| ObjectiveTo investigate the level of functional factors secreted from CD8+HLA-DR+effector T cells in peripheral blood (PB) of the patients with severe aplastic anemia(SAA), the damage on hematopoietic cells by this group of cells, and which were the target cells attacked by CD8+HLA-DR+effector T cells. Thus, we investigated the function of CD8+HLA-DR+cells, which were considered to be activated CTL, in peripheral blood to further explore the pathogenesis of SAA. Also, we explored the heterogenic proteins in myeloid dentritic cells between SAA patients and normal controls, which may be the pathogenic antigen in SAA.MethodsUntreated SAA patients, remission paitents, as well as normal controls were enrolled in this study. Part Ⅰ The CD8+HLA-DR+cells were sorted from the PB of24untreated SAA patients and23normal controls by immunomagnetic separation. The mRNA expressions of perforin, granzyme B, tumor necrosis factor-β (TNF-β) and FasL of sorted cells were analyzed by RT-PCR. Part ⅡCD8+HLA-DR+T cells (effector cells) from untreated SAA patients, remission patients and normal controls were co-cultured with bone marrow mononuclear cells (with CD3+T cells depleted) from remission patients and normal controls (target cells). The effector T cells of untreated SAA were cocultured with the target cells of remission patients and normal controls, which were designated as SAA groups1and2, respectively. There were two control groups:one was the remission SAA group (effector cells from untreated SAA patients and target cells of remission patients), and the other was the normal control (effector cells from untreated SAA patients and target cells of normal controls). Apoptosis of target cells was detected by flow cytometry after staining with AnnexinⅤ. The levels of lactate dehydrogenase (LDH) in the supernatant were determined by automatic biochemistry analyzer. Part ⅢCD8+HLA-DR+T cells from untreated SAA patients, and normal controls were co-cultured with bone marrow mononuclear cells (with CD3+T cells depleted) from normal controls. Apoptosis of CD14+、CD33+、CD34+、GlycoA+cells was detected by flow cytometry after staining with AnnexinV. Meanwhile, the expression of Fas on the CD14+、CD33+、CD34+、 GlycoA+cells was detected by flow cytometry in untreated patients and normal controls respectively. Part Ⅳ Bone marrow mononuclear cells were isolated and inoculated in cell culture flask,2hours later the suspended cells were given up. The cells adhering to the wall cultured for7days and became myeloid dentritic cells. After that, the cells were purified by flow cytometry, and extracted total protein from them. Furtherly, explore the difference of the protein among untreated SAA, remission SAA and normal controls by two-dimensinal electrophoresis.ResultsPart I The mRNA of perforin, granzyme B of CD8+HLA-DR+T cells were (0.66±0.25)、(0.56±0.26) in untreated group, higher than these of controls (0.53±0.14)、(0.40±0.13)(P=0.042、0.012). The mRNA of FasL in CD8+HLA-DR+T cells of untreated SAA patients was (0.77±0.24),higher than that of controls (0.61±0.16)(P=0.011). The mRNA of TNF-β in CD8+HLA-DR+T cells of untreated SAA patients was (0.58±0.16),also higher than that of controls (0.46±0.15)(P=0.011).Part II The apoptosis values in SAA group1(CD8+HLA-DR+T cells from untreated SAA patients and CD3-bone marrow mononuclear cells from remission patients), SAA group2(CD8+HLA-DR+T cells from untreated SAA patients and CD3-bone marrow mononuclear cells from normal controls), the remission group (CD8+HLA-DR+T cells and CD3-bone marrow mononuclear cells from remission patients) and the normal control (CD8HLA-DR+T cells and CD3-bone marrow mononuclear cells from normal controls) were41.12±24.84%,45.81±20.47%,35.03±22.09%,20.95±13.82%. There were no significant differences between SAA group1, SAA group2, and the remission group (P>0.05). However, the apoptosis in each of these groups was higher than in the normal control (P<0.05).The LDH levels in SAA group1, SAA group2, the remission group and normal control were74.56±49.13U/L,62.61±31.76U/L,61.06±28.41U/L, and28.60±8.91U/L. There were no significant differences between SAA group1, SAA group2, and the remission group (P>0.05). However, the level of LDH in these groups was significantly higher than in the normal control (P<0.05). Part Ⅲ The ratios of Fas in the CD34+cells from SAA groups was46.59±27.60%, significantly higher than that of normal control (8.89%±7.28%, P<0.01). The ratio of Fas in the CD14+、CD33+、GlycoA+cells from SAA groups were29.29±9.23%、46.88±14.30%，15.15±9.26%, significantly lower than that of normal control (51.25±38.36%，72.06±39.88%、50.38±39.88%, P<0.05). The apoptosis values of CD34+、CD33+、CD14+cells in SAA group (CD8+HLA-DR+T cells from untreated SAA patients and CD3-bone marrow mononuclear cells from normal contros) were55.43±20.50%、38.13±20.10%、61.87±21.65%, significantly higher than that in the normal control (35.02±13.95%、23.44±10.33%、37.04±22.41%,P<0.05). However, there were no significant differences of CD45-cells between SAA group and normal control (P>0.05).Part IV Our studies about intracellular proteins in mDC have demonstrated that the expression of apoptotic protein (p53,caspase3,Fas), spliceosome-related protein, and ubiquitin-related protein were higher in SAA than normal controls, which seem to related to mDC activation.Conclusions(1) By RT-PCR, we found that the expression of perforin, granzyme B were elevated in the CD8+HLA-DR+T cells, suggested that CD8+HLA-DR+T cells and their functional products might contribute to bone marrow failure in SAA. We also found that the expression of FasL of CD8+HLA-DR+T cells were higher in the SAA than normal controls. Thus, FasL might take part in the pathogenesis of SAA, however, it might have no effect on CD8+HLA-DR+T cells. Moreover, the expression of tumor necrosis factor-beta (TNF-β) were elevated in the CD8+HLA-DR+T cells, which might be the important functional factor in the CD8+HLA-DR+T cells of SAA patients.(2) The CD8+HLA-DR+T cells from untreated SAA and remission patients were more cytotoxic than those cells from healthy controls. These observations suggest that the induction of apoptosis of hematopoietic cells by CD8+HLA-DR+T cells might play a critical role in the bone marrow failure of SAA. In our research, the cytotoxicity of CD8+HLA-DR+T cells was reduced after IST treatment, but it did not reach the level of the control subjects as the normal state. Thus, in order to decrease the risk of relapse, it is important for SAA patients to maintain the IST treatment for a longer period of time, and to taper cyclosporine very slowly.(3) The apoptosis of CD34+、CD33+、CD14+cells from the normal persons increased after coculture with the CD8+HLA-DR+T cells from SAA patients, which indicated that the CD8+HLA-DR+T cells from SAA patients may lead to the excessive apoptosis of hematopoietic cells in normal controls. The expression of Fas on the CD34+、CD33+、CD14+、GlycoA+cells were all positive, which may induce the damage by CD8+HLA-DR+T cells. And the expression of Fas on the CD34+cells markly increased, which may be the main target cell in the pathogenesis of SAA.(4) Abnomal expression of apoptotic protein, spliceosome-related protein,and ubiquitin-related protein in SAA might be involved in the activation of mDC. |
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