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The Journal of Immunology, 2005, 174: 3658-3667.
Copyright © 2005 by The American Association of Immunologists

Helicobacter pylori Disrupts NADPH Oxidase Targeting in Human Neutrophils to Induce Extracellular Superoxide Release1

Lee-Ann H. Allen2, Benjamin R. Beecher, Jeffrey T. Lynch, Olga V. Rohner and Lara M. Wittine3

Department of Medicine and Inflammation Program, University of Iowa, Coralville, IA 52241; and the Veterans Affairs Medical Center, Iowa City, IA 52246


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Helicobacter pylori (Hp)4 is a Gram-negative bacterium that has colonized the gastric mucosa of half the world’s population. Once acquired, Hp are not eliminated by the host immune response, and the infection persists for a lifetime in the absence of antibiotic treatment. In all cases, Hp induces gastritis, and a subset of infections progress to peptic ulceration or gastric cancer (1, 2). The majority of bacteria reside in the mucus layer over the gastric epithelium, and from this locale, Hp stimulates a novel type of chronic inflammation that is characterized by a massive influx of neutrophils (polymorphonuclear leukocyte neutrophils (PMNs)) into the gastric mucosa (1, 2). Neutrophils traverse the epithelium in large numbers and encounter bacteria in the mucus layer and at ulcer margins (2). Nevertheless, killing is inefficient in vivo, and in vitro studies suggest that Hp is controlled only when neutrophils are present in vast excess (2, 3, 4).

Although Hp reside in a PMN-rich environment, relatively little is known about Hp-neutrophil interactions at the molecular level. The receptors and adhesins that modulate Hp phagocytosis have not been defined, and how these organisms evade phagocytic killing is unclear. At the same time, the results of several studies indicate that Hp can activate neutrophils in vitro (3, 4, 5, 6, 7, 8). Moreover, neutrophil density in vivo correlates directly with the ability of Hp to cause severe disease (9, 10, 11), and the available data suggest that neutrophils and bacteria act in concert to induce gastric ulceration (8, 10, 12, 13).

Synthesis of toxic reactive oxygen species (ROS) by human neutrophils is an essential component of innate defense. Conversion of molecular oxygen into superoxide anions is catalyzed by the multicomponent NADPH oxidase (14). Because ROS can damage host tissue, NADPH oxidase activity is tightly controlled. In resting neutrophils, the enzyme is unassembled and inactive with subunits segregated in the membrane and cytosol. In response to an activating stimulus, the soluble subunits p47phox, p67phox, and p40phox translocate en bloc to the membrane where they bind flavocytochrome b558. Rac2 translocates separately and is also required for activity. Importantly, the site of oxidant generation depends on the stimulus. In general, soluble agonists such as fMLP promote oxidase assembly at the plasma membrane, and superoxide is generated primarily in the extracellular milieu, whereas microbes target the NADPH oxidase to form phagosomes, and superoxide is generated in the phagosome lumen (15, 16, 17, 18, 19, 20, 21, 22).

Although it is clear that neutrophils are key players in Hp pathogenesis and that bacteria can activate neutrophils in vivo and in vitro several important questions remain unanswered. Notably lacking is a direct measurement of the Hp-induced respiratory burst. Previous studies have used luminol chemiluminescence (CL) to assess PMN activation by Hp (3, 4, 5, 6, 7, 8). Although this assay is very sensitive, it detects oxidants generated in the presence of myeloperoxidase and as such cannot be used to measure NADPH oxidase activity (18). Indeed, neutrophils from persons with inherited myeloperoxidase deficiency contain a fully functional NADPH oxidase but are inactive as judged by luminol CL (23, 24). Furthermore, no study to date has localized NADPH oxidase subunits in Hp-infected cells, and where oxidants are generated is unclear. In this study, we used biochemical approaches (O2 consumption, reduction of ferricytochrome c, isoluminol CL, NBT staining, and dichlorodihydrofluorescein fluorescence) along with confocal microscopy to quantify and localize NADPH oxidase activity in Hp-infected neutrophils and compare Hp-induced responses to other particulate and soluble stimuli. In this process, we tested the hypothesis that Hp alters NADPH oxidase activity and targeting in PMNs. Our data demonstrate for the first time that Hp induces a robust respiratory burst in neutrophils while simultaneously disrupting NADPH oxidase targeting such that superoxide anions accumulate in the extracellular space and not inside phagosomes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

HBSS, HEPES-RPMI 1640, L-glutamine, and Dulbecco’s PBS were obtained from BioWhittaker. Phosphate-free RPMI 1640 was purchased from Invitrogen Life Technologies, FBS from HyClone, PMA from LC Laboratories, and pertussis toxin (PTx) from Calbiochem. 2',7'-Dichlorodihydrofluorescein diacetate (DCF) was obtained from Molecular Probes and [32P]orthophosphate (10 mCi/ml) from PerkinElmer Life and Analytical Sciences. Rabbit polyclonal Abs (pAb) to p47phox and p67phox and murine mAb to gp91phox (54.1 and 7D5) and p22phox (44.1) have been described (15, 25). Rabbit anti-lactoferrin (Lf) pAb was obtained from Dr. B. Britigan (University of Iowa, Coralville, IA). Rabbit anti-Hp pAb were purchased from Accurate Chemical & Scientific and affinity-purified goat anti-Hp pAb from Kirkegaard and Perry Laboratories. Affinity-purified FITC- or rhodamine-conjugated donkey anti-rabbit, donkey anti-goat, and goat anti-mouse IgG F(ab')2 were obtained from Jackson ImmunoResearch Laboratories. The peptide Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) was synthesized and HPLC purified by SynPep. Other reagents were purchased from Sigma-Aldrich.

