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The Journal of Immunology, 2002, 168: 6002-6006.
Copyright © 2002 by The American Association of Immunologists

Cutting Edge

Cutting Edge: Urease Release by Helicobacter pylori Stimulates Macrophage Inducible Nitric Oxide Synthase1

Alain P. Gobert*,{ddagger}, Benjamin D. Mersey*,{ddagger}, Yulan Cheng*,{ddagger}, Darren R. Blumberg*,{ddagger}, Jamie C. Newton* and Keith T. Wilson2,*,{dagger},{ddagger}

* Department of Medicine, Division of Gastroenterology, and {dagger} Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201; and {ddagger} Veterans Affairs Maryland Health Care System, Baltimore, MD 21201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inducible NO synthase (iNOS) expression and production of NO are both up-regulated with Helicobacter pylori infection in vivo and in vitro. We determined whether major pathogenicity proteins released by H. pylori activate iNOS by coculturing macrophages with wild-type or mutant strains deficient in VacA, CagA, picB product, or urease (ureA-). When filters were used to separate H. pylori from macrophages, there was a selective and significant decrease in stimulated iNOS mRNA, protein, and NO2- production with the ureA- strain compared with wild-type and other mutants. Similarly, macrophage NO2- generation was increased by H. pylori protein water extracts of all strains except ureA-. Recombinant urease stimulated significant increases in macrophage iNOS expression and NO2- production. Taken together, these findings indicate a new role for the essential H. pylori survival factor, urease, implicating it in NO-dependent mucosal damage and carcinogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Helicobacter pylori is a Gram-negative, microaerophilic bacterium, which selectively colonizes the mammalian stomach, causes gastritis and peptic ulcers, and has been implicated in gastric carcinoma. H. pylori expresses several major proteins that are critically important in the pathogenicity of the bacterium. These include unique virulence factors, such as: VacA, which induces apoptosis of epithelial cells (1); CagA, which is injected into eukaryotic cells and induces cell signal transduction events (2, 3); and picB product, which appears to be required for the induction of IL-8 by gastric epithelial cells (4). In addition, urease, consisting of the two subunits, UreA and UreB (5), neutralizes gastric acid by generating ammonium from urea and is therefore required for in vivo colonization of H. pylori, because strains deficient in ureA (6) or lacking urease activity (7) fail to infect the mammalian stomach. The release of H. pylori proteins facilitates survival of the bacterium in the gastric mucosa but also results in a vigorous innate and adaptive immune response.

A major part of innate immunity is the up-regulation of inducible NO synthase (iNOS)3 (3). Increased iNOS expression has been demonstrated in H. pylori gastritis tissues, with localization in both epithelium and lamina propria (8). This expression has been linked to epithelial cell injury (9) and apoptosis (10). Moreover, we have previously demonstrated that H. pylori directly stimulates murine macrophage NO production through the induction of iNOS expression (11, 12). NO generation by macrophages is activated by intact bacteria or by soluble components of H. pylori and does not require the presence of H. pylori LPS (11, 13). In addition, H. pylori medium filtrates cause macrophage NO synthesis (14), supporting the hypothesis that factors released from H. pylori are activators of iNOS.

The aim of this study was to determine whether any of the major proteins released by H. pylori induce iNOS expression and activity. For this purpose, we used mutant strains deficient in virulence and survival factors of H. pylori. When intact bacteria were separated from macrophages, or when water-soluble extracts were used, a mutant strain of H. pylori lacking urease failed to induce iNOS mRNA expression, iNOS protein, or NO production, whereas mutant strains deficient in vacA, cagA, or picB had no loss of iNOS induction compared with wild-type (WT) strains. In addition, activation of iNOS-derived NO synthesis by macrophages was reproduced by recombinant urease. Therefore, we suggest that the induction of iNOS by H. pylori urease represents a new and important immunologic role for this enzyme in the pathogenesis of H. pylori gastritis.


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

All the reagents for cell culture and RNA analysis were obtained from Life Technologies (Gaithersburg, MD). Recombinant H. pylori urease was obtained from Orovax (Cambridge, MA) and used as described (15). Endotoxin level in the urease preparation was measured by Limulus amebocyte lysate testing at BioWhittaker (Walkersville, MD); there were 6.5 pg of LPS per microgram of urease. All other chemicals were from Sigma (St. Louis, MO).

