| Background and Objectives:Rheumatoid arthritis(RA)is an autoimmune disease characterized by chronic invasive synovial inflammation and bone destruction.Nowadays,patients with RA account for 0.5%-1%of the world's population,and RA prevalence in China is about 0.32%-0.36%.Generally,in the natural course of RA,the disability rate is up to 15%.Thus,RA has became a serious threat to human life and health,and the early diagnosis and treatment for RA is of most significance.So far,the etiology of RA has not been revealed,which may be related to many factors,such as genetic factors,infection,environmental factors,and immune status.The immune injury mediated by the autoimmune response is observed throughout the entire course of RA.Previous studies have also shown that in RA patients,there are high expression of autoantibodies(including rheumatoid factor,anti-citrullinated peptide antibody,etc.)and a large number of T lymphocytes infiltration in the synovial tissues,indicating the abnormal activation of both B cell and T cell immunity.The number of naive T cells in the RA patients is very low,and most T cells are activated,with high expression of CD69,CD28,CTLA-4,CD40 and other co-stimulatory molecules.Studies have shown that these factors are involved in the progress of inflammatory pathology by regulation of the lymphocyte activation,adhesion molecule up-regulation and cytokine/chemokine secretion.And the absence of these co-stimulatory molecules can always lead to the immune tolerance and lymphocyte activation disorders.Thus,searching for the new lymphocyte activation markers provides not only insight into the pathogenesis of RA,but also new targets for RA disease prophylaxis and treatment.Transmembrane glycoprotein CD3 8 molecule is widely expressed on the cell surface of lymphocytes,and it is a surface marker for the lymphocyte activation.CD3 8 has enzyme activity and participates in the intracellular signal transduction,thus regulating the lymphocyte proliferation,apoptosis,differentiation and migration.In recent years,studies have found that the function loss of CD38 were closely related to the immune response changes and CD38 may be involved in the pathogenesis of a variety of autoimmune diseases.Studies have shown that in CD38 knockout mouse model with collagen-induced arthritis,there were lymphocyte migration disorders,less inflammatory cell infiltration in the synovial tissues,and reduced inflammation in the joints.Previously,we found that CD38 mRNA expression in the synovial tissue of RA patients was significantly higher than that of the normal subjectsCD56 is a marker of neuronal cell adhesion molecules and NK cells.Our previous study showed that CD38 and CD56 co-expressed in synovial tissue of RA.All these finds suggested that CD38 might play a role in RA pathogenesis.In this study,the expression,effect and mechanism of CD38 in RA patients were investigated.Our findings may provide further understanding the relationship between CD38 and RA.for the further exploration of the pathogenesis of RA.This may provid us a theoretical basis with new strategy for targeting CD38 molecules in the treatment of RA.PART ONEObjective:To observe the CD38 expression in different lymphocyte subsets of RA patients and the influencing factors of CD38 expression in the peripheral blood of RA patients.To understand the distribution and activation status of different lymphocyte subsets in the RA patients,and especially to study the changes of NK cells and its phenotype changes in the peripheral blood of RA patients.Methods:Totally,139 cases of RA patients were enrolled in this study,and 120 cases of healthy people with matched age and gender were used as the control group.The clinical data of patients,including gender,age,duration of RA,number of swollen joints(SJC),tender joint count(TJC),VAS score,rheumatoid factor(RF)and anti cyclic citrullinated peptide antibody(anti-CCP),erythrocyte sedimentation rate(ESR),C reactive protein(CRP),the disease activity index(DAS28-CRP),and the grading of bone erosion,were all collected.The proportion of NK cells(CD3-CD56+),T cells(CD3+),and B cells(CD 19+)and the proportion of CD3 8 positive NK,T,and B cells were measured by multicolor flow cytometry.After that,the factors that influence the proportion of NK cells in peripheral blood of RA were analyzed by the general linear model.The relationship of CD3 8-positive lymphocyte proportion and clinical index was analyzed by the Pearson and Spearman rank correlation analysis.The correlationof CD3 8-positive