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The Journal of Immunology, 2004, 173: 586-593.
Copyright © 2004 by The American Association of Immunologists

Helicobacter pylori Arginase Inhibits T Cell Proliferation and Reduces the Expression of the TCR {zeta}-Chain (CD3{zeta})1

Jovanny Zabaleta*,§, David J. McGee{dagger}, Arnold H. Zea§, Claudia P. Hernández§, Paulo C. Rodriguez§, Rosa A. Sierra*, Pelayo Correa* and Augusto C. Ochoa2,{ddagger},§

* Department of Pathology, Louisiana State University Health Sciences Center, New Orleans, LA 70112; {dagger} Department of Microbiology and Immunology, University of South Alabama, Mobile, AL 36688; {ddagger} Department of Pediatrics, Louisiana State University Health Science Center, and § Tumor Immunology Laboratory, Stanley S. Scott Cancer Center, New Orleans, LA 70112


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Helicobacter pylori infects approximately half the human population. The outcomes of the infection range from gastritis to gastric cancer and appear to be associated with the immunity to H. pylori. Patients developing nonatrophic gastritis present a Th1 response without developing protective immunity, suggesting that this bacterium may have mechanisms to evade the immune response of the host. Several H. pylori proteins can impair macrophage and T cell function in vitro through mechanisms that are poorly understood. We tested the effect of H. pylori extracts and live H. pylori on Jurkat cells and freshly isolated human normal T lymphocytes to identify possible mechanisms by which the bacteria might impair T cell function. Jurkat cells or activated T lymphocytes cultured with an H. pylori sonicate had a reduced proliferation that was not caused by T cell apoptosis or impairment in the early T cell signaling events. Instead, both the H. pylori sonicate and live H. pylori induced a decreased expression of the CD3{zeta}-chain of the TCR. Coculture of live H. pylori with T cells demonstrated that the wild-type strain, but not the arginase mutant rocF(–), depleted L-arginine and caused a decrease in CD3{zeta} expression. Furthermore, arginase inhibitors reversed these events. These results suggest that H. pylori arginase is not only important for urea production, but may also impair T cell function during infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Helicobacter pylori infection has been associated with diseases ranging from gastritis to gastric cancer and MALT lymphomas (1, 2, 3). Low income, overcrowding, and other factors characteristic of lower socioeconomic status are related to the high prevalence of infection (4, 5, 6). All infected individuals present histological signs of gastritis (7, 8), but many do not develop clinical symptoms of the disease (9, 10, 11). Gastric pathology appears to be closely associated with H. pylori virulence genes and with the immune response of the infected host against the bacterium (12, 13, 14, 15). In murine models a Th1 response is associated with damage to the mucosa, whereas a Th2 response appears to be protective (16). In addition, IFN response factor 1 knockout mice, which are unable to establish a Th1 response, do not develop damage of the gastric mucosa after exposure to H. pylori (17).

In humans, a Th1 response is observed in some patients with active gastritis and duodenal ulcer; however, it does not appear to confer protection against H. pylori (18, 19, 20). Therefore, it is possible that H. pylori, like other bacteria, has mechanisms to escape the immune response. Virulent strains of H. pylori carrying the cag pathogenicity island (PAI)3 delay phagocytosis by macrophages in vitro and are killed less efficiently than those without the PAI element (21, 22, 23). Recent reports have also shown that cytotoxin-associated protein A (CagA) binds to Src homology protein tyrosine phosphatase 2, which could impair signal transduction in cells (24).

L-arginine is a key factor in the activation and function of T cells. Its depletion results in T cell dysfunction (25). We therefore studied the possibility that H. pylori arginase could modulate T cell function. To survive the stomach environment, H. pylori produces arginase that hydrolyzes L-arginine to urea and ornithine. Urea is then converted by urease to ammonia that neutralizes the acidic pH in the stomach. The data presented in this study suggest that the depletion of L-arginine by H. pylori may impair T cell function. Jurkat T cells and freshly isolated human normal T lymphocytes cultured in the presence of an H. pylori sonicate or cocultured with live H. pylori in Transwells (Boyden chamber) showed a significant decrease in proliferation, which was paralleled by a reduced expression of the CD3{zeta}-chain of the TCR. Inhibition of arginase or culture of T cells with an isogenic arginase mutant strain of H. pylori prevented the molecular and functional alteration in T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
H. pylori sonicate

H. pylori ATCC 43504 strain (American Type Culture Collection, Manassas, VA) was cultured on blood agar (BD Biosciences, Cockeysville, MD) for 5 days using the BBL Campy Pouch System (BD Biosciences) to generate a microaerobic environment. After 5 days the cells were collected and washed twice in PBS. The bacteria were resuspended in PBS and subjected to six rounds of 30-s sonication at 200 W using a cell disruptor (model 450; Branson, Danbury, CT). The sonicated bacteria were centrifuged at 20,000 x g for 30 min at 4°C, and the supernatant was collected and filtered through a 0.2-µm pore size filter. The protein concentration of the supernatant was determined by bicinchoninic acid assay, following the manufacturer’s instructions (Pierce, Rockford, IL).