Neutrophil isolation

Heparinized venous blood was obtained from healthy adults in accordance with a protocol approved by the Institutional Review Board for Human Subjects at the University of Iowa. PMNs were isolated using dextran sedimentation and density gradient separation on Ficoll-Hypaque followed by hypotonic lysis of erythrocytes (16). The purity of the isolated cells was ≥95%. PMNs were resuspended in PBS containing 10 mM glucose or 0.9% saline and then diluted into appropriate media as indicated.

Bacterial growth and viability

Hp strains 11637, 60190, DT61A, and 11916 and a urease mutant derived from Hp 60190 (strain 4) (26) were obtained from the American Type Culture Collection and cultured as described (27). All Hp strains used in this study contain the cag pathogenicity island and secrete an active form of VacA (28, 29, 30) and as such are ulcerogenic (type I) organisms (31). Salmonella enterica serovar Typhimurium 14028s and the rough variant J5541 (from Dr. A. Aderem, Institute for Systems Biology, Seattle, WA) and S. aureus ALC 1435 (32) (from J. Femling, University of Iowa, Coralville, IA) were grown overnight with shaking at 37°C in Luria Bertani or tryptic soy broth, respectively. Washed bacteria were dispersed in HEPES-RPMI 1640 containing 10% heat-inactivated FBS or the indicated buffer. Where noted, bacteria or zymosan particles were opsonized with 50% fresh autologous serum. Multiplicity of infection (MOI) was 1:1–50:1 as indicated, and phagocytosis was synchronized by centrifugation (27). Phagocytic killing of Hp by PMNs was quantified by enumeration of CFU (27).

Respiratory burst

Oxygen consumption by stirred PMN suspensions was quantified using a Clark oxygen electrode (YSI Instruments). Basal O2 consumption by PMNs (5 x 106/ml) in 37°C HBSS containing 1 mM CaCl2, 1.5 mM MgCl2, and 10 mM glucose was followed for 10–15 min. Stimuli were added (PMA, bacteria (MOI 5:1–25:1) or opsonized zymosan (OpZ, MOI 2.5:1–10:1)), and O2 consumption was measured for another 10–30 min. O2 consumed by bacteria in the absence of PMNs was also quantified. Following O2 consumption measurements, aliquots of the cell suspension were chilled on ice, attached to coverslips using a Shandon Cytospin, fixed and permeabilized with methanol, and stained with Wright-Giemsa or rabbit anti-Hp pAb and FITC-conjugated secondary Abs. Association indices (total bacteria or OpZ per PMN) were counted for 50–65 cells per sample in triplicate using light or fluorescence microscopy.

All other assays used PMNs attached to serum-coated microtiter wells or glass coverslips (16). Extracellular release of superoxide anions was quantified by measuring the superoxide dismutase-inhibitable reduction of ferricytochrome c as described (16) or using isoluminol CL (18). For isoluminol experiments, PMNs (1 x 106/ml) were added to coated microtiter wells in HBSS containing 1 mM CaCl2, 1.5 mM MgCl2, 10 mM glucose, 1% human serum albumin, 50 µM isoluminol, and 4 U/ml HRP. After warming to 37°C, stimuli were added, and CL was followed for 60 min using a Victor2 Multilabel counter (PerkinElmer Life and Analytical Sciences). All assays were run in triplicate. Intracellular ROS were measured using the DCF assay (16). Fluorescence of duplicate samples was followed for 90 min at 37°C using a BMG Fluostar 403 microplate fluorometer (BMG Labtechnologies). To visualize superoxide anions generated by PMNs, cells attached to serum-coated glass coverslips were incubated with bacteria (MOI 5–10:1), OpZ (MOI 3:1), or 200 nM PMA in HEPES-RPMI 1640 containing 1 mg/ml NBT. After 60 min at 37°C, samples were washed with PBS, fixed in methanol, and stained as described above. Samples were examined by light and fluorescence microscopy for the presence of a blue-black formazan precipitate (17). At least 300 cells and/or phagosomes were scored per experiment in triplicate samples.

Immunofluorescence and confocal microscopy

We used established methods to localize NADPH oxidase subunits in Hp-infected PMNs (15, 16, 17). Briefly, PMNs were plated onto serum-coated glass coverslips in RPMI 1640 + 10% heat-inactivated FBS and then infected with Hp at an MOI of 10:1. Phagocytosis was synchronized by centrifugation at 12°C and after 1–60 min at 37°C samples were washed with PBS, fixed in 10% formalin, permeabilized in –20°C acetone, and then blocked in buffer containing PBS, 0.5 mg/ml NaN3, 5 mg/ml BSA, and 10% horse serum (15). Cells were double-stained with mAb to gp91phox (54.1) or p22phox (44.1) and rabbit anti-Hp pAb. Similarly, affinity-purified rabbit pAb to p47phox, p67phox, or Lf were used with goat anti-Hp pAb. Secondary Abs were conjugated to FITC or rhodamine. Images were obtained using an LSM-510 confocal microscope (Carl Zeiss).

To follow recruitment of flavocytochrome b558 to the cell surface, neutrophils on serum-coated coverslips were exposed to Hp (MOI 10:1) or 200 nM PMA for 2 min at 37°C. Cells were fixed in 4% paraformaldehyde (but not permeabilized), blocked, and then stained with mAb 7D5 and anti-Hp pAb. The amount of cytochrome b558 at distinct points on the cell surface was measured as relative fluorescence intensity using the Profile function of the LSM-510 software. All images were captured using identical settings and care was taken to ensure that signals in PMA-treated cells were not saturated. At least 100 cells per sample were quantified in triplicate. Similarly, gp91phox at the phagosome was assessed in fixed and permeabilized cells stained with mAb 54.1. In other experiments, differential staining was used to quantify the percentage of cell-associated Hp that were ingested by PMNs after incubation at 37°C for 1–30 min (33).

