Xiao-juan Zhu

Elevated profile of Th17, Th1 and Tc1 cells in patients with immune thrombocytopenic purpura

T-lymphocyte abnormalities are considered important in the pathogenesis of chronic immune thrombocytopenic purpura (ITP). Both CD4+ (Th) and CD8+ (Tc) T lymphocytes can be functionally divided into type 1 (T1) and type 2 (T2) subsets based on the secretion of cytokines. Since Semple[1][1] discovered

Profile of Th17 cytokines (IL-17, TGF-β, IL-6) and Th1 cytokine (IFN-γ) in patients with immune thrombocytopenic purpura

The data on polarization of the immune system towards T helper 1 (Th1) or T helper 2 (Th2) cells in immune thrombocytopenic purpura (ITP) are limited and contradictory. Th17 characterized by the production of Interleukin 17 (IL-17) has been shown to play a crucial part in the induction of autoimmune diseases. To further investigate the role of Th cytokines in the pathogenesis of ITP, we measured the plasma concentration of three Th17-associated cytokines [IL-17, transforming growth factor-ß (TGF-ß), IL-6] and Th1 cytokine interferon-γ (IFN-γ) in ITP patients, and evaluated their clinical relevance. The concentration of IL-17, TGF-ß, IL-6, and IFN-γ in plasma specimens from 29 adults with chronic ITP and 38 controls was analyzed by enzyme-linked immunosorbent assay method. No significant differences of Th17 cytokines (IL-17, TGF-ß, and IL-6) and Th1 cytokine (IFN-γ) concentration were observed between patients with active ITP and the control group. And the IFN-γ/IL-17 ratio representing Th1/Th17 cytokine profile was not significantly different between ITP patients and control, either. However, significantly positive correlation between IL-6 and IFN-γ in ITP patients was observed (r = 0.48, P = 0.01). Among the ITP patients, Plasma IL-17 levels in male were marginally higher than those in female, while similar for TGF-ß, IL-6 or IFN-γ. There was a significantly positive correlation between age and IL-6 concentration in ITP patients (r = 0.56, P = 0.0002), while no statistical significance between age and the other three cytokines. No significant correlation between cytokine concentrations and platelets or megakaryocytes number was found in ITP patients. In summary, ITP may not be associated with changes of plasma Th17 and Th1 cytokine concentrations relative to control population.

Elevated Interleukin-21 Correlated to Th17 and Th1 Cells in Patients with Immune Thrombocytopenia

BackgroundInterleukin-21 (IL-21) is critical in the development of autoimmune diseases. The role of IL-21 in the pathogenesis of immune thrombocytopenia (ITP) remains unknown.Materials and MethodsWe examined the expression of IL-21, IL-17, and interferon (IFN)-γ in ITP patients and controls by enzyme-linked immunosorbent assay and flow cytometry. Detection of specific anti-platelet GPIIb/IIIa and/or GPIb/IX autoantibodies was measured by modified monoclonal antibody specific immobilization of platelet antigens.ResultsIL-21 was expressed on both CD3+CD8− T cells and CD3+CD8+ T cells by flow cytometry. Plasma IL-21 level and the percentage of CD3+CD8−IL-21+ T cells and CD3+CD8+IL-21+ T cells were significantly elevated in ITP patients compared to controls. The percentage of CD3+CD8−IL-17+ T (Th17), CD3+CD8−IFN-γ+ T (Th1), and CD3+CD8+IFN-γ+ T (Tc1) cells also significantly increased in ITP patients. Moreover, we found a significant positive correlation between CD3+CD8−IL-21+ T cells and Th17 cells. In addition, a positive correlation between CD3+CD8−IL-21+ T cells and Th1 cells was also found.ConclusionTogether, our results indicated a possible role of IL-21 in ITP patients correlated to Th17 and Th1 cells, and blockade of IL-21 may be a reasonable therapeutic strategy for ITP especially those with active disease.