H. pylori arginase mutant

The plasmid pBS-rocF::aphA3, containing the disrupted rocF gene was electroporated into H. pylori strain ATCC 43504 as previously described (26). Mutants were selected on blood agar containing 20 µg/ml kanamycin. One mutant was selected for further analysis as previously reported (26). The kanamyin-resistant cassette used is a nonpolar cassette to minimize premature termination of rocF transcription, as shown by McGee et al. (26). The rocF gene is not in an operon-like structure based on genomic analysis. Sonicates were prepared as described above, and arginase activity was measured as described below.

Proliferation assays

Unless otherwise stated the cells were always cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 1.1 mM L-arginine, 2.5 mM L-glutamine, (Life Technologies), 25 mM HEPES (Life Technologies), 100 µg/ml penicillin/streptomycin (Life Technologies), and 10% heat-inactivated FCS (HyClone, Logan, UT). Jurkat cells were maintained at 0.5 x 106/ml. For proliferation assays, Jurkat cells or PBMC were placed at 2 x 105/well in round-bottom, 96-well plates (Corning Glass, Corning, NY) and incubated for 2 h with increasing concentrations of the H. pylori sonicate ranging from 0.01–100 µg/ml. After this, 30 ng/ml anti-CD3 (Ortho Diagnostics, Raritan, NJ) and 100 ng/ml anti-CD28 (BD Biosciences, Palo Alto, CA) were added to the cells. Jurkat cells were incubated for an additional 24 h, and PBMC were incubated for 48 h. One microcurie of [3H]thymidine (NEN, Boston, MA) was added per well for the last 20 h. The cells were collected onto glass-fiber filters (Unifilter GF/B; Packard Instrument, Meriden, CT), and radioactivity was counted in a beta counter (Microplate Scintillation and Luminescence Counter, TopCount; Packard).

CD3{zeta} chain expression

Jurkat cells and activated normal T cells were cultured for 24 h in medium with the H. pylori sonicate at 50 and 20 µg/ml, respectively, or with purified recombinant Ags CagA (2 µg/ml; Austral Biologicals, San Ramon, CA), VacA (4 µg/ml; Austral), H. pylori urease A (2 µg/ml; Austral), H. pylori urease B (2 µg/ml; Austral), and Escherichia coli LPS (500 ng/ml; Sigma-Aldrich, St. Louis, MO). Jurkat and T cells were counted and stained with FITC-conjugated anti-human CD3 (Beckman Coulter, Miami, FL) for 20 min in the dark. After washing with PBS, the cells were stained for 8 min with PE-conjugated, anti-human CD3{zeta} (Beckman Coulter) in PBS containing 50 µg/ml digitonin. The cells were washed twice in PBS and analyzed in an EPICS XL flow cytometer (Beckman Coulter). The results compare the mean fluorescence intensity of CD3{zeta} expression. The expressions of other cell surface receptors, including CD4 and CD69 (BD Pharmingen, San Diego, CA), were also tested.

Apoptosis

Jurkat cells and normal T lymphocytes were treated with the H. pylori sonicate for 24 h and stained with the nonisotopic stain annexin V as recommended by the manufacturer (Oncogene Research Products, La Jolla, CA). Briefly, 5 x 105 Jurkat cells were washed once in 0.5 ml of binding buffer (supplied by the manufacturer) and resuspended in 0.5 ml of binding buffer containing 1.25 µl of annexin V. The cells were incubated for 15 min at room temperature in the dark and centrifuged at 1000 x g. Ten microliters of propidium iodide (supplied by the manufacturer) were added per sample. Fluorescence was measured using an EPICS XL flow cytometer (Beckman Coulter). The percentage of cells undergoing either necrosis (propidium iodide-positive cells) or apoptosis (annexin V-positive cells) was determined. In experiments designed to determine whether cells with reduced CD3{zeta} expression were also undergoing apoptosis, staining for annexin V was followed by staining for CD3{zeta} as described above. Cells treated with actinomycin D (0.5 µg/ml) for 12 h were used as positive controls for apoptosis.

