Studies Of Immunologic Mechanism In The Pathogenesis Of Paroxysmal Nocturnal Hemoglobinuria

OBJECTIVE: Paroxysmal nocturnal hemoglobinuria(PNH) is an acquired disease of the pluripotent hematopoitec stem cell. Although the pathogenesis is speculative, there is incontrovertible evidence that somatic mutation of a specific gene(PIG-A) is a necessary component of the disease process. Clinically, PNH is characterized by intermittent, recurrent episodes of hemoglobinuria, thrombophilia and abnormal hematopoiesis. The hemoglobinuria is a consequence of intravascular hemolysis that is due to the abnormal sensitivity of PNH erythrocytes to the lytic action of complement. The increased sensitivity to complement results from membrane deficiencies of complement regulatory proteins such as CD55 and CD59. The absence of these proteins is the most reliable diagnostic criterion of the disease and is responsible for many of the clinical manifestations of PNH. Nevertheless, the mechanism by which PNH clonal cells gain proliferative advantage over normal hematopoitic cells is still obscure. It is not entirely clear whether this defect is sufficient to make the disease manifest, though these GPI-deficient cells can explain many of the clinical symptoms. Peripheral blood T cells in patients with PNH comprise a mixture of residual normal and glycosylphosphatidylinositol (GPI)-deficient PNH cells. PNH frequently occurs in association with bone marrow failure (BMF), including idiopathic aplastic anemia (AA), which is responsive to immunosuppressive therapy. The possible explanation may be that some GPI-anchored proteins are critical targets recognized by immune effectors cells. PNH clones not possessing these critical GPI-anchored proteins will survive because they are selectively resistant to the autoimmune assault that eliminates most normal clones. Therefore, the underlying pathophysiology of the disease may relate to an immunologic disorder. The aim of our study is to assess the cellular immunology status of patients with PNH and to investigate immunologic mechanisms in the pathogenesis of PNH.METHODS: 1. In order to determine the proportions of various lymphocytes in PNH, the peripheral blood mononuclear cells (PBMC) from 18 patients and 20 controls were separated into two subpopulations using an anti-CD59 monoclonal antibody combined with goat-anti-mouse IgG immunomagnetic beads. Lymphocyte subsets CD3, CD4 and CD8 were detected by the immunofluorescence technique of flow cytometry in CD59+/CD59- and unsorted populations. In 6 selected patients, other phenotypic analyses associated with T cell activation such as CD28+ of CD4+ cells or CD8+ cells, CD8+ CD38+ of CD8+ cells, and CD8+HLA-DR+ of CD8+ cells as well as NK cell phenotype were done in the same way. 2. MTT[3-(4,5-dimethylthiazol-2-yl)-2] assay for detection of proliferation of lymphocytes as well as their anti-tumor effect was performed to delineate T cell reactive function. 3. To testify the hypothesis that PNH hematopoitic stem cells (HSCs) are resistant to the cytotoxic effect of T cells because they lack surface expression of one or more glycosylphosphatidylinositol (GPI)-linked molecules, the sensitivities of PNH patients bone marrow to autologous lymphocytes was tested as fellows. Unsorted, CD34- and CD34+ bone marrow cells (prepared by immunomagnetic methods) were incubated in a liquid culture system with autologous CD59+ and CD59- cells,...