β2 Integrin-mediated Cell-Cell Contact Transfers Active Myeloperoxidase from Neutrophils to Endothelial Cells*

Background: How endothelial cells (ECs) could acquire exogenous neutrophil myeloperoxidase (MPO) is unknown. Results: ECs acquired enzymatically active MPO directly from neutrophils, via β2 integrin-mediated cell-cell contact independent of extracellular MPO release. Conclusion: Neutrophils directly transfer MPO to ECs by cell-cell contact. Significance: Direct delivery of MPO to ECs may contribute to the vascular pathology and dysfunction seen in atherosclerosis and vasculitides. Atherosclerosis and vasculitis both feature inflammation mediated by neutrophil-endothelial cell (EC) contact. Neutrophil myeloperoxidase (MPO) can disrupt normal EC function, although the mechanism(s) by which MPO is transferred to ECs are unknown. We tested the hypothesis that close, β2 integrin-dependent neutrophil-EC contact mediates MPO transfer from neutrophils to ECs. We used sensitive MPO assays and flow cytometry to detect MPO in ECs and demonstrate that ECs acquired MPO when contacted by neutrophils directly but not when ECs and neutrophils were separated in Transwells. The transfer was dependent on neutrophil number, exposure time, and incubation temperature. Transfer occurred in several EC types, increased with endotoxin, was not accompanied by MPO release into the medium, and was not abrogated by inhibiting degranulation to secretagogues. Confocal microscopy showed MPO internalization by ECs with cytoplasmic and nuclear staining. Neutrophils and ECs formed intimate contact sites demonstrated by electron microscopy. Blocking CD11b or CD18 β2 integrin chains, or using neutrophils from CD11b gene-deleted mice, reduced MPO transfer. EC-acquired MPO was enzymatically active, as demonstrated by its ability to oxidize the fluorescent probe aminophenyl fluorescein in the presence of a hydrogen peroxide source. The data suggest an alternative to EC uptake of soluble MPO, namely the cell contact-dependent, β2 integrin-mediated transfer from neutrophils. The findings could be of therapeutic relevance in atherosclerosis and vasculitis.