Bacteria

H. pylori strains 3401 and 26695 were used. H. pylori 3401, and the isogenic vacA, cagA, picB, and ureA mutants were obtained from M. J. Blaser, Vanderbilt University (16). H. pylori 26695 and the ureA-deficient mutant (17) were provided by H. L. T. Mobley (University of Maryland, Baltimore, MD). It has been shown that ureA deletion results in elimination of total urease protein (17). WT or mutant bacteria were grown on Brucella agar plates containing 10% sheep blood under microaerobic conditions for an equal number of passages. For the experiments, H. pylori were harvested from plates, washed twice, and suspended in PBS. Bacteria concentrations were then evaluated by the determination of OD600. Water extracts of bacteria were obtained, and protein concentration was determined as reported (12).

Mice, cells, and culture conditions

The murine macrophage cell line RAW 264.7 was maintained and cocultured with H. pylori as previously reported (18). H. pylori was added to macrophages with a multiplicity of infection (MOI) from 10 to 100. To separate bacteria from macrophages, filter supports (0.4-µm pore size; Transwell; Corning, Corning, NY) were used. Cultures were also performed in the presence or absence of water extracts of H. pylori and recombinant urease. Peritoneal macrophages from C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were isolated as previously described (19) and cultured in complete DMEM.

mRNA analysis

RAW 264.7 macrophages were cocultured with H. pylori, with or without filter supports. After incubations, cells were washed twice with PBS, and total RNA was isolated using TRIzol reagent. Reverse transcription, PCR cycle conditions, and primer sequences were as described (18). PCR products were run on 2% agarose gels. Ethidium bromide-stained bands were visualized under UV light and photographed with a digital gel documentation system (EDAS 290 and 1D software; Kodak Digital Science, Rochester, NY). Northern blot analysis of total RNA (10 µg/lane) was performed as previously described (11), using a 32P random primer-labeled cDNA probe for murine iNOS (Cayman Chemical, Ann Arbor, MI) and a cDNA probe (1.1 kb) for GAPDH (Clontech, Palo Alto, CA). Densitometric analysis of band intensity was determined using NIH Image version 1.62 (http://rsb.info.nih.gov/nih-image/).

Western blot analysis

After coculture with H. pylori, macrophages were washed and lysed in 250 µl of PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml PMSF, 50 µg/ml aprotinin, and 1 mM sodium orthovanadate. Soluble proteins from macrophages (100 µg/lane) or from H. pylori water extracts (25 µg/lane) were separated by SDS-PAGE on 10% gels, and transferred onto Immobilon-P membranes (Millipore, Bedford, MA) by electroblotting. Membranes were blocked using PBS containing 0.1% Tween and 5% nonfat dry milk overnight at 4°C. Polyclonal Ab to murine iNOS (1/2,000; BD Transduction Laboratories, Lexington, KY) or to H. pylori UreB (1/50,000; H. L. T. Mobley), and a donkey anti-rabbit Ab conjugated to HRP (1/2,000; Amersham Pharmacia Biotech, Piscataway, NJ) were used, each for 1 h at room temperature. Chemiluminescent detection was performed using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and exposure to Hyperfilm ECL (Amersham, Little Chalfont, U.K.).

Measurement of NO concentration

The concentration of the oxidized product of NO, nitrite (NO2-), was assessed by the Griess reaction, as described (11, 12).

Statistical analysis

Quantitative data are shown as the mean ± SEM. For comparisons between multiple groups the Student-Newman-Keuls test was used, and for single comparisons between two groups the Student’s t test was used.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of different H. pylori mutants on iNOS induction

To determine the potential roles of the proteins VacA, CagA, PicB, and urease on iNOS activation in RAW 264.7 cells, different strains of H. pylori were added to macrophages, with or without Transwell filters. When there was contact between macrophages and bacteria (Fig. 1Go, left), there was no difference in the degree of up-regulation of iNOS mRNA (Fig. 1Go, A and B), iNOS protein (Fig. 1GoC), and NO production (Fig. 1GoD) between 3401 WT, vacA-, cagA-, ureA-, or picB- strains.



