Research On Quantities And Function Of NK Cells In The Immune Thrombocytopenia

Immune thrombocytopenia(ITP)is a clinically common autoimmune disease charactererized by bleeding,which makes up about a third of hemorrhagic disease.It is characterized by peripheral blood platelet reduction,bone marrow megakaryocyte number increased or normal accompanied by mature obstacles and detected antiplatelet autoantibodies in the body?In recent years,NK cells as a kind of immune regulatory cells in autoimmune diseases are catching more and more attention.Study[1] in SLE patients with peripheral blood NK cell number,cell killing activity significantly reduced,and the combination of arthritis and thrombocytopenia in patients with more significant reduction in the number of NK cells,thinking that NK cells mainly play a protective role in the disease.In addition rheumatoid arthritis[2],Graves disease [3] have similar findings.Studies on NK cells in patients with ITP is less,in this study we analyze different disease states of ITP patients and healthy controls peripheral blood NK cell number,subgroup,and the difference of function,to explore the effect of NK cells in the pathogenesis of ITP.Part1 Research on quantities and function of NK cells in the immune thrombocytopeniaObjective To study NK cell quantities and function of patients with immune thrombocytopenia(ITP).Methods A total of 66 ITP patients(32 in remission and 34 newly diagnosed)were collected from september 2015 to april 2016 in Tianjin Medical Uniwersity General Hospital,and 30 healthy volunteers were recruited as controls.The percentage of CD3-CD56+NK cell in peripheral blood lymphocytes,the expression of activating receptor(NKp44),inhibitory receptor(NKG2A),perforin and granzyme ? were detected by flow cytometry.The correlation between the above parameters and patients' clinical profile were evaluated.Results(1)The percentage of CD3-CD56+NK cell in new diagnosed(10.99 ±4.89)% and remission(9.73 ± 6.75)% ITP patients were significantly lower than that in healthy control(14.67 ± 7.24)%,(p=0.023,0.003);In addition,the level of CD3-CD56+NK cell at initial diagnosed ITP patients responding to treatment(11.71±5.5)%was higher than that in the refractory patients(7.97±3.07)%(p=0.045).(2)The ratio of CD3-CD56 bright CD16-NK cell/total NK cells in new diagnosed ITP patients(6.48 ± 4.33)% were significantly higher than that in healthy control(4.21 ±2.70)%(p=0.020);The ratio of CD3-CD56 dim CD16-NK cell/total NK cells in new diagnosed ITP patients(93.51 ± 4.33)% were significantly lower than that in healthy control(95.79 ± 2.70)%,(P=0.020)(3)The expression of activating receptor NKp44 in new diagnosed ITP patients(0.28(0.95))%was significantly lower than that in remission group(0.61(2.05))% and healthy controlls(0.92(0.90))%,(p=0.047,0.048);The expression of inhibitory receptor NKG2 A in new diagnosed ITP patients(42.34 ± 23.86)%was significantly higher than that in healthy control(29.25 ± 12.83)%,(P=0.009);(4)The expression of perforin in new diagnosed ITP patients(87.52(25.29)% were significantly lower than that in healthy controls(91.55(8.29))%,(P=0.025);The expression of granzyme ? in ITP patients and controls make no difference(p=0.560,0.426,0.179)(5)The level of NK cells in ITP patients was negatively correlated with CD3+CD8+ T cells(n=41,r=-0.387,p=0.012)and CD5+CD19+ B cells(n=66 r=-0.273,p=0.028);The level of NK cells in ITP patients was positively correlated with the ratio of CD3+CD4+/CD3+CD8+(n=41,r=0.358,p=0.028)and peripheral platelet count(n=66,r=0.314,p=0.011).Conclusion Deceased quantities and impaired total NK function might play a role in the pathogenesis of ITP.Part 2Research on the negative immune regulation of NK cells in patients with immune thrombocytopeniaObjective To test the leves of NK cells and relevant cytokines(IL-10,TGF-? and IFN-?)in patients with primary immune thrombocytopenia(ITP)Methods All samples were taken from 42 patients(22 newly diagnosed and 20 inremission)and 20 healthy volunteers.The levels of IL-10 and IFN-? in blood serum were detected by enzyme-linked immuno sorbent assay(ELISA).The percentage of CD3-CD56+ NK cell,CD3-CD56 bright CD16-NK cell,CD3-CD56 dim CD16+NK cell in peripheral blood lymphocyte were detected by flow cytometry,The NK cells were extracted by immunomagnetie microbeads.The m RNA expression levels of IL-10,TGF-?,IFN-? in NK cells were detected by real-time fluorescent quantitative PCR.Correlation between the above measured results was analysedResults(1)The blood serum level of IFN-? in new diagnosed ITP patients [(653.0±221.6)ng/L] was higher than that in remission ITP patients [(484.4±219.5)ng/L]and healthy control [(390.9 ±253.5)ng/L],(p=0.022,p=0.001).The blood serum level of IL-10 in new diagnosed ITP patients was lower than that in healthy control [(52.09±26.66)ng/L vs(79.44±38.43)ng/L,P=0.007].(2)The percentage of NK cell in new diagnosed ITP patients and remission ITP patients[(9.53 ± 3.93)%,(9.03 ±3.78)%] were significantly lower than that in healthy control[(13.72 ±7.42)% ](P=0.013,P=0.007).The ratio of CD3-CD56 bright CD16-NK cell/total NK cells in new diagnosed ITP patients was higher than that in healthy control [(6.85 ±4.43)% vs(4.05 ± 2.81)%,P=0.032].The ratio of CD3-CD56 dim CD16-NK cell/total NK cells in new diagnosed ITP patients was lower than that in healthy control [(93.14±4.43)% vs(95.94±2.81)%,P=0.032].(3)The m RNA expression level of IFN-? in NK cells of ITP patients and healthy control make no difference(all P>0.05).The m RNA expression levels of IL-10 and TGF-? in NK cells in new diagnosed ITP patients were significantly higher than that in healthy control(1.82±1.32 vs 1.02±1.03,P=0.023;2.80±2.31 vs 1.46±1.37,P=0.028).The ratio of CD3-CD56 bright CD16-NK cell/total NK cells was positively correlated with the m RNA expression levels of IL-10,TGF-? in NK cells(r=0.424,0.432,all P<0.05).Conclusion NK cells may compensate for the deficiency of the number by enhancing the secretion of negative regulation cytokines,acting as “protective” roles in the disease.