Protein tyrosine phosphorylation

Four million Jurkat cells were cultured overnight in serum-free medium and treated with 50 µg/ml H. pylori sonicate for periods ranging from 1–4 h, after which the cells were treated for 7 min with 100 µM pervanadate as previously reported (27, 28). The cells were washed in PBS containing 100 µM Na3VO4 and lysed in a buffer containing 50 mM HEPES, 150 mM NaCl, 5 mM EDTA, 100 µM Na3VO4, and 0.5% Triton X-100, pH 7.5. In addition, the buffer contained 100 µg/ml each of aprotinin, leupeptin (Roche, Indianapolis, IN), 100 µg/ml trypsin-chemotrypsin inhibitor, and 2 mM PMSF (Sigma-Aldrich). Proteins were separated by SDS-PAGE, transferred to Immobilon-P membranes (Millipore, Bedford, MA), and immunoblotted with anti-phosphotyrosine 4G10 Ab (Upstate Biotechnology, Lake Placid, NY). Protein bands were visualized using ECL (Amersham Pharmacia Biotech, Piscataway, NJ) and X-OMAT AR films (Eastman Kodak, Rochester, NY). When freshly isolated T cells were used to study phosphorylation, they were stimulated with anti-CD3 plus anti-CD28 in the presence of H. pylori sonicate. After 20 h, the cultures were treated with pervanadate and processed as described above for Jurkat cells.

Arginase inhibition with N-hydroxy-L-arginine (NOHA)

The H. pylori sonicate was preincubated overnight with the arginase inhibitor NOHA (Sigma-Aldrich) at 100 µg/ml and added to the cell cultures at a final concentration of 50 µg/ml. The cells were cultured for an additional 24 h, and the expression of CD3{zeta} was detected by flow cytometry.

Arginase activity in H. pylori

To determine arginase activity in the H. pylori sonicate we used a modified version of the methodology described previously (29), which measures the conversion of L-arginine to L-ornithine. Briefly, 25 µl of the H. pylori sonicate was mixed with 25 µl of 5 mM CoCl2 and incubated at 56°C for 20 min to activate the enzyme. One hundred and fifty microliters of prewarmed 100 mM Tris (pH 7.4) containing 5 mM L-arginine (Sigma-Aldrich) was added to the mixture. The sample was incubated at 37°C for 1 h. The reaction was stopped by adding 750 µl of glacial acetic acid. Two hundred and fifty microliters of ninhydrin solution (2.5 g of ninhydrin dissolved in 40 ml of 6 M phosphoric acid and 60 ml of glacial acetic acid) was added to the sample and heated at 90°C for 1 h. After cooling, 200 µl of the mixture was plated on flat-bottom, 96-well plates, and absorbance was read at 515 nm using a Benchmark Plus Microplate spectrophotometer (Bio-Rad, Hercules, CA). The concentration of L-ornithine present in the sample was estimated using a standard L-ornithine curve ranging from 2–250 nmol.

L-arginine detection by HPLC coupled to electrochemical detection (HPLC-ECD)

HPLC-ECD was performed as previously reported (30) using an ESA-CoulArray (model 540; ESA, Chelmsford, MA) with an 80 x 3.2-mm column with 120-A pore size. Briefly, supernatants were deproteinized by methanol. After centrifugation at 6000 x g for 10 min at 4°C, the supernatant was derivatized with 0.2 M OPA/BME (o-phthaldialdehyde containing 2-ME). Fifty microliters of the sample was injected per sample. The retention time for L-arginine was 10.2 min. Standards of L-arginine were prepared in methanol.