Inhibitor studies

Where indicated, PMNs in HBSS containing 10 mM glucose were preincubated for 30 min at 37°C in the presence of PTx (200–800 ng/ml) before addition of stimuli, and superoxide release was followed using isoluminol or ferricytochrome c. To down-regulate the formyl peptide receptor (FPR) and FPR-like receptor 1 (FPRL1), PMNs were incubated with fMLP (0.1–1 µM) and/or WKYMVm (0.1 µM) at 37°C (34) and then stimulated with Hp (MOI 5:1) or a second dose of fMLP or WKYMVm.

32P labeling and subcellular fractionation

PMNs (2 x 107/ml) were incubated in phosphate-free RPMI 1640 containing 0.5 mCi/ml [32P]orthophosphate for 60 min at room temperature. Cells were washed twice to remove unincorporated label and resuspended in phosphate-free RPMI 1640 containing Hp 11637 or DT61A (MOI 25:1). After 0–60 min at 37°C, cells were placed on ice and centrifuged at 600 x g (2 min, 4°C). Cell pellets were resuspended in 4°C radioimmunoprecipitation assay lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS) containing protease and phosphatase inhibitors (0.1 U/ml aprotinin, 1 mM PMSF, 0.4 µg/ml leupeptin, 3.5 mM diisopropyl fluorophosphate, and 200 µM orthovanadate). Incorporation of 32Pi into p47phox was assessed by phosphor imager analysis after immunoprecipitation and SDS-PAGE (16).

To assess translocation of p47phox to the membrane, neutrophils in RPMI 1640 were infected with Hp (MOI 25:1) for 0–60 min at 37°C. Cells were collected by centrifugation, resuspended in relaxation buffer containing the protease and phosphatase inhibitors noted above, and disrupted by nitrogen cavitation (16). Cytosol and plasma membrane/phagosome-enriched fractions were isolated by differential centrifugation from postnuclear supernatants (16). Samples were resolved by 10% SDS-PAGE and p47phox was detected by immunoblotting (16, 33).

Statistics

Paired samples were compared by Student’s t test and groups were compared by ANOVA (p < 0.05, significant).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Hp induces a strong respiratory burst in human neutrophils

In our initial experiments, we compared the ability of Hp strains 60190, 11637, DT61A, and 11916 to activate PMNs. As shown in Table I, Hp consumed very little O2 and 5 x 106 resting PMNs consumed 14.6 ± 1.4 nmol of O2 over a 10-min period. However, when bacteria were mixed with PMNs, all Hp strains tested stimulated a robust, dose-dependent respiratory burst. At an MOI of 15:1, Hp increased neutrophil O2 consumption 7.1 ± 0.3-fold, an effect comparable to the potent neutrophil activator PMA (Table I, p > 0.325). Enumeration of total cell-associated bacteria demonstrated that an MOI of 15:1 resulted in 12–14 Hp per cell (Table I).


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  		Table I. Neutrophil oxygen consumptiona

 
We also compared cell activation by Hp to other particulate stimuli including OpZ, Salmonella, and S. aureus. As shown in Table I, these particles differed in their ability to bind to and activate PMNs. Neutrophils readily ingested OpZ, and cells containing four large OpZ particles consumed as much O2 as cells containing three Hp 11637 or four Hp 60190. Wild-type Salmonella (14028s) were more strongly activating than a variant with rough LPS (J5541), and S. aureus triggered an oxidative burst after opsonization with fresh serum. However, on a per organism basis, none of these bacteria increased PMN O2 consumption to the same extent as Hp.

ROS do not accumulate inside Hp phagosomes

The site of ROS generation in activated PMNs depends on the nature of the stimulus. In general, soluble stimuli target the NADPH oxidase to the plasma membrane and ROS are released into the extracellular medium whereas particulate stimuli such as OpZ, Neisseria meningitidis serogroup B (NMB), and other bacteria target the enzyme to forming phagosomes, and ROS accumulate in the phagosome lumen (16, 18, 19, 20, 21, 22). As a first approach to localizing ROS in Hp-infected cells we used NBT, which forms a blue-black formazan precipitate in the presence of superoxide (17). By this assay superoxide anions accumulated inside 70 ± 7% of OpZ and 74 ± 5% of opsonized S. aureus phagosomes and were distributed diffusely on PMNs stimulated with PMA (Fig. 1, A and B). The vast majority of Hp-infected cells were also NBT positive (77 ± 9%), but the staining pattern differed markedly from PMNs containing other particles. Superoxide anions were detected inside only 15–27% of Hp phagosomes (Fig. 1, A and B), and formazan deposits were distributed diffusely on Hp-infected cells in a manner comparable to cells treated with PMA (Fig. 1A, black arrows). Combining NBT staining with confocal imaging confirmed that Hp phagosomes were NBT negative and distinct from large formazan deposits (Fig. 1A, far right).



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  		FIGURE 1. Oxidants do not accumulate inside Hp phagosomes. A, NBT staining of resting PMNs and cells exposed to PMA, Hp 11637, OpZ, opsonized S. aureus (OpSa), or opsonized Hp 11637 (OpHp). Black arrows, diffuse formazan deposits on PMA-treated or Hp-infected cells; black arrowheads, NBT-negative phagosomes; white arrowheads, NBT-positive phagosomes; asterisks, nuclear lobes. Far right panels, confocal images show that Hp (upper panel, green) are not associated with formazan deposits (lower panel, black). B, Percentage of phagosomes containing OpZ, Hp strains 60190, 11637, or DT61A, opsonized Hp 11637 (OpHp), or opsonized S. aureus (OpSa) that accumulated superoxide anions as judged by NBT staining. Data are the average ± SD of three experiments. *, p < 0.01 vs other particles. C, Intracellular ROS production. DCF-loaded PMN were left uninfected or exposed to OpZ (5:1), Hp 11637, or Hp 60190 (each at an MOI 15:1). Data indicate the rate of change of DCF fluorescence for each 5-min interval and are the mean ± SD of four determinations performed in duplicate. p ≤ 0.001 for OpZ vs Hp.

