Senior R M, Gresham H D, Griffin G L, Brown E J, Chung A E
Department of Medicine, Jewish Hospital, Washington University Medical Center, St. Louis, Missouri 63110.
J Clin Invest. 1992 Dec;90(6):2251-7. doi: 10.1172/JCI116111.
Entactin is an integral component of basement membranes that plays a major role in basement membrane assembly through its ability to bind avidly to both laminin and type IV collagen. Because neutrophil (PMN) interactions with entactin have not been examined, we investigated the ability of natural and recombinant entactin to mediate PMN adhesion and chemotaxis. With both forms of entactin, we observed that entactin-coated surfaces promoted PMN adhesion and that entactin stimulated PMN chemotaxis. The increase in adhesion to entactin over control was two to threefold whereas the chemotactic response to 15 ng/ml (1 x 10(-10) M) entactin was equivalent to the chemotactic response elicited with 1 x 10(-8) M formyl-methionyl-leucyl-phenylalanine (fMLP). HL-60 cells, after differentiation with dimethylsulfoxide, also demonstrated adhesion and chemotaxis to entactin. A synthetic peptide of the Arg-Gly-Asp (RGD) domain in entactin, SIGFRGDGQTC (S-RGD), mediated PMN adhesion and chemotaxis, and preexposure of PMN to S-RGD blocked PMN adhesion and chemotaxis induced by entactin without diminishing the adhesive and chemotactic activities of fMLP. In contrast, preexposure to peptides SIGFRGEGQTCA or SIGFKGDGQTCA had no effect. The findings with synthetic peptides were confirmed with a recombinant entactin mutant in which aspartic acid at residue 674 was replaced with glutamic acid, thus converting the RGD sequence of entactin to RGE. RGE-entactin was neither adhesive nor chemotactic for neutrophils. Monoclonal antibodies to the leukocyte response integrin (LRI) and the integrin-associated protein blocked entactin-mediated adhesion and chemotaxis whereas monoclonal antibodies to beta 1 and beta 2 integrins had no effect and PMN from an individual with leukocyte-adhesion deficiency adhered normally to entactin-coated surfaces. These data demonstrate that entactin mediates biologically and pathologically important functions of PMN through its RGD domain and that LRI, which has been shown previously to mediate RGD-stimulated phagocytosis, is also capable of mediating RGD-stimulated PMN adhesion and chemotaxis.
巢蛋白是基底膜的一个组成部分，通过其与层粘连蛋白和IV型胶原的紧密结合能力，在基底膜组装中起主要作用。由于尚未研究中性粒细胞（PMN）与巢蛋白的相互作用，我们研究了天然和重组巢蛋白介导PMN粘附和趋化作用的能力。使用两种形式的巢蛋白，我们观察到包被巢蛋白的表面促进了PMN的粘附，并且巢蛋白刺激了PMN的趋化作用。与对照相比，对巢蛋白的粘附增加了两到三倍，而对15 ng/ml（1×10⁻¹⁰ M）巢蛋白的趋化反应与1×10⁻⁸ M甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）引发的趋化反应相当。经二甲基亚砜分化后的HL-60细胞也表现出对巢蛋白的粘附和趋化作用。巢蛋白中精氨酸-甘氨酸-天冬氨酸（RGD）结构域的合成肽SIGFRGDGQTC（S-RGD）介导了PMN的粘附和趋化作用，并且PMN预先暴露于S-RGD可阻断巢蛋白诱导的PMN粘附和趋化作用，而不会降低fMLP的粘附和趋化活性。相反，预先暴露于肽SIGFRGEGQTCA或SIGFKGDGQTCA则没有效果。用重组巢蛋白突变体证实了合成肽的研究结果，其中第674位残基的天冬氨酸被谷氨酸取代，从而将巢蛋白的RGD序列转化为RGE。RGE-巢蛋白对中性粒细胞既无粘附性也无趋化性。针对白细胞反应整合素（LRI）和整合素相关蛋白的单克隆抗体可阻断巢蛋白介导的粘附和趋化作用，而针对β1和β2整合素的单克隆抗体则没有作用，并且来自白细胞粘附缺陷个体的PMN能正常粘附于包被巢蛋白的表面。这些数据表明，巢蛋白通过其RGD结构域介导PMN的生物学和病理学重要功能，并且先前已证明能介导RGD刺激的吞噬作用的LRI，也能够介导RGD刺激的PMN粘附和趋化作用。
