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Am J Physiol Gastrointest Liver Physiol 293: G1004-G1012, 2007. First published September 13, 2007; doi:10.1152/ajpgi.00096.2007
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INFLAMMATION/IMMUNITY/MEDIATORS

Toll-like receptor (TLR) 2 induced through TLR4 signaling initiated by Helicobacter pylori cooperatively amplifies iNOS induction in gastric epithelial cells

Kaname Uno,1,2 Katsuaki Kato,2,3 Tomoaki Atsumi,2 Takehito Suzuki,1,4 Jun Yoshitake,1 Hidetoshi Morita,4 Shuichi Ohara,2 Yashige Kotake,5 Tooru Shimosegawa,2 and Tetsuhiko Yoshimura1

1Research Project of Biofunctional Reactive Species, Yamagata Promotional Organization for Industrial Technology, Yamagata; 2Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai; 3Cancer Detection Center, Miyagi Cancer Society, Sendai; and 4School of Veterinary Medicine, Azabu University, Sagamihara, Japan; and 5Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma

Submitted 23 February 2007 ; accepted in final form 30 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell-surface Toll-like receptors (TLRs) initiate innate immune responses, such as inducible nitric oxide synthase (iNOS) induction, to microorganisms' surface pathogens. TLR2 and TLR4 play important roles in gastric mucosa infected with Helicobacter pylori (H. pylori), which contains lipopolysaccharide (LPS) as a pathogen. The present study investigates their physiological roles in the innate immune response of gastric epithelial cells to H. pylori-LPS. Changes in the expression of iNOS, TLR2, and TLR4, as well as downstream activation of mitogen-activated protein kinases and nuclear factor-{kappa}B (NF-{kappa}B), were analyzed in normal mouse gastric mucosal GSM06 cells following stimulation with H. pylori-LPS and interferon-{gamma}. Specific inhibitors for mitogen-activated protein kinases, NF-{kappa}B, and small interfering RNA for TLR2 or TLR4 were employed. The immunohistochemistry of TLR2 was examined in human gastric mucosa. H. pylori-LPS stimulation induced TLR2 in GSM06 cells, but TLR4 was unchanged. TLR2 induction resulted from TLR4 signaling that propagated through extracellular signal-related kinase and NF-{kappa}B activation, as corroborated by the decline in TLR4 expression on small interfering RNA treatment and pretreatment with inhibitors. The induction of iNOS and the associated nitric oxide production in response to H. pylori-LPS stimulation were inhibited by declines in not only TLR4 but also TLR2. Increased expression of TLR2 was identified in H. pylori-infected human gastric mucosa. TLR4 signaling initiated by H. pylori-LPS and propagated via extracellular signal-regulated kinase and NF-{kappa}B activation induced TLR2 expression in gastric epithelial cells. Induced TLR2 cooperated with TLR4 to amplify iNOS induction. This positive correlation may constitute a mechanism for stimulating the innate immune response against various bacterial pathogens, including H. pylori-LPS.

lipopolysaccharide; nitric oxide; stomach

HELICOBACTER PYLORI (H. pylori) is the causative agent for a variety of pathologies in the human stomach (29). H. pylori-infected gastric mucosa is characterized by chronic and active inflammation of extended duration. This microorganism thrives on human gastric mucosa, and gastric epithelial cells play essential roles in the first line of defense and in the initiation of the innate immune response against the virulent factors of this organism.

The innate immune response is initiated by cell surface Toll-like receptors (TLRs) that recognize widely conserved molecular patterns on the microorganism's surface (1, 2). Such pathogen-associated pattern recognition is essential for innate immune cells to discriminate between self and microbial nonself, and TLRs play an important role in host defense and tissue repair responses, thus maintaining mucosal homeostasis (6). To date, 11 mammalian TLRs have been isolated based on their divergence of molecular structure and their binding affinity for specific ligands. A variety of pathogen cell surface molecules, such as lipopolysaccharide (LPS), peptidoglycan (PGN), lipoteichoic acid, lipoarabinomannan, lipoprotein, and unmethylated DNA with a CpG motif, are recognized as pathogen-associated patterns by their specific TLRs (31).

H. pylori is a gram-negative bacterium, and, therefore, it possesses LPS as a virulent factor in its outer membrane (20–22). Microbial LPS is specifically recognized by TLR4, which induces various innate immune responses (6, 31). The activation of TLR4, in cooperation with the adaptor molecule MyD88, signals the downstream mitogen-activating protein kinase (MAPK) pathway, which subsequently activates the nuclear factor-{kappa}B (NF-{kappa}B) system (6, 31). Inducible nitric oxide synthase (iNOS) is a target molecule for TLR4 signaling activated by microbial LPS (3, 7), and iNOS-derived nitric oxide (NO) is well-known to play important roles in various inflammatory responses (4, 5). Gastric mucosal cells express TLR4 on their cell surface membrane (11, 27), and the induction of iNOS is also reported in gastric epithelial cells following exposure to E. coli-LPS (32). Therefore, it is conceivable that H. pylori-LPS can activate the innate immune response of gastric epithelial cells via pattern recognition by the TLR4 system (13, 15, 17, 27). In contrast, recent studies also suggest that H. pylori-induced innate immune responses are (at least partially) mediated through TLR2 in vitro and in vivo (8, 17, 18, 28), although TLR2 is generally considered to respond to PGN and lipoproteins derived from gram-positive bacteria and mycobacteria (25).

