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The Journal of Immunology, 2005, 175: 7162-7169.
Copyright © 2005 by The American Association of Immunologists

Helicobacter pylori Activates NF-{kappa}B via the Alternative Pathway in B Lymphocytes

Tomoya Ohmae1,*, Yoshihiro Hirata*, Shin Maeda*,{dagger}, Wataru Shibata*, Ayako Yanai*,{dagger}, Keiji Ogura*, Haruhiko Yoshida*, Takao Kawabe* and Masao Omata*

* Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan and {dagger} Division of Gastroenterology, The Institute for Adult Diseases, Asahi Life Foundation, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Helicobacter pylori causes various gastroduodenal diseases including gastric MALT lymphoma, but the mechanism underlying H. pylori-induced carcinogenesis is not known. The alternative pathway for NF-{kappa}B activation, which involves the processing of NF-{kappa}B2/p100 to p52, has been implicated in lymphocyte survival, attenuated apoptosis, and secondary lymphoid tissue development. In this study, we investigated H. pylori-induced activation of NF-{kappa}B through the alternative pathway in B lymphocytes. In immunoblot and EMSA, H. pylori induced NF-{kappa}B2/p100 processing to p52 and subsequent nuclear accumulation in IM-9 (human B cell line) cells and human peripheral blood B cells, but not in AGS (human gastric cancer cell line) cells. The activation of the alternative pathway was LPS-dependent but not cag pathogenicity island-dependent. Alternative pathway activation by H. pylori was associated with attenuated apoptosis. The expression levels of B lymphocyte chemoattractant, EBI-1 ligand chemokine, and stromal cell-derived factor-1{alpha} mRNAs were up-regulated in cocultured human B cells and in infected human gastric mucosa. In the infected mucosa, NF-{kappa}B2/p100 and p52 were detected immunohistochemically in the cytoplasm and nuclear compartments of lymphocytes, but not in epithelial cells. In summary, H. pylori activates the alternative NF-{kappa}B pathway in B lymphocytes. The effects on chemokine production and antiapoptosis mediated by H. pylori-induced processing of NF-{kappa}B2/p100 to p52 may drive lymphocytes to acquire malignant potential.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Gram-negative Helicobacter pylori is a bacterium that infects the human gastric mucosa (1). The infection is strongly associated with gastroduodenal diseases, such as chronic active gastritis, gastroduodenal ulcers, gastric adenocarcinoma (2, 3, 4, 5, 6), and gastric mucosa-associated lymphoid tissue (MALT) lymphoma, which is a low-grade B cell lymphoma. The etiological association between H. pylori infection and gastric MALT lymphoma was established in the early 1990s (7, 8). Subsequently, it has been shown that 50–80% cases of gastric MALT lymphoma regress after H. pylori eradication by antibiotics (9, 10, 11), and bacterial eradication is now considered to be the first-line therapy for this neoplasm. However, the mechanism by which H. pylori contributes to the development of MALT lymphoma remains unclear.

The transcription factor NF-{kappa}B regulates the expression of genes that are involved in inflammation, cell proliferation, and apoptosis (12, 13). Five members have been identified in mammals: NF-{kappa}B1/p105, NF-{kappa}B2/p100, c-Rel, RelA, and RelB. In the classical pathway, NF-{kappa}B activation is tightly controlled by the I{kappa}B kinase (IKK)2 complex, which consists of two catalytically active kinases, IKK{alpha} and IKK{beta}, and an inactive compound, IKK{gamma} (14, 15). Activation of the IKK complex leads to phosphorylation, ubiquitination, and proteolytic degradation of the inhibitory NF-{kappa}B{alpha} (I{kappa}B{alpha}), which allows NF-{kappa}B homodimers or heterodimers to translocate to the nucleus. Various cell surface receptors, such as TNF-{alpha} receptor and IL-1-like/TLR family members, can activate the classical pathway (16).

Recently, it has been reported that the alternative pathway for NF-{kappa}B activation contributes to the development, survival, and attenuation of apoptosis of B cells. The alternative NF-{kappa}B pathway involves the processing and cleavage of NF-{kappa}B2/p100 precursor to p52, which are triggered by p100 phosphorylation by NF-{kappa}B-inducing kinase (NIK) and IKK{alpha} (17). This pathway is also known to be activated by stimulation of the lymphotoxin (LT){beta} receptor, BAFF (B cell activating factor belonging to the TNF family) receptor, or CD40, but not by the TNF-{alpha} receptor (18, 19, 20). The p52 molecule, which forms a heterodimer with another NF-{kappa}B subunit, translocates to the nucleus and binds to the NF-{kappa}B sites. The activation of the alternative NF-{kappa}B pathway and subsequent up-regulation of target genes is reportedly necessary for the secondary development of lymphoid organs, such as the spleen, lymph nodes, and Peyer’s patches (21).

