Mansfield P J, Suchard S J
Department of Pediatrics, University of Michigan School of Medicine, Ann Arbor 48109.
J Immunol. 1993 Mar 1;150(5):1959-70.
Differentiation of the human promyelocytic leukemia cell line HL-60 to polymorphonuclear leukocyte (PMN)-like cells with DMSO provides an important model for studying the acquisition of PMN functional responses that accompany differentiation. We showed previously that the extracellular matrix (ECM) protein thrombospondin (TSP) binds to PMN surface receptors and promotes adhesion and motility. Undifferentiated HL-60 cells did not adhere and were not motile in response to TSP, whereas cells differentiated toward PMN-like cells demonstrated both TSP-mediated adhesion and chemotaxis, with chemotaxis evident by day 2 of induction. With differentiation, a maximal response was obtained with 100 to 300 nM TSP, 10-fold lower than required for maximal PMN chemotaxis. Checkerboard analysis confirmed the directional nature of motility. mAb recognizing different domains of TSP inhibited chemotaxis, suggesting the involvement of multiple sites on TSP. Although both the NH2-terminal heparin-binding domain (HBD) and 140 kDa COOH-terminal fragment supported chemotaxis in PMN-like cells, neither fragment was as potent as intact TSP. Both pertussis and cholera toxin inhibited TSP-mediated chemotaxis, suggesting the involvement of GTP-binding proteins. The toxin effects did not indirectly result from elevated cAMP levels because high concentrations of either 8-bromo-cAMP or dibutyryl cAMP did not inhibit chemotaxis. TSP bound to nitrocellulose filters induced the directed migration (haptotaxis) of PMN-like cells rather than the random motility observed with PMN. Haptotaxis was stimulated by either the HBD or 140-kDa fragment and was inhibited by mAb against these two domains. Haptotaxis rather than random migration was confirmed by checkerboard analysis. Our results demonstrate that PMN-like HL-60 cells respond differently to TSP than human peripheral blood PMN. These differences may reflect 1) an aberration in HL-60 differentiation reflecting their leukemic phenotype 2) differentiation of HL-60 cells to a cell type characteristic of "activated" PMN.
用二甲基亚砜（DMSO）将人早幼粒细胞白血病细胞系HL-60诱导分化为多形核白细胞（PMN）样细胞，为研究分化过程中PMN功能反应的获得提供了一个重要模型。我们先前表明，细胞外基质（ECM）蛋白血小板反应蛋白（TSP）与PMN表面受体结合并促进其黏附与运动。未分化的HL-60细胞对TSP不发生黏附且无运动反应，而向PMN样细胞分化的细胞则表现出TSP介导的黏附和趋化作用，诱导第2天趋化作用明显。随着分化，100至300 nM的TSP可获得最大反应，这一浓度比PMN最大趋化所需浓度低10倍。棋盘分析证实了运动的方向性。识别TSP不同结构域的单克隆抗体抑制趋化作用，提示TSP上多个位点参与其中。虽然NH2末端肝素结合结构域（HBD）和140 kDa COOH末端片段均支持PMN样细胞的趋化作用，但二者的效力均不及完整的TSP。百日咳毒素和霍乱毒素均抑制TSP介导的趋化作用，提示GTP结合蛋白参与其中。毒素作用并非由cAMP水平升高间接导致，因为高浓度的8-溴-cAMP或二丁酰-cAMP均不抑制趋化作用。结合到硝酸纤维素滤膜上的TSP诱导PMN样细胞的定向迁移（趋触性），而非PMN所观察到的随机运动。HBD或140-kDa片段均可刺激趋触性，且针对这两个结构域的单克隆抗体可抑制趋触性。棋盘分析证实是趋触性而非随机迁移。我们的结果表明，PMN样HL-60细胞对TSP的反应与人外周血PMN不同。这些差异可能反映出：1）HL-60分化异常，反映其白血病表型；2）HL-60细胞分化为“活化”PMN特征的细胞类型。
