Abstract Number: PB1963
Meeting: ISTH 2020 Congress
Background: The αMβ2 integrin (or Mac-1) controls neutrophil mobilization during inflammation by binding to multiple ligands, including ICAM-1, fibrinogen and CD40-L. Thiol oxidoreductase PDI binds to Mac-1 and promotes neutrophil trafficking in animal models, however the mechanism is not understood.
Aims: We hypothesized that PDI regulates neutrophil trafficking by reduction and/or oxidation of disulphide bonds in Mac-1.
Methods: The effect of PDI on the redox state of Mac-1 disulphides was determined by differential cysteine alkylation-coupled mass spectrometry. The binding of ligands to Mac-1 was measured by flow cytometry and plate adhesion using BHK cells expressing wild type or disulphide bond mutant Mac-1 (Cys to Ser substitutions). Neutrophil adhesion to endothelial cells and ligands under shear was measured by microfluidics. Neutrophil trafficking in vivo was measured using a tail-wounding model in Tg(lyzC:gfp) zebrafish.
Results: PDI reduced 2 of the 28 disulphide bonds in the β2 subunit of Mac-1; Cys169-Cys176 and Cys224-Cys264. Under static conditions, ablation of both disulphides Cys169-Cys176 and Cys224-Cys264 resulted in decreased adhesion to fluorescently-labelled fibrinogen, however only the absence of Cys224-Cys264 decreased adhesion to ICAM-1 detected by flow cytometry with anti-ICAM-1 Ab (Figure 1A, 1B). PDI was detected at the leading edge and trailing end of mobilizing neutrophils and colocalised with β2 integrin. PDI inhibitory antibody RL90 decreased adhesion of fMLF-primed neutrophils under shear (0.35 dynes/cm2) to ICAM-1 but not to fibrinogen (Figure 1C). The PDI small molecule inhibitor isoquercetin decreased adhesion of neutrophils to endothelial cells stimulated with TNF-α (Figure 1D) and neutrophil migration to the site of zebrafish tail injury (Figure 1E).
Conclusions: PDI decreases the affinity of Mac-1 for fibrinogen and ICAM-1 by reducing disulphide bonds in the β2 subunit. The consecutive reduction-oxidation of Mac-1 disulphide bonds may facilitate cycles of adhesion-de-adhesion to substrate, facilitating neutrophil mobilization. Blocking of PDI function may inhibit neutrophil mobilization to sites of inflammation.
To cite this abstract in AMA style:Dupuy A, Oehlers S, Hagimola L, Gomez C, Zheng S, Hogg P, Chiu J, Passam F. Redox Regulation of Neutrophil Mac-1 Function by Protein Disulphide Isomerase [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/redox-regulation-of-neutrophil-mac-1-function-by-protein-disulphide-isomerase/. Accessed January 28, 2022.
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