| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
This study is focused on the functional significance of neutrophil lactosylceramide (LacCer)-enriched microdomains, which are involved m the initiation of signal transduction pathway leading to superoxide generation. Treatment of neutrophils with anti-LacCer antibody, T5A7 or Huly-m13, induced superoxide generation from the cells, which was blocked by PP1, a Src kinase inhibitor, wortomannin, a PI3K inhibitor, SB203580 a p38-MAPK. inhibitor, and H7, an inhibitor for protein kinase C. When promyelocytic leukemia HL-60 cells were differentiated into neutrophilic lineage by dimethylsulfoxide (DMSO)-treatment, they acquired superoxide generating activity, but did not respond to anti-LacCer antibodies. Density gradient centrifugation revealed that LacCer and Lyn were recovered in detergent-insoluble membrane (DIM) of neutrophils and DMSO-treated HL-60 cells. However, immunoprecipitation experiments indicated that LacCer was associated with Lyn in neutrophils but not in DMSO treated HL 60 cells. Interestingly T5A7 induced the phosphorylation of Lyn in neutrophils but not in DMSO treated HL 60 cells. Moreover, T5A7 induced the phosphorylation of p38 MAPK in neutrophils.
Collectively, these data suggest that neutrophils are characterized by the presence of cell surface LacCer-enriched glycosphingolipid signaling domain coupled with Lyn, and that the ligand binding to LacCer induces the activation of Lyn/PI3K/p38 MAPK/protein kinase C signal transduction pathway leading to superoxide generation.
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