The purpose of the present study is to elucidate the implication of TLR2 and TLR4 in gastric epithelial cells in the innate immune response against H. pylori infection. Our in vitro study investigated the influence of H. pylori-LPS on the expression of TLR2 and TLR4 and on the pathway of signal propagation using a mouse normal gastric epithelial GSM06 cell line (30). The innate immune response to H. pylori-LPS in GSM06 was monitored with the induction of iNOS and its associated NO production. In vivo TLR2 expression was assessed in human gastric mucosa, with or without H. pylori infection by immunohistochemistry.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
H. pylori-LPS. H. pylori, cytotoxin-associated gene-A-positive standard species ATCC no. 43504 (the American Type Culture Collection, Manassas, VA), was grown using a large-scale cultivation technique kindly provided by Dr. Suzuki (Keio University). Briefly, H. pylori was inoculated and grown in Brucella broth (BD Diagnostic Systems, Franklin Lakes, NJ) containing 5% heat-inactivated horse serum (56°C for 30 min), under a microaerophilic condition at 37°C for 18–20 h, with rotary shaking at 60 rpm using the AnaeroPack system (Mitsubishi Gas Chemical, Tokyo, Japan), which contains oxygen-absorbing and carbon dioxide-producing agents to provide a microaerophilic environment of ~14% carbon dioxide and 6% oxygen, ideal for most Campylobacter species and H. pylori. Thereafter, H. pylori was harvested at a concentration of ~1 x 107 colony-forming units/ml.

Microbial LPS was extracted using Moran's hot phenol-water method (20). Briefly, the bacteria were dried with cold acetone and treated with pronase (Nacalai Tesque, Kyoto, Japan), and the LPS was extracted using 45% aqueous phenol at 68°C for 30 min. The crude LPS was purified by treating with RNase A, DNase II (Sigma-Aldrich, St. Louis, MO), and proteinase K (Nacalai Tesque), followed by centrifugation at 100,000 g for 18 h. The purity of the LPS was confirmed by SDS-PAGE, and the activity was determined colorimetrically using a standard endotoxin CSE-H kit, an Endospecy ES-24S kit, and a Toxicolor DIA kit (Seikagaku, Tokyo, Japan), in accordance with the manufacturer's instructions. The data of the purity and activity of the H. pylori-LPS sample employed in the present study corresponded to the previous report (20) (data not shown).

Cell culture. GSM06 cell line (Riken Cell Bank, Tsukuba, Japan) was cultured in Dulbecco's modified Eagle's medium F-12 supplemented with 10% low endotoxin fetal bovine serum, antibiotics, antimycotics, and insulin-transferrin selenium X (Invitrogen, Carlsbad, NM). When the cultures were 80–90% confluent, the cells were cultured in freshly prepared serum-free medium containing H. pylori-LPS and/or recombinant interferon (IFN)-{gamma} (Peprotech, London, UK), with or without pretreatment for 30 min with specific inhibitors for MAPKs and NF-{kappa}B, i.e., PD98059 (Biomol Research Laboratories, Plymouth Meeting, PA) for extracellular signal-related kinase (ERK), SP600125 (Biomol Research Laboratories) for c-Jun NH2-terminal kinases (JNK), SB203580 (Biomol Research Laboratories) for p38, and pyrrolidine dithiocarbamate (PDTC) (Sigma-Aldrich) for NF-{kappa}B. The inhibitors alone did not affect cell viability.

RT-PCR. Total RNA samples were prepared from cells treated with H. pylori-LPS (10–50 ng/ml) and/or IFN-{gamma} (10–1,000 U/ml) for 3 h using Rapid Total RNA Purification System kit (Marligen Biosciences, Ijamsville, MD), in accordance with the manufacturer's instructions. Following treatment with DNase (Nippon Gene, Tokyo, Japan), one-step reverse transcription (RT)-PCR was performed with 100 ng of total RNA using SuperScript One-Step RT-PCR with Platinum kit (Invitrogen). The primer pair sets for murine TLR2, TLR4, iNOS, and beta-actin were as follows: TLR2 for the 321-bp PCR product, 5'-TCTGGGCAGTCTTGAACATTT-3' and 5'-AGAGTCAGGTGATGGATGTCG-3'; TLR4 for the 406-bp, 5'-GCAATGTCTCTGGCAGGTGTA-3' and 5'-CAAGGGATAACGCTGAGA-3'; iNOS for the 559-bp, 5'-GACAAGAGGCTGCCCCCC-3' and 5'-GCTGGGAGTCATGGAGCCG-3'; and beta-actin for the 540-bp, 5'-TAAAACGCAGCTCAGTAACAGTCGG-3' and 5'-TGCAATCCTGTGGCATCCATGAAC-3' (14, 34). The PCR reactions were performed under the following condition: 27 cycles at 94°C for 15 s, 57°C for 30 s, and 72°C for 30 s. The PCR products were segregated by 2% agarose gel electrophoresis and were visualized with ethidium bromide staining. Densitometric analysis of the PCR products in three separate experiments was performed using National Institutes of Health (NIH) image software (version 1.63, National Institutes of Health, Bethesda, MD). The results were expressed as the mean ratio of the densitometric value for a specific mRNA compared with that for beta-actin mRNA.

