Protein Disulfide Isomerase Plays An Important Role in Beta2 Integrin-Mediated Neutrophil Recruitment During Vascular Inflammation

Abstract 17

Cell surface protein disulfide isomerase (PDI), a prototypic thiol isomerase, plays an important role in regulating integrin-mediated cellular functions. Although surface PDI has been reported to regulate L-selectin shedding on neutrophils, its role in neutrophil recruitment during vascular inflammation has not been explored. In this paper, we present novel findings on how neutrophil PDI regulates neutrophil recruitment into the site of vascular inflammation. Using real-time microscopy with cell-impermeable inhibitors for PDI (bacitracin or function-blocking antibodies), we demonstrated that inhibition of PDI reduces adhesion of human blood neutrophils to TNF-alpha-stimulated human umbilical vein endothelial cells (HUVECs) under venous shear. There was no additive effect on neutrophil adhesion in a combination of blocking anti-PDI and anti-beta2 antibodies, suggesting that surface PDI regulates beta2 integrin-mediated neutrophil adhesion to TNF-alpha-activated HUVECs under shear. Further, bacitracin and blocking anti-PDI antibodies diminished alphaMbeta2 integrin-mediated neutrophil adhesion to intercellular adhesion molecule-1 (ICAM-1)-coated surfaces under static conditions. When PDI gene is knocked down in neutrophil-like HL60 cells by shRNA, alphaMbeta2 activation and cell adhesion to TNF-alpha-activated HUVECs under shear were significantly reduced. High resolution confocal microscopy and immunoprecipitation assay revealed that surface PDI interacts with alphaMbeta2 integrin and that such interaction was enhanced by neutrophil activation and inhibited by bacitracin, a thiol isomerase inhibitor. Exogenous PDI equivalently bound to CHO cells stably expressing alphaMbeta2 or alphaLbeta2 when those cells were treated with Mn2+, whereas PDI binding was significantly reduced when the active site CGHC residues on PDI were mutated. Using multi-channel fluorescence intravital microscopy, we demonstrated that inhibition of PDI by infusion of PDI inhibitors impairs stable adhesion of neutrophils to the TNF-alpha-inflamed cremaster muscle venule wall in living mice. These findings suggest the potentially fundamental but poorly understood role for surface PDI in the regulation of beta2 integrin function in neutrophils. Therefore, our results provide the first evidence that inhibition of PDI may be a therapeutic target for the neutrophil-mediated vascular inflammation.