Study Of Autoantigens Targeted By Autoantibodies On The Membrane Of Bone Marrow Cells Of The Patients With Immuno-related Pancytopenia

Objective:1.To investigate the auto-antigens targeted by auto-antibodies IgG on the membrane of bone marrow hematopoietic cells of the patients with immuno-related pancytopenia(IRP),for further purifying and cloning them.2. To investigate the expression of erythropoietin receptor (EPOR), which is a previously discovered auto-antigen, on dendritic cells (DCs) for elucidating the antigen presenting procedure in IRP patients.Method:1.32newly diagnosed IRP patients and15aplastic anemia patients were enrolled in the study, as well as15healthy donors as controls. The antibody IgG was examined by flow cytometry, and then the total membrane protein was extracted. At the same time, the IgG in the marrow supernatant was purified by organic acid and salting out method. The IgG in supernatant was also examined by western blot. And the bone marrow cell membrane auto-antigens targeted by IgG in IRP were identified from membrane protein extracts by SDS-PAGE, Western blot and liquid chromatography-mass spectrography/mass spectrography. Furthermore, the abnormal expression of a newly discovered auto-antigen on the membrane of bone marrow nucleared erythroid cells was investigated.2.39newly diagnosed GlycoA+IRP patients and30GlycoA-IRP patients were enrolled in the study, as well as17healthy donors as controls. The expression of EPOR on DCs (both on cytomembrane and in cytoplasm) was investigated, and then statistically analyzed. Furthermore, the mRNA expression of EPOR in bone marrow cells without erythroid cells was examined by PT-PCR.Result:1. Autoantibody IgG reacting with bone marrow cell membrane antigens could be found in bone marrow supernatant of IRP patients in a positive rate of50%(16/32), which was significantly higher than those of aplastic anemia(AA)(0%) or normal controls(13%,2/15)(P<0.01). Autoantibody IgG in IRP could react with several auto-antigens with approximate MWs of72-95kDa及55kDa. Positive rates of each auto-antigen were56.25%(9/16) 和31.25%(5/16) respectively. The positive bands on the gel were cut, and identified by liquid chromatography-mass spectrography/mass spectrography; the result suggested that the two proteins were lactoferrin (Lf) and WD repeat-containing protein. Furthermore, part of the discovered lactoferrin had the point replaced mutation in the amino acid of Arg121, which replaced by Ser or Asp. The10-25kDa proteins in normal controls were failed to be identified. And then, the expression of lactoferrin on the membrane of marrow nucleared erythroid cells was examined. The expression rate in GlycoA+group patients is13.44±3.53%, which is significantly higher than that in the GlycoA-group patients’3.22±2.32%(P<0.001) or controls’0.54±0.48%(P<0.001), and the later two also has significant difference.2. The expression of EPOR in DCs.a) Comparison between GlycoA+group and GlycoA-group,ⅰ. The expression rate of EPOR on the membrane of DC1was1.45±5.45%in the GlycoA+group,9.89±4.03%in the GlycoA-group, and1.06±2.06%in control group, except the later two had significant difference (P=0.016), no difference between other groups. And the expression rate of EPOR in the cytoplasm of DC1was24.67±31.15%in the GlycoA+group, which is significantly higher than that in GlycoA-group (8.45±3.04%, P<0.05) and control group (12.45±28.46%, P=0.001), there is no significant difference between the later two groups.ⅱ. The expression rate of EPOR on the membrane of DC2was10.47±5.26%in the GlycoA+group, which is significantly higher than that in control group (0.94±2.67%, P<0.05), but compared with the GlycoA+group’s13.23±7.27%, there is no significant difference. And the expression rate of EPOR in the cytoplasm of DC2was30.85±11.20%in the GlycoA+group, which is significantly higher than that in GlycoA-group (15.44±6.53%, P <0.05) and control group (10.34±4.88%, P=0.001), the later two also has significant difference (P<0.05) b) According to the date above, the expression comparisons were done in each group.i. In GlycoA+group, on the membrane of both DC1and DC2, the expression rate of EPOR is significantly lower than that in cytoplasm (P<0.05). The expression in DC2is significantly higher than that in DC1, both on the membrane and in cytoplasm,ii. In GlycoA-group, there is no difference between each group.c) The mRNA expression of EPOR in bone marrow cells without erythroid cells are all negative in the three groups.Conclusion:1. There are auto-antibodies in the bone marrow supernatant of IRP patients, which can target several antigens on hematopoietic cells’ membrane, and lactoferrin and WD repeat-containing protein just two probable candidates. But part of the lactoferrin had the point replaced mutation in the amino acid of Arg121. Furthermore, there is abnormal expression of lactoferrin on the bone marrow erythroid membrane of GlycoA+IRP patients.2. The expression of EPOR in the cytoplasm of DC2was abnormally higher in GlycoA+patients group than that in other groups. However, the EPOR expression on the membrane of DC2and in the cytoplasm of it is not match, suggesting that DC2may be a critical factor in the early stage of EPOR antigen presenting.