Mechanisms of pertussis toxin‐induced myelomonocytic cell adhesion: role of Mac‐1(CD11b/CD18) and urokinase receptor (CD87)

Abstract

Stimulation of monoblastic U937 cells with transforming growth factor β1 and 1,25-(OH)₂ vitamin D₃ (TGF-β1/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-β1/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 α-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-NNN ′,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.