 
Next, we assessed the effect of Hp on production of intracellular oxidants using the DCF assay (35, 36). Consistent with the results of previous studies (16, 18), phagocytosis of OpZ triggered production of substantial intracellular ROS (Fig. 1C). On the other hand, responses to Hp 11637 and 60190 at an MOI of 15:1 were significantly lower (p < 0.010; Fig. 1C) and barely exceeded the background fluorescence of uninfected cells. Collectively, the NBT and DCF data demonstrate that ROS do not accumulate inside Hp phagosomes.

Hp-infected PMNs release large amounts of superoxide

The failure of ROS to accumulate inside Hp phagosomes despite the strong respiratory burst suggested that Hp infection might stimulate production of extracellular oxidants. To test this hypothesis, we quantified the amount of superoxide released from Hp-infected PMNs using ferricytochrome c. By this assay, both PMA and Hp (MOI of 10:1) activated neutrophils to a similar extent and extracellular superoxide anions were generated at a rate of 1.5–2.2 nmol/min/106 cells (Fig. 2A; p = 0.050). In contrast, very little superoxide was released by PMNs during phagocytosis of other types of particles including OpZ (0.34 ± 0.12 nmol/min/106 PMN) (Fig. 2A; p = 0.010), and we have shown previously that superoxide release is inefficient in PMNs infected with NMB (16). Thus, Hp differs from other bacteria and the yeast cell wall particle zymosan in its ability to trigger substantial superoxide release from PMNs.



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  		FIGURE 2. Hp-infected PMN release large amounts of superoxide. A, PMNs were treated with 200 nM PMA, OpZ (MOI 5:1) or Hp strains 61090, 11637, and DT61A (MOI 10:1) and the rate of generation of extracellular superoxide was determined using the ferricytochrome c assay. Superoxide release by uninfected cells was 0.10 ± 0.02 nmol/min/106 PMN. Data are the average ± SD from six experiments. *, p < 0.010 vs other stimuli. B, Time course of superoxide release from PMNs as judged by isoluminol CL. PMN were left untreated or stimulated with 200 nM PMA, OpZ (MOI 5:1), or Hp 60190 (MOI 15:1). Data are the average ± SD (n = 3). Comparable data were obtained for other strains of Hp (not shown).

 
Interestingly, we found that the kinetics of superoxide release differed for neutrophils stimulated with PMA and Hp even though the magnitude of the response was similar. In cells treated with phorbol esters, superoxide was detected in the medium after a lag of 2.0 ± 0.5 min (n = 8), and maximal superoxide release occurred at 4.5 ± 1.1 min (n = 7). For cells infected with Hp, extracellular superoxide was detected after a lag of 5.6 ± 3.2 min (n = 7) and peaked at 16.6 ± 3.6 min (n = 7). Comparable data were obtained when production of extracellular superoxide was assessed using isoluminol (18) rather than ferricytochrome c. In this case, CL peaked ~4 min after addition of PMA and ~16 min after addition of Hp (Fig. 2B). Moreover, the CL generated by PMNs ingesting OpZ was 5-fold lower than the response to Hp or PMA (Fig. 2B).

To assess whether superoxide release resulted from incomplete internalization of bound Hp, we used our established differential staining protocol to quantify attached and ingested bacteria (33). Accordingly, we found that 88 ± 6% (n = 3) of neutrophil-associated Hp were engulfed within 5 min. Therefore, Hp-induced superoxide release does not occur secondary to "frustrated phagocytosis".

Nascent Hp phagosomes acquire flavocytochrome b558 but not p47/67phox

The biochemical data demonstrate that Hp strongly activated PMNs and that large amounts of superoxide were generated in the extracellular space but not inside phagosomes. These data suggest that Hp might disrupt NADPH oxidase targeting. To test this hypothesis, we used confocal microscopy to localize NADPH oxidase subunits in infected cells and compared Hp compartments with phagosomes containing OpZ and other microbes (15, 16, 17). Flavocytochrome b558 is a heterodimer of gp91phox and p22phox and constitutes the integral membrane component of the NADPH oxidase. Low levels of flavocytochrome b558 are present in the plasma membrane of resting PMNs, and the majority of the protein is located in the membranes of specific and gelatinase granules that are mobilized in response to an activating stimulus (37). We followed up-regulation of flavocytochrome b558 at the surface of unstimulated, PMA-treated, and Hp-infected PMNs using mAb 7D5, which binds to an extracellular epitope in gp91phox (25). As shown in Fig. 3A, gp91phox accumulated in the plasma membrane subjacent to attached Hp and increased throughout the plasma membrane of PMNs treated with PMA. Measurement of fluorescence intensity at specific points in the membrane demonstrated that gp91phox increased 7.2 ± 1.7-fold directly beneath attached Hp and increased 8.2 ± 0.7-fold throughout the membrane in response to PMA. The gp91phox also increased 8.1 ± 0.4-fold directly beneath attached OpZ (not illustrated).



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  		FIGURE 3. Impaired recruitment of NADPH oxidase subunits to Hp phagosomes. A, gp91phox (green) associated with the surface of resting, PMA-treated or Hp 11637-infected PMNs (2-min time point). B, Accumulation of gp91phox (top panels) and p47phox (bottom panels) on 5- to 60-min OpZ phagosomes. Arrows indicate phagosomes. C, Association of gp91phox with 5- and 60-min Hp phagosomes. Data are single confocal sections that show gp91phox (green) and Hp (red). Arrows indicate phagosomes. D, Association of p47phox (red) with 5- and 10-min phagosomes containing Hp (green). Data are single confocal sections. Arrows and arrowheads indicate circumferential and patchy p47phox staining of Hp phagosomes, respectively. E, Time course of p47phox and gp91phox association with OpZ and Hp phagosomes. Data are the average ± SD of four experiments performed in triplicate. Positive phagosomes include weak/patchy staining. *, p ≤ 0.020 for Hp p47phox vs OpZ. **, p < 0.010 for Hp gp91phox and p47phox vs OpZ.