Western blotting. Protein samples (20 µg/well) from cells treated with H. pylori-LPS (50 ng/ml) and/or IFN-{gamma} (1,000 U/ml) for 8 h were separated by 4–12% SDS-PAGE and transferred to polyvinylidene difluoride membranes, as described previously (32). The membranes were probed with primary goat polyclonal antibodies against TLR2 and TLR4 (optimal dilution at 1:400; S-16 and M-16, Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal antibodies against total-ERK, p38, and JNK (1:1,000; nos. 9102, 9212, and 9252, Cell Signaling Technology, Beverly, MA); and rabbit monoclonal antibodies against phospho-ERK, p38, and JNK (1:400; nos. 9101, 9211, and 9251, Cell Signaling Technology) at 4°C overnight, followed by horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia, Piscataway, NJ). An enhanced chemiluminescence detection system (Amersham Pharmacia) was used for protein detection, according to the manufacturer's instructions. Subsequently, the blots were stripped off the first probe using ReBlot Plus stripping buffer (Chemicon, Temecula, CA) and were reprobed with beta-actin (1:5,000, Santa Cruz Biotechnology) to monitor protein loading. Densitometric analyses of changes in the protein expressions of TLR2 and TLR4 with or without H. pylori-LPS and/or IFN-{gamma} treatment were performed using NIH image software (version 1.63). The results were expressed as the mean ratios of the densitometric values in three separate experiments for TLR2 and TLR4 protein in the treated cells vs. those in the untreated control.

Quantitative analyses of NF-{kappa}B activation. Quantification of NF-{kappa}B activity was performed using the ELISA-based Trans-AM NF-{kappa}B p65 kit (Active Motif, Carlsbad, CA). This ELISA measures free p65 NF-{kappa}B protein contained in a nuclear protein sample on the basis of its ability to bind to oligo-DNAs composed of an NF-{kappa}B binding motif and a specific antibody for p65 NF-{kappa}B protein. Cells treated with H. pylori-LPS (50 ng/ml) and/or IFN-{gamma} (1,000 U/ml) for 60 min, with or without pretreatment with PDTC (10, 100 µM), PD98059 (10, 100 µM), SB203580 (1, 10 µM), and SP600125 (1, 10 µM), were used in the analyses. Nuclear protein samples were extracted using the Nuclear Extract Kit (Active Motif), in accordance with the manufacturer's instructions, as was briefly summarized, i.e., the cells were separated into the cytoplasmic fraction (supernatant) and nuclear fraction (pellet) by treatment with a hypotonic buffer and subsequent centrifugation at 14,000 g. Thereafter, nuclear protein samples were isolated from nuclear pellets by incubation with a complete lysis buffer containing 1 mM dithiothreitol. ELISA was performed using the nuclear protein samples in accordance with the manufacturer's instructions, and the free p65 NF-{kappa}B binding activity was expressed as the mean ratio of absorbance at 450 nm in untreated cells vs. that in cells treated with inhibitors. Recombinant p65 protein (Active Motif) was employed as a control for effective quantification (data not shown).

Diminishing TLR2 and TLR4 by the small interfering RNA technique. Recent advances in small interfering RNA (siRNA) techniques facilitate functional analyses of target genes by the loss of constituent mRNAs in transfected cells. SuperArray SureSilencing siRNA kits for mouse TLR2 and TLR4 (SuperArray Bioscience, Frederick, MD) were employed. GSM06 cells, seeded at a concentration of 5 x 106 per well, were incubated for 24 h. Oligofectamine 2000 reagent (Invitrogen) and Opti-MEM 1 reduced-serum medium (Invitrogen) were used for transfection in accordance with the manufacturer's instructions. The negative control siRNA (siNC) targets green fluorescent protein, a protein not normally expressed in mammalian cells. Following incubation with siRNAs [siNC, small interfering TLR2 (siTLR2), and siTLR4] for 24 h, the medium was replaced with freshly prepared serum-free medium containing H. pylori-LPS (50 ng/ml) and IFN-{gamma} (1,000 U/ml), and the cells were subjected to RT-PCR and NO assay after 3 and 48 h, respectively. The functional declines in TLR2 and TLR4 were assessed by monitoring NO production induced by ligands specific for individual receptors, i.e., PAM3CSK4 (50 ng/ml, Invitrogen) for TLR2 and E. coli-LPS (serotype 055:B5, 50 ng/ml, Sigma-Aldrich) for TLR4 in the presence of IFN-{gamma}.

NO assay. The NO levels in the culture medium were measured indirectly as hydrolyzed NO derivatives (NOx: nitrites and nitrates) using the Griess reagent kit (Dojindo Molecular Technologies, Kumamoto, Japan), in accordance with the manufacturer's instructions. The amount of culture medium per well was normalized to the total cell number in individual samples. The NOx concentration in the medium was determined by measuring absorbance at 540 nm and using a standard curve.

Immunohistochemistry of TLR2. Gastric mucosal specimens, with or without H. pylori infection (6 cases each), obtained by endoscopic biopsy from the gastric antrum with patient consent were examined by immunohistochemistry. The specimens were fixed in 10% buffered formalin and embedded in paraffin. The sections were incubated with the primary antibody for TLR2 (1:100; H-175, Santa Cruz Biotechnology) overnight at 4°C, followed by reaction with the secondary antibody and detected using a modified streptoavidin-biotin method (Vectastain ABC kit, Vector, Burlingame, CA). The antigen-antibody complexes were visualized by immersion in 3,3'-diaminobenizidine solution (0.001 M 3,3'-diaminobenizidine, 0.05 M Tris·HCl buffer, pH 7.6, 0.01 M sodium azide, and 0.006% hydrogen peroxidase). The specificity of immunohistochemical staining was monitored in sections processed without the primary antibody (data not shown). This study protocol was approved by the ethical committee of Tohoku University School of Medicine.