Two major chromosomal translocations, t(11;18)(q21;q21) and t(1;14)(p22;q32), are known to be associated with MALT lymphoma; the former is more common, being observed in approximately one-third of cases (22, 23). Dierlamm et al. (24) have reported that t(11;18)(q21;q21) results in the fusion of API2 (apoptosis inhibitor gene 2) on 11q21 with MALT1 (MALT lymphoma-associated translocation gene 1) on 18q21, which is known as API2-MALT1. The fusion protein produced by API2-MALT1 strongly enhances classical NF-{kappa}B pathway activation (25). In contrast, t(1;14)(p22;q32) leads to overexpression of Bcl-10 protein with a frame shift mutation in the CARD (caspase recruitment domain), which also leads to constitutive activation of NF-{kappa}B (26, 27, 28). Thus, it is now generally recognized that enhanced activation of the classical NF-{kappa}B pathway is closely linked to the pathogenesis of MALT lymphoma (29, 30).

Although virulent strains of H. pylori are known to activate NF-{kappa}B through the classical pathway in epithelial cells (31, 32), NF-{kappa}B activation in lymphocytes has not been well defined. In this study, we analyzed the activation of NF-{kappa}B in lymphocytes following stimulation with H. pylori, and focused on the contribution of the alternative pathway and subsequent phenotypic changes. We demonstrate that H. pylori induces the processing of NF-{kappa}B2/p100 to p52 and subsequently the target gene up-regulation in B lymphocytes. H. pylori also induces the attenuation of apoptosis via the alternative pathway activation in B lymphocytes.


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

The TN2 strain, which is positive for cytotoxin-associated gene A (CagA) and for vacuolating cytotoxin A (VacA), was generously provided by Dr. Masafumi Nakao (Takeda Chemical Industries, Osaka, Japan). The TN2-isogenic cagA-negative and cagE-negative mutants (TN2{Delta}cagA and TN2{Delta}cagE, respectively) were constructed by insertion of the kanamycin-resistant gene, as previously described (33). In the coculture experiments, H. pylori was cultured for 24 h in Brucella broth that contained 7.5% FBS, centrifuged, washed with PBS, resuspended in RPMI 1640 that contained 10% FBS, and used immediately thereafter in the assays. The bacterium to cell ratio was 5:1 in all experiments, except in the TUNEL to detect cell death. Heat-killed H. pylori were prepared by heating the bacteria at 80°C for 60 min.

Preparation of H. pylori LPS

H. pylori (TN2) LPS was prepared by the method of Galanos et al. (34). Briefly, 20 mg (dry-weight) of H. pylori cells were washed in PBS, suspended in cold distilled water (10 mg/ml), and then poured into 10 volumes of cold acetone (–20°C). The sediments were dried under vacuum, ground, and suspended in water (6% w/v at 65°C). An equal volume of phenol solution (90% w/v) was added and, after incubation, the mixture was centrifuged at 10,000 x g for 10 min at 4°C. After removing the upper aqueous layer, the LPS was precipitated by pouring the solution into 10 volumes of cold acetone (–20°C). The precipitate was collected, resuspended in a small amount of distilled water, and ultracentrifuged (100,000 x g) three times for 6 h. The LPS was used in 20 µg/ml.

Cell lines and primary mammalian B lymphocytes

The human B lymphoblastoid cell line IM-9 was purchased from the Japanese Collection of Research Bioresources Cell Bank/National Institute of Health Sciences (Tokyo, Japan) and maintained in RPMI 1640 that contained 10% FBS. The human gastric cancer cell line AGS was purchased from American Type Culture Collection and maintained in Ham’s F12 medium, which contained 10% FBS.

Human peripheral blood B lymphocytes were obtained from heparinized 50 ml blood samples drawn from healthy volunteers. Each blood sample was mixed with an equal volume of HBSS (Sigma-Aldrich), and the tube was inverted gently several times. Each sample was layered on 25 ml of Ficoll-Paque Plus (Amersham Biosciences) and centrifuged at 400 x g for 40 min at 20°C. The lymphocyte layer was transferred to a clean tube, centrifuged, washed twice with PBS, and incubated with anti-human CD22 microbeads (Miltenyi Biotec) for 15 min at 10°C, centrifuged at 400 x g for 10 min, and washed with PBS. Then the suspensions were sorted by passage through an MS magnetic column (Miltenyi Biotec) and resuspended in RPMI 1640 that contained 10% FBS. Approximately 5 x 106 B cells were obtained from one 50-ml blood sample.