Purified T cell cultures

After isolating PBMC by Ficoll-Paque Plus (Amersham Pharmacia Biotech), T cells were purified by negative selection using an affinity column (R&D Systems, Minneapolis, MN). The purity was always >90% T cells, as measured by the expression of CD3{epsilon}. H. pylori extracts or live H. pylori have no effect on resting T cells; therefore, T cells were stimulated by cross-linking anti-CD3 and anti-CD28. For this, 24-well plates were coated with 0.3 ml of goat anti-mouse Ab (Kirkegaard & Perry Laboratories, Gaithersburg, MD) at 10 µg/ml in HBSS for 1 h. After washing twice with HBSS, 0.5 x 106 purified T cells were added per well in 1 ml of RPMI 1640 without L-arginine containing 1 µg/ml anti-human CD3 (Ortho Diagnostics, Raritan, NJ) and 100 ng/ml anti-human CD28 (BD Biosciences) and were cultured for 24 h. After this time, L-arginine was added to the wells to give a final concentration of 0.4 mM. H. pylori sonicate (20 µg/ml) from either the wild-type strain ATCC 43504 or its rocF(–) isogenic mutant was also added to the wells. After 24 h under these conditions, the cells were recovered and stained for CD3{zeta}. Six different donors were tested. Using the same system, purified T cells were cultured with recombinant H. pylori Ags at the concentrations mentioned above and stained for the expression of CD3{zeta}.

Coculture of live H. pylori with Jurkat cells

Jurkat cells (0.5 x 106/well) were plated in 0.7 ml of RPMI 1640 without L-arginine. Live H. pylori was plated in a Transwell insert (0.4-µm pore size; BD Biosciences) in RPMI 1640 containing 400 µM L-arginine. The final bacteria:cell ratio was 400:1. The cells were then cultured at 37°C for 24 h, after which the cells were stained for CD3{zeta} and annexin V as described previously.

Statistical analysis

Differences between groups were determined using either paired or unpaired Student’s t test. All statistical analysis was performed using PRISM 3.0 (GraphPad, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
H. pylori sonicate decreases T cell proliferation without increasing apoptosis

To test the effect of H. pylori on T cell proliferation, Jurkat cells and PBMC from normal donors stimulated with anti-CD3 and anti-CD28 were cultured with increasing concentrations of a sonicate derived from the ATCC 43504 strain of H. pylori. Jurkat cells were used as a model to study changes in proliferation and signal transduction, similar to those seen in anergic T cells (25, 31). All data were confirmed in normal T lymphocytes. As shown in Fig. 1A, there was a dose-dependent decrease in the proliferation of Jurkat cells and activated PBMC. The Ab-stimulated PBMC appeared to be more sensitive to the effects of the H. pylori sonicate, as shown by the fact that concentrations as low as 10 µg/ml caused a >90% decrease in proliferation (Fig. 1A). The effect of H. pylori sonicate on proliferation was not reversed by time in culture, as demonstrated in Fig. 1B, where Jurkat cells cultured for up to 96 h in the presence of the H. pylori sonicate failed to recover their proliferative capacity. Cell viability was not affected by culture with the H. pylori sonicate (data not shown).



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  		FIGURE 1. H. pylori sonicate impairs the proliferation of Jurkat cells and PBMC. Jurkat cells and PBMC (2 x 105/well) were cultured with increasing concentrations of H. pylori sonicate for 2 h. After this time, 30 ng/ml anti-CD3 and 100 ng/ml anti-CD28 were added to the PBMC. One microcurie of [3H]thymidine was added for the last 20 h of culture. A, Proliferation of Jurkat cells and PBMC after 24 and 48 h of culture, respectively. The results show the average cpm ± SEM of three experiments. B, Jurkat cells were cultured with (50 µg/ml) or without the H. pylori sonicate for up to 96 h to study its effects on proliferation over time. The results show the average cpm ± SEM of two experiments.

 
Changes in T cell signal transduction induced by H. pylori

To further explore possible mechanisms leading to a decreased T cell response induced by the H. pylori sonicate, we studied various aspects of T cell signal transduction. For these experiments, H. pylori sonicate was added to Jurkat cells cultures at 50 µg/ml and to PBMC or T cells at 20 µg/ml, concentrations shown to significantly impair cell proliferation. H. pylori sonicate did not impair the early stages of T cell signal transduction, such as Ca2+ flux (data not shown). However, it did induce a moderate reduction in the intensity of tyrosine phosphorylation in Jurkat cells and normal T cells (Fig. 2) compared with that induced by pervanadate (100 µM) (27, 28), which may provide some insight into the possible mechanisms by which H. pylori impairs T cell function.



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  		FIGURE 2. H. pylori induce a moderate down-regulation in tyrosine phosphorylation in T cells. A, Four million Jurkat cells were cultured in serum-free medium overnight and treated with H. pylori sonicate for up to 4 h before stimulation with 100 µM pervanadate to induce tyrosine phosphorylation. The cells were washed and lysed, and the proteins were run on SDS-PAGE and immunoblotted with 4G10 Ab. The proteins were visualized by ECL. B, Four million normal T cells were cross-linked with anti-CD3 plus anti-CD28 in the presence of 20 µg/ml H. pylori sonicate for 20 h before being treated with 100 µM pervanadate for 7 min. The proteins were isolated and processed as described for Jurkat cells.