 
We have shown previously that nascent phagosomes containing OpZ or NMB accumulate large amounts of gp91phox and p22phox (see Ref.16 and Fig. 3B). Confocal analysis of 5-min Hp phagosomes demonstrated the presence of gp91phox in the phagosome membrane (Fig. 3C). However, a direct comparison of neutrophils containing Hp and OpZ demonstrated that the gp91phox signal was 2.5-fold lower on Hp phagosomes compared with OpZ compartments (relative fluorescence intensity 97 ± 30 vs 243 ± 13 arbitrary units (n = 150), p = 0.033). Moreover, OpZ phagosomes retained high levels of gp91phox and p22phox for at least 60 min (Fig. 3, B and E and Ref.16), but Hp phagosomes did not (Fig. 3, C and E). At best, only trace amounts of gp91phox were detected on 30- and 60-min Hp phagosomes. Similar data were obtained using mAb to p22phox (not illustrated).

Formation of functional NADPH oxidase complexes requires translocation of the soluble NADPH oxidase subunits p47phox and p67phox to the membrane where they bind to flavocytochrome b558. Our published data indicate that p47phox and p67phox rapidly accumulate on forming phagosomes containing OpZ or NMB, are retained for over 30 min, and then dissociate upon termination of the respiratory burst (Refs.15 and 16 and Fig. 3, B and E). By contrast, p47phox was detected on only one-third of 5-min Hp phagosomes (Fig. 3, D and E). Moreover, the p47phox signal at the phagosome was often weak or patchy and did not completely encircle ingested bacteria (Fig. 3D, arrowheads). As infection progressed, the amount of p47phox at the Hp phagosome rapidly declined, and by 20 min, phagosomal p47phox was rarely observed (Fig. 3, D and E). The limited and weak accumulation of NADPH oxidase complexes at the Hp phagosome contrasts sharply with the intense and sustained accumulation of p47phox on virtually all OpZ phagosomes (Fig. 3, B and E, and Refs.15 and 16). Comparable data were obtained using pAb to p67phox (not illustrated). Taken together, the data demonstrate that nascent Hp phagosomes acquire gp91phox/p22phox heterodimers but do not efficiently recruit or retain p47phox and p67phox. We conclude that ROS are not found inside Hp phagosomes due to the failure of these organelles to accumulate functional NADPH oxidase complexes.

NADPH oxidase accumulates in patches at the surface of Hp-infected neutrophils

In the course of our microscopy studies, we noted that although p47phox was rarely observed on Hp phagosomes, this protein accumulated in patches at the surface of infected cells (Fig. 4A) as did gp91phox (Fig. 4B). Importantly, these structures were not generated during Hp engulfment but appeared after bacterial internalization. Specifically, plasma membrane patches of gp91phox and p47phox were detected 10 min into infection, became prominent at 15–30 min, and as shown in Fig. 4, A and B, were often located in close proximity to Hp phagosomes. In contrast, plasma membrane NADPH oxidase complexes were detected in <3% of resting PMNs and <4% of cells containing OpZ (Fig. 4C). The absence of p47phox in the plasma membrane of PMNs containing OpZ is indicated by arrowheads in Fig. 3B. Collectively, the data suggest that Hp alters the trafficking of both membrane and cytosolic NADPH oxidase subunits in infected neutrophils, and as a result, NADPH oxidase complexes accumulate at the cell surface and not on bacterial phagosomes. Our microscopy findings are in good agreement with the biochemical data demonstrating that Hp-induced superoxide release peaked at ~16 min. To our knowledge, Hp are unique in their ability to disrupt NADPH oxidase targeting to phagosomes while simultaneously promoting enzyme assembly at heterologous sites.



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  		FIGURE 4. Surface NADPH oxidase complexes in Hp-infected PMNs. A and B, PMNs infected with Hp for 20 min. A, Confocal sections show p47phox (red) and Hp (green). Arrows indicate plasma membrane patches of p47phox. B, Confocal sections show gp91phox (green) and Hp (red). Note that gp91phox is enriched at the cell surface (arrowhead) but is barely detectable on Hp phagosomes (arrows). C, Percentage of resting PMNs or cells containing Hp 11637 or OpZ with plasma membrane patches of p47/67phox colocalized with flavocytochrome b558. Data are the average ± SD of four experiments performed in triplicate (*, p = 0.001 vs Hp).

 
Mechanism of Hp-induced superoxide release

Having established that Hp trigger release of ROS from infected PMNs, we began to dissect the mechanism. Surprisingly, formalin-fixed Hp retained their activating potential but heat-killed or ethanol-fixed bacterial Hp did not (Fig. 5A, p = 0.103, 0.004, and 0.027, respectively). Moreover, opsonization of Hp with 50% fresh serum (but not serum heated to inactivate complement) reduced superoxide release by 86 ± 4% (p = 0.002; Fig. 5A).