Murine splenic B cells were prepared from 6- to 8-wk-old C57BL/6JJcl and ALY/NscJcl-aly/aly male mice (CLEA Japan). The experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals in our division, and were approved by the Ethical Committee. Murine splenic cells were isolated and resuspended, and the erythrocytes were lysed with ACK lysis buffer. The cell suspension was incubated with rat anti-mouse CD45R/B220 mAb (1/100 dilution; BD Pharmingen) for 30 min on ice, centrifuged at 400 x g for 10 min, and washed with 10 ml of PBS. Finally, the sample was incubated with MACS goat anti-rat IgG microbeads (Miltenyi Biotec) for 15 min at 10°C, washed with PBS, and sorted by passage through an MS magnetic column (Miltenyi Biotec). Approximately 1 x 107 splenic B cells were obtained using this protocol.

Abs and reagents

The anti-human NF-{kappa}B2/p100 and anti-p52 Ab was purchased from Upstate Biotechnology, the anti-mouse NF-{kappa}B2/p100 and anti-p52 Ab was from Santa Cruz Biotechnology, the anti-human and anti-mouse phospho-I{kappa}B{alpha} Abs were from Cell Signaling Technology, and the anti-human and anti-mouse actin Abs were from Sigma-Aldrich. To stimulate the cells, we used TNF-{alpha} (10 ng/ml; Pharma-Biotechnologie), BAFF (2 µg/ml; PeproTech), Salmonella typhimurium LPS (20 µg/ml; List Biological Laboratories) and Escherichia coli LPS (20 µg/ml; Sigma-Aldrich). Ammonium pyrrolidine dithiocarbamate (APDC, 100 µM; Dojindo Laboratories) was used to inhibit the classical NF-{kappa}B pathway.

Whole cell, cytoplasmic, and nuclear protein extracts

Cells were washed with PBS and lysed in ice-cold 1% Triton X-100 (Sigma-Aldrich) and Complete Mini EDTA-free (Roche Diagnostics). The lysate was centrifuged at 10,000 x g for 5 min at 4°C, and the supernatant was used as the whole cell protein extract. Cytoplasmic and nuclear extracts were prepared from IM-9 cells using the PARIS kit (Ambion) according to the manufacturer’s instructions.

RNA interference

For small interfering RNA targeted to IKK{alpha}, we generated 21-base complementary RNAs with two thymidine residues (dTdT) at the 3' end, i.e., 5'-(AA)GCAGGCUCUUUCAGGGACA-3', as previously described (35). The nonsilencing RNA sequence 5'-(AA)TTCTCCGAACGTGTCACGT-3' was also used. The NF-{kappa}B2/p100 small interfering (si)RNA was purchased from Santa Cruz Biotechnology. These siRNA were transfected with Lipofectamine 2000 (Invitrogen Life Technologies) into 1 x 105 IM-9 cells in 3.5-cm dishes. Three hours after the addition of the siRNA, the IM-9 cells were washed, cocultured with H. pylori, and used in the following assays.

Immunoblotting

The whole cell, cytoplasmic, and nuclear extracts were electrophoresed on SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Amersham Biosciences) and blocked for 1 h with TBST plus 5% dry milk. The membrane was probed overnight at 4°C with the primary Abs, and subsequently washed and incubated with the secondary peroxidase-conjugated Ab. The immunocomplexes were detected with the Enhanced Chemiluminescence Detection kit (ECL Advance; Amersham Biosciences).

EMSA

NF-{kappa}B DNA-binding activity was analyzed in the nuclear extracts (10 µg) using the EMSA kit (Panomics) according to the manufacturer’s instructions. For the Ab supershift analysis, the nuclear extracts were preincubated for 30 min with rabbit antisera against human NF-{kappa}B2/p100 and p52 (Upstate Biotechnology).