The effects of BAFF and BAFF-R-Fc fusion protein in immune thrombocytopenia

Elevated level of B-cell activating factor (BAFF) has been implicated in the pathogenesis of some autoimmune diseases. Blockade of receptor and ligand binding by decoy receptor has demonstrated a clinical benefit in both oncologic and immunologic diseases. In this report, we have detected plasma BAFF and BAFF mRNA expression in immune thrombocytopenia (ITP) patients by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR). The effects of recombinant human BAFF (rhBAFF) and BAFF-R-Fc fusion protein (BR3-Fc) on B cells, T cells, platelets, secretion of interferon gamma (IFNgamma), and interleukin-4 (IL-4) were measured by flow cytometry and ELISA. Patients with active disease had higher levels of plasma BAFF and BAFF mRNA than patients in remission and controls. In in vitro assays, rhBAFF promoted the survival of CD19(+) and CD8(+) cells, and increased the apoptosis of platelets and the secretion of IFN-gamma. BR3-Fc successfully corrected the effects of rhBAFF on lymphocytes, platelets, and cytokines. These findings suggest that BAFF may play a pathogenic role in ITP by promoting the survival of CD19(+) and CD8(+) cells, and increasing the apoptosis of platelets and the secretion of IFN-gamma. Blockade of BAFF by BR3-Fc might be a promising therapeutic approach for ITP.

Contributions of TRAIL-mediated megakaryocyte apoptosis to impaired megakaryocyte and platelet production in immune thrombocytopenia

Recent in vitro studies provide evidence for autoantibody-induced suppression of megakaryocytopoiesis and show a reduction in megakaryocyte production and maturation in the presence of immune thrombocytopenia (ITP) plasma. Here, we present CD34(+) cells from healthy umbilical cord blood mononuclear cells cultured in medium containing thrombopoietin, stem cell factor, interleukin-3, and 10% plasma from either ITP patients or healthy subjects. The quantity, quality, and apoptosis of megakaryocytes were measured. We observed that most ITP plasma boosted megakaryocyte quantity but impaired quality, resulting in significantly less polyploidy cells (N ≥ 4) and platelet release. In these megakaryocytes, we found a lower percentage of cell apoptosis, a lower expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and a higher expression of Bcl-xL. Furthermore, there was a decrease of sTRAIL in ITP plasma and in cell culture supernatants of this group compared with the control group. Our findings suggest that decreased apoptosis of megakaryocytes also contributes to in vitro dysmegakaryocytopoiesis and reduced platelet production. The abnormal expression of sTRAIL in plasma and TRAIL and Bcl-xL in megakaryocytes may play a role in the pathogenesis of impaired megakaryocyte apoptosis in ITP.

Interleukin 18 and interleukin 18 binding protein in patients with idiopathic thrombocytopenic purpura

To evaluate the balance of interleukin IL18 and its endogenous antagonist IL18 binding protein (IL18BP) in patients with idiopathic thrombocytopenic purpura (ITP), plasma IL18, IL18BP, interferon gamma (IFNG) and IL4 levels, as well as platelet counts were measured in patients with active ITP (n = 23), ITP in remission (n = 21) and in healthy subjects (n = 24) by enzyme linked immunosorbent assay (ELISA). Using real‐time quantitative polymerase chain reaction, the mRNA expression of IL18, IL18BP, IFNG, IL4, T‐box (TBX21) and GATA‐binding protein 3(GATA3) were studied in all subjects. The results showed that IL18 and IFNG protein and mRNA levels were significantly increased in patients with active ITP than in control subjects, but that IL18BP were not significantly elevated in ITP patients, which resulted in an elevated ratio of IL18/IL18BP in patients with active disease. During remission stages, the levels of these cytokines were comparable to those of healthy controls. The elevated levels of IL18/IL18BP in plasma during active stages of disease suggest a possible role in the pathogenesis and course of ITP.

High-Dose Dexamethasone Inhibits BAFF Expression in Patients with Immune Thrombocytopenia

IntroductionB-cell activating factor belonging to the TNF family (BAFF) is elevated in several autoimmune diseases including immune thrombocytopenia (ITP). High-dose dexamethasone (HD-DXM) has shown its clinical efficacy in ITP patients. Materials and MethodsThe plasma BAFF concentration and BAFF mRNA were measured in ITP patients before and after oral administration of 40 mg/day DXM for four consecutive days by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR. Moreover, we evaluated the effects of DXM on BAFF expression and proliferation of lymphocytes by ELISA, real-time quantitative PCR and cell proliferation respectively in in vitro experiment.ResultsBoth plasma BAFF concentration and BAFF mRNA were significantly increased in active ITP patients at pretherapy when compared with controls (P < 0.001). After 4-day treatment with HD-DXM, the BAFF and BAFF mRNA were decreased, and lower than that for controls. In in vitro assays, we found DXM-inhibited BAFF, IFN-γ expression, and the proliferation of lymphocytes in a dose-dependent manner.ConclusionThese results suggest that BAFF expression is increased in ITP patients with active disease, and DXM is an effective inhibitor of BAFF production. As immunosuppressant, DXM may play its role in ITP treatment partly through regulating BAFF expression.