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  		FIGURE 1. Effect of WT and mutant strains of H. pylori on macrophage iNOS expression and NO production. RAW 264.7 cells were cocultured in contact with WT and mutants of H. pylori 3401 at an MOI of 10 (left) or with H. pylori above filter supports at an MOI of 100 (right). At 18 h, iNOS mRNA in macrophages was assessed by RT-PCR (A) and Northern blot analysis (B). At 24 h, iNOS protein (130 kDa) was detected by Western blotting (C). NO2- concentration was measured in macrophage supernatants (D); n = 5 separate experiments performed in duplicate. Ctrl, Control. **, p < 0.01 vs control, and §§, p < 0.01 vs WT, vacA-, cagA-, or picB-.

 
When RAW 264.7 cells were separated from the bacteria by Transwell filters (Fig. 1Go, right), iNOS up-regulation was observed at the same level in macrophages cocultured with H. pylori WT, vacA-, cagA-, and picB- strains (Fig. 1Go, A and B). However, in those same conditions, the level of iNOS mRNA in macrophages stimulated by H. pylori ureA- was decreased by 76 ± 3% compared with the WT strain (by densitometric analysis of Northern blots, p < 0.05, n = 3) and by a similar amount when compared with the other mutant strains. These results were confirmed when iNOS protein levels were compared by Western blotting (Fig. 1GoC). Similarly, NO2- production was decreased by 68 ± 6% in macrophages activated by H. pylori deficient in ureA (Fig. 1GoD), when compared with H. pylori WT, whereas the other mutant strains of H. pylori had no loss of ability to induce NO2- release. The levels of stimulated NO2- production were similar to those we have previously observed with other WT strains of H. pylori (12, 18). The effect of urease deletion was confirmed by using the 26695 strain of H. pylori; NO2- generation by macrophages cocultured with the ureA- mutant of this strain for 24 h was decreased by 56 ± 12% compared with the WT strain (p < 0.01, n = 3 experiments performed in duplicate; results not shown).

Lack of induction of macrophage NO2- generation with ureA- H. pylori at varying MOI

To further assess differences in iNOS stimulation by WT and ureA- H. pylori, we analyzed the effects of bacterial concentration and incubation time on macrophage NO2- generation. At a time point of 24 h (Fig. 2GoA), a concentration-dependent effect of bacterial MOI on macrophage NO2- release was observed with H. pylori WT stimulation. There was a significant decrease in stimulated NO2- release by the ureA- vs WT strain at each MOI of 25 or greater. After 48 h, high levels of NO2- were detected in the cocultures with H. pylori WT, even at an MOI of 10 (Fig. 2GoB). There was a significant decrease of stimulated NO2- by the ureA- strain vs the WT at each MOI tested from 10 to 100. As an example, NO2- concentration in supernatants was inhibited by 7.8- ± 0.2-fold at an MOI of 50.



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  		FIGURE 2. H. pylori concentration-dependent NO production by RAW 264.7 cells stimulated by H. pylori 3401 WT ({square}) vs ureA- ({blacksquare}) strains. Macrophages were cocultured with H. pylori above filter supports, with different MOI shown. [NO2-] was assessed at 24 h (A) and 48 h (B). n = 3, each in duplicate. *, p < 0.05, ***, p < 0.01, and ***, p < 0.001 vs ureA-.

 
Decreased stimulation of macrophage NO production by water extracts of ureA- H. pylori

A concentration-dependent increase of NO2- generation was observed when macrophages were stimulated by water extracts of WT, vacA-, cagA-, or picB- strains (Fig. 3GoA). Consistent with the results in the Transwell experiments in Figs. 1Go and 2Go, the water extract of the ureA- strain did not induce any NO2- production at 2.5 or 5.0 µg/ml. At a protein concentration of 10 µg/ml, NO2- levels were significantly attenuated by 68, 61, 51, and 64% when compared with WT, vacA-, cagA-, or picB- mutants, respectively. Similarly, there was a marked induction of NO2- release by resident peritoneal macrophages in response to WT water extract, which was abolished when the ureA- water extract was used (Fig. 3GoB). Fig. 3GoC demonstrates that urease protein was detected in the water extract of WT and of vacA-, cagA-, or picB- mutants, but not in the soluble fraction of the ureA- mutant.



