Clinical And Basic Studies On The Immune Pathogenesis Of ITP

Idiopathic thrombocytopenic purpura (ITP) belongs to an autoimmune disease. Its immunologic abnormality is related to platelet-associated immunoglobulin (PAIg), which results in the over-destruction of platelet in reticuloendothelial system (RES) or complement system. Patients with ITP manifest thrombocytopenia and clinical bleeding symptom. With the development of research, it has been discovered that the cellular immune dysfunction especially activated self-reactive T cells in response to glucoprotein on platelet membrane might play critical roles in the pathogenesis of ITP, which results in autoimmune intolerance. Both a new type of regulatory T cells-CD4+CD25+ T cells that was firstly reported by Sakaguchi et al. in 1956 and dendritic cells (DCs) play important roles in the maintenance of immunological self-tolerance. Natural CD4+CD25+ regulatory T cells (Treg) are generated in the thymus, which suppress the activation of self-reactive T cells and maintain autoimmune tolerance. Known as the strongest antigen-presenting cell (APC), DCs stimulate immune response. Mature DCs present antigen and have an increased T-cell stimulatory capacity, whereas immature DCs are considered to induce immune tolerance. Therefore, DCs also regulate immune response. The activation of B cells depends on T cells help. Meanwhile, B cells can directly activate T cells as APC. B cells also play vital roles in the activation of self-reactive T cells.In our study, basic and clinical study was carried out to explore CD4+CD25+ Treg cells, DC, and B cells effects on the immune pathogenesis of ITP. We seek to clarify the immune mechanism of CD4+CD25+ Treg cells in ITP and the relevantly immune effect of high dose dexamethasome (HD-DXM) by investigating the circulating number and the immunologic suppression of CD4+CD25+ Treg cells, the changes of CD4+CD25+ Treg cells number and anti-inflammatory cytokine IL-10 or TGF-β1 after the treatment with HD-DXM in ITP patients; To clarify the immune mechanism of DCs by investigating the expression of co-stimulatory molecules on circulating DCs, the changes of DCs subsets number after treatment with HD-DXM in ITP patients, and the proliferation of CD4+ T cells; To clarify the immune mechanism of B cells by investigating the circulating number of B cells and the expression of co-stimulatory molecules on B cells.1. The immune dysfunction of CD4+CD25+ Treg cells in patients with ITP.To study the effect of CD4+CD25+ Treg cells in the pathogenesis of ITP, we used flow cytometry and found the proportion of CD4+CD25+ T cells in the peripheral blood of patients with ITP [(15.64±5.82)%] was significantly higher than that in normal control group ([9.30±3.95)%(]P﹤0.001); There was no significantly difference in the percentage of CD4+CD25high T cells between ITP patients and control [(1.53±0.66)% versus (1.36±0.55)%( P=0.317)]; But the number of CD4+ FOXP3+ T cells ([1.82±1.42)% versus (3.90±1.37)% (P﹤0.001)]and CD4+CD25+ FOXP3+ T cells[(1.25±0.94)% versus (2.65±0.92)% ( P﹤0.001)] in patients was significantly lower than that in control group. The expression of Foxp3 mRNA in patients was reduced (P﹤0.001) by using real-time PCR. The suppressive activity of CD4+CD25 high T cells was lower than that of healthy controls (P﹤0.001).The results implied that patients with ITP show a decreased circulating number and reduced suppressive activity of CD4+CD25+ Treg cells.2. The changes of CD4+CD25+ Treg cells after the treatment with HD-DXM in ITP patients and the relationship between CD4+CD25+ Treg cells and anti-inflammatory cytokine IL-10 or TGF-β1.To study the relationship between CD4+CD25+ Treg cells and anti-inflammatory cytokine IL-10 or TGF-β1 and the effects of high dose dexamethasome (HD-DXM) on them contained CD4+CD25+ Treg cells, IL-10 and TGF-β1. A good initial response to HD-DXM occurred in 24 of the 26 patients with chronic ITP (92.3%): the mean platelet count was (84.9±30.4)×109/L [range, (20～150)×109/L]] one week after the initiation of treatment with HD-DXM. We used flow cytometry and found the number of CD4+ CD25+ T cells (P﹤0.0001), CD4+CD25high T cells ( P﹤0.0001) , and CD4+FOXP3+ T cells ( P﹤0.0001) in patients with chronic ITP was significantly increased after the treatment with HD-DXM. Meanwhile the TGF-β1 and IL-10 in the serum of