Signaling via b 2 Integrins Triggers Neutrophil-dependent Alteration in Endothelial Barrier Function

Activation of polymorphonuclear leukocytes (PMNs) and adhesion to the endothelial lining is a major cause of edema formation. Although known to be dependent on the function of b 2 integrins (CD11/CD18), the precise mechanisms by which adherent PMNs may impair endothelial barrier capacity remain unclear. Here, the role of transmembrane signaling by b 2 integrins in PMN-induced alterations in tight junctional permeability of cultured endothelial cell (EC) monolayers was investigated. PMN activation, in the absence of proinflammatory stimuli, was accomplished through antibody cross-linking of CD11b/CD18, mimicking adhesion-dependent receptor engagement. CD18 cross-linking in PMNs added to the EC monolayer provoked a prompt increase in EC permeability that coincided with a rise in EC cytosolic free Ca 2 1 and rearrangement of actin filaments, events similar to those evoked by chemoattractant PMN activation. Cell-free supernatant obtained after CD18 cross-linking in suspended PMNs triggered an EC response indistinguishable from that induced by direct PMN activation, and caused clear-cut venular plasma leakage when added to the hamster cheek pouch in vivo preparation. The PMN-evoked EC response was specific to b 2 integrin engagement inasmuch as antibody cross-linking of l -selectin or CD44 was without effect on EC function. Our data demonstrate a causal link between outside-in signaling by b 2 integrins and the capacity of PMNs to induce alterations in vascular permeability, and suggest a paracrine mechanism that involves PMN-derived cationic protein(s) in the cellular crosstalk between PMNs and ECs. light microscopy 15 min after induction of CD18 cross-linking. In Vivo Determination of Microvessel Permeability Changes. To study the effect of PMN secretion products on microvessel permeability in vivo, intravital microscopy of the hamster cheek pouch microcirculation was applied. As described previously in detail (19), the left cheek pouch of anesthetized Syrian golden hamsters was everted and prepared for microscopic observation under continuous superfusion with a bicarbonate buffer maintain-ing physiological levels of temperature, pH, and gas tensions. FITC-conjugated dextran (mol wt: 150,000) injected intravenously (250 mg/kg body weight) was used as plasma tracer to permit visualization of changes in vascular permeability to macromolecules. The hamsters were treated with fucoidin (10 mg/kg intravenously; Sigma-Aldrich) to prevent leukocyte adhesion in the microvessels (20) and in that way eliminate influences of potential activation of endogenous leukocytes. The microvascula-ture in the exposed cheek pouch was observed under low magni-fication in fluorescent light with a Leitz Orthoplan microscope (Leica) and recorded on video. After control recordings, superfusion was stopped and 1 ml of medium containing CD18 cross-linking–evoked PMN secretion at 37 8 C was topically applied to the cheek pouch. In parallel experiments, medium of anti-CD18–treated PMNs not subjected to cross-linking was used. The animal experiments were approved by the regional ethical committee for animal experimentation. the that stimulus-induced initiates secretion to b 2 integrin signaling, which capacitates changes in endothelial barrier function that are independent of PMN transmigration. These provide novel information with regard to the role of b 2 integrins in PMN-evoked effects on vessel wall permeability in conjunction with neutrophil