Mechanisms for leukocyte-mediated adjustment of endothelial barrier function

Sammanfattning: Extravasation of polymorphonuclear leukocytes (PMN) and associated plasma exsudation are key events in the inflammatory process. The mechanism by which activated PMN may induce increased vascular permeability for macromolecules is not fully understood but crucially dependent on an intact leukocytic beta2 integrin (CD11/CD18) adhesion receptor function. In this study, a method based on measurement of electrical resistance across confluent monolayers of endothelial cells (EC), cultured on permeable membranes and mounted in a two-compartment diffusion chamber, was utilized to analyze PMN activationinduced effects on endothelial junction integrity. Adding PMN to the upper compartment and a chemotactic stimulus (fMLP or LTB4) to the lower compartment resulted in a rapid decline in transendothelial electrical resistance (TEER), followed by an increase in protein flux and PMN migration across the EC monolayer. Addition of both PMN and the chemotactic stimulus to the upper compartment similarly induced a decline in transendothelial electrical resistance and increase in protein flux together with a rise in EC [Ca2+]i, but no PMN transmigration. When non-activated PMN or chemotactic stimulus alone was added to monolayers, no change in TEER, was observed, indicating that the presence of both PMN and chemoattractant was required to induce the EC response. Furthermore, inhibition of PMN adhesion to the EC monolayer by pretreatment with anti-CD18 mAb prevented all EC responses to chemotactic PMN stimulation. These findings suggest that activated PMN via CD 11 /CD I 8-dependent adhesion but irrespective of PMN transmigration, may directly affect EC barrier capacity. The role of transmembrane signaling by beta2 integrins in PMN-induced alterations in junctional permeability of cultured EC monolayers was investigated next. PMN activation, in the absence of proinflammatory stimuli, was accomplished through antibody cross-linking of CD I 1b/CD 18, mimicking adhesion-dependent receptor engagement. Activation of PMN through antibody cross-linking of CD18 provoked a prompt increase in EC permeability that coincided with a rise in EC cytosolic free Ca 2' and rearrangement of actin filaments, events similar to those evoked by chemoattractant PMN activation. Cellfree supernatant obtained after CD18 cross-linking in suspended PMN 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 beta2 integrin engagement inasmuch as antibody cross-linking of distinct cell surface molecules (L-selectin or CD44) was without effect on EC function. The data demonstrate a causal link between outside-in signaling by beta2 integrins and the capacity of PMW to induce alterations in vascular permeability and suggest a paracrine mechanism in the crosstalk between activated PMN and EC. Investigation of the suggested paracrine mechanism revealed that, consequent to neutrophil adhesion to the endothelial lining, leukocytic beta2 integrin signaling triggers the release of heparin binding protein (HBP), also known as CAP37/azurocidin, a member of the serprocidin family of neutrophil cationic proteins. Adding recombinant human HBP to the luminal side of the EC monolayer elicited Ca dependent cytoskeletal rearrangement and gap formation, and a dose-dependent increase in protein flux. Further, HBP provoked leakage of plasma from postcapillary and small venules when added to the hamster cheek pouch microvasculature in vivo. Removal of HBP from the secreted material by specific immunoadsorption rendered the PMN secretion totally inactive with regard to its potency to cause EC hyperpermeability. These data suggest a fundamental role of neutrophil-derived HBP in mediating the alteration in vessel wall permeability in response to PMN trafficking in inflammation. Bradykinin (BK) derived from the contact phase system, is a potent vasoactive peptide that, induces EC cytoskeletal rearrangement and paracellular gap formation at sites of inflammation. It was found that a BK receptor antagonist and a mAb against BK largely attenuated the increase in permeability of cultured EC monolayers evoked by stimulation with either HBP or activated PMN, suggesting a coupling between the EC response to PMN stimulation and activation of the contact system. Displacement of the major components of the contact system, plasma prekallikrein and H-kininogen, from the endothelial surface prevented HBP-induced hyperpermeability. Similarly, this treatment prevented changes in EC permeability induced by activated PMN. These data suggest a novel pathway involving the contact system by which activated PMN, via the release of HBP, act to regulate the endothelial barrier function at sites of inflammation.