Real-time PCR analysis and RT-PCR

Total cellular RNA samples were isolated using ISOGEN (Nippon Gene) from human peripheral blood B cells or endoscopic gastric biopsy specimens, which were obtained from healthy volunteers and patients with H. pylori-induced gastritis. The cDNAs were generated by reverse transcription from 1 µg of total RNA using the ImProm-II Reverse Transcription System (Promega). The mRNA expression levels of B lymphocyte chemoattractant (BLC), EBI-1 ligand chemokine (ELC), stromal cell-derived factor-1{alpha} (SDF-1{alpha}), and GAPDH were analyzed by quantitative real-time PCR or RT-PCR. Real-time PCR amplification was performed according to the SYBR Green PCR Master Mix (Applied Biosystems) protocol. Relative quantification of gene expression was obtained using GAPDH mRNA as an internal standard. To perform RT-PCR, the PCR mixtures were incubated for 10 min at 94°C, followed by 30 cycles of 30 s at 96°C, 1 min at 54°C, and 1 min at 72°C. The PCR products were analyzed by electrophoresis on 1.5% agarose gels. All primer sequences are available upon request.

Immunohistochemistry

Formalin-fixed paraffin-embedded gastric biopsy specimens, which were obtained endoscopically from human healthy volunteers and patients with H. pylori-induced gastritis, were examined immunohistochemically for NF-{kappa}B2/p100 and p52. Sections cut at a thickness of 3 µm were deparaffinized and rehydrated, and endogenous peroxidase activity was blocked using 3% H2O2. Immunohistochemistry was performed using the anti-human NF-{kappa}B2/p100 and anti-p52 Ab (1/100 dilution) and the avidin-biotin method (DAKO LSAB2 System; DAKO). In immunofluorescent staining, anti-human NF-{kappa}B2/p100 and anti-p52 Ab and anti-CD20 Ab (1/100 dilution; DAKO) were used as primary Ab. Fluorochrome Alexa Fluor 488 and 555 (Molecular Probes) were used as secondary Abs and the sections were observed under a confocal microscope (Leica Microsystems).

Apoptosis detection by TUNEL staining

IM-9 cells that were pretreated for 4 h with the control (nonsilencing), IKK{alpha}, or NF-{kappa}B2/p100 siRNA were plated in a 12-well plate (1 x 105 cells/well) in RPMI 1640 medium that contained 5% FBS, and then stimulated with H. pylori for 48 h. The ratio of bacteria to cancer cells was set at 100:1. Apoptosis was analyzed using the ApopTag Plus Fluorescein In Situ Apoptosis Detection kit (Chemicon International) according to the manufacturer’s instructions. Briefly, cells were fixed with 1% paraformaldehyde in PBS (pH 7.4) for 10 min at room temperature. Then each 20 µl of cell suspension was grounded, dried and postfixed with precooled ethanol and acetic acid (2:1) for 5 min at –20°C on a microscope slide. After washing, the glass slides were incubated with a reaction mixture containing TdT for 1 h at 37°C. Anti-digoxigenin conjugate solution was added, and the slides were incubated for 30 min at room temperature, washed with PBS, and mounted with propidium iodide containing mounting medium (0.5 µg/ml). Each slide was observed under a confocal microscope (Leica Microsystems) and the number of apoptotic cells in a total of 1000 nuclei was counted. Three sample slides were examined for each experiment, and the average number of apoptotic cells was calculated.

Statistical analysis

The data are expressed as the means ± SD. Statistical analysis was performed using the Student’s t test, two-sided. Differences were considered statistically significant with values of p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
H. pylori activates both the classical and alternative NF-{kappa}B pathways in B lymphocytes

Initially, we examined whether H. pylori activated NF-{kappa}B through the classical pathway in B lymphocytes. As determined by immunoblotting, H. pylori strain TN2 induced the phosphorylation of I{kappa}B{alpha} in IM-9 cells and human peripheral blood B cells within 15–20 min; this was followed by rapid degradation over 2 h (Fig. 1, A and B), which indicates that H. pylori activates NF-{kappa}B through the classical pathway in B cells.



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  		FIGURE 1. H. pylori activates NF-{kappa}B via the classical and alternative pathway in lymphocytes. IM-9 cells (A) and human peripheral B cells (B) were infected with H. pylori TN2 strain for the indicated time periods. The cell lysates were immunoblotted with the anti-phospho-I{kappa}B{alpha} and anti-actin Abs. IM-9 cells (C) and human peripheral B cells (D) were treated with H. pylori, BAFF, or TNF-{alpha} for the indicated time periods. The cell lysates were analyzed by immunoblotting with the anti-p100/p52 and anti-actin Abs. Data shown are representative results of three independent experiments.

