|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1998 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1997 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Platelets play an important role in inflammatory and allergic processes. Activated platelets are capable of releasing a number of inflammatory mediators, i.e., melanocyte growth-stimulating activity (GRO), and regulated on activation with normal T cell expressed and secreted (RANTES). However, little is known about the mechanisms whereby human megakaryocytes produce these chemokines.
In a serum-free liquid culture, thrombopoietin (TPO) selectively stimulated the growth of megakaryocytic cells from CD34-positive cord blood cells. Using these cultured cells, we investigated cytokine production by human megakaryocytes. Two x 10^5 Day l0-megakaryocytes secreted more than 1,000 pg/mL of interleukin (IL)-8. The megakaryocyte-conditioned medium had the chemotactic potential of polymorphonuclear leukocytes, which was abrogated by the addition of anti-IL-8 antibody, suggesting the secretion of biologically active IL-8. Direct evidence for IL-8 synthesis in megakaryocytes was provided by reverse
transcription-polymerase chain reaction on purified CD41b^+ cells and by the detection of intracellular LL-8 in CD41b^+ cells.
Next, we examined the effects of IL-1. the common mediator of the inflammatory process, on the development and secretory functions of megakaryocytes generated from CD34^+ cord blood cells under stimulation with TPO.The addition of IL-1alpha did not influence the generation, endomitosis or expression of surface markers of megakaryocytes, as compared with TPO alone. However, IL-1 enhanced the ability of megakaryocytes to produce IL-8 and growth regulating oncogene-alpha (GRO) in the presence of TPO.In contrast, the production of RANTES, platelet factor 4 (PF4) and thromboglobulin (TG) were not potentiated. A flow cytometric analysis and a reverse transcription-polymerase chain reaction analysis revealed IL-1 receptor type I (IL-lR I) expression of megakaryocytes generated by TPO.Moreover, the addition of an anti-IL- 1 R I monoclonal antibody significantly decreased the TPO + IL-1-induced secretion of IL-S by the cultured megakaryocytes, to the level obtained by TPO alone. These results suggest that the production of IL-8 and GRO, but not RANTES, PF4 and TG, by megakaryocytes is potentiated by signaling through IL-1R I with the aid of TPO.Thus, megakaryocytes and platelets may play an important role in the development of inflammation via chemokine release. Less