Helicobacter pyloriDisrupts NADPH Oxidase Targeting in Human Neutrophils to Induce Extracellular Superoxide Release

Abstract

Helicobacter pylori (Hp) infection triggers a chronic influx of polymorphonuclear leukocyte neutrophils (PMNs) into the gastric mucosa. Although Hp reside in a neutrophil-rich environment, how these organisms evade phagocytic killing is largely unexplored. We now show that live Hp (strains 11637, 60190, DT61A, and 11916) are readily ingested by PMNs and induce a rapid and strong respiratory burst that is comparable to PMA. Relative to other particulate stimuli, Hp are more potent activators of PMNs than opsonized zymosan, Staphylococcus aureus, or Salmonella. Strikingly, biochemical and microscopic analyses demonstrate that Hp disrupt NADPH oxidase targeting such that superoxide anions are released into the extracellular milieu and do not accumulate inside Hp phagosomes. Specifically, nascent Hp phagosomes acquire flavocytochrome b558 but do not efficiently recruit or retain p47phox or p67phox. Superoxide release peaks at 16 min coincident with the appearance of assembled oxidase complexes in patches at the cell surface. Oxidant release is regulated by formalin-resistant and heat-sensitive bacterial surface factors distinct from urease and Hp(2–20). Following opsonization with fresh serum, Hp triggers a modest respiratory burst that is confined to the phagosome, and ingested bacteria are eliminated. We conclude that disruption of NADPH oxidase targeting allows unopsonized Hp to escape phagocytic killing, and our findings support the hypothesis that bacteria and PMNs act in concert to damage the gastric mucosa.

Immunofluorescence and Confocal Microscopy of Neutrophils

“…The coating agent will spread evenly over the acid-washed glass, and surface tension holds the domes of liquid in place. 8 We described previously the phenotype of PMNs attached loosely to serum-or fibrinogen-coated coverslips [6][7][8][9][10]12]. Here, we extended these results to show that the ability of PMNs to digest or bleach and migrate along surfaces coated with extracellular matrix proteins can also be analyzed (Figs.…”

“…Summarized here are methods we developed originally for analysis of macrophages [4,5] and then optimized for immunofluorescence and confocal analysis of human neutrophils [6][7][8][9][10][11][12]. Our approaches address general issues such as methods of cell fixation and permeabilization that are also relevant to studies of other cell types.…”

“…We find that Sigma-Aldrich horse serum works well and is cost effective (as compared with FBS, goat serum, or donkey serum (see also Note 6)). 5 We routinely use the following antibodies for immunofluorescence microscopy of human neutrophils [6][7][8][9][10]. To detect NADPH oxidase components, antibodies specific for p22 phox (mAb 44.1) and gp91 phox (mAb 54.1) [20,21] are now available from Santa Cruz Biotechnology (Santa Cruz, CA); mAb 7D5 detects an epitope of gp91 phox present in mature flavocytochrome b 558 [22] and can be used on intact PMNs to detect protein upregulation at the cell surface [7]; a rabbit mAb specific for human p40 phox (Epitomics, Burlingame, CA); an antibody that specifically detects active Rac (NewEast Biosciences, Malvern, PA); and rabbit antisera from William Nauseef (University of Iowa) specific for p47 phox and p67 phox [6].…”

“…2) or coronin [6] may give superior results in neutrophils for detection of forming phagosomes, as actin filaments in this cell type are less robust than in macrophages. 7 We prefer the polyvinyl alcohol base of gelvatol mounting medium because it hardens rapidly and completely. A common alternative to gelvatol is buffered glycerol (supplemented with an anti-fading agent).…”

“…We prefer phase contrast objectives for analysis of intraphagosomal superoxide accumulation revealed by nitroblue tetrazolium staining, as we find that color images obtained in this manner can reveal details and subtleties that are more difficult to discern in similar grey-scale NBT-DIC merged images that we obtain by confocal microscopy [7,[9][10][11].…”