High-dose dexamethasone regulates interleukin-18 and interleukin-18 binding protein in idiopathic thrombocytopenic purpura

Idiopathic thrombocytopenic purpura is no longer seen as a simple antibody mediated disorder and the role of T cells and T-regulatory cytokines such as IFN-Á and IL-18 are now known to play an important role. This information should help better understanding of why some treatments are effective and in this study a down regulation of IL18 is reported in response to high dose dexamethasone. To evaluate the effects of high-dose dexamethasone (HD-DXM) on the balance of interleukin-18 (IL-18) and its endogenous antagonist IL-18 binding protein (IL-18BP) in ITP patients, IL-18, IL-18BP as well as IFN-γ, IL-4 plasma levels and platelet counts were determined in 17 ITP patients receiving DXM 40 mg/day for four consecutive days and in 24 healthy subjects. Using RT-PCR, the mRNA expression of IL-18, IL-18BP, IFN-γ, IL-4, T-box (T-bet) and GATA-binding protein 3(GATA-3) were studied in all subjects. The in vitro effects of DXM on IL-18BP and IL-18 of peripheral blood mononuclear cells (PBMCs) were studied by ELISA. HD-DXM administration increased IL-18BP and reduced IL-18 expression significantly (p<0.05), which resulted in a downregulation of IL-18/IL-18BP ratio p<0.05). In vitro, DXM had a significant effect on secretion of IL-18BP while diminishing IL-18 release from cultures of PBMCs. These results suggest that downregulation of IL-18/IL-18BP might account for its clinical efficacy of HD-DXM in active ITP.

In vitro recovery of Th1/Th2 balance in PBMCs from patients with immune thrombocytopenia through the actions of IL-18BPa/Fc

To determine the effects of IL-18BPa/Fc on the cytokine production and survival of peripheral blood mononuclear cells (PBMCs) of immune thrombocytopenia (ITP), PBMCs isolated from patients with ITP and healthy donors were treated with or without of IL-18BPa/Fc. The production of IFN-γ, IL-2, tumor necrosis factor (TNF)-α, IL-4, IL-5 and IL-10 was measured by ELISA, and mRNA expression of IFN-γ and IL-18R was evaluated by RT-PCR. Besides, flow cytometric analysis of cell apoptosis was performed by staining with annexin V-FITC/ Propidium Iodide (PI). The proliferation rate of PBMCs was examined by CCK-8 assay. IL-18BPa/Fc at 10 ng/ml significantly stimulated IL-10 secretion from PBMCs in patients with ITP and healthy donors, while it decreased IFN-γ release. Further, IL-18BPa/Fc enhanced dexamethason(DEX) reduction of PHA-induced IFN-γ production by an additional 38.9%(DEX 20 nmol/l) and 49.9%(DEX 50 nmol/l) in ITP patients. Interestingly, the treatment of PBMCs with IL-18BPa/Fc increased the percentage of early apoptotic cells in patients with ITP. In conclusion, IL-18BPa/Fc, via neutralizing the biologic activity of mature IL-18, accelerates lymphocyte apoptosis and downregulates IFN-γ, while permitting the production of Th2 cytokine IL-10. These observations suggest a role of IL-18BPa/Fc in the recovery of Th1/Th2 balance, as well as its therapeutic potential in the treatment of ITP.

Aberrant expression of Notch signaling molecules in patients with immune thrombocytopenic purpura

To investigate the role of Notch signaling pathway in immune thrombocytopenic purpura (ITP), we measured the expression of 11 Notch pathway molecules in ITP patients and evaluated their clinical relevance. Real-time reverse transcriptase polymerase chain reaction results showed there was aberrant expression of some Notch molecules in ITP. Notch1 and Notch3 expression elevated, while Notch2 decreased statistically in ITP patients. As for Notch ligands, only DLL1 was found downregulated in ITP. The expression of Notch target gene, Hes1, was also upregulated. In accordance with the mRNA level, Notch1 and Hes1 protein expression was also found elevated by Western blot. Immunocytochemistry showed that Notch1 expressed highly in the cytomembrane, cytoplasm, and part of cellular nucleus for ITP while weak in cytomembrane for controls, and Hes1 of ITP was found expressed higher in cellular nucleus than that of controls. Our findings suggest that the aberrant expression profile of Notch pathway may be involved in ITP, and blockage of Notch1 pathway is likely a promising therapeutic concept.