Autoimmune rheumatic disease IgG has differential effects upon neutrophil integrin activation that is modulated by the endothelium

Abstract

The importance of neutrophils in the pathogenesis of autoimmune rheumatic diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), is increasingly recognised. Generation of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs) by activated neutrophils are both thought to contribute to pathology; although the underlying mechanisms, particularly the effects of IgG autoantibodies upon neutrophil function, are not fully understood. Therefore, we determined whether purified IgG from patients with SLE or RA have differential effects upon neutrophil activation and function. We found that SLE- and RA-IgG both bound human neutrophils but differentially regulated neutrophil function. RA- and SLE-IgG both increased PMA-induced β1 integrin-mediated adhesion to fibronectin, whilst only SLE-IgG enhanced αMβ2 integrin-mediated adhesion to fibrinogen. Interestingly, only SLE-IgG modulated neutrophil adhesion to endothelial cells. Both SLE- and RA-IgG increased ROS generation and DNA externalisation by unstimulated neutrophils. Only SLE-IgG however, drove DNA externalisation following neutrophil activation. Co-culture of neutrophils with resting endothelium prevented IgG-mediated increase of extracellular DNA, but this inhibition was overcome for SLE-IgG when the endothelium was stimulated with TNF-α. This differential pattern of neutrophil activation has implications for understanding SLE and RA pathogenesis and may highlight avenues for development of novel therapeutic strategies.

Identification of a novel HIF-1α-αMβ2Integrin-NETosis axis in fibrotic interstitial lung disease

“…Neutrophils were isolated as previously described 73 . In brief, neutrophils were isolated by Percoll density centrifugation from sodium citrate anticoagulated blood obtained by informed consent from healthy volunteers.…”

“…NETs were stained for immunofluorescence microscopy as described 73 . In brief, 5x10 5 neutrophils were added to coverslips, stimulated, and then fixed with 4% PFA.…”

“…24 hours prior to experimentation, HUVEC were subjected to normoxia or hypoxia in the absence or presence of 10ng/ml TNF-α. Neutrophil adhesion in response to 20nM PMA or 100ng/ml LPS were measured as previously described 73…”

“…Trans-endothelial migration assays were performed as previously described 73 . In brief, HUVEC were grown on transwell inserts (Millipore, UK).…”

“…2 generation was measured as previously described73 . Briefly, neutrophils were cultured under normoxia or hypoxia for 1 hour before addition of HRP (Sigma, UK) and Amplex® UltraRed (Invitrogen, UK).…”