Statistical analysis. The data are expressed as the means ± SD of a minimum of three separate samples and analyzed by Fisher's protected least squares difference test. A P value <0.05 was defined to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of TLR2 and TLR4 in GSM06 cells and influence of treatment with H. pylori-LPS and/or IFN-{gamma}. The levels of TLR2 and TLR4 mRNA in GSM06 cells were assessed in the presence or absence of H. pylori-LPS and IFN-{gamma} by RT-PCR. Untreated cells exhibited very low expression of TLR2 mRNA, but H. pylori-LPS caused cells to increase TLR2 expression in a dose-dependent manner (Fig. 1A). IFN-{gamma} (10–1,000 U/ml) alone did not affect the expression of TLR2 mRNA; nevertheless, this cytokine dose dependently exhibited synergism with H. pylori-LPS to induce TLR2 mRNA (Fig. 1A). In contrast, TLR4 mRNA was constitutively expressed such that neither H. pylori-LPS nor IFN-{gamma} influenced its expression level compared with control cells (Fig. 1A). Changes in protein levels were consistent with the mRNA data showing that IFN-{gamma} alone did not affect the expression of TLR2 protein, but that cotreatment with H. pylori-LPS synergistically induced the expression of TLR2 protein (Fig. 1B). The observation that the expression level of TLR2 protein significantly increased in the cells treated with H. pylori-LPS and IFN-{gamma} but not with IFN-{gamma} alone was confirmed by the densitometric analyses of blots (P < 0.0001, Fig. 1C). TLR4 protein was unchanged by these treatments, as was the mRNA (Fig. 1, B and C).


Figure 1
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  		Fig. 1. Expression levels of Toll-like receptor (TLR)2 and TLR4 and influence of H. pylori (Hp)-LPS and/or interferon (IFN)-{gamma} treatment. A: changes in mRNA expression levels of TLR2 and TLR4 in GSM06 cells treated with Hp-LPS (0, 10, 50 ng/ml) and/or IFN-{gamma} (0, 10, 100, 1,000 U/ml) were assessed by RT-PCR. B: TLR2 and TLR4 protein levels in cells treated with Hp-LPS (50 ng/ml) and/or IFN-{gamma} (1,000 U/ml) were assessed by Western blotting. C: their changes were quantified as densitometric ratios of TLR2 or TLR4/beta-actin protein. TLR2 expression was significantly increased by treatment with Hp-LPS alone or Hp-LPS + IFN-{gamma}, although IFN-{gamma} alone did not affect it. TLR4 was unchanged regardless of treatment. Values are expressed as means ± SD from three independent experiments. ns, Not significant. *P < 0.0001: untreated vs. treated cells.

 
Participation of TLR4 signaling initiated by H. pylori-LPS in TLR2 induction. The expression levels of TLR2 and TLR4 diminished following transfection of GSM06 cells with siRNAs specific for mouse TLR2 and TLR4, respectively. Under unstimulated conditions, the cells transfected with TLR2- and TLR4-siRNAs exhibited declines in mRNA expression to 79.2 ± 2.3 and 76.4 ± 4.9% of the control values by 24 h posttransfection, and to 66.1 ± 4.2 and 62.9 ± 3.6% by 96 h, respectively (data not shown). The control siNC transfection did not affect the expression levels of TLR2 or TLR4 from 24 to 96 h posttransfection (data not shown).

TLR4 mRNA expression remained unchanged in TLR2-diminished cells vs. control siNC cells, but was significantly reduced in TLR4-diminished cells, regardless of treatment with H. pylori-LPS and IFN-{gamma} (Fig. 2A). TLR2 mRNA expression was low in control siNC cells under the unstimulated condition and significantly increased in response to H. pylori-LPS and IFN-{gamma} stimulation (Fig. 2B). Such increase in TLR2 mRNA expression induced by H. pylori-LPS and IFN-{gamma} treatment was significantly inhibited in TLR2-diminished cells as a consequence of effective interference of TLR2 mRNA transcription by siTLR2 (Fig. 2B). Furthermore, TLR4-diminished cells also exhibited a significant reduction of TLR2 induction by H. pylori-LPS and IFN-{gamma} treatment to nearly untreated control level (Fig. 2B), suggesting that TLR4 signaling may be essential for TLR2 induction by H. pylori-LPS and INF-{gamma} stimulation.


Figure 2
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  		Fig. 2. Influences of declines in TLR2 and TLR4 expression on the induction of TLR2 by Hp-LPS and IFN-{gamma} treatment. The negative control small interfering RNA (siNC) cells, small interfering TLR (siTLR) 2-, and siTLR4-diminished cells were treated with Hp-LPS (50 ng/ml) + IFN-{gamma} (1,000 U/ml). Changes in mRNA expression were assessed by RT-PCR for TLR4 and TLR2 and were quantified as densitometric ratios of TLR2 and TLR4/beta-actin mRNA. A: TLR4 expression was unchanged in control and TLR2-diminished cells, but was significantly low in TLR4-diminished cells, regardless of treatment. B: TLR2 induction by the treatment was significantly inhibited in TLR2- and TLR4-diminished cells. Values are expressed as the means ± SD from three independent experiments. **P < 0.01: siNC with Hp-LPS + IFN-{gamma} treatment vs. TLR2- or TLR4-diminished cells.

 
Possible signaling pathways of TLR2 induction by H. pylori-LPS stimulation. The activation of ERK, p38, and JNK in GSM06 treated with H. pylori-LPS and IFN-{gamma} was investigated by Western blotting using total and phosphorylation-specific antibodies. IFN-{gamma} alone did not affect the phosphorylation levels of MAPKs compared with untreated cells (Fig. 3). In cells treated with H. pylori-LPS and IFN-{gamma}, phosphorylated ERK protein, but no others, was clearly observed (Fig. 3).


