Mechano-Redox Control of Mac-1 De-Adhesion by PDI Promotes Directional Movement Under Flow

Abstract

BACKGROUND: Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMβ2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. METHODS: Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. RESULTS: PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the βI domain of the β2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the βI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. CONCLUSIONS: Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

Not all (cells) who wander are lost: Upstream migration as a pervasive mode of amoeboid cell motility

“…Association of PDI with the α M chain, but not cleavage, lead to significant upstream migration when compared to unbound control, similar to the antibody blocking studies designed by Buffone et al (Buffone et al, 2019). In both studies (Buffone et al, 2019;Dupuy et al, 2023), globally blocking Mac-1 with oxidized PDI or monoclonal antibodies, lead to over 65% of cells crawling upstream. Dupuy and coworkers' result indicates that the engagement of PDI alone can promote upstream migration in neutrophils.…”

“…We believe the difference between our results and the results from the Theodoly laboratory is due to differences in ICAM-1 density; we used lower densities of ICAM-1 which seems to have enhanced the mobility of neutrophils (Valignat et al, 2013;Buffone et al, 2019) A recent paper by Dupuy et al expanded upon the interplay between Mac-1 and LFA-1 in coordinating neutrophil upstream migration (Dupuy et al, 2023). By treating neutrophils with Protein disulfide Isomerase (PDI), Dupuy et al demonstrated that PDI localizes specifically to Mac-1 at the trailing edge of the cell and selectively cleaves disulfide bonds, leading to Mac-1 release of ICAM-1.…”

Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment

“…In support of our hypothesis, the roles of extracellular thiol isomerase in the regulation of Mac-1 activity were previously documented, though with discrepancies. 27,28 Using gene conditional knockout mice, Hahm et al 27 showed that PDI increases the binding affinity of Mac-1 by regulating thiol exchange in CD11b and the clustering of Mac-1 without affecting its conformation. A more recent study showed that PDI cleaves the C224-C264 bond of CD18 to induce mechanical stress in the βI domain, which allosterically alters the exposure of the αI domain and decreases the binding affinity of Mac-1.…”

“…27 A more recent study showed that cleavage of a disulfide bond in the βI domain by extracellular PDI selectively regulates the disengagement of Mac-1 with ICAM-1 under shear. 28 In addition to PDI, ERp72 (endoplasmic reticulum–resident protein 72) was also reported on the surface of neutrophils and implicated in neutrophil activation. 29 It was reported that a polyclonal antibody against ERp72 suppressed neutrophil adhesion on activated endothelial cells.…”

Immune Thrombosis: Exploring the Significance of Immune Complexes and NETosis

“…Endothelial inflammation (thromboinflammation) involves the upregulation of adhesion molecules including E-selectin, P-selectin, and intracellular adhesion molecule-1 (ICAM-1) [84], which are critical in the development of thrombotic diseases including stroke and cardiovascular events. Neutrophils are attracted to damaged endothelium, attach via integrins such as Mac-1 [85], and stimulate further inflammation, the formation of neutrophil-platelet aggregates and promote atherosclerosis [86]. NETs stimulate endothelial inflammation through the activity of NETs components such as cathepsin G [87].…”