Expression Of Mitogen-activated Protein Kinases In CD8~+T Cells Of Patients With Severe Aplastic Anemia

ObjectiveAt present,many studies have suggested that aplastic anemia is an autoimmune disease caused by T cell hyperfunction.The most important part of pathogenesis which is the CD8~+ cytotoxic T cells lead to bone marrow hematopoietic cells apoptosis.However,there are few studies on CD8~+ T cells signaling pathway in patients with AA.This study aims at exploring the expression level of Mitogen-activated protein kinases(MAPKs)in peripheral blood CD8~+T cells and the relationship between the MAPKs and function molecules of CD8~+T cells from patients with severe aplastic anemia.Meanwhile,this study further clarifies the abnormal immunity mechanism of bone marrow hematopoiesis and provides the theoretical basis for the diagnosis and treatment.MethodsThe SAA patients who were untreated,convalescent and normal controls in Tianjin Medical University General Hospital Department of Hematology and Oncology from November 2015 to June 2016 were included in this study.Part I Extracellular regulated protein kinases(ERK)of peripheral blood CD8~+ T cells from untreated SAA patients(n=20),convalescent SAA patients(n=20)and normal controls(n=20)was detected by flow cytometry and Real-Time PCR.Function molecules of CD8~+ T cells from untreated SAA patients,convalescent SAA patients and normal controls was detected by flow cytometry as well.Correlation of ERK with function molecules and clinical parameters were analyzed.Part II c-Jun N-terminal kinases(JNK)of peripheral blood CD8~+ T cells from untreated SAA patients(n=20),convalescent SAA patients(n=20)and normal controls(n=20)was detected by flow cytometry and Real-Time PCR.Correlation of JNK with function molecules of CD8~+ T cells and clinical parameters were analyzed.Part III P38 MAPK of peripheral blood CD8~+ T cells from untreated SAA patients(n=20),convalescent SAA patients(n=20)and normal controls(n=20)was detected by flow cytometry and Real-Time PCR.Correlation of P38 MAPK with function molecules of CD8~+ T cells and clinical parameters were analyzed.ResultsPart I The MFI % of p-ERK1/2 in peripheral blood CD8~+ T cells was detected by FCM.MFI% =(MFI of PMA stimulated p-ERK1/2)/(MFI of PMA unstimulated p-ERK1/2)× 100%.The MFI% of p-ERK1/2 in SAA untreated patients,convalescent SAA patients and normal controls were(333.50±92.83)%,(328.28±116.11)% and(254.35±63.63)% respectively.The expression of p-ERK1/2 in SAA untreated and convalescent patients were higher than that in normal controls(P<0.05).The expression of p-ERK1/2 was positively associated with the expression of Granzyme B in CD8~+ T cells.Meanwhile,the expression of p-ERK1/2 was negatively associated with the count of PLT.The mRNA levels of ERK in SAA untreated patients(n=10),convalescent SAA patients(n=10)and normal controls(n=8)were(1.98±0.85),(0.43±0.59)and(0.59±0.64)severally.The mRNA levels of ERK in SAA untreated patients was higher than that in convalescent SAA patients and normal controls(P<0.05).Part II The MFI% of p-JNK in peripheral blood CD8~+ T cells was detected by FCM.MFI% =(MFI of PMA stimulated p-JNK)/(MFI of PMA unstimulated p-JNK)× 100%.The MFI% of p-JNK in SAA untreated patients,convalescent SAA patients and normal controls were(132.15±24.64)%,(120.10±25.16)% and(120.00±24.21)% respectively.The comparison between the three groups didn't show any statistical significance.The expression of phospho-JNK wasn't associated with the expression of perforin,granzyme B in CD8~+ T cells.Meanwhile,the expression of phospho-JNK was negatively associated with the clinical parameters.The mRNA levels of JNK in SAA untreated patients(n=10),convalescent SAA patients(n=10)and normal controls(n=8)were(5.47±4.65),(3.83±3.81)and(0.59±0.64)severally.Part III The MFI% of p-P38 MAPK in peripheral blood CD8~+ T cells was detected by FCM.MFI% =(MFI of PMA stimulated p-P38 MAPK)/(MFI of PMA unstimulated p-P38 MAPK)× 100%.The MFI% of p-P38 MAPK in SAA untreated patients,convalescent SAA patients and normal controls were(121.90±15.90)%,(114.98±15.02)% and(105.98±7.76)% respectively.The expression of p-P38 MAPK in SAA untreated and convalescent SAA patients were higher than that in normal controls(P<0.05).The expression of p-P38 MAPK was positively associated with the expression of perforin,granzyme B in CD8~+ T cells.Meanwhile,the expression of phospho-P38 MAPK was negatively associated with the clinical parameters.The mRNA levels of P38 MAPK in SAA untreated patients(n=10),convalescent SAA patients(n=10)and normal controls(n=8)were(22.11±28.15),(12.70±17.69)and(2.31±3.22)severally.Conclusions1.The expression level of ERK and P38 MAPK in peripheral blood CD8~+ T cells from SAA patients were higher than that in normal controls.Besides,the expression level of JNK and P38 MAPK were positively associated with the expression of perforin,granzyme B in CD8~+ T cells.This results reveals that ERK and P38 MAPK may play a important role in the activication of CD8~+ T cells signal path from SAA patients,participate in the toxicity of CD8~+ T cells.2.Meanwhile,the expression of ERK and P38 MAPK were negatively associated with the clinical parameters.This results reveals that ERK and P38 MAPK may participate in the abnormal immunity mechanism of bone marrow hematopoiesis.3.The expression level of JNK in peripheral blood CD8~+ T cells from SAA patients didn't show any statistical significance with normal controls.Besides,the expression level of JNK weren't associated with the expression of perforin,granzyme B in CD8~+ T cells.Meanwhile,the expression of JNK have no correlation with the clinical parameters.This results reveals that JNK may participate in other pathophysiology in SAA patients.4.In summary,this study found that the expression of MAPKs subfamily in peripheral blood CD8~+T cells of SAA patients is not consistent.MAPK signaling pathway may be involved in CD8~+T cell hyperfunction in SAA patients,provide a new theoretical basis for future targeted therapy.But it still need to be confirmed by further studies,such as inhibition of MAPKs activity in the peripheral blood CD8~+T cells of SAA patients.In addition,we can also continue to explore the other biological role of MAPKs in SAA patients.