Abstract 39: Protein Disulfide Isomerase Plays an Important Role in αMβ2 Integrin-Mediated Neutrophil Recruitment During Vascular Inflammation

Abstract

Introduction: Protein disulfide isomerase (PDI), a cell-surface localized oxidoreductase, plays a crucial role in regulating integrin-mediated platelet functions. Although PDI is detected on the neutrophil surface, its role in neutrophil recruitment during vascular inflammation remains unknown. Objectives: To determine the physiological role of neutrophil surface PDI in integrin-mediated neutrophil recruitment during vascular inflammation. Methods and Results: Using real-time fluorescence intravital microscopy in myeloid-specific PDI knockout mice, we have demonstrated that neutrophil PDI is required for neutrophil recruitment into the site of TNF-alpha-induced cremaster venular inflammation. Infusion of wild-type PDI but not functionally-inactive PDI rapidly rescued the defect of neutrophil recruitment in the PDI knockout mice. Cell-impermeable PDI inhibitors also perturbed neutrophil recruitment to the site of TNF-alpha-inflamed venular inflammation in wild-type mice. Neutrophil recruitment was further inhibited when blocking anti-PDI antibodies were infused into alphaLbeta2 but not alphaMbeta2 knockout mice, suggesting that extracellular PDI regulates alphaMbeta2-mediated neutrophil recruitment. To further investigate the role of neutrophil surface PDI in integrin-mediated neutrophil adhesion during vascular inflammation, adhesion assays were performed using human neutrophils treated with cell-impermeable PDI inhibitors. PDI inhibition impaired human neutrophil adhesion to TNF-alpha-activated endothelial cells under venous shear and to ICAM-1-coated surfaces under static conditions. Neutrophil adhesion was further inhibited when anti-PDI was combined with anti-alphaL but not anti-alphaM antibodies. PDI inhibitors also perturbed fibrinogen binding to activated alphaMbeta2 integrin. Using PDI shRNA in neutrophil-like HL60 cells, we found that knockdown of PDI gene diminished alphaMbeta2-mediated cell adhesion to activated endothelial cells under shear. Immunoprecipitation assays and confocal microscopy revealed that surface PDI-alphaMbeta2 interaction is enhanced upon neutrophil activation and regulated by the isomerase activity. Conclusion: Our results indicate that neutrophil surface PDI regulates the alphaMbeta2-ligand interaction, thereby playing an important role in neutrophil recruitment to the activated endothelium during vascular inflammation.