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  		FIGURE 5. Mechanism of NADPH oxidase activation. A, Superoxide release was quantified using the ferricytochrome c assay, and data were normalized to PMNs infected with Hp 60190 (MOI 5:1). HK, EtOH, and Frm indicate bacteria killed by heating to 65°C or fixation in 70% ethanol or 10% formalin. Op and HI-Op indicate bacteria opsonized with 50% fresh or heat-inactivated serum. Ure indicates a urease-deficient mutant derived from Hp 60190. Data are the average ± SD of five to eight experiments. *, p < 0.050 vs Hp. B, Cells pretreated with diluent (DMSO), 0.1 µM fMLP (F), and/or 0.1 µM WKYMVm (W) were infected with Hp 60190 (MOI 5:1), and superoxide release was measured. Data are the average ± SD of four experiments (p = 0.091). C, Confocal image shows that Lf (red) does not accumulate in Hp phagosomes (green). D, In vivo phosphorylation of p47phox. PMN were labeled with [32P]orthophosphate and then infected with Hp 11637 for 0–60 min. *, p ≤ 0.023 for 5 min vs 10–30 min. Inset, Time course of p47phox translocation to PMN membranes as judged by Western blotting (p = 0.010 for 5 vs 20 min). a.u., arbitrary units. For both panels data are the average ± SD (n = 4).

 
Several components of Hp extracts and sonicates can activate phagocytes including LPS, urease, Hp neutrophil-activating protein (HP-NAP) and the cecropin-like peptide Hp(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) (1, 34, 38, 39). Important in this regard is the fact that urease, HP-NAP, and Hp(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) are cytosolic proteins that are released by bacterial autolysis, and the amount of each factor that adsorbs to the surface of live Hp is controversial (40, 41, 42). In our system, a urease mutant derived from Hp strain 60190 (26) was not impaired in its ability to trigger superoxide release from infected PMNs (Fig. 5A). Cell activation by HP-NAP and Hp(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) is sensitive to PTx and Hp(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) acts via FPRL1 (34). Notably, the fMLP-triggered respiratory burst was efficiently blocked by PTx (4.5 ± 7.8% of control (n = 6)), but responses to Hp were unaffected (103 ± 6% of control (n = 6)). In other experiments, FPR and FPRL1 were down-regulated by treating neutrophils with fMLP or the peptide WKYMVm before infection with Hp. Down-regulation of FPR and/or FPRLI prevented cell activation by a second dose of fMLP or WKYMVm (97.0 ± 1.0% and 95.4 ± 7.4% reduction, respectively (n = 3)). In contrast, prior exposure to fMLP, WKYMVm, or both reduced Hp-triggered superoxide release by only 12–19%, which was not statistically significant (p = 0.091; Fig. 5B). Collectively, these findings suggest a role for formalin-resistant bacterial surface factors, but not urease, HP-NAP, or Hp(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), in NADPH oxidase activation by Hp.

The transient enrichment of gp91phox on Hp phagosomes suggested that disruption of granule targeting might be central to the ability of Hp to prevent oxidase assembly on bacterial phagosomes. In support of this notion, <5% of Hp phagosomes accumulated the specific granule marker Lf (37) (Fig. 5C) compared with 72 ± 6% of OpZ phagosomes (not illustrated).

Phosphorylation of p47phox

Phosphorylation of p47phox is essential for translocation of cytosolic phox complexes to the membrane, and the onset of phosphorylation coincides with activation of the respiratory burst (14). To measure phosphorylation of p47phox in vivo we labeled PMNs with [32P]orthophosphate and quantified phosphorylation of p47phox 0–60 min after addition of Hp (Fig. 5D). In parallel experiments, we also assessed p47phox translocation to the membrane (Fig. 5D, inset). In uninfected PMNs, p47phox was minimally phosphorylated and was not associated with cell membranes. Within 5 min of Hp binding, p47phox phosphorylation increased ~10-fold. Phosphorylation of p47phox declined to a distinct intermediate level at 10–30 min (p ≤ 0.023) and was near basal levels by 60 min. In contrast, the amount of p47phox associated with cell membranes was modest at 5 min, peaked at ~20 min, and then declined (Fig. 5D, inset). In view of these data and our microscopy findings, we conclude that maximal phosphorylation of p47phox parallels weak protein targeting to phagosomes, whereas moderate phosphorylation accompanies NADPH oxidase assembly at the cell surface.

Effect of serum opsonins on NADPH oxidase activity and targeting

As shown in Fig. 5A, we found that opsonized Hp 60190 were severely impaired in their ability to trigger superoxide release from infected PMNs. To explore the effects of opsonins in more detail, we infected neutrophils with opsonized or unopsonized bacteria and measured O2 consumption, intracellular and extracellular ROS, and total cell-associated bacteria. Opsonization had no effect on the efficiency of infection by Hp (12 ± 4 Hp/PMN vs 14 ± 3 OpHp/PMN (MOI 15:1), p = 0.691) yet markedly reduced the overall oxidative burst and nearly ablated superoxide release from cells infected with Hp 60190 or 11637 (Fig. 6A, p ≤ 0.022). At the same time, intracellular ROS increased 2.5- to 3.3-fold (p ≤ 0.034) and OpHp phagosomes accumulated superoxide as judged by NBT staining (Fig. 1, A and B). Confocal analysis demonstrated that OpHp phagosomes recruited and retained p47phox, and oxidase complexes were not detected at the cell surface (Fig. 6B). Thus, NADPH oxidase activity and targeting were controlled under these conditions by opsonins deposited on the surface of Hp.



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  		FIGURE 6. Effect of opsonization on NADPH oxidase activity and targeting. A, Effect of opsonins on ROS generated by PMNs infected with Hp 11637 or 60190. O2 consumption (total O2), intracellular ROS (ROS in, DCF assay), and extracellular ROS (ROS out, ferricytochrome c assay) were measured. Data are the average ± SD (n = 4) and indicate opsonized Hp/unopsonized Hp (p ≤ 0.034 for all Op vs Unop). B, Enrichment of p47phox on phagosomes containing opsonized Hp 11637. C, Phagocytic killing. Neutrophils were infected with Hp 11637 (MOI 20:1). Total cell-associated bacteria were scored at 30 min (association index) and viable Hp were scored at 3 h (CFU, *, p < 0.010). Data indicate the average ± SD (n = 3).