lymphocyte proportion with the disease activity parameters of CRP and DAS28-CRP were evaluated by the linear correlation analysis.Results:1.There was statistical difference in the proportions of CD3+T cells and CD3+CD4+ T helper cells between the RA patients and the healthy controls(P= 0.0065;P=0.0038)whereas there was no significant difference in the proportions of CD3+CD8+ Ts cells and B cells between the two groups(P=0.613;P=0.587).The proportion of NK cells(CD3-CD56+)in RA patients was significantly lower than that in healthy controls(P=0.033).Besides,the decrease of NK cells in the peripheral blood was prominent in RA patients with moderate and high activity.Contrast to healthy subjects,the proportions of CD38 positive all lymphocytes was dramaticly high inRA(P=1.91E-09).The proportions of CD38 positive cells in Th lymphocytes and NK cells were significantly higher than that in the healthy controls(P<0.05),while the ratios of CD38+ B lymphocytes in RA and healthy subjects were not significantly different between the two groups(P>0.05).2.There was no correlation between the proportion.of NK cells in the peripheral blood of RA patients and the clinical parameters,like age,duration of disease and the degree of joint bone erosion.However,the NK cell proportion was affected by the status of the disease,which was shown by the general linear model analysis.3.The frequency of CD38 positive lymphocyte in the peripheral blood of RA patients showed a positive linear relationship with the CRP and disease activity(DAS28-CRP)(R2=0.255,R2=0.288 respectively).However,it was no relationship between CD38 and the serum markers of RA(RF,anti-CCP),age,course of disease and the grading of bone erosion using Pearson and Spearman correlational analysis.Conclusions:1.In the peripheral blood of RA patients,the helper T cells and NK cells are highly activated,which are characterized by the high CD38 expression on the cell surface.2.In the peripheral blood of RA patients,the number of NK cells decreases compared with the healthy people,and the NK cell ratio is closely related to the disease process.3.CD38 expression in the lymphocytes is positively correlated with CRP and DAS28-CRP in the RA patients.PART TWOObjectives:To study the distribution of NK cells and the CD38 positive NK subpopulations in the peripheral blood,synovial fluid and synovial tissues in RA patients.Methods:Totally,36 cases of RA patients with single or bilateral knee joint effusion were enrolled,and 29 cases of healthy people were enrolled as control.The synovial tissues from RA patients were collected,and then the single cell suspension were prepared by enzymatic digestion.The lymphocytes were obtained by density gradient centrifugation.The levels of NK cells,CD38 positive subpopulation and NK cell surface chemokine receptors(CXCR3,CCR5),as well as granzyme B and perforin were detected by flow cytometry in peripheral blood and knee synovial fluid.Expression levels of CD38 and CD56 in tissues from 35 RA patients and 25 patients undergoing arthroscopy or joint replacement were detected with immuno-histochemical staining.Results:1.The proportion of NK cells in the peripheral blood of RA patients was significantly lower than that in the synovial fluid(P<0.05).The expression of CCR5 and CXCR3 in NK cells was significantly higher than that in normal healthy subjects(P<0.05).And the two chemokine receptors were mainly expressed by the CD3 8+ subgroups of NK cells.2.The expression of granzyme B and perforin in synovial NK cells of RA patients was higher than that in the peripheral NK cells and NK cells from the normal healthy subjects(P<0.05).3.The expression of CD38 in NK cells of peripheral blood and synovial fluid were both higher than that in normal healthy subjects(P<0.05).4.Immunohistochemical staining results showed that the CD38 expression was observed on the inflammatory cells and fibroblasts that infiltrated the synovial lining layer and the lower layer,and its staining intensity was moderate with a massive or homogeneous distribution.However,CD56 was mainly expressed surrounding vascular endothelial cells tissues.Conclusions:1.In the RA patients,there is abnormal infiltration of NK cells in the inflammatory sites(both synovial fluid and synovium).2.NK cells in the peripheral blood are of highly expressed CC-family chemokines,especially on the surface of CD38+ NK cells,while the synovial NK cells are of highly expressed granzyme B and perforin.These indicated that the synovial NK cells are more lethal than the