 
We also examined the alternative pathway, which involves the cleavage of NF-{kappa}B2/p100 to p52. The level of p52 protein increased in IM-9 cells and human peripheral blood B cells (Fig. 1, C and D), which indicates the involvement of the alternative NF-{kappa}B pathway in B cells. In contrast to BAFF stimulation, H. pylori stimulation did not decrease the level of NF-{kappa}B2/p100. We speculate that this is due to the up-regulation of NF-{kappa}B2/p100 through the classical pathway (20). Therefore, we investigated whether H. pylori increased the level of p52 in the presence of APDC, which is an inhibitor of the classical NF-{kappa}B pathway. APDC suppressed the increase and the subsequent degradation of phospho-I{kappa}B{alpha} in H. pylori-induced IM-9 cells (Fig. 2A), whereby the level of p52 still increased, but that of NF-{kappa}B2/p100 did not (Fig. 2B). These results suggest that H. pylori activates both the classical and alternative pathways in B lymphocytes. As shown in Fig. 2C, activation of the alternative pathway was not observed in AGS cells.



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  		FIGURE 2. H. pylori activates the alternative NF-{kappa}B pathway in a manner that is independent of the classical pathway. A, IM-9 cells were treated with (+) or without (–) APDC, the NF-{kappa}B classical pathway inhibitor, and subsequently infected with H. pylori for the indicated time periods. I{kappa}B{alpha} phosphorylation and actin was assessed by immunoblotting. B, IM-9 cells were pretreated with APDC and then stimulated with H. pylori or BAFF. The level of p100, p52, and actin protein was assessed by immunoblotting. C, IM-9 cells or AGS cells were infected with H. pylori for 2, 8, or 24 h. The cell lysates were analyzed for p100, p52, and actin.

 
H. pylori induces nuclear translocation of p52

We assessed the nuclear translocation of p52 by immunoblotting cytoplasmic and nuclear extracts. As shown in Fig. 3A, H. pylori induced p52 accumulation in the nuclear extracts of IM-9 cells within 8–24 h. In contrast, there was no increase in the level of p52 in the cytosolic fraction, which confirms the translocation of p52 into the nucleus. We also analyzed the DNA-binding activity of the nuclear NF-{kappa}B complex by EMSA. As shown in Fig. 3B, although the nuclear extract from untreated IM-9 cells showed low NF-{kappa}B DNA-binding activity, this activity was strongly enhanced by stimulation with H. pylori or BAFF. Binding was confirmed by supershifting with an Ab directed against p52. The blc, elc, and sdf-1{alpha} genes are reportedly up-regulated by LT{beta}1 stimulation via the alternative NF-{kappa}B pathway, but not through the classical pathway in splenocytes (20). Indeed, blc, elc, and sdf-1{alpha} mRNA were up-regulated by H. pylori TN2 by 5.4 ± 1.0-fold, 6.8 ± 0.4-fold, and 23.1 ± 3.4-fold, respectively, in human peripheral blood B cells at 12 h (Fig. 3C).



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  		FIGURE 3. Functional analysis of p52 in H. pylori-infected lymphocytes. A, Nuclear and cytoplasmic extracts of H. pylori-infected IM-9 cells were analyzed by immunoblotting for p100 and p52. TATA box binding protein (TBP) and Sos1 were used as the nuclear and cytoplasmic loading controls, respectively. B, Nuclear extracts from H. pylori- or BAFF-treated IM-9 cells were prepared. Equivalent amounts of the nuclear extracts were incubated with the NF-{kappa}B probe and anti-p52 Ab, and EMSA was performed. Unstimulated cells (first lane), H. pylori-stimulated cells (second lane), H. pylori-stimulated cells (third lane) in the presence of the anti-p52 Ab, and BAFF-stimulated cells (last lane). C, Real-time PCR analysis for the expression of blc, elc, sdf-1{alpha}. Human peripheral blood B cells were stimulated with H. pylori or BAFF for 12 h, and total RNA was prepared. The gapdh gene was used as the control for mRNA expression.

 
Alternative pathway activation does not depend on cag pathogenicity island (cagPAI) gene

We used the isogenic mutants TN2{Delta}cagA and {Delta}cagE to investigate whether the activation of the alternative pathway is dependent on cagPAI of H. pylori. Changes in the p52 levels were assessed by immunoblotting. As shown in Fig. 4A, the p52 levels in IM-9 cells were increased by coculture with {Delta}cagA or {Delta}cagE, as well as with the wild-type strain. Stimulation of the IM-9 cells with heat-inactivated TN2 bodies or purified LPS enhanced the production of p52, as well as LPS from S. typhimurium and E. coli (Fig. 4, B and C). These findings indicate that H. pylori activates the alternative pathway independently of cagPAI, and that LPS is one of the effectors for B lymphocytes.