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  		FIGURE 3. A, NO2- generation by RAW 264.7 cells stimulated by soluble proteins of H. pylori 3401. Water extracts were added to macrophages for 48 h, and [NO2-] was assessed in the culture supernatants. n = 3, each in duplicate. *, p < 0.05, and **, p < 0.01 vs unstimulated macrophages (Ctrl) for WT, vacA-, cagA-, and picB-; #, p < 0.05 vs unstimulated macrophages for ureA-; and §, p < 0.05 and §§, p < 0.01 for ureA- compared with WT, vacA-, cagA-, or picB-. B, NO2- generation by mouse peritoneal macrophages stimulated with 5 µg/ml water extracts of H. pylori 3401 WT and ureA- H. pylori for 48 h. **, p < 0.01 vs both unstimulated control and ureA-. n = 2, each performed in duplicate. C, Western blot analysis of urease in H. pylori water extracts using an anti-UreB polyclonal Ab. Urease was detected at ~61 kDa.

 
Recombinant urease stimulates macrophage iNOS expression and NO production

Because of our findings of reduced iNOS inducing activity by the ureA- mutant strain, we tested the ability of H. pylori urease to activate macrophage iNOS. In RAW 264.7 cells exposed to recombinant urease, iNOS mRNA expression was up-regulated (Fig. 4GoA). A concentration-dependent increase in macrophage NO2- generation induced by H. pylori urease is shown in Fig. 4GoB. This increase was not attributable to LPS contamination of the recombinant urease, because Escherichia coli LPS added at the amount of LPS in the urease preparation (see Materials and Methods) failed to stimulate NO2- production (data not shown).



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  		FIGURE 4. Stimulation of iNOS-derived NO by recombinant urease. RAW 264.7 cells were cocultured with different concentrations of recombinant urease. A, iNOS expression was investigated by RT-PCR after 6 h. B, NO2- concentrations were measured in the macrophage supernatants after 24 h. n = 3, each performed in duplicate. *, p < 0.05, and **, p < 0.01 compared with unactivated macrophages.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As a major part of innate immunity, iNOS is induced in macrophages in numerous diseases related to pathogenic microorganisms (20). Most bacteria activate iNOS in macrophages after invasion (21) or by an LPS-mediated pathway (22). Herein, we demonstrate that soluble H. pylori-released proteins cause NO generation by macrophages. When there is contact between macrophages and H. pylori, our data indicate that iNOS is expressed via a nonspecific response, which may involve multiple pathways of activation. However, when the bacteria are separated from the cells, which is the predominant case in vivo, a urease-dependent induction of macrophage iNOS was observed. Our results indicate that at an MOI of <100, which mimics the conditions occurring in vivo in the mammalian stomach (11, 23), urease is the main inducer of iNOS. Also, urease may be the only iNOS inducer when H. pylori protein concentrations are low (<=5 µg/ml in our experimental conditions), but at higher concentrations other factors may be capable of inducing iNOS.

H. pylori secretes or releases numerous proteins in the extracellular environment. Some of them, such as catalase and superoxide dismutase, are directly involved in the defense of the bacteria against reactive oxygen species (24). In culture medium, H. pylori secretes VacA rapidly by a specific transporter (25). When H. pylori is in contact with gastric epithelial cells, CagA protein is injected into the host cells by a type IV secretion system (3). However, there is no evidence of secretion of CagA into the extracellular environment. Similarly, no specific secretion mechanism has been described for the picB product. In contrast, there is evidence for a specific secretion system for urease (26). Additionally, it should be recognized that release of H. pylori cytoplasmic proteins can occur by autolysis of the bacteria (27). Urease has been detected in the lamina propria of gastric antrum from patients with H. pylori gastritis (28), indicating that urease release occurs in vivo and is capable of reaching lamina propria immune cells beneath the epithelium, as we have modeled in the current study. In a previous report, it was suggested that contact was required for murine macrophage J774A.1 iNOS activation (13), because when well inserts were used, no iNOS induction was observed. However, iNOS mRNA expression was assessed only at 4 h after H. pylori stimulation, and at this time point no expression was found in our experiments (data not shown), even at an MOI of 100. It is likely that a longer time for coculture is required to allow urease to be released and then activate the macrophages.

Although the main role of H. pylori urease is to protect the bacteria from acidic conditions through the formation of ammonia, urease possesses other properties. H. pylori urease exhibits chemotactic activity for human monocytes (28) and is a strong activator of production of the Th1 cytokines IFN-{gamma} and IL-12p40 (15) as well as IL-1{beta}, IL-6, and TNF-{alpha} (29) by PBMC.

