition,the CI of CD4~+T cells from the patients with pSS was found to be related to the ESSDAI(r=0.172,p=0.018),but not to the SSDDI(r=0.011,p=0.564).The data implied that the elevated expression of CCR7 on CD4~+T cells enhanced the chemotactic migration of such cells to the inflammatory site,thus promoting the disease development of pSS.5.The expressions of p-JNK and p-p38MAPK in lymphocytesThe expressions of p-JNK and p-p38MAPK in the CD4~+T lymphocytes from pSS patients were higher than that from normal controls(P<0.05).The lymphocytes in both normal controls and pSS patients expressed more p-JNK and p-p38MAPK at the presence of CCL21 than in the absence of it(P<0.05).After the treatment of CCR7 mAb,the expressions of p-JNK and p-p38MAPK in pSS patients decreased significantly(P<0.01).The results indicated that CCR7 played an important role on the activation of JNK and p38MAPK signaliing pathways induced by CCL21.6.The influence of JNK and p38MAPK on lymphocyte chemotaxisSP600125 and SB203580,as the specific blockers of JNK and p38MAPK pathways,could partially inhibit the lymphocyte migration in pSS.Furthermore,the CI of pSS p38MAPK-blocked group was lower than that of pSS JNK-blocked group(P<0.05).The antagonist against p38MAPK showed more effect on CD4~+T cells migration.When CD4~+T cells were treated by the two blockers at the same time,the CI decresed more sibnificantly,but they couldn't inhibit the migration of CD4~+T cells completely.It indicated that there were other transductive pathways regulating the cells trafficking mediated by CCL21/CCR7.Conclusion:1.The expression of CCR7 on pSS T lymphocytes increased prominently,especially on pSS CD4+T cells.This indicated that CCR7 maybe participate the pathogenesis and exocrine glands damage in SS.2.The levels of CCR7 on CD4+T from pSS were positively correlated with ESR,serum IgG and ESSDAI.This showed that CCR7 might be used as an index to evaluate the pSS activity and direct the clinical treatment.3.The CD4~+T lymphocyte chemotaxis mediated by CCL21 in pSS was higher than that in normal controls.This was related to the high expression of CCR7 on CD4~+T lymphocyte in pSS.After treatment with CCR7 mAb,the CI of CD~+T cells decreased significantly,which proved that CCR7 was one of the important factors mediating the cells migration.CCL21/CCR7 interaction induced the cell trafficking in pSS patients and played an important role in the glands damage process due to the infiltration of massive lymphocytes.CCL21/CCR7 might become to a new target for pSS theropy.4.As the exocellular signal molecules,CCL21/CCR7 can activate JNK and p38MAPK pathways.If the two pathways were blocked,the migrating lymphocytes would decrease obviously.This indicated that JNK and p38MAPK pathways act important roles in the signal transduction of lymphocyte chemotaxis mediated by CCL21/CCR7.It developed a new idea to control the disease progress of pSS.