 
We then tested for possible changes in the TCR and specifically in the expression of the CD3{zeta}-chain, the main signaling element of the TCR. As shown in Fig. 3, A and B, culture of Jurkat cells with the H. pylori sonicate decreased the expression of the CD3{zeta}-chain, but did not alter the expression of other surface molecules, such as CD4 and CD69 (Fig. 3C). H. pylori sonicate did not affect CD3{zeta} expression in resting T cells; however, it markedly diminished CD3{zeta} expression in activated T lymphocytes. Normally, stimulation of T cells through the TCR causes its internalization and the degradation of {zeta}-chain. Within 24–48 h, {zeta}-chain is synthesized, and the TCR is re-expressed on the cell membrane (32). Normal T cells stimulated with anti-CD3 plus anti-CD28 showed low CD3{zeta} expression (Fig. 3D, first column). Cells cultured for an additional 24 h (total 48 h) in tissue culture medium re-expressed CD3{zeta}-chain. However, cells cultured in the presence of H. pylori failed to re-express CD3{zeta} (Fig. 3D, third column, and Fig. 3E). Furthermore, cells treated with the H. pylori sonicate expressed similar levels of CD3{zeta} as cells cultured in the absence of L-arginine (compare first and third columns in Fig. 3D; see also the dotted line in Fig. 3E).



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  		FIGURE 3. H. pylori sonicate reduces the expression of CD3{zeta}-chain in T cells. A, Jurkat cells incubated with H. pylori sonicate for 24 h were stained for CD3{zeta}. Control cells were left untreated (labeled as 0). The mean ± SEM of at least six experiments is shown. *, p = 0.01 compared with cells without H. pylori sonicate. B, Representative histogram showing CD3{zeta} expression in Jurkat cells in the presence of WT derived-H. pylori sonicate (darker line). Son, sonicate; Iso, isotype. C, CD4 (top, dotted line) and CD69 (bottom, dotted line) expression in Jurkat cells treated with H. pylori sonicate. Jkt, Jurkat cells. D, Normal T cells were stimulated with anti-CD3 and anti-CD28, which induces the down-regulation of CD3{zeta}, as described in Materials and Methods. The first column shows the level of CD3{zeta} expression after stimulation-induced down-regulation during 24 h. Cells cultured in medium with L-arginine (400 µM) recover the expression of CD3{zeta} (middle column). Instead, cells cultured for 24 h in medium with L-arginine and 20 µg/ml H. pylori sonicate did not (third column). The recovery of the CD3{zeta} was monitored after 48 h. **, p = 0.0006; ***, p = 0.02 (compared with A, +). E, Representative histogram of CD3{zeta} down-regulation in normal T cells showing that T cells cultured in the presence of H. pylori (continuous thin line) express levels of CD3{zeta} similar to those cultured in the absence of L-arginine (dotted line). L-arg, L-Arginine.

 
We then tested whether the cells showing a decreased expression of CD3{zeta}-chain were undergoing apoptosis (33). As shown in Table I, although there was an increase in the percentage of annexin V-positive Jurkat cells, this was <5% (range, 3–5%) and would most likely not account for the drop in proliferation induced by the H. pylori sonicate. Furthermore, the cells that lost CD3{zeta}-chain were not the ones undergoing apoptosis. Contrary to Jurkat cells, normal T cells cultured in the presence of the H. pylori sonicate exhibited a reduction in the percentage of apoptosis. Double staining of Jurkat cells with annexin V and CD3{zeta} showed that the decreased expression of CD3{zeta}-chain was not seen in the small percentage undergoing apoptosis (Fig. 4).


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  		Table I. Apoptosis and culture with H. pylori sonicatea

 


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  		FIGURE 4. Apoptosis is not responsible for the reduction of CD3{zeta}. Jurkat cells and activated normal T cells were treated with H. pylori sonicate for 20 h and double stained for annexin V and CD3{zeta} as described in Materials and Methods. The percentages of cells undergoing apoptosis (annexin V positive) as well as the mean fluorescence intensity (MFI) for CD3{zeta} were determined. The figure is the representative of at least three different experiments. A control of apoptosis is shown at the right panel in which Jurkat cells were treated with 0.5 µg/ml actinomycin-D. NS, Nonstimulated.

