Mac-1 Receptor Clustering Initiates Production of Pro-Inflammatory, Antibacterial Extracellular Vesicles From Neutrophils

Abstract

Depending on the prevailing environmental conditions, neutrophilic granulocytes release extracellular vesicles (EV) which have either anti-inflammatory effects on other neutrophils or pro-inflammatory and antibacterial effects. In the present study we investigated the molecular mechanisms underlying the biogenesis of functionally heterogenic EVs. We show that selective stimulation of Mac-1 integrin (complement receptor 3) by specific ligands initiates the generation of EVs which are able to impair bacterial growth and to induce the secretion of the pro-inflammatory cytokine IL-8 (aEV). However, direct Mac-1 stimulation results in aEV release only if neutrophils were activated on ligand coated surfaces whereas soluble ligands are ineffective. Using total internal reflection fluorescence (TIRF) microcopy, an increased clustering of Mac-1 molecules could be visualized in neutrophils added to C3bi coated surfaces; moreover antibody induced cluster formation triggers aEV release as well. Mac-1 induced production of aEV apparently necessitates a strong calcium signal as it fully depends on the presence of extracellular calcium. However, initiation of a strong calcium signal by an ionophore only results the generation of EV devoid of any antibacterial or pro-inflammatory effect. Our results thus demonstrate that stimulation and clustering of Mac-1 is necessary and sufficient for initiation of aEV biogenesis. In contrast, an intracellular calcium signal is necessary but by itself not sufficient for the production of antibacterial and pro-inflammatory EVs.

The roles of extracellular vesicles in the immune system

“…Neutrophils release a broad spectrum of EVs 79 , the composition of which depends on the environmental conditions at the time of EV production 80 . Of note, the same population of neutrophils can secrete either pro-inflammatory or anti-inflammatory EVs, with clustering of the complement receptor Mac1 (CD11B–CD18) on neutrophils functioning as a switch from anti-inflammatory to pro-inflammatory EV production 81 . Stimulation of neutrophils by opsonized particles induces the release of bacteriostatic EVs, which form large aggregates with bacteria 82 .…”

The human factor H protein family – an update

“…FH can play a role in the recruitment of leukocytes to the site of damage/inflammation by having a chemotactic effect on monocytes and neutrophils on its own or when bound to Candida albicans or endothelial cells ( 195 , 287 , 293 , 314 – 316 ). In addition, surface-bound but not soluble FH was shown to enhance the release of extracellular vesicles from neutrophils ( 317 ). The combination of FH with other stimuli may have different effect, since upon LPS mediated activation of monocyte-derived DCs (MoDCs), the FH treatment induced downregulation of CCR7 and reduced MoDC migration toward the chemokine CCL21, pointing to a tolerogenic, anti-inflammatory role of FH ( 318 ).…”

Beyond host defense and tissue injury: the emerging role of neutrophils in tissue repair

“…Neutrophil microvesicles typically express CD66b, CD11b, CD18, MPO and to varying extent phosphatidylserine on their surface ( 157 ), but are heterogeneous in composition ( 158 , 159 ), likely reflecting the activation state of the parent cell. For example, microvesicles generated from adherent neutrophils express the anti-inflammatory proresolution protein AnxA1 on their surface ( 160 ), whereas Mac-1 ligation and clustering evoke the release of proinflammatory, antibacterial microvesicles ( 161 ). Consistent with the heterogeneity in composition, neutrophil microvesicles likely exert divergent and cell type-selective actions.…”