Anoxia/reoxygenation-induced neutrophil adherence to cultured endothelial cells.

Abstract

Previous studies have shown enhanced neutrophil adhesion to endothelial cells exposed to anoxia and then reoxygenated (A/R). To define the molecular basis for these observations, we evaluated the relative roles of CD11/CD18 determinants (CD11a and CD11b) of neurtrophils and the endothelial adhesion proteins intercellular adhesion molecule 1 (ICAM-1) and endothelial-leukocyte adhesion molecule 1 (ELAM-1). Human umbilical vein endothelial cell (HUVEC) monolayers were exposed to anoxia for 30 min, reoxygenated, and then reacted with 51Cr-labeled neutrophils in adhesion assays. Neutrophil adhesion to HUVEC exposed to A/R was significantly increased (2.7-fold) as compared with that observed with normoxic (control) HUVEC. This A/R-induced hyperadherence was significantly diminished by monoclonal antibodies (MAb) directed at CD11a, CD11b, CD18 or ICAM-1, but not by MAb directed at ELAM-1. The inhibitory effects of anti-CD11a and anti-CD11b were additive and equivalent to that of anti-CD18 MAb. A/R did not elicit increased levels of ICAM-1 or ELAM-1 mRNA or surface protein. However, immunofluorescence flow cytometry indicated that incubation of neutrophils in supernatants of A/R-conditioned HUVEC elicited an increase of surface CD11b and CD18, but not CD11a. Supernatants from A/R-conditioned HUVEC promoted neutrophil adherence to naive HUVEC, and this hyperadhesivity was diminished by a platelet-activating factor (PAF) receptor antagonist and catalase but not by a 5-lipoxygenase inhibitor, a leukotriene B4 receptor antagonist, or superoxide dismutase. These studies indicate that A/R promotes neutrophil adherence via CD11a/CD18- and CD11b/CD18-dependent interactions with ICAM-1 that appear to be mediated by hydrogen peroxide and PAF.

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