 
Several H. pylori proteins implicated in the pathogenesis of infection have also been shown to alter the immune response in vitro (34, 35). We therefore tested whether recombinant purified H. pylori proteins could reduce CD3{zeta} expression in Jurkat cells and purified normal T lymphocytes. The recombinant proteins CagA, VacA, urease A (UreA), and urease B (UreB) were titrated up to 4.0 µg/ml. None of the recombinant H. pylori proteins significantly decreased the expression of CD3{zeta} in Jurkat cells or prevented the re-expression of CD3{zeta} in normal activated T lymphocytes (Fig. 5) compared with the H. pylori sonicate. Furthermore, various combinations of these proteins or LPS (derived from E. coli) also failed to decrease the expression of CD3{zeta}-chain (data not shown).



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  		FIGURE 5. Purified recombinant H. pylori proteins do not change CD3{zeta} expression in T cells. Cells were cultured in the presence of recombinant purified H. pylori proteins CagA (2.5 µg/ml), VacA (4.0 µg/ml), UreA (2.0 µg/ml), and UreB (2.0 µg/ml) or in the presence of WT H. pylori sonicate (50 µg/ml for Jurkat cells and 20 µg/ml for normal T cells). Cells were stained for CD3{zeta}, and the mean fluorescence intensity (MFI) was tested by flow cytometry. A, Jurkat cells. The mean CD3{zeta} MFI ± SEM of three experiments is shown. *, p = 0.01 compared with nonstimulated (NS) Jurkat cells. B, Purified normal T cells stimulated with anti-CD3 plus anti-CD28 in the presence of recombinant H. pylori proteins or H. pylori sonicate for additional 48 h after which the CD3{zeta} MFI was determined. *, p = 0.0009 compared with nonstimulated cells.

 
H. pylori arginase reduces CD3{zeta} expression and proliferation of T cells

Previously described in vitro models have shown that arginase produced by macrophages may deplete arginine and cause the reduction of CD3{zeta} (36). To test whether H pylori arginase might be responsible for reducing CD3{zeta} in T cells, we used an isogenic H. pylori arginase mutant of H. pylori strain ATCC 43504. The arginase gene rocF, which, based on genomic analysis, is not in an operon-like structure, was inactivated by the insertion of a kanamycin resistance gene as previously described (26). The kanamyin-resistant cassette used is a nonpolar cassette, to minimize premature termination of rocF transcription (26). This inactivation probably did not affect genes flanking rocF in H. pylori, because the upstream gene is transcribed in the opposite orientation, and the downstream gene is >0.5 kb away from the 3' end of rocF (37, 38). Similarly, rocF inactivation did not appear to alter the function of other enzymes involved in nitrogen metabolism, including urease (26). As expected, the arginase mutant H. pylori rocF(–) had no detectable arginase activity, as measured by the ability to convert L-arginine to L-ornithine, nor was the L-arginine concentration reduced in the culture medium (data not shown). Furthermore, sonicate from the rocF(–) H. pylori strain failed to reduced the proliferation of Jurkat cells compared with that of the wild-type strain (Fig. 6A). In addition, the rocF(–) H. pylori did not decrease the expression of CD3{zeta} in Jurkat cells (Fig. 6, B and C) or in stimulated T lymphocytes (Fig. 6, D and E), further supporting the possibility that H. pylori arginase could play an important role in the induction of T cell dysfunction. However, as stated by McGee et al. (26), the inactivation of the rocF gene in H. pylori can have pleiotropic effects, and therefore experiments with a complemented strain of H. pylori would be important for a final proof of this hypothesis. Unfortunately, complementation of this bacterium has been exceedingly difficult (26). However, to further support the concept that arginase caused the decrease in CD3{zeta}-chain expression, Jurkat cells and activated normal T cells were cultured with NOHA, an arginase inhibitor (36), and the WT H. pylori sonicate. The inhibition of arginase by NOHA resulted in the recovery of CD3{zeta} in normal T cells (Fig. 7A) and a partial recovery in Jurkat cells (Fig. 7B). A similar effect on proliferation was also observed (data not shown). Finally, a decrease in the expression of CD3{zeta} was also induced when live H. pylori were cocultured with Jurkat cells using a Transwells (Boyden chambers) that kept bacteria separated from the cells (Fig. 8), suggesting that bacteria-T cell contact is not requires for the induction of these molecular changes.