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  		FIGURE 4. The H. pylori virulence factor cagPAI does not play a significant role in the alternative NF-{kappa}B pathway. A, IM-9 cells were infected with the TN2 wild-type, cagA mutant, and cagE mutant for 24 h. The levels of p100, p52, and actin were assessed by immunoblot analysis. IM-9 cells were stimulated with heat-killed TN2 (B) or LPS (C) (H. pylori, S. typhimurium, or E. coli) for indicated hours, and immunoblotting was performed.

 
Activation of the alternative NF-{kappa}B pathway via NIK and IKK{alpha}

We investigated the roles of NIK and IKK{alpha} in the activation of the alternative pathway by H. pylori. Murine splenic CD45R/B220-positive B cells from C57BL/6JJcl and ALY/NscJcl-aly/aly mice, the latter having an NIK point mutation (36), were stimulated with H. pylori or BAFF. As shown in Fig. 5A, the level of p52 was increased by H. pylori or BAFF in B cells from C57BL/6JJcl mice but not in those from ALY/NscJcl-aly/aly mice. In addition, we transfected siRNA for IKK{alpha} into IM-9 cells. H. pylori treatment increased the level of p52 in IM-9 cells that were transfected with nonsilencing siRNA but not in cells that contained the IKK{alpha} siRNA (Fig. 5B). These results suggest that H. pylori activates the alternative NF-{kappa}B pathway via NIK and IKK{alpha} in B cells.



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  		FIGURE 5. H. pylori activates the alternative NF-{kappa}B pathway through NIK and IKK{alpha}. A, Splenic B cells were prepared from C57BL/6 mice and NIK point mutation aly/aly mice. The cells were treated with H. pylori or BAFF for 8 h, and protein extracts were subjected to immunoblotting for p100, p52, and actin. B, IM-9 cells were transfected with control siRNA and IKK{alpha} siRNA for 6 h, and then infected with H. pylori for the indicated time periods. Immunoblotting was performed for IKK{alpha}, NF-{kappa}B2/p100, p52, and actin.

 
H. pylori inhibits B cell apoptosis via the alternative pathway

We investigated the potential linkage between alternative NF-{kappa}B pathway activation by H. pylori and lymphocyte apoptosis. First, we used siRNA for IKK{alpha} and NF-{kappa}B/p100 to inhibit the alternative pathway, and performed immunoblotting to confirm the RNA interference effects. The siRNA for IKK{alpha} effectively suppressed the levels of the IKK{alpha} and p52 proteins, and the siRNA for NF-{kappa}B/p100 reduced the p100 and p52 protein level (Fig. 6A). Second, we tested apoptosis induction by H. pylori using TUNEL staining. As shown in Fig. 6B, apoptotic cells induced by H. pylori were further increased by IKK{alpha} or NF-{kappa}B2/p100 silencing. Indeed, H. pylori induced apoptosis in 3.0 ± 0.3%, 7.4 ± 1.3%, and 8.6 ± 0.6% of the nonsilencing, IKK{alpha}, and NF-{kappa}B2/p100 siRNA affected cells, respectively. Similar results were obtained by cell death detection ELISA analysis (data not shown). These results suggested that the H. pylori-induced alternative NF-{kappa}B pathway activation was associated with antiapoptotic effects on B lymphocytes.



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  		FIGURE 6. H. pylori inhibits B cell apoptosis via the alternative pathway. A, Nonsilencing, IKK{alpha}-specific, and NF-{kappa}B2/p100-specific siRNAs were transfected into IM-9 cells. The levels of IKK{alpha}, NF-{kappa}B2/p100, and p52 were analyzed by immunoblotting. The siRNA for IKK{alpha} decreases the levels of both IKK{alpha} and p52 proteins, whereas the siRNA for NF-{kappa}B2/p100 reduces the level of p100 and p52 protein. B, IM-9 cells, which were transfected with siRNAs as described in A, were incubated with H. pylori for 48 h. The cells were analyzed for apoptosis by TUNEL staining. TUNEL positive cells were visualized by fluorescein (green) and nuclei were counterstained with propidium iodide (red). C, Apoptotic cells per total cell ratio was calculated. The values shown represent the mean ± S.D. from three independent experiments. Statistically significant differences, compared with nonsilencing siRNA and IKK{alpha} or NF-{kappa}B2/p100 siRNA, are defined at p < 0.05.