 
Resistance to phagocytic killing

Previous studies have shown that unopsonized Hp are not killed efficiently by PMNs (2, 3, 4). Because opsonization redirected NADPH oxidase assembly to the phagosome, we assessed the effects of opsonins on Hp survival in neutrophils. As shown in Fig. 6C, opsonization markedly enhanced the ability of neutrophils to kill ingested bacteria, and 3 h after engulfment, ~80% of Hp 11637 remained viable compared with <10% of opsonized organisms (p < 0.010).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Hp is a major human pathogen that thrives in a neutrophil-rich environment and causes a broad spectrum of disease that includes gastritis, peptic ulceration, and gastric cancer. Although it is well documented that Hp can activate PMNs, the infection is not controlled. The reasons for this host defense defect are unclear, and we know very little about Helicobacter-neutrophil interactions at the molecular level. In this study we examined specifically the oxidative responses of Hp-infected human neutrophils, and our findings are significant in three respects. First, we show that Hp induce a strong dose-dependent respiratory burst in PMNs that surpasses responses to other bacteria and is comparable in magnitude with cell activation by phorbol esters. Second, biochemical analyses and confocal microscopy demonstrate that Hp disrupt NADPH oxidase targeting such that ROS are generated in the extracellular space and do not accumulate inside Hp phagosomes. Third, our data indicate that urease, HP-NAP, and Hp(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) are not required for neutrophil activation. Rather, superoxide release is triggered by formalin-resistant and heat-sensitive bacterial surface components that can be bypassed by serum opsonins. Altogether, our data indicate that Hp disrupt neutrophil function by a unique mechanism that allows these organisms to evade killing and, at the same time, promote host tissue damage.

Measurement of oxygen consumption is the preferred method for studies of the respiratory burst because it is quantitative and is not limited by spatial constraints or production of specific ROS. Accordingly, we show here for the first time that neutrophils infected with four different strains of Hp undergo a strong, dose-dependent respiratory burst, and cells containing as few as 12 organisms consume as much O2 as PMNs activated with PMA. Moreover, our data demonstrate that Hp is a more potent activator of human neutrophils than Salmonella or S. aureus and, particle for particle, is comparable with the much larger OpZ.

Under normal circumstances, tight spatial and temporal control of NADPH oxidase activity minimizes host tissue damage during the immune response and promotes killing of ingested microbes. To subvert oxidative killing, pathogenic microbes must either prevent oxidant generation or evade toxic ROS. For example, virulent Yersinia species block the signaling pathways that control phagocytosis and NADPH oxidase assembly (43), and Coxiella enters PMNs without triggering a respiratory burst (44). The results of this study demonstrate that Hp differs from other pathogens in its ability to disrupt NADPH oxidase assembly not only to prevent ROS accumulation inside bacterial phagosomes, but also to induce the accumulation of extracellular oxidants.

Typically, established biochemical assays such as reduction of ferricytochrome c, isoluminol CL, DCF fluorescence, and NBT staining have been used to determine whether a given stimulus results in the production of intracellular or extracellular oxidants by activated neutrophils. Using this approach, we and others have demonstrated that the vast majority of ROS generated by cells infected with Neisseria gonorrhoeae, NMB, Escherichia coli, Bordetella pertussis, S. aureus, or OpZ accumulate inside phagosomes (16, 21, 22, 45) (this study). Thus, ingestion of each of these particles by PMNs results in significant DCF fluorescence and deposition of formazan inside phagosomes with little or no extracellular superoxide production as measured by ferricytochrome c or isoluminol. In marked contrast, the results of this study indicate that Hp-infected neutrophils have the opposite phenotype, and toxic ROS accumulate in the extracellular milieu but not inside Hp compartments.

Using synchronized phagocytosis and confocal microscopy, we show here that trafficking of NADPH oxidase subunits in Hp-infected cells differs markedly from PMNs containing OpZ or NMB (15, 16, 17). Our findings are in good agreement with the biochemical data and further suggest that diversion of NADPH oxidase subunits away from Hp phagosomes may be linked to alterations in granule targeting. Phagosomes containing OpZ or NMB rapidly acquire gp91phox/p22phox from specific and gelatinase granules and retain high levels of these proteins for at least 60 min (16) (this study). Although Hp binding to PMNs caused local up-regulation of flavocytochrome b558 as judged by 7D5 staining, the amount of gp91phox and p22phox associated with nascent (5 min) Hp phagosomes was 2.5-fold lower than on OpZ compartments. As infection progressed, gp91/p22phox staining at the phagosome was not sustained and NADPH oxidase complexes appeared in patches at the cell surface. These data suggest that gelatinase and specific granules are inefficiently recruited to, or actively diverted away from, Hp phagosomes. This notion is supported by the fact that Hp compartments also lack the specific granule marker Lf. Clearly, a complete analysis of the effects of Hp on granule mobilization is needed.

Phosphorylation of p47phox is an essential step in NADPH oxidase assembly that triggers translocation of p47/67/40phox complexes to the membrane (14). We show here that soluble phox proteins are not recruited efficiently to Hp phagosomes. At the same time, our in vivo labeling data indicate that this defect in protein targeting cannot be explained by blockade of p47phox phosphorylation. 32P-Labeled p47phox levels increase rapidly in response to Hp and remain elevated for the duration of the respiratory burst. Although phosphorylation triggers translocation of p47/67phox complexes to the membrane, it is not sufficient, and soluble oxidase subunits are not retained at the membrane in the absence of flavocytochrome b558 (15, 46, 47, 48). Therefore, we favor a model in which the paucity of gp91phox and p22phox on Hp phagosomes impairs retention of p47/67phox and thereby inhibits the accumulation of functional NADPH oxidase complexes and generation of intraphagosomal ROS.