Figure 3
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  		Fig. 3. Activation of mitogen-activating protein (MAP) kinases by treatment with Hp-LPS and IFN-{gamma}. The activation of MAP kinases in GSM06 cells treated with IFN-{gamma} (1,000 U/ml) alone (left column) or Hp-LPS (50 ng/ml) + IFN-{gamma} (1,000 U/ml) (right column) was analyzed by Western blotting using anti-phosphorylation-specific and total extracellular signal-regulated kinase (ERK) (top panels), p38 (middle panels), and c-Jun NH2-terminal kinase (JNK) (bottom panels) antibodies. Each pair of blots was run side by side. The marked phosphorylation of ERK, but neither p38 nor JNK, was identified in the cells treated with Hp-LPS + IFN-{gamma}, while INF-{gamma} alone did not phosphorylate MAP kinases. The results represent three independent experiments.

 
Next, the activation of NF-{kappa}B was assessed by ELISA-based quantification of the DNA-binding p65 NF-{kappa}B molecule. The cells were pretreated with specific inhibitors for MAPKs and NF-{kappa}B, i.e., PD98059 (for ERK), SP600125 (for JNK), SB203580 (for p38), and PDTC (for NF-{kappa}B) before treatment with H. pylori-LPS and IFN-{gamma}. The cells treated with H. pylori-LPS and IFN-{gamma} in the absence of inhibitor pretreatment showed a significant increase in the level of activated NF-{kappa}B compared with the control value (Fig. 4). On the one hand, when cells were pretreated with PDTC and PD98059, NF-{kappa}B activation significantly diminished in a dose-dependent manner (Fig. 4). On the other hand, there was no significant inhibition following pretreatment with SB203580 or SP600125 (Fig. 4).


Figure 4
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  		Fig. 4. Activation of nuclear factor-{kappa}B (NF-{kappa}B) by treatment with Hp-LPS and IFN-{gamma} and effects of inhibitors for NF-{kappa}B and MAP kinases. NF-{kappa}B activation in GSM06 cells, with or without pretreatments with pyrrolidine dithiocarbamate (PDTC) (10, 100 µM), PD98059 (10, 100 µM), SB203580 (1, 10 µM), and SP600125 (1, 10 µM) before Hp-LPS + IFN-{gamma} stimulation, was quantitatively assessed as free p65 NF-{kappa}B binding activity. Treatment with Hp-LPS + IFN-{gamma} significantly increased free p65 NF-{kappa}B binding activity. Pretreatment with either PDTC or PDTC inhibited NF-{kappa}B activation in a dose-dependent fashion. Neither SB203580 nor SP600125 elicited an inhibitory effect on NF-{kappa}B activation. A450, absorbance at 450 nm. Values are expressed as means ± SD from four independent experiments. *P < 0.05 and **P < 0.01 vs. Hp-LPS + IFN-{gamma}. ¥P < 0.01: untreated control vs. Hp-LPS + IFN-{gamma}. #P < 0.05: PDTC 10 vs. 100 µM. $P < 0.05: PD98059 10 vs. 100 µM.

 
In addition, the influence of pretreatment with the inhibitors on H. pylori-LPS- and IFN-{gamma}-induced TLR2 mRNA was investigated by RT-PCR. The induction of TLR2 was blocked to nearly control levels by PDTC and was reduced in a dose-dependent fashion by PD98059 (Fig. 5). However, neither SB203580 nor SP600125 attenuated TLR2 induction in the treated cells (Fig. 5).


Figure 5
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  		Fig. 5. Effects of NF-{kappa}B and MAP kinase inhibitors on TLR2 induction upon treatment with Hp-LPS and IFN-{gamma}. TLR2 expression in GSM06 cells pretreated with PDTC (10, 100 µM), PD98059 (10, 100 µM), SB203580 (1, 10 µM), and SP600125 (1, 10 µM) before Hp-LPS (50 ng/ml) + IFN-{gamma} (1,000 U/ml) treatment was assessed. PDTC blocked TLR2 induction to nearly control levels, and PD98059 also reduced it in a dose-dependent fashion. Neither SB203580 nor SP600125 attenuated TLR2 induction. The results represent three independent studies.

 
Roles of TLR2 and TLR4 in iNOS induction as an innate immune response against H. pylori-LPS. The innate immune response in GSM06 cells treated with H. pylori-LPS and/or INF-{gamma} was monitored by changes in iNOS mRNA level and its associated NO production. The treatment with H. pylori-LPS alone was very weak in iNOS induction and NO production, whereas INF-{gamma} alone significantly induced these responses in GSM06 cells (P < 0.0001, Fig. 6A). Furthermore, treatment with H. pylori-LPS together with INF-{gamma} significantly multiplied the increment of iNOS mRNA and NO production compared with their individual treatments (P < 0.0001, Fig. 6A). This induction of iNOS mRNA was inhibited by PDTC in a dose-dependent manner (Fig. 6B).


Figure 6
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  		Fig. 6. Induction of inducible nitric oxide synthase (iNOS) mRNA and the associated nitric oxide (NO) production by treatment with Hp-LPS and IFN-{gamma}. A: the nitrite (NOx) contents (top) and iNOS mRNA (bottom) were assessed in GSM06 cells. These were significantly increased by respective treatments with Hp-LPS (50 ng/ml) alone, IFN-{gamma} (1,000 U/ml) alone, and Hp-LPS (50 ng/ml) + IFN-{gamma} (1,000 U/ml) in increasing order. Values are expressed as means ± SD from four independent experiments. *P < 0.0005 and **P < 0.0001 vs. control. #P < 0.0001 vs. Hp-LPS alone. $P < 0.01 vs. IFN-{gamma} alone. B:the increased expression of iNOS mRNA by Hp-LPS + IFN-{gamma} treatment was dose-dependently inhibited upon pretreatment with PDTC (10, 100 µM). The results represent three independent studies.