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  		FIGURE 6. H. pylori arginase reduces the expression of the CD3{zeta} chain and proliferation of Jurkat cells. A, Proliferation of Jurkat cells with sonicate from either H. pylori WT or rocF(–) bacteria. The mean cpm ± SEM of two experiments is shown. *, p = 0.03 compared with unstimulated (NS) Jurkat cells. B, Expression of CD3{zeta} in Jurkat cells cultured with 50 µg/ml sonicate from either WT or rocF(–) mutant H. pylori. The mean fluorescence intensity (MFI) ± SEM of six experiments is shown. *, p = 0.03; **, p = 0.002 (compared with control cells). NS, nonstimulated. C, Representative histogram showing CD3{zeta} expression in Jurkat cells cultured in the presence of either WT or rocF(–)-derived H. pylori sonicate. D, Normal T cells stimulated with anti-CD3 and anti-CD28 reduced expression of CD3{zeta} (first column). These cells were then cultured in medium with L-arginine (A+, second column; 400 µM) or medium with L-arginine plus 20 µg/ml WT or rocF(–) H. pylori sonicate for additional 24 h (48 h total). CD3{zeta} expression was measured by flow cytometry. The mean MFI ± SEM of six different normal subjects is shown in the graph. *, p = 0.01 compared with T cells treated with H. pylori WT-derived sonicate. E, Representative histogram showing CD3{zeta} reduction in normal T cells treated with sonicate from either the WT or the arginase mutant H. pylori.

 


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  		FIGURE 7. Arginase inhibitor NOHA recovers CD3{zeta} expression in T cells. A, Normal T cells were incubated with either 50 µg/ml WT sonicate or WT sonicate plus 10 µg/ml arginase inhibitor NOHA in the presence of 400 µM L-arginine, and CD3{zeta} mean fluorescence intensity (MFI) was determined by flow cytometry. Data shows the mean MFI ± SEM of at least three experiments. *, p = 0.003 compared MFI of cells treated with WT H. pylori sonicate. B, Expression of CD3{zeta} in Jurkat cells treated with the H. pylori sonicate in the presence of NOHA. The mean MFI ± SEM of the mean fluorescence intensity for CD3{zeta} of three different experiments is shown. *, p = 0.004; **, p = 0.01 (compared with nonstimulated (NS) Jurkat cells). An increase in CD3{zeta} expression was seen in all experiments of Jurkat cells treated the mixture sonicate plus NOHA (n = 3); however, there was no statistical difference (p = 0.3).

 


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  		FIGURE 8. Live WT H. pylori reduces the expression of CD3{zeta} in Jurkat cells. Jurkat cells were exposed to either WT or arginase mutant H. pylori in a Transwell system (400 bacteria/Jurkat cell). The mean ± SEM of CD3{zeta} mean fluorescence intensity (MFI) of two experiments is shown. *, p = 0.02 compared with nonstimulated (NS) cells. No statistical differences were observed between nonstimulated cells and those treated with the rocF(–) sonicate (p = 0.7).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
H. pylori infection induces an inflammatory response characterized by infiltrating polymorphonuclear leukocytes, macrophages, and lymphocytes and the production of several inflammatory cytokines, including TNF-{alpha}, IFN-{gamma}, and IL-8 (39, 40, 41, 42, 43). However, this strong immune response appears to confer little or no protection against H. pylori infection. In vitro models show that virulent strains of H. pylori (carrying the PAI) can impair phagocytosis by delaying actin rearrangement (21). Once phagocytosed, these strains of H. pylori cause the fusion of phagosomes into megasomes, decreasing the killing ability of macrophages (21). In doing so, H. pylori not only delays its own phagocytosis, but also that of other particles and bacteria (23). However, little is known about how H. pylori affects T cells.

