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Extracellular protein disulfide isomerase regulates ligand-binding activity of ?M?2 integrin and neutrophil recruitment during vascular inflammation.

ABSTRACT: ?2 integrins play a crucial role during neutrophil recruitment into the site of vascular inflammation. However, it remains unknown how ligand-binding activity of the integrin is regulated. Using fluorescence intravital microscopy in mice generated by crossing protein disulfide isomerase (PDI) floxed mice with lysozyme-Cre transgenic mice, we demonstrate that neutrophil PDI is required for neutrophil adhesion and crawling during tumor necrosis factor-?-induced vascular inflammation in vivo. Rescue experiments show that the isomerase activity of extracellular PDI is critical for its regulatory effect on neutrophil recruitment. Studies with blocking anti-PDI antibodies and ?L?2 or ?M?2 null mice suggest that extracellular PDI regulates ?M?2 integrin-mediated adhesive function of neutrophils during vascular inflammation. Consistently, we show that neutrophil surface PDI is important for ?M?2 integrin-mediated adhesion of human neutrophils under shear and static conditions and for binding of soluble fibrinogen to activated ?M?2 integrin. Confocal microscopy and biochemical studies reveal that neutrophil surface PDI interacts with ?M?2 integrin in lipid rafts of stimulated neutrophils and regulates ?M?2 integrin clustering, presumably by changing the redox state of the integrin. Thus, our results provide the first evidence that extracellular PDI could be a novel therapeutic target for preventing and treating inappropriate neutrophil sequestration.

SUBMITTER: Hahm E

PROVIDER: S-EPMC3650702 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Extracellular protein disulfide isomerase regulates ligand-binding activity of αMβ2 integrin and neutrophil recruitment during vascular inflammation.

Hahm Eunsil E Li Jing J Kim Kyungho K Huh Sungjin S Rogelj Snezna S Cho Jaehyung J

Blood 20130304 19

β2 integrins play a crucial role during neutrophil recruitment into the site of vascular inflammation. However, it remains unknown how ligand-binding activity of the integrin is regulated. Using fluorescence intravital microscopy in mice generated by crossing protein disulfide isomerase (PDI) floxed mice with lysozyme-Cre transgenic mice, we demonstrate that neutrophil PDI is required for neutrophil adhesion and crawling during tumor necrosis factor-α-induced vascular inflammation in vivo. Rescu ...[more]

PMID: 23460613

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Project description:BackgroundPlatelet-neutrophil interactions contribute to vascular occlusion and tissue damage in thromboinflammatory disease. Platelet glycoprotein Ibα (GPIbα), a key receptor for the cell-cell interaction, is believed to be constitutively active for ligand binding. Here, we established the role of platelet-derived protein disulfide isomerase (PDI) in reducing the allosteric disulfide bonds in GPIbα and enhancing the ligand-binding activity under thromboinflammatory conditions.MethodsBioinformatic analysis identified 2 potential allosteric disulfide bonds in GPIbα. Agglutination assays, flow cytometry, surface plasmon resonance analysis, a protein-protein docking model, proximity ligation assays, and mass spectrometry were used to demonstrate a direct interaction between PDI and GPIbα and to determine a role for PDI in regulating GPIbα function and platelet-neutrophil interactions. Also, real-time microscopy and animal disease models were used to study the pathophysiological role of PDI-GPIbα signaling under thromboinflammatory conditions.ResultsDeletion or inhibition of platelet PDI significantly reduced GPIbα-mediated platelet agglutination. Studies using PDI-null platelets and recombinant PDI or Anfibatide, a clinical-stage GPIbα inhibitor, revealed that the oxidoreductase activity of platelet surface-bound PDI was required for the ligand-binding function of GPIbα. PDI directly bound to the extracellular domain of GPIbα on the platelet surface and reduced the Cys4-Cys17 and Cys209-Cys248 disulfide bonds. Real-time microscopy with platelet-specific PDI conditional knockout and sickle cell disease mice demonstrated that PDI-regulated GPIbα function was essential for platelet-neutrophil interactions and vascular occlusion under thromboinflammatory conditions. Studies using a mouse model of ischemia/reperfusion-induced stroke indicated that PDI-GPIbα signaling played a crucial role in tissue damage.ConclusionsOur results demonstrate that PDI-facilitated cleavage of the allosteric disulfide bonds tightly regulates GPIbα function, promoting platelet-neutrophil interactions, vascular occlusion, and tissue damage under thromboinflammatory conditions.

Dataset Information

Extracellular protein disulfide isomerase regulates ligand-binding activity of ?M?2 integrin and neutrophil recruitment during vascular inflammation.

Publications

Extracellular protein disulfide isomerase regulates ligand-binding activity of αMβ2 integrin and neutrophil recruitment during vascular inflammation.

Similar Datasets

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