 
Next, the influence of declines in TLR2 and TLR4 on iNOS induction by H. pylori-LPS and INF-{gamma} stimulation was investigated. siNC cells responded to treatment with H. pylori-LPS and INF-{gamma} with a significant increase in iNOS expression (Fig. 7). As anticipated, such iNOS induction by H. pylori-LPS and INF-{gamma} stimulation was significantly inhibited in TLR4-diminished cells (P < 0.01, Fig. 7). Surprisingly, TLR2-diminished cells, which expressed TLR4 at the same level as siNC cells, as shown in Fig. 2A, also exhibited a significant reduction of iNOS induction by H. pylori-LPS and INF-{gamma} stimulation (P < 0.01, Fig. 7). In other words, declines in not only TLR4 but also TLR2 attenuated responsiveness to treatment with H. pylori-LPS and INF-{gamma}. These data were corroborated by changes in NO production. Following treatment with H. pylori-LPS and IFN-{gamma}, NOx content as well as iNOS mRNA markedly increased in siNC cells, but such increase in NO production was significantly inhibited in both TLR2- and TLR4-diminished cells (P < 0.0001, Fig. 8).


Figure 7
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  		Fig. 7. Changes in iNOS induction by treatment with Hp-LPS and IFN-{gamma} in TLR2- and TLR4-diminished cells. The expression of iNOS mRNA was assessed by RT-PCR in TLR2- and TLR4-diminished cells treated with Hp-LPS (50 ng/ml) + IFN-{gamma} (1,000 U/ml). The treatment induced iNOS in control siNC cells, but TLR2- and TLR4-diminished cells exhibited significantly low responses. The densitometric ratios of iNOS to beta-actin mRNA are expressed as means ± SD from three independent experiments. **P < 0.01: siNC vs. TLR2- and TLR4-diminished cells treated with Hp-LPS + IFN-{gamma}.

 

Figure 8
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  		Fig. 8. iNOS-associated NO production induced by Hp-LPS, E. coli (Ec)-LPS, and PAM3CSK4 in TLR2- and TLR4-diminished cells. NOx contents following treatments with Hp-LPS (50 ng/ml), Ec-LPS (50 ng/ml), or PAM3CSK4 (50 ng/ml) in the presence of IFN-{gamma} (1,000 U/ml) were assessed in siNC cells and TLR2- and TLR4-diminished cells. An increase in NOx content by treatment with Hp-LPS + IFN-{gamma} was identified in siNC cells, but was significantly inhibited in both TLR2- and TLR4-diminished cells. Ec-LPS significantly increased NO production in siNC cells and TLR2-diminished cells, but not in TLR4-diminished cells; whereas PAM3CSK4 significantly increased NO production in siNC cells and TLR4-diminished cells, but not in TLR2-diminished cells. NO production in siNC cells significantly increased upon respective treatments with PAM3CSK4, Hp-LPS, and Ec-LPS in increasing order (P < 0.0001). Every treatment significantly increased NO production compared with the untreated control (P < 0.0001). Values are expressed as means ± SD from three independent experiments. **P < 0.0001 vs. siNC cells stimulated with Hp-LPS + IFN-{gamma}, ##P < 0.0001 vs. siNC cells stimulated with Ec-LPS + IFN-{gamma}, and $$P < 0.0001 vs. siNC cells stimulated with PAM3CSK4 + IFN-{gamma}.

 
To investigate whether TLR2 responsiveness to H. pylori-LPS was particular to this pathogen, the ligand-specific reaction for individual receptors was also assessed in TLR2- and TLR4-diminished cells by treatment with PAM3CSK4 (for TLR2) and E. coli-LPS (for TLR4) in the presence of IFN-{gamma}. On the one hand, an increase in NO production (NOx content) following E. coli-LPS stimulation was observed in both siNC cells and TLR2-diminished cells, but was significantly inhibited in TLR4-diminished cells (P < 0.0001, Fig. 8). On the other hand, PAM3CSK4 increased NO production in TLR4-diminished cells as well as siNC cells, but not in TLR2-diminished cells (P < 0.0001, Fig. 8); the observations indicated that declines in TLR2 or TLR4 by siRNA treatments resulted in the loss of functions specific to these receptors. NO induction in siNC cells was significantly low upon H. pylori-LPS treatment compared with E. coli-LPS treatment, despite the same concentration used (P < 0.0001, Fig. 8). PAM3CSK4 was also weak in NO induction (P < 0.0001, Fig. 8), reflecting low TLR2 expression in the cells (Fig. 2B). Nevertheless, NO production was significantly high upon every treatment compared with the untreated control (P < 0.0001, Fig. 8), as cotreatment with IFN-{gamma} may synergistically enhance NO production in addition to ligand-specific reaction, as shown in Fig. 6A.

Increased expression of TLR2 in human gastric mucosa infected with H. pylori. An immunohistochemical analysis of TLR2 expression was performed using biopsy specimens obtained from human gastric mucosa, with or without H. pylori infection. H. pylori-infected gastric mucosa showed an intense reaction on the apical surface of the epithelial cells and on invading inflammatory cells (Fig. 9, A, B, and C). However, gastric mucosa without H. pylori infection exhibited very little immune reaction against TLR2 antibody (Fig. 9D).