chronic ITP patients were also measured by Enzyme-linked immunosorbent assay (ELISA), the results showed the expression level of TGF-β1 was lower than that of healthy controls([305.90±202.35)pg/ml versus (1479.00±182.70)pg/ml (P﹤0.0001)] and HD-DXM could significantly increase TGF-β1 levels in chronic ITP patients(1414.70±270.35) pg/ml,(P﹤0.0001). There was no significantly difference in the expression level of IL-10 between chronic ITP patients and control [(441.10±239.26)pg/ml versus (349.88±21.27)pg/ml ( P﹥0.05)] and there was no remarkably changes of IL-10 in patients after HD-DXM treatment (395.95±149.95) pg/ml,(P﹥0.05). Using the methods of real-time PCR examine the expression of gene Foxp3, IL-10, TGF-β1 in the CD4+ T cells . Both expressions of Foxp3 and TGF-β1 gene were lower than that of controls (P﹤0.05 and P﹤0.05); HD-DXM administration significantly increased the expressions of Foxp3 and TGF-β1 gene (P﹤0.05 and P﹤0.0001)that were even higher than that of controls(P﹤0.05 and P﹤0.05); There was a positive correlation between the expressions of Foxp3 and TGF-β1 gene after treatment with HD-DXM (r =0.403, P=0.041). The expression rate of Foxp3 protein in spleen T cells was observed using immunohistochemistry. The Foxp3 protein level in refractory ITP patients who received DXM therapy before spleen surgery was higher as compared with that in splenic rupture group; whereas lower than that in HBV hepatits group. Together, the results suggested that both CD4+CD25+ Treg cells and TGF-β1 play roles in the pathogenesis of ITP; Glucocorticosteroid may inhibit immune response by affecting CD4+CD25+ Treg cells and TGF-β1.3. The changes of circulating DC number after the treatment with HD-DXM and the expression level of co-stimulatory molecule on DC in ITP patients.To study the effect of DCs in the pathogenesis of ITP, flow cytometry was used to analyze the absolute number of circulating DCs subsets including mDCs and pDCs in 26 chronic ITP patients, meanwhile the co-stimulatory molecule on total DC. No statistically significant difference was found between patients and controls in the absolute number of circulating mDCs or pDCs (P﹥0.05 and P﹥0.05). HD-DXM therapy suppressed pDCs by 75.5% (P<0.0001), whereas stimulated mDCs by 24.3% (P<0.05); Interestingly, the median fluorescence intensity (MFI) of CD11c in mDCs was remarkable reduced from 340±30 to 199±21 (P<0.0001). The MFI of CD86 on DCs was higher in ITP patients than in healthy controls (P<0.05), while the proportion of CD86, CD40, CD80 and the MFI of CD40, CD80 in ITP patients were normal (P﹥0.05). Monocyte-derived DCs were propagated in granulocyte-macrophage colony stimulating factor (GM-CSF) combined with IL-4. Briefly, autologous CD4+ T cells and monocyte-derived DCs were cultured with autologous or allogeneic normal fresh platelets in 96-well plates. After 6 days of incubation, 3H-TdR was used to assay the proliferation of CD4+ T cells. In ITP patients DCs, cultured with autologous or allogeneic platelets, were highly efficient in stimulating autologous CD4+ T cells proliferaton as compared to DCs derived from health donors(P﹤0.05 and P﹤0.05). Our results indicated that DCs also play an important role in the pathogenesis of ITP , and DXM may affect immune response by decreasing the number of circulating pDCs and inhibiting the maturation of mDCs.4.The circulating number of B cells and the expression level of co-stimulatory molecule on B cells in ITP patients.To study the effect of B cells in the pathogenesis of ITP, flow cytometry was used to analyze the proportion of circulating CD19+ B cells, the expression level of co-stimulatory molecule on CD19+ B cells in ITP patients, the expression of PAIgG. The proportion of CD86 on CD19+ B cells was higher in untreated group of ITP patients than in healthy controls (P<0.05); while the proportion of CD19+ cells , CD19+ CD40+ cells, CD19+CD80+ cells and the MFI of CD86, CD40, CD80 in untreated group of ITP patients were normal (P﹥0.05). The proportion of CD80 and CD86 on CD19+ B cells in treated group of ITP patients was lower than that in untreated group (P<0.05 and P<0.05). The high level of PAIgG in 12 of the 18 untreated patients with ITP and 4 of the 15 treated patients with ITP was detected. No association was observed between high level PAIgG and the expression level of CD40, CD80, CD86 on CD19+ B cells.