Wong W S, Simon D I, Rosoff P M, Rao N K, Chapman H A
Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
Immunology. 1996 May;88(1):90-7. doi: 10.1046/j.1365-2567.1996.d01-646.x.
Stimulation of monoblastic U937 cells with transforming growth factor beta 1 and 1,25-(OH)2 vitamin D3 (TGF-beta 1/D3) upregulates urokinase receptor (uPAR) and confers urokinase-dependent adhesiveness to the cells for serum- or vitronectin-coated surfaces. Recent studies show that uPAR itself is a high-affinity adhesion receptor for vitronectin and that urokinase (uPA) is an activator of this adhesive function. In the course of exploring possible G-protein involvement in this adhesion it was observed that TGF-beta 1/D3-primed U937 cells became adhesive to vitronectin in an uPAR-dependent manner when exposed to pertussis toxin (PTX). The adherent response is concentration- and time-dependent, and was not due to the ADP-ribosyltransferase activity of the toxin because the purified B-subunit of PTX was equally effective. Although promoting adhesion to serum- or vitronectin-coated surfaces, PTX blocked spontaneous cell adhesion to fibrinogen, an endogenous ligand for the Mac-1 receptor (CD11b/CD18). Flow cytometry study showed that expression of the alpha-subunit of Mac-1 (CD11b) on primed cells was increased by nearly threefold. Monoclonal antibody to CD11b abolished the PTX-induced cell adhesion and the binding of the primed cells to PTX-coated plates. Activation of Mac-1 receptor by its endogenous ligand fibrinogen induced cell adherent response similar to PTX. PTX, but not uPA, triggered a rapid rise in [Ca2+]i in primed U937 cells, and PTX-induced adhesion was significantly attenuated by 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy-methyl ester (BAPTA/AM), a selective membrane-permeant [Ca2+]i chelator. PTX-induced cell adhesion was also prevented by antibodies to uPAR and by conditioned medium containing soluble uPAR. Together these data indicate that PTX B-subunit may bind to Mac-1 integrin, which leads to a rapid rise in [Ca2+]i and subsequent activation of uPAR for adherence to vitronectin, suggesting a functional link between Mac-1 and activation of uPAR important to cellular trafficking and host defence in response to Bordetella pertussis infection.
用转化生长因子β1和1,25 -（OH）2维生素D3（TGF -β1/D3）刺激单核细胞U937细胞可上调尿激酶受体（uPAR），并赋予细胞对血清或玻连蛋白包被表面的尿激酶依赖性黏附性。最近的研究表明，uPAR本身是玻连蛋白的高亲和力黏附受体，尿激酶（uPA）是这种黏附功能的激活剂。在探索G蛋白可能参与这种黏附的过程中，观察到当暴露于百日咳毒素（PTX）时，经TGF -β1/D3预处理的U937细胞以uPAR依赖性方式黏附于玻连蛋白。黏附反应呈浓度和时间依赖性，且不是由于毒素的ADP -核糖基转移酶活性，因为纯化的PTX B亚基同样有效。尽管PTX促进细胞黏附于血清或玻连蛋白包被的表面，但它阻断细胞对纤维蛋白原（Mac -1受体（CD11b/CD18）的内源性配体）的自发黏附。流式细胞术研究表明，预处理细胞上Mac -1（CD11b）α亚基的表达增加了近三倍。抗CD11b单克隆抗体消除了PTX诱导的细胞黏附以及预处理细胞与PTX包被平板的结合。其内源性配体纤维蛋白原对Mac -1受体的激活诱导了类似于PTX的细胞黏附反应。PTX而非uPA引发预处理的U937细胞中[Ca2+]i的快速升高，并且1,2 -双 -（2 -氨基苯氧基）乙烷 - N,N,N',N' -四乙酸/乙酰氧甲基酯（BAPTA/AM，一种选择性膜渗透性[Ca2+]i螯合剂）显著减弱了PTX诱导的黏附。抗uPAR抗体和含有可溶性uPAR的条件培养基也可阻止PTX诱导的细胞黏附。这些数据共同表明，PTX B亚基可能与Mac -1整合素结合，这导致[Ca2+]i快速升高以及随后uPAR的激活以黏附于玻连蛋白，提示Mac -1与uPAR激活之间的功能联系对百日咳博德特氏菌感染时的细胞转运和宿主防御很重要。
