Iwabuchi Kazuhisa, Nagaoka Isao
Department of Biochemistry, Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo, Japan.
Blood. 2002 Aug 15;100(4):1454-64.
This study is focused on the functional significance of neutrophil lactosylceramide (LacCer)-enriched microdomains, which are involved in the initiation of a 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; wortmannin, a phosphatidylinositol-3 kinase inhibitor; SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor; and H7, an inhibitor for protein kinase C. When promyelocytic leukemia HL-60 cells were differentiated into neutrophilic lineage by dimethyl sulfoxide (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. T5A7-induced Lyn phosphorylation in neutrophil DIM fraction was significantly enhanced by cholesterol depletion or sequestration with methyl-beta-cyclodextrin or nystatin. 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, which may be suppressibly regulated by cholesterol, leading to superoxide generation through the phosphatidylinositol-3 kinase-, p38 MAPK-, and protein kinase C-dependent signal transduction pathway.
本研究聚焦于富含中性粒细胞乳糖神经酰胺（LacCer）的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，可诱导细胞产生超氧化物，而Src激酶抑制剂PP1、磷脂酰肌醇 - 3激酶抑制剂渥曼青霉素、p38丝裂原活化蛋白激酶（MAPK）抑制剂SB203580以及蛋白激酶C抑制剂H7均可阻断这一过程。当用二甲基亚砜（DMSO）处理早幼粒细胞白血病HL - 60细胞使其分化为中性粒细胞系时，它们获得了产生超氧化物的活性，但对抗LacCer抗体无反应。密度梯度离心显示，LacCer和Lyn存在于中性粒细胞以及经DMSO处理的HL - 60细胞的去污剂不溶性膜（DIM）中。然而，免疫沉淀实验表明，LacCer在中性粒细胞中与Lyn相关联，而在经DMSO处理的HL - 60细胞中则不然。有趣的是，T5A7可诱导中性粒细胞中Lyn的磷酸化，但在经DMSO处理的HL - 60细胞中则不能。此外，T5A7可诱导中性粒细胞中p38 MAPK的磷酸化。用甲基 - β - 环糊精或制霉菌素进行胆固醇耗竭或螯合可显著增强T5A7诱导的中性粒细胞DIM组分中Lyn的磷酸化。总体而言，这些数据表明，中性粒细胞的特征是存在与Lyn偶联的富含细胞表面LacCer的糖鞘脂信号结构域，并且与LacCer结合的配体可诱导Lyn的激活，而Lyn的激活可能受到胆固醇的抑制调节，进而通过磷脂酰肌醇 - 3激酶、p38 MAPK和蛋白激酶C依赖性信号转导途径导致超氧化物生成。
