Study about structure and function of neutrophil-glycosignaling domain

本研究的重点是中性粒细胞乳糖神经酰胺（LacCer）富集微结构域的功能意义，这些微结构域参与了导致超氧化物生成的信号转导途径的启动。用抗laccer抗体T5A7或Huly-m13处理中性粒细胞，诱导细胞产生超氧化物，这被PP1，一种Src激酶抑制剂，wortomannin，一种PI3K抑制剂，SB203580 a p38-MAPK阻断。当早幼粒细胞白血病HL-60细胞通过二甲亚砜（DMSO）处理分化为中性粒细胞谱系时，它们获得了超氧化物生成活性，但对抗laccer抗体没有反应。密度梯度离心发现，中性粒细胞和dmso处理的HL-60细胞的洗涤剂不溶性膜（DIM）中可回收LacCer和Lyn。然而，免疫沉淀实验表明LacCer在中性粒细胞中与Lyn相关，而在DMSO处理的HL 60细胞中与Lyn无关。有趣的是，T5A7在中性粒细胞中诱导Lyn磷酸化，而在DMSO处理的HL 60细胞中则没有。此外，T5A7诱导中性粒细胞p38 MAPK磷酸化。综上所述，这些数据表明中性粒细胞的特征是细胞表面存在与Lyn偶联的富含LacCer的鞘糖脂信号域，并且与LacCer结合的配体诱导Lyn/PI3K/p38 MAPK/蛋白激酶C信号转导通路的激活，导致超氧化物的产生。

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.