Macrophage-derived NO can be a potent inhibitor of H. pylori growth (12), and it can also contribute to mucosal damage. Both bacterial killing and intense tissue injury are events that should limit the ultimate survival of H. pylori in the stomach of the host. Thus, an intriguing question is raised: "Why should the essential survival factor, urease, stimulate macrophage iNOS expression?" One possibility is that this is a necessary price for the bacteria to pay, because the bacteria must produce and release large quantities of urease, and the host can have both nonspecific and adaptive responses to this protein. Thus, there is a balance between the host and the pathogen. In addition, H. pylori has elaborated numerous strategies to escape the effects of NO (12) and/or peroxynitrite (30). Because NO and its metabolites have been implicated in gastric carcinogenesis (31), it is important to realize that the chronic expression of iNOS due to urease released from persistent H. pylori infection in the stomach may be an important contributor to the risk for H. pylori-associated gastric cancer.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants DK53620, DK 02469, and DK56938 and by the Office of Medical Research, Department of Veterans Affairs. Back

2 Address correspondence and reprint requests to Dr. Keith T. Wilson, University of Maryland School of Medicine, 22 South Greene Street, Room N3W62, Baltimore, MD 21201. E-mail address: kwilson{at}umaryland.edu Back

3 Abbreviations used in this paper: iNOS, inducible NO synthase; WT, wild type; MOI, multiplicity of infection. Back

Received for publication March 29, 2002. Accepted for publication April 29, 2002.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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E. J. Beswick, I. V. Pinchuk, K. Minch, G. Suarez, J. C. Sierra, Y. Yamaoka, and V. E. Reyes
The Helicobacter pylori Urease B Subunit Binds to CD74 on Gastric Epithelial Cells and Induces NF-{kappa}B Activation and Interleukin-8 Production
Infect. Immun., February 1, 2006; 74(2): 1148 - 1155.
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Y. Cheng, R. Chaturvedi, M. Asim, F. I. Bussiere, H. Xu, R. A. Casero Jr., and K. T. Wilson
Helicobacter pylori-induced Macrophage Apoptosis Requires Activation of Ornithine Decarboxylase by c-Myc
J. Biol. Chem., June 10, 2005; 280(23): 22492 - 22496.
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F. I. Bussiere, R. Chaturvedi, Y. Cheng, A. P. Gobert, M. Asim, D. R. Blumberg, H. Xu, P. Y. Kim, A. Hacker, R. A. Casero Jr., et al.
Spermine Causes Loss of Innate Immune Response to Helicobacter pylori by Inhibition of Inducible Nitric-oxide Synthase Translation
J. Biol. Chem., January 28, 2005; 280(4): 2409 - 2412.
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R. Chaturvedi, Y. Cheng, M. Asim, F. I. Bussiere, H. Xu, A. P. Gobert, A. Hacker, R. A. Casero Jr., and K. T. Wilson
Induction of Polyamine Oxidase 1 by Helicobacter pylori Causes Macrophage Apoptosis by Hydrogen Peroxide Release and Mitochondrial Membrane Depolarization
J. Biol. Chem., September 17, 2004; 279(38): 40161 - 40173.
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S. Duleu, P. Vincendeau, P. Courtois, S. Semballa, I. Lagroye, S. Daulouede, J.-L. Boucher, K. T. Wilson, B. Veyret, and A. P. Gobert
Mouse Strain Susceptibility to Trypanosome Infection: An Arginase-Dependent Effect
J. Immunol., May 15, 2004; 172(10): 6298 - 6303.
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S. Tan and D. E. Berg
Motility of Urease-Deficient Derivatives of Helicobacter pylori
J. Bacteriol., February 1, 2004; 186(3): 885 - 888.
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A. P. Gobert, J.-C. Bambou, C. Werts, V. Balloy, M. Chignard, A. P. Moran, and R. L. Ferrero
Helicobacter pylori Heat Shock Protein 60 Mediates Interleukin-6 Production by Macrophages via a Toll-like Receptor (TLR)-2-, TLR-4-, and Myeloid Differentiation Factor 88-independent Mechanism
J. Biol. Chem., January 2, 2004; 279(1): 245 - 250.
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F. Meyer, K. S. Ramanujam, A. P. Gobert, S. P. James, and K. T. Wilson
Cutting Edge: Cyclooxygenase-2 Activation Suppresses Th1 Polarization in Response to Helicobacter pylori
J. Immunol., October 15, 2003; 171(8): 3913 - 3917.
[Abstract] [Full Text] [PDF]

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