β2 integrin-mediated cell-cell contact transfers active myeloperoxidase from neutrophils to endothelial cells

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 EC are unknown. We tested the hypothesis that close, beta2-integrin-dependent neutrophil-EC contact mediates MPO transfer from neutrophils to EC. We used sensitive MPO assays and flow cytometry to detect MPO in EC and demonstrate that EC acquired MPO when contacted by neutrophils directly but not when EC 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 EC with cytoplasmic and nuclear staining. Neutrophils and EC formed intimate contact sites demonstrated by electron microscopy. Blocking CD11b or CD18 beta2-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, {beta}2-integrin-mediated transfer from neutrophils. The findings could be of therapeutic relevance in atherosclerosis and vasculitis

Supplementary Material for: Methionine Sulfoxide Reductases Protect against Oxidative Stress in <b><i>Staphylococcus aureus</i></b> Encountering Exogenous Oxidants and Human Neutrophils

To establish infection successfully, <i>Staphylococcus aureus</i> must evade clearance by polymorphonuclear neutrophils (PMN). We studied the expression and regulation of the methionine sulfoxide reductases (Msr) that are involved in the repair of oxidized staphylococcal proteins and investigated their influence on the fate of <i>S. aureus </i>exposed to oxidants or PMN. We evaluated a mutant deficient in <i>msrA1 </i>and <i>msrB </i>for susceptibility to hydrogen peroxide, hypochlorous acid and PMN. The expression of <i>msrA1</i> in wild-type bacteria ingested by human PMN was assessed by real-time PCR. The regulation of <i>msr </i>was studied by screening a library of two-component regulatory system (TCS) mutants for altered <i>msr</i> responses. Relative to the wild-type bacteria, bacteria deficient in Msr were more susceptible to oxidants and PMN. Upregulation of staphylococcal <i>msrA1 </i>occurred within the phagosomes of normal PMN and PMN deficient in NADPH oxidase activity. Furthermore, PMN granule-rich extract stimulated the upregulation of <i>msrA1.</i> Modulation of <i>msrA1</i> within PMN was shown to be partly dependent on the VraSR TCS. Msr contributes to staphylococcal responses to oxidative attack and PMN. Our study highlights a novel interaction between the oxidative protein repair pathway and the VraSR TCS that is involved in cell wall homeostasis

BK channels in innate immune functions of neutrophils and macrophages

Oxygen-dependent antimicrobial activity of human polymorphonuclear leukocytes (PMNs) relies on the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to generate oxidants. As the oxidase transfers electrons from NADPH the membrane will depolarize and concomitantly terminate oxidase activity, unless there is charge translocation to compensate. Most experimental data implicate proton channels as the effectors of this charge compensation, although large-conductance Ca2+-activated K+ (BK) channels have been suggested to be essential for normal PMN antimicrobial activity. To test this latter notion, we directly assessed the role of BK channels in phagocyte function, including the NADPH oxidase. PMNs genetically lacking BK channels (BK(-/-)) had normal intracellular and extracellular NADPH oxidase activity in response to both receptor-independent and phagocytic challenges. Furthermore, NADPH oxidase activity of human PMNs and macrophages was normal after treatment with BK channel inhibitors. Although BK channel inhibitors suppressed endotoxin-mediated tumor necrosis factor-alpha secretion by bone marrow-derived macrophages (BMDMs), BMDMs of BK(-/-) and wild-type mice responded identically and exhibited the same ERK, PI3K/Akt, and nuclear factor-kappaB activation. Based on these data, we conclude that the BK channel is not required for NADPH oxidase activity in PMNs or macrophages or for endotoxin-triggered tumor necrosis factor-alpha release and signal transduction BMDMs

A structurally dynamic N-terminal region drives function of the staphylococcal peroxidase inhibitor (SPIN)

The heme-containing enzyme myeloperoxidase (MPO) is critical for optimal antimicrobial activity of human neutrophils. We recently discovered that the bacterium Staphylococcus aureus expresses a novel immune evasion protein, called SPIN, that binds tightly to MPO, inhibits MPO activity, and contributes to bacterial survival following phagocytosis. A co-crystal structure of SPIN bound to MPO suggested that SPIN blocks substrate access to the catalytic heme by inserting an N-terminal β-hairpin into the MPO active-site channel. Here, we describe a series of experiments that more completely define the structure/function relationships of SPIN. Whereas the SPIN N terminus adopts a β-hairpin confirmation upon binding to MPO, the solution NMR studies presented here are consistent with this region of SPIN being dynamically structured in the unbound state. Curiously, whereas the N-terminal β-hairpin of SPIN accounts for ∼55% of the buried surface area in the SPIN-MPO complex, its deletion did not significantly change the affinity of SPIN for MPO but did eliminate the ability of SPIN to inhibit MPO. The flexible nature of the SPIN N terminus rendered it susceptible to proteolytic degradation by a series of chymotrypsin-like proteases found within neutrophil granules, thereby abrogating SPIN activity. Degradation of SPIN was prevented by the S. aureus immune evasion protein Eap, which acts as a selective inhibitor of neutrophil serine proteases. Together, these studies provide insight into MPO inhibition by SPIN and suggest possible functional synergy between two distinct classes of S. aureus immune evasion proteins