peripheral NK cells in the RA patients.3.High expression of CD38 in NK cells both in peripheral blood and synovial fluid,indicated that CD38 maybe involved in the mechanism of chemotaxis and killing capability of NK cells.PART THREEObjectives:To investigate the effect and mechanism of CD38 agonist on the function of the lymphocytes both in peripheral blood and synovial fluid in RA patients.Methods:A total of 6 patients with synovial effusion of single and bilateral knee joints were selected.The peripheral blood were collected with heparin sodium anticoagulant.The synovial fluid of the knee joint was collected.The lymphocytes in the the peripheral blood and synovial fluid were separated by gradient density centrifugation.The isolated cells were then cultured in vitro and divided into 3 groups,namely blank control group,IB4 stimulation group(Anti-CD38 mAb),IL-2 and IB4 co-stimulation group.Then the expression of granzyme B and perforin in NK cells was measured by multicolor flow cytometry.And the concentration of TNF and IL-6 in culture supernatants was determined by ELISA.Liposome CD38-siRNA was transfected into peripheral blood lymphocytes of RA patients,and transfection efficiency was detected by RT-PCR.The effect of CD38 and CD38-siRNA silencing on the expression of phospholipase C(PLC-gamma 1)protein in the peripheral blood lymphocytes of RA was detected by Western blot.Results:1.The expression of granzyme B and perforin in NK cells slightly increased after the peripheral blood lymphocytes cells and synovial lymphocytes cells were stimulated by the CD38 agonist(IB4)in RA patients.But,they were not significantly different from those in the blank control group.However,the levels of granzyme B and perforin in NK cells were significantly increased after peripheral blood lymphocytes cells and synovial lymphocytes cells were stimulated by the combination of IB4 and IL-2,which were significantly different from those of blank control group and IB4 stimulation group(P<0.05).2.Compared with the blank control group,IB4 stimulation and IL-2 and IB4 co-stimulation significantly increased the secretion of IL-6 and TNF-a in supernatant of peripheral blood lymphocytes cells and synovial lymphocytes cells(P<0.05).However,there are no significant difference between IB4 stimulation group and combined stimulation group(P>0.05).3.The levels of granzyme B and perforin in synovial lymphocytes cells were significantly higher than those in peripheral blood lymphocytes,both in the blank control group and the stimulated groups(P<0.05).Nevertheless,the secretion of IL-6 and TNF-a in synovial and peripheral blood lymphocytes cells existed significant difference only in IL-2 and IB4 co-stimulation group(P>0.05).3.The levels of PLC-yl protein in the peripheral blood lymphocytes of RA patients was significantly decreased after the treatment with CD38-siRNA.Beside,after treated with IB4,the expression of PLC-yl protein in the peripheral blood lymphocytes of RA patients was significantly higher than that in the blank control group(P<0.05).There was statistical difference between the three groups(P<0.05).Conclusions:1.CD38 agonist could significantly promote the secretion of proinflammatory cytokines in the peripheral blood and synovial fluid of RA patients,thus enhancing the lethality of the NK cells.2.CD38 agonist could significantly promote the capablity of killing of the NK cells both in peripheral blood and synovial fluid.With the addition of IL-2,this enhancement is markedly magnified.3.CD38 might mediate the phosphorylation of intracellular proteins via the PKC(Protain kinase C)pathway.Blocking CD38 molecules can reduce the phosphorylation level of intracellular proteins.Statistical analysis:SPSS 16.0 statistical software was used to analyze the data,and all the data were expressed as meanąstandard deviation(MeanąSD).K-S tests were performed to test if the data were in normal distribution.The difference of mean value between two groups were compared with the Student's t test or paired t test.The comparisons among multiple groups were performed with one-way ANOVA.The factors affecting the NK cell proportion in the peripheral blood of RA patients was tested by the general linear model analysis.Pearson and Spearman grade correlation analysis were used to test the correlations between the CD38 expression and the corresponding clinical parameters of patients.All the experiments were performed three times.Significance level a=0.05,P<0.05 was considered statistically significant. |
PDF Full Text Request