One question raised by this study is whether phagocytosis is a necessary prerequisite for superoxide release. The results of ferricytochrome c and isoluminol assays indicate PMA-induced superoxide release occurs after a lag of ~2 min and peaks at ~5 min. In contrast, there is a delay of ~5 min between Hp binding and appearance of superoxide in the extracellular medium and oxidant generation peaks at ~16 min. As Hp uptake is complete in <5 min, superoxide release clearly occurs after Hp engulfment. Nevertheless, whether phagocytosis is essential for superoxide release is not yet known since many of the signaling pathways that regulate NADPH oxidase assembly are also required for Hp internalization (our unpublished observations).

Factors released from dying bacteria such as urease, HP-NAP, and Hp(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) promote chemotaxis and activation of monocytes and PMNs in vitro and recruit phagocytes to the gastric mucosa in vivo (1, 34, 39). However, the results of this study argue against a role for these virulence factors in PMN responses to intact Hp. Oxidant generation is not impaired by deletion of urease, exposure of neutrophils to PTx, or down-regulation of FPR and FRPL1. Moreover, cell activation by Hp(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) is short-lived (34) but responses to whole Hp are sustained. In contrast, our findings suggest that the neutrophil respiratory burst is regulated by formalin-resistant bacterial surface factors that can be bypassed by serum opsonins. In this manner Hp is reminiscent of Legionella pneumophila, which uses formalin-resistant surface components to block phagosome maturation in macrophages (49). Collectively, the data support a model in which urease, HP-NAP, and Hp(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) recruit neutrophils to the stomach and other unidentified determinants associated with whole organisms trigger NADPH oxidase activation during phagocytosis. Candidate activating factors include LPS, as well as other surface lipids and proteins. This model is also supported by the findings of Danielsson and colleagues (3, 6, 7, 8, 50), which suggest that neither the secreted cytotoxin VacA nor the type IV secretion system effector CagA is required for neutrophil activation.

Compared with macrophages, relatively few microbes are ingested by PMNs in the absence of opsonins. The results of this study and published data (7, 51, 52) demonstrate that human neutrophils phagocytose unopsonized Hp in vitro. More importantly, although both specific Abs and C3b are present in the infected gastric mucosa, the available data suggest that Hp are not opsonized efficiently in vivo (53, 54). Neither IgG nor IgA are detected on Hp in biopsy samples or antral brushings, and only 20% of bacteria bind Abs upon incubation with patient sera (53). The reasons for this host defense defect have not been determined definitively, but low pH or other aspects of the local environment (such as mucins) may play a role since Hp bind IgG more readily following in vitro cultivation (53, 55). In addition, a dominant Ag, CagA, is primarily intracellular (56); Abs generated against Lewis-X and Lewis-Y motifs of Hp endotoxin cross-react with host tissue (56), and VacA impairs B cell Ag presentation (57). The fact that protective immunity can be achieved in mice lacking B cells (58) and the observation that human IgA deficiency does not alter the prevalence or severity of Hp infection (56) also suggest that Abs do not play a major role in Hp-phagocyte interactions. At the same time, urease retards deposition of active C3 on the microbe surface in vivo and in vitro (55). Therefore, high concentrations of nonimmune serum are required to opsonize Hp in the laboratory setting (3, 55, 59, 60). Taken together, these findings are significant since mechanism of entry has profound effects on microbe fate (61, 62). Indeed, we show here that Hp trigger a robust respiratory burst in human neutrophils, disrupt NADPH oxidase targeting, and resist elimination. In contrast, opsonized Hp stimulate a modest respiratory burst that is confined to the phagosome, and bacterial killing is enhanced. Thus, it is likely that signaling downstream of complement receptors modulates both NADPH oxidase targeting and the magnitude of the respiratory burst during opsonophagocytosis. In future studies, it will be important to identify the adhesins and receptors that mediate binding and uptake of unopsonized Hp, to determine how these components interact with PMN-activating factors of the organism, and to define how these molecules are affected by opsonins.

In summary, we show here for the first time that Hp have the unique ability to disrupt the innate immune response via their effects on NADPH oxidase targeting. Because large amounts of ROS are released by infected PMNs, the results of this study support the hypothesis that Hp and neutrophils collaborate to induce host tissue damage and ulceration (8, 10, 12, 13). Moreover, it has been suggested that tissue damage allows Hp to acquire critical nutrients (63). Consequently, it is tempting to speculate that manipulation of the respiratory burst may be essential for bacterial survival.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Acknowledgments
 
We thank Dr. William Nauseef for many helpful discussions and for Abs to NADPH oxidase subunits, Dr. Bradley Britigan for Abs to Lf, Dr. Alan Aderem for Salmonella, and Jon Femling for S. aureus.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by a Veterans Affairs Merit Review Grant and National Institutes of Health R01-AI43617 to L.-A.H.A. Back

2 Address correspondence and reprint requests to Dr. Lee-Ann H. Allen, Inflammation Program, University of Iowa, 2501 Crosspark Road, MTF-D154, Coralville, IA 52241. E-mail address: lee-ann-allen{at}uiowa.edu Back

3 Current address: Department of Medicine, Johns Hopkins University, Baltimore, MD 21287. Back

4 Abbreviations used in this paper: Hp, Helicobacter pylori; PMN, polymorphonuclear leukocyte neutrophil; ROS, reactive oxygen species; CL, chemiluminescence; PTx, Pertussis toxin; pAb, polyclonal Ab; Lf, lactoferrin; DCF, 2',7'-dichlorodihydrofluorescein diacetate; MOI, multiplicity of infection; OpZ, opsonized zymosan; FPR, formyl peptide receptor; FPRL1, FPR-like receptor 1; HP-NAP, Hp neutrophil-activating protein; NMB, Neisseria meningitidis serogroup B. Back

Received for publication November 8, 2004. Accepted for publication December 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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