Figure 9
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  		Fig. 9. Immunohistochemistry of TLR2 in human gastric mucosa, with or without Hp infection. A: immunohistochemistry of TLR2 with formalin-fixed and paraffin-embedded specimens from human gastric antral mucosa with Hp infection. A positive reaction of TLR2 staining was identified at the apical surface of the epithelial cells and on invading inflammatory cells. Black squares in A indicate enlarged images of TLR2 expression corresponding to the apical surface of gastric epithelial cells (B) and invading inflammatory cells (C). D: noninflamed gastric mucosa without Hp infection exhibited little expression of TLR2. Original magnifications of A and D are at x40.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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TLRs are thought to play important roles in the first line of defense against H. pylori infection in gastric epithelial cells; however, their pathophysiological roles have not been completely resolved. In the present study, we investigated innate immune responses to H. pylori-LPS through the action of TLR2 and TLR4 using the mouse normal gastric epithelial GSM06 cell line (30). This cell line is derived from transgenic mice harboring the temperature-sensitive SV40 large T-antigen gene and exhibits 1) nontransformed cell characteristics similar to gastric mucosal cells, and 2) significant homologous sequences in TLR2 and TLR4 proteins with humans (NIH Blast2). IFN-{gamma} was used in the present study, since this Th1 cytokine is known to play a crucial role in H. pylori-associated gastritis, i.e., IFN-{gamma} mediates the induction of iNOS by H. pylori in mouse gastric epithelial cells (24).

GSM06 cells exhibited constitutive expression of TLR4 such that neither H. pylori-LPS nor IFN-{gamma} influenced the level of TLR4 mRNA or protein compared with control cells. While IFN-{gamma} alone had no effect on TLR2 levels, there was a small but significant increase in TLR2 expression upon treatment with H. pylori-LPS. Furthermore, IFN-{gamma}, together with H. pylori-LPS, synergistically enhanced TLR2 induction, suggesting that this cytokine may play a role in the posttranscriptional stabilization of TLR2 mRNA induced by H. pylori-LPS. In comparison, the transcriptional activation of iNOS by IFN-{gamma} results from the IFN-{gamma}-responding element located in the promoter region of the iNOS gene (12).

Participation of TLR4 signaling in the induction of TLR2 by H. pylori-LPS stimulation was corroborated in TLR2- and TLR4-diminished cells using the siRNA technique. TLR2 induction was significantly inhibited in TLR4-diminished cells compared with control siNC cells, although TLR4 siRNA did not interfere with the expression of TLR2 mRNA directly. In contrast, TLR4 expression was unchanged in TLR2-diminished cells. The facts suggest that TLR2 induction in GSM06 cells possibly results from the interaction between H. pylori-LPS and TLR4. Previous studies reported TLR2 induction via the interaction of a microbial LPS with TLR4 in mouse macrophages (18) and endothelial cells (9), an observation that is consistent with the present data. Furthermore, TLR2 induction by LPS stimulation is reported to be severely impaired in splenic macrophages isolated from TLR4-deficient C3H/HeJ mice (18), suggesting that TLR4 plays an essential role in the process. Controversially, TLR4-independent TLR2 induction by exposure to high concentrations of LPS is also reported in peritoneal macrophages from C3H/HeJ mice (16).

In general, the activation of TLR4, in cooperation with the adaptor molecule MyD88, signals the downstream MAPK pathway, which subsequently activates the NF-{kappa}B system (6, 31). TLR4 signaling for TLR2 induction initiated by H. pylori-LPS stimulation is possibly propagated through phosphorylation of the ERK pathway and, subsequently, NF-{kappa}B activation. Several lines of evidence support this notion: 1) phosphorylation of ERK but not p38 or JNK was identified in cells treated with H. pylori-LPS and INF-{gamma}; 2) the level of activated p65 NF-{kappa}B was significantly increased in treated cells; 3) the activated NF-{kappa}B level significantly declined by pretreatment with the ERK inhibitor PD98059; and 4) TLR2 induction by H. pylori-LPS and INF-{gamma} stimulation was inhibited by pretreatment with PD98059 and the NF-{kappa}B inhibitor PDTC. Neither p38 (SB208530) nor JNK (SP600125) inhibitors elicited such inhibitory effects. Thus TLR4 signaling through ERK-mediated NF-{kappa}B activation is crucial for TLR2 induction by H. pylori-LPS in gastric epithelial cells. Musikacharoen et al. (23) reported that the DNA element that binds activated NF-{kappa}B molecules is located in the promoter region of the mouse TLR2 gene, which is in agreement with our data.

In the present study, the innate immune response to H. pylori-LPS in GSM06 was monitored with the induction of iNOS and its associated NO production. iNOS is a target molecule for TLR4 signaling initiated by microbial LPS, not only in inflammatory cells (3, 7), but also in gastric epithelial cells (32). The induced iNOS in gastric epithelial cells exerts antimicrobial activity by NO attacking microorganisms (5). While iNOS-derived NO is possibly causative of gastric mucosal damages, it also works for exclusion of injured gastric epithelial cells by promoting apoptosis and eventually contributes to maintenance of gastric mucosa; therefore, it seems presumable that persistent inflammation without apoptosis in iNOS-deficient mice with H. pylori infection may be rather linked to pre-neoplastic transformation (19). Thus iNOS induction and its associated NO production play crucial roles in the formation of various pathological features in H. pylori-infected gastric mucosa.

H. pylori-LPS, with the synergistic assistance of IFN-{gamma} and NF-{kappa}B activation, induced iNOS expression and increased NO production in GSM06 cells. In sharp contrast to TLR2 induction by H. pylori-LPS and/or INF-{gamma} stimulation, iNOS induction and its associated NO production were also facilitated by treatment with INF-{gamma} alone. Such difference is possibly accounted by the fact that transcriptional activation of the iNOS gene may occur via direct interaction of IFN-{gamma} with the promoter region of this gene (12). TLR4-diminished cells significantly deteriorated the responsiveness to H. pylori-LPS stimulation, suggesting that TLR4 signaling initiated by H. pylori-LPS causes this response as well as TLR2 induction. Furthermore, TLR2-diminished cells also exhibited a low response to H. pylori-LPS stimulation, although TLR4 expression was unchanged in these cells. The observations suggest that induced TLR2 cooperates with TLR4 to amplify the innate immune response to H. pylori-LPS in GSM06 cells.

