Background/Purpose: Dysregulation of neutrophil activation is important in the pathogenesis of various inflammatory and autoimmune rheumatic diseases (ARDs), including RA, SLE and APS. Neutrophils are exquisitely sensitive to their environment and neutrophil function can be modulated by numerous factors including cytokines, circulating IgG and localised tissue hypoxia (0.5-2.7% O₂). One mechanism of neutrophil activation results in the release of a meshwork of chromatin fibres decorated with antimicrobial proteins, called neutrophil extracellular traps (NETs). Aberrant NETosis has been described in various ARDs. We have previously shown that purified IgG and hypoxia promotes NETosis, however the underlying mechanism is unknown. Integrin engagement modulates several aspects of neutrophil function including: adhesion, cytokine production, NETosis and reactive oxygen species (ROS) generation. Therefore, we investigated the effects of hypoxia upon neutrophil integrin expression, adhesion to immobilised ligands and endothelial cells and trans-endothelial migration (TEM) to better understand the effects of hypoxia upon the stages preceding NETosis.

Methods: Neutrophils were isolated from whole blood donated by healthy controls (HC) and cultured under normoxia (21% O₂) or hypoxia (1% O₂). NETosis was visualised by immunofluorescence (IF) staining for histone H3. Levels of surface integrin expression of α₁ (CD49a), α₄ (CD49d), α₅ (CD49e), α_L (CD11a), α_M (CD11b), α_X (CD11c), β₁ (CD29) and β₂(CD18) were measured by flow cytometry. Assessment of 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM)-labelled neutrophil adhesion to immobilised fibrinogen, fibronectin and intercellular adhesion molecule (ICAM)-1 and human umbilical cord endothelial cells (HUVEC) was measured using a fluorescent plate reader. Neutrophil TEM across HUVEC monolayers was measured by flow cytometry.

Results: IF studies found that NETosis was inhibited by EDTA treatment, indicating cation-dependent integrin involvement. Moreover, NETosis could be induced by integrin activation with either Mn²⁺ or macrophage-1 antigen (Mac-1, α_Mβ₂)-specific activation with leukadherin-1. Hypoxia significantly increased expression of α_M (p<0.001) and α_X(p=0.038) in HC neutrophils compared to normoxia (n=7). Adhesion to immobilised fibrinogen, fibronectin and ICAM-1 were all significantly reduced under hypoxia (p<0.01, p<0.01 and p<0.01 respectively). In contrast, hypoxia enhanced both unstimulated (p<0.01) and LPS-stimulated (p<0.001) adhesion to and TEM across HUVEC monolayers (p<0.05) compared to normoxic controls.

Conclusion: Hypoxia significantly increased neutrophil: expression of α_M and α_X integrin subunits; adhesion to HUVEC but not immobilised integrin ligands; and TEM across HUVEC monolayers. Further work is currently underway to dissect the molecular and signalling mechanisms that regulate neutrophil function under hypoxia and whether ARD-IgG further modulate these responses.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/hypoxia-modulates-neutrophil-integrin-expression-adhesion-and-trans-endothelial-migration/