 
Alternative NF-{kappa}B pathway activation in the human gastric mucosa

We examined human gastric biopsy specimens obtained from subjects with or without H. pylori infection (as determined by culture) using immunohistochemistry for NF-{kappa}B2/p100 and p52 (Fig. 7A). NF-{kappa}B2/p100 and p52 were detected in infiltrating inflammatory cells in the H. pylori-infected mucosa. Some of the gastric epithelial cells in the H. pylori-infected mucosa were also positive for NF-{kappa}B2/p100 or p52, although these cells were stained exclusively in the cytoplasm and were so to a lesser extent than in the lymphocytes. To confirm these finding, we performed immunofluorescent double staining for NF-{kappa}B2/p100 and CD20 B cell surface Ag. The infiltrating inflammatory cells are positive for both NF-{kappa}B2/p100 and CD20 (Fig. 7B). Furthermore, in high magnification image, NF-{kappa}B/p100 or p52 was positive in both cytoplasm and nucleus of the cell (Fig. 7C). In contrast, in H. pylori uninfected mucosa, B cells in the mucosa were positive for NF-{kappa}B2/p100 only in cytoplasm (Fig. 7D), suggesting that the alternative pathway of the NF-{kappa}B was not activated. These findings are consistent with the in vitro findings that p52 was increased by H. pylori in nucleus of human B cells. The levels of blc, elc, and sdf-1{alpha} mRNA expression in the gastric tissues were assessed by RT-PCR and found to be markedly up-regulated in H. pylori-infected mucosa, but were very low in uninfected mucosa (Fig. 7E). These findings indicated that NF-{kappa}B alternative pathway activation and its downstream chemokine expression were actually induced in vivo as well as in vitro experiments.



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  		FIGURE 7. Alternative pathway activation in the human gastric samples. A, Immunohistochemistry of NF-{kappa}B2/p100 and p52 in H. pylori noninfected (left) and infected (right) gastritis tissue. B, Immunofluorescent staining of NF-{kappa}B/p100 (red), CD20 (green), and their merge image of H. pylori-infected gastric mucosa. Serial section of right panel in A was used. C, High magnification image of B. D, High magnification image of B cells in H. pylori uninfected gastric mucosa. p52 was not stained in the nucleus of the cells. E, Total RNA was extracted from the gastric mucosa samples of individuals who were either infected (n = 3) or not infected (n = 3) with H. pylori, and RT-PCR for blc, elc, sdf-1{alpha}, and gapdh mRNAs was performed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report, we have shown that H. pylori activates NF-{kappa}B in lymphocytes through both the classical and alternative pathways. As for the classical pathway, chromosomal abnormalities that cause API2-MALT1 fusion protein or Bcl-10 overexpression are reported to activate the NF-{kappa}B classical pathway continuously and to contribute to the MALT lymphoma (25, 26, 27, 28). This finding may be one of the reasons why the eradication therapy of H. pylori is not effective in patients with these chromosomal abnormalities. Otherwise, it has been reported that mice with a homozygous deletion of the C-terminal ankyrin repeat of NF-{kappa}B2/p100, which induces constitutive p52 formation, have marked gastric hyperplasia with lymphocytic infiltration, resulting in early postnatal death (37). Thus, the activation of the alternative pathway may also play a role in lymphocytic infiltration and proliferation in H. pylori-infected gastric mucosa.

We also showed the up-regulation of blc, elc, and sdf-1{alpha} expression following exposure to H. pylori. A previous immunohistochemical study has shown that BLC (BCA-1) is expressed in H. pylori-infected gastric MALT and MALT lymphoma tissues (38). BLC, ELC, and SDF-1{alpha} are all chemokines that play roles in lymphocytic chemotaxis, recruitment, and lymphoid tissue development (39, 40, 41). Thus, these H. pylori-induced chemokines may promote B cell infiltration through autocrine or paracrine mechanisms, leading to the development of MALT lymphoma.

Interestingly, in the present study, NF-{kappa}B activation through the alternative pathway by H. pylori was found to attenuate lymphocyte apoptosis. However, in the BrdU ELISA, no advantageous effects on cell proliferation were associated with alternative pathway activation (data not shown). Thus, H. pylori may induce lymphocyte proliferation or malignant transformation by enhancing cell survival through antiapoptotic effects that are mediated by the alternative NF-{kappa}B pathway, rather than by direct stimulation of cell proliferation.