Such TLR2 responsiveness to H. pylori-LPS was particular to this pathogen, as demonstrated by the NO production assay: 1) TLR4-diminished cells lost the responsiveness to E. coli-LPS, but not to PAM3CSK4, and 2) TLR2-diminished cells conversely responded to E. coli-LPS, but not to PAM3CSK4. Thus declines in TLR2 or TLR4 expressions, respectively, deteriorated specific responses to corresponding ligands PAM3CSK4 or E. coli-LPS; therefore, the reduced responsiveness to H. pylori-LPS in TLR2-diminished cells may indicate a possible interaction between TLR2 and H. pylori-LPS. So far, no proof has been provided that TLR2 specifically binds to H. pylori-LPS; nevertheless, the possibility remains controversial, i.e., Smith et al. (28) reported with in vitro transfectants of gastric epithelial cell lines that TLR2 and TLR5, but not TLR4, are required for H. pylori-LPS-induced NF-{kappa}B activation. Differently, since repurification of LPS may eliminate signaling through both human and murine TLR2 (10), it seems possible that H. pylori-LPS employed in the present study may still retain other H. pylori components that react with TLR2 during the extraction process. Regardless of what pathogen contained in the H. pylori organism is able to interact with TLR2, this receptor is considered to play important roles in innate immune responses to H. pylori infection in gastric mucosa (17, 18, 28).

H. pylori-LPS itself is weak in pathogenic activity, with 1,000- to 10,000-fold less potency than other enterobacterial LPS (20–22); nevertheless, the interaction between H. pylori-LPS and TLR4 plays a significant role in the initiation and development of various pathological statuses in H. pylori-infected gastric mucosa, i.e., mitogen oxidase 1, which is known to play crucial roles in innate immune responses of gastric epithelium, was upregulated in guinea pig gastric pit cells by H. pylori-LPS-initiated TLR4 signaling (13). Furthermore, a previous in vivo study demonstrated that TLR4-intact C3H/He mice showed severe atrophic gastritis in response to H. felis infection, but that TLR4-deficient C3H/HeJ mice showed minimal atrophic gastritis with much reduced macrophage infiltration in the gastric mucosa, despite a heavy colonization of the bacteria (26). In contrast, although H. pylori-LPS can activate macrophages in a TLR4-dependent manner, Mandell et al. (17) pointed out the possibility that TLR2 might act as a dominant innate immune receptor for recognition of whole H. pylori organisms, because H. pylori may contain abundant components with a high affinity for TLR2, of which expression levels are regulated by cag pathogenicity island-encoded genes. Considering all of these observations together, the induction of TLR2 by TLR4 signaling initiated by H. pylori-LPS possibly indicates a positive correlation between TLR2 and TLR4 to intensify the innate immune responses of gastric epithelial cells against H. pylori infection.

Finally, our immunohistochemical data demonstrate that the expression of TLR2 was very weak in noninflamed gastric mucosa, but clearly increased on the apical surfaces of gastric mucosal epithelial cells in H. pylori-infected patients. Concerning TLR4 expression in human gastric mucosa, Schmausser et al. (27) reported that TLR4 expression was identified at the apical and basolateral pole of human gastric epithelium and that its intensity tended to be strong in H. pylori-infected gastric mucosa. Controversially, a previous study with a cDNA microarray system reported that only TLR2 was not upregulated in H. pylori-infected gastric mucosa, suggesting a selective expression pattern of TLRs against this gram-negative bacterium (33). The difference from our data may be derived from the fact that increased TLR2 expression is limited to the apical surface of H. pylori-infected gastric mucosa or the fact that there are controversial results of TLR2 regulation in response to H. pylori-LPS, potentially reflecting differences in bacterial strains and a variety of LPS structures affected by growing environments (15, 17, 20). Nevertheless, an increase of TLR2 expression in H. pylori-infected gastric mucosa may be advantageous, since gastric epithelial cells can acquire a broad susceptibility to a variety of H. pylori pathogens. Moreover, such adaptation of the TLR system is beneficial as a defense system against the combined infection of gram-positive bacteria, those that likely invade via salivary or dietary contamination and survive under a low-acidic environment in atrophic gastric mucosa caused by chronic H. pylori infection.

In summary, the present study showed that H. pylori-LPS stimulation of normal mouse gastric epithelial GSM06 cells induced TLR2 through TLR4 signaling, which was mediated by the ERK pathway and NF-{kappa}B activation. The induced TLR2 cooperated with TLR4 to activate iNOS transcription. Furthermore, these results translate to humans as increased expression of TLR2, which was also identified immunohistochemically in H. pylori-infected human gastric mucosa. This apparent positive correlation between TLR2 and TLR4 expression may yield a viable mechanism for stimulating the innate immune response against bacterial pathogens, including H. pylori-LPS. However, many things still remain unknown about the innate immune response to H. pylori infection in human gastric mucosa, warranting further studies.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by a Grant-in-Aid to T. Yoshimura for Scientific Research (17550146) from the Japan Society for the Promotion of Science and for Scientific Research on Priority Areas, Application of Molecular Spins, and Grant 15087212 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Kato, Cancer Detection Center, Miyagi Cancer Society, 5-7-30 Kamisugi, Aoba-ku, Sendai, Miyagi 980-0011, Japan (e-mail: kkato{at}cat-v.ne.jp)

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


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