Cell Mediated Cytotoxity Toward Platelet In Chronic Idiopathic Thrombocytopenic Purpura

Idiopathic thrombocytopenic purpura (ITP) is a common hematologic disorder manifested by immune-mediated thrombocytopenia. It is usually a persisting disease, which relapses frequently and requires a long-term treatment. In some cases, it progresses rapidly and even threatens the patients' survival. The severe side effects of routine treatment lead to a poor prognosis.The pathogenic mechanism of ITP is not completely clear yet. It was long believed that thrombocytopenia is mediated by autoantibodies. Platelets from 50%-60% of ITP patients are coated with IgG antibodies that recognize one or more platelet surface glycoproteins. The antibody-coated platelets are recognized by tissue macrophage Fc γ receptors, resulting in their phagocytosis. Other studies suggested either inhibition of platelet production or intramedillary destruction of nascent platelets. Recent in vitro studies, showing reduced megakaryocyte production and maturation in the presence of ITP plasma, provide evidence for autoantibody-induced suppression of megakryocytopoiesis.However these mechanisms cannot account for all observations made in this disorder. Some ITP patients' platelets are absent of detectable antigen-specific autoantibody and remission can occur despite the presence of platelet autoantibodies. These phenomena indicate the presence of other mechanisms.Recently, Olsson B reported several cytotoxic genes, such as Apo-1/Fas,granzyme B and perform , together with genes involve in the Thl cell response, such as interferon- Y and interleukin-2 receptor- 3 , showed increased expression in the ITP patients. With a modified UIIn release assay, they noted the destruction of autologous platelet by CD 14" and CD 19' mononuclear cells.We have developed a novel system by using CD8+ or CD 56+ cells to study the cytotoxity of specific cell towards patient's autologous platelet, and we adopted annexin V and flow cytometry for measuring CTL and NK cell-mediated cytotoxicity. Furthermore, we investigated the expression ratio of FasL, TNF a and TRAIL on the CD8+ cells by flow cytometry. Perform and granzyme B mRNA in CD8+ cells were measured by a semiquantatative reverse transcriptase polymerase chain reaction (RT-PCR) procedure.1 Cell-mediated lysis of autologous platelets in chronic idiopathic thrombocytopenic purpura.Objective: To investigate the contribution of cell-mediated cytotoxity to the pathogenesis of ITP, we established an in vitro assay measuring the destruction of autologous platelets by CD8+ cells (cytotoxity T cells) or CD56+ cells (nature killer cells).Methods: * 40 ml blood from 14 cases of ITP and 10 cases of health control were collected respectively.* Mononuclear cells and platelets were prepared from peripheral blood.* CTLs were positively selected using CD8+ magnetic microbeads and NK cells were selected using CD8+ magnetic microbeads, according to the manufacture's recommendations (Miltenyi Biotec).* The CTLs and NK cells were used as effector cells respectively and autologous platelets were used as target cells. All cells applied in the assay were washed free from plasma. To stimulate cytolytic effector T-cells, anti-CD3-antibody was added. To measure NK cell activity, target and effector cells were co-cultured without the addition of anti-CD3-antibody. Effector cells, target cells and anti-CD3-antibody wereincubated for 4 hours.* The cell suspension was incubated with FITC-Annexin V monoclonal antibody. Analysis was performed on a flow cytometer. Logarithmic amplifiers were used for fluorescence signals and 10000 events were collected. The data were analyzed using the WinMDI software. A platelet gate was set in the forward light scatter (FSC ) and side-scatter(SSC) dotplot. The fraction of platelets expressing FITC was determined by setting a quadrant gate in the fluorescence dotplot for platelets. The results are expressed in percent.* Comparing positive expression ratio on platelet between the ITP group and the control group.Results: FITC positive ratio of platelets in ITP group(7.56%±2.80%) in which the CD8+ cells were used as effector cell was higher than normal controls(3.61%± 0.90%). There was a significant difference(P<0.05).When CD56+ cells were used as effector cell , FITC positive ratio of ITP plateIets(3.50%±l.ll%) was the same as normal controls( 3.63%+0.95%).Conclusion: The cell-mediated cytotoxic lysis of platelets by CTLs may be involved in the pathogenesis of ITP. The NK cells have no direct cytotoxic effect towards platelets.2 Measurement of the molecules and genes involved in CTL mediated cytotoxity.Objective: The principal mechanism of CTL mediated cytotoxicity towards autolougus platelets is believed to involve 1) the activation of the apoptosis receptor in the target cell membrane. 2) By the release of secretory granules on the surface of a target cell. These secretory granules contain at least two distinct classes of cytotoxins that are expressed selectively in T cytotoxic cells, perform and granzyme .To study the mechanism through which cytotoxic effector CD8+ cell destroy platelet, measurement of the molecules and genes involved in CTL mediated cytotoxity was performed.Methods: * Separating CD8+ cells from peripheral blood as described in section* The cell suspension were incubated with combination of phycoerythrin or FITC conjugated MoAbs: anti-FasL-PE,anti-TNF a -FITC and anti-TRAIL-PE. Appropriated isotype matched control antibodyes were used. Analysis was performed on a flow cytometer. Logarithmic amplifiers were used for fluorescence signals and 10000 events were collected. The fraction of CD8+ cells expressing FasL,TNFa and TRAIL were determined by setting a quadrant gate in the fluorescence dotplot for CD8+ cells. The results are expressed in percent. Comparing positive expression ratio between the ITP group and the control group.* RNA was isolated from the CD8+ cells preparation. The expression levels of perform and granzyme B mRNA in CD8+ cells were measured by a semiquantatative reverse transcriptase polymerase chain reaction (RT-PCR) procedure. Comparing the expression level between the ITP group and control group.Results: * Expression ratio (17.51% ±4.44%) of FasL on CD8+ cells was remarkably higher than in control(8.92%± 1.53%)(P<0.05) .* Expression ratio(11.91%±4.93%) of TNFaon CD8+ cells was remarkably higher than in control(6.41%±2.08%) (i><0.05).* Expression ratio(16.06%±3.84%) of TRAIL on CD8+ cells in ITP was similar with that in control(13.95%±3.22%)(/>>0.05).* The perform and granzyme B expression on CD8+ cells in the ITP group were significantly higher than the control group (PO.05).Conclusion: CTLs are activated in ITP and might be involved in the pathogenesis of ITP. Perform & granzyme-mediated Cytotoxicity and Fas/FasL-mediated cytotoxicity are two important pathways through which CTLs destruct platelets.