In this study, we also discovered that cagPAI does not contribute to the activation of NF-{kappa}B in lymphocytes, which is similar to the situation in monocytes, as we have previously reported (42). Recently, it has been reported that ectopic expression of CagA in IL-3-dependent B cells inhibits cell proliferation by suppressing JAK-STAT signaling (43). It seems likely that CagA does not enhance antiapoptotic or proliferative responses in lymphocytes. LPS derived from E. coli, as well as BAFF, LT{beta}1, and CD40L, reportedly activate the alternative pathway in splenocytes (44). In the current study, we show that LPS derived from H. pylori also activates the alternative pathway, as well as LPS from E. coli or S. typhimurium. This suggests bacteria expressing LPS may activate this pathway as well as H. pylori activation in vitro. However, because H. pylori is the only bacterium that infects in human gastric lumen continuously, we consider that LPS derived from other bacteria cannot be responsible for gastric MALT lymphoma development. Otherwise, H. pylori does not activate the alternative pathway in epithelial cells, as shown in AGS cells and confirmed in the immunohistochemical studies. The different roles of the alternative pathway in epithelial cells and lymphocytes may reflect the differential expression of cell surface receptors, especially the LPS receptor TLR4. Indeed, TLR4, which is indispensable for classical pathway activation in lymphocytes, is expressed poorly in gastric epithelial cells (45), whereas it is expressed strongly in lymphocytes and monocytes. Further studies on TLR4 may help us to understand the mechanisms of H. pylori-induced alternative pathway activation.

NF-{kappa}B activation through the alternative pathway is slower than through the classical pathway. In general, signal-induced processing of p100 required the new or continued synthesis of a protein, which could explain the slow onset of processing upon stimulation. For example, it was previously reported that the processing of p100 to p52 induced by BAFF (18), LPS (44), LT{beta} (44), or CD40L (19) was inhibited when cycloheximide was pretreated. We also analyzed whether H. pylori-induced this pathway was inhibited by cycloheximide and found that H. pylori could not induce p100 processing in cycloheximide pretreated IM-9 cells (data not shown). Thus, we consider that H. pylori could activate this pathway via new or continued synthesis of a protein as well as other factors, which could activate the alternative pathway such as BAFF. We cannot exclude a possibility that some indirectly factors such as cytokines or chemokines induced by H. pylori are associated with the processing of p100. This possibility has not analyzed even in the case of BAFF or LT{beta}. Further examination is needed to resolve this query.

In the normal gastric mucosa, B lymphocytes are rarely in direct contact with bacteria in the gastric lumen. H. pylori often contacts with gastric epithelial cells and once H. pylori infects the mucosa, it induces lymphocytic chemotaxis and infiltration, which facilitate interactions between B cells and H. pylori-specific T cells (46, 47), and may be involved in NF-{kappa}B activation. So it may occur that these cells involved in H. pylori infection also contribute to the NF-{kappa}B activation. However, H. pylori also causes gastric erosion or ulcer, then it may occur that H. pylori contact directly with infiltrating inflammatory cells including B lymphocytes in the lamina propria. Thus it is likely that H. pylori, and its related components such as dead bacterial bodies and LPS, activates NF-{kappa}B classical and alternative pathway in human B cells in the gastric mucosa. In our current study, we focused on the H. pylori-induced activation of NF-{kappa}B alternative pathway. We have not assessed whether the direct involvement of Bcl-10 or MALT1 proteins in dysregulated NF-{kappa}B activation by classical and alternative pathway in B cells may promote malignant transformation.

In conclusion, H. pylori activates NF-{kappa}B in B lymphocytes not only through the classical pathway but also through the alternative pathway. H. pylori LPS is a candidate effector for the alternative pathway activation. The alternative pathway activation by H. pylori may contribute to the development of gastric MALT lymphoma, possibly by suppressing B cell apoptosis.


   Acknowledgments
 
We are grateful to Mitsuko Tsubouchi for excellent technical assistance.


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


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

1 Address correspondence and reprint requests to Dr. Tomoya Ohmae, Department of Gastroenterology, University of Tokyo, Hongo 7–3-1, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail address: tohmae-tky{at}umin.ac.jp Back

2 Abbreviations used in this paper: IKK, I{kappa}B kinase; siRNA, small interfering RNA; MALT, mucosa-associated lymphoid tissue; NIK, NF-{kappa}B-inducing kinase; APDC, ammonium pyrrolidine dithiocarbamate; PAI, pathogenicity island; BLC, B lymphocyte chemoattractant; ELC, EBI-1 ligand chemokine; SDF stromal cell-derived factor. Back

Received for publication March 7, 2005. Accepted for publication September 19, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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