Several reports have shown that coculture of T cells with H. pylori or H. pylori-derived products decreases their response to mitogens (44, 45, 46). Our data confirm this effect with an H. pylori sonicate that reduced the proliferation of Jurkat cells and stimulated T cells in a dose- and time-dependent manner, an effect that was not reversible by extended incubation time in culture. Jurkat cells were used as an indicator of arginase effects, because in the absence of L-arginine they undergo a reduction in proliferation and CD3{zeta} expression similar to that seen in anergic cells (25, 31). However, most findings were also tested and confirmed in normal T cells. The reduced CD3{zeta} expression and the diminished proliferation did not appear to be mediated by apoptosis induced by H. pylori as previously reported (47), because, on the average, <10% of cells became annexin V positive in all experiments. Instead, we found a decreased expression of CD3{zeta}-chain, the principal signal transduction protein in the TCR. T cell activation is initiated by the binding of Ag to the {alpha}{beta}-chains of the TCR, which triggers the phosphorylation of the CD3{zeta}-chain. This protein has three ITAM sequences, which in turn phosphorylate other tyrosine kinases, including ZAP-70, and eventually lead to T cell activation. A decreased expression of CD3{zeta} has been demonstrated in various chronic infections, such as leprosy, tuberculosis, and AIDS (48, 49, 50, 51), and appears to partially explain the T cell anergy that characterizes some of these diseases (52). Changes in other signal transduction molecules have also been described, including a decreased Jak-3 tyrosine kinase and abnormal expression or function of NF-{kappa}B p65 nuclear transcription factor (52), although these additional changes have not been studied in H. pylori infections.

The mechanisms by which L-arginine depletion causes alterations in T cell signal transduction in these infectious diseases is still unclear. Our laboratory has previously shown that Jurkat cells or Ag-stimulated T cells cultured in medium without L-arginine undergo a rapid reduction of CD3{zeta} and have a decreased proliferation and a diminished production of cytokines such as IFN-{gamma} (25). The diminished expression of CD3{zeta}-chain appears to be caused by a decrease in CD3{zeta}-chain mRNA stability (31).

L-arginine is essential for H. pylori survival. L-arginine is metabolized by arginase into L-ornithine and urea, providing a substrate for urease to synthesize ammonia and carbon dioxide, thereby protecting the bacteria from the acidic environment of the stomach. H. pylori arginase activity was initially described by Mendz and Hazell (53) in experiments using L-arginine as the sole carbon source and measuring the accumulation of L-ornithine and urea by nuclear magnetic resonance. They suggested that H. pylori arginase activity was associated with the inner cell membrane and that its activity was dependent on cobalt as a cofactor (29). The latter characteristic differentiates H. pylori arginase from arginase produced by human macrophages, which uses Mn2+ as its main cofactor (54). Gobert et al. (55) recently reported that H. pylori also induces arginase II in macrophages, a process that was linked to an increased macrophage apoptosis. In addition to its role in H. pylori acid resistance (26), H. pylori arginase can impair the bactericidal activity of macrophages by inhibiting the production of NO via L-arginine depletion (56). The data using arginase-mutant H. pylori suggest that arginase activity from H. pylori can also alter the expression of CD3{zeta} and T cell proliferation by decreasing L-arginine availability. However, as previously reported by McGee et al. (26), inactivation of the rocF gene in H. pylori can have pleiotropic effects, as shown by reduced serine dehydratase activity. Complementation studies are required to prove the role of H. pylori arginase in regulating the immune system of the host. The development of a highly efficient H. pylori complementation system is currently underway.

Our results suggest that the enzymatic pathway used by H. pylori for the production of L-ornithine and urea could also serve as a mechanism for impairing macrophage and T cell responses. It is possible that this mechanism may in part explain the lack of protective effect of the immune response and the chronicity of H. pylori infection. It is also possible that, as shown by Gobert et al. (55), H. pylori Ags can translocate into the gastric mucosa and induce the production of arginase by host macrophages, which could further limit the availability of L-arginine and induce T cell dysfunction. Preliminary data suggest that these changes occur in patients with H. pylori infection (J. Zabaleta, P. Correa, and A. C. Ochoa, unpublished observations). However, the impact of this mechanism on the development and outcome of H. pylori infection has yet to be determined.


   Acknowledgments
 
We thank Sandra Lee for assistance with the preparation of the manuscript.


   Footnotes
 
1 This work was supported in part by a grant from the Louisiana State Board of Regents, National Institutes of Health Grants RO1CA082689 and RO1CA101931 (to D.J.M.), and National Cancer Institute Grant PO1CA028842. Back

2 Address correspondence and reprint requests to Dr. Augusto C. Ochoa, Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar Street, Room 455, New Orleans, LA 70112. E-mail: aochoa{at}lsuhsc.edu Back

3 Abbreviations used in this paper: PAI, cag pathogenicity island; CagA, cytotoxin-associated protein A; CD3{zeta}, {zeta}-chain of the TCR/CD3 complex; ECD, electrochemical detection; NOHA, N-hydroxy-L-arginine; UreA, urease A; UreB, urease B; VacA, vacuolating cytotoxin; WT, wild type. Back

Received for publication January 27, 2004. Accepted for publication April 28, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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