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. Author manuscript; available in PMC: 2010 Feb 23.
Published in final edited form as: Cell Microbiol. 2007 May 21;9(10):2457–2469. doi: 10.1111/j.1462-5822.2007.00973.x

Helicobacter pylori environmental interactions: effect of acidic conditions on H. pylori-induced gastric mucosal interleukin-8 production

Il Ju Choi 1,†, Saori Fujimoto 1,‡, Kazuyoshi Yamauchi 1,§, David Y Graham 1, Yoshio Yamaoka 1,*
PMCID: PMC2827486  NIHMSID: NIHMS178115  PMID: 17517062

Summary

To explore the interactions between the host, environment and bacterium responsible for the different manifestations of Helicobacter pylori infection, we examined the effect of acidic conditions on H. pylori-induced interleukin (IL)-8 expression. AGS gastric epithelial cells were exposed to acidic pH and infected with H. pylori [wild-type strain, its isogenic cag pathogenicity island (PAI) mutant or its oipA mutant]. Exposure of AGS cells to acidic pH alone did not enhance IL-8 production. However, following exposure to acidic conditions, H. pylori infection resulted in marked enhancement of IL-8 production which was independent of the presence of the cag PAI and OipA, indicating that H. pylori and acidic conditions act synergistically to induce gastric mucosal IL-8 production. In neutral pH environments H. pylori-induced IL-8 induction involved the NF-κB pathways, the extracellular signal-regulated kinase (ERK)→ c-Fos/c-Jun→activating protein (AP-1) pathways, JNK→c-Jun→AP-1 pathways and the p38 pathways. At acidic pH H. pylori-induced augmentation of IL-8 production involved markedly upregulated the NF-κB pathways and the ERK→c-Fos→AP-1 pathways. In contrast, activation of the JNK→c-Jun→AP-1 pathways and p38 pathways were pH independent. These results might explain the clinical studies in which patients with duodenal ulcers had higher levels of IL-8 in the antral gastric mucosa than patients with simple H. pylori gastritis.

Introduction

Acid has long been known to be a major factor in peptic ulcer disease (e.g. Schwarz’s dictum ‘no acid – no ulcer’) (Schwarz, 1910). The discovery of Helicobacter pylori temporarily changed the focus of research away from acid but did not negate a century of elegant research in gastric secretion and its role in the pathogenesis of gastroduodenal diseases. Fasting gastric contents are extremely acidic compared with intracellular pH and gastric acidity varies throughout the day and with different gastroduodenal diseases. Duodenal ulcer disease is associated with higher than normal acid secretion, with the mean pH over a 24 h period being approximately 1.4. A number of overlapping mechanisms protect the gastric epithelium from its corrosive contents including the presence of a thick mucus gel layer that limits diffusion of acid to the mucosa, bicarbonate secretions of the surface cells that tend to neutralize the acid, and a luxurious mucosal blood flow. Overall, these protective mechanisms result in the pH at the surface of the gastric epithelial layer being typically higher than that of the gastric contents (Chu et al., 1999; Baumgartner and Montrose, 2004).

Helicobacter pylori have also developed mechanisms that allow the bacteria to survive and remain motile in an acidic milieu. After the motile organism is ingested, it migrates through the gastric contents, penetrates through and beneath the mucus layer, and attaches to the gastric mucosal surface. Attachment to the gastric mucosal surface results in bacterial–host interaction leading to a marked inflammatory response with infiltration of neutrophils, lymphocytes, monocytes and plasma cells. The initial migration and activation of inflammatory cells into the gastric mucosa is believed to depend on the production of various pro-inflammatory cytokines, especially interleukin (IL)-8, a potent neutrophil chemotactic and activating peptide produced by gastric epithelial cells (Crabtree et al., 1994; Yamaoka et al., 1996; 2004). Mucosal IL-8 levels are also increased in gastric cancer tissues and are thought to play a role in increasing angiogenesis and metastatic potential, to an extent that IL-8 levels have been shown to be a prognostic factor in gastric cancer (Kitadai et al., 1998; Kido et al., 2001).

Mammalian cells respond to acid in a variety of ways including enhanced IL-8 secretion (Shi et al., 1999; 2000; Xu and Fidler, 2000; Karashima et al., 2003). Recent studies using a variety of non-gastric cell lines have suggested that nuclear factor (NF)- κB and/or activating protein (AP-1) are involved in IL-8 gene expression in response to acidic pH (Shi et al., 2000; Xu and Fidler, 2000; Karashima et al., 2003). Low extracellular pH also activates three main mitogen-activated protein kinase (MAPK) signal transduction pathways in a host cell-dependent manner (Xue and Lucocq, 1997; Xu et al., 2002; Owen et al., 2005; Martinez et al., 2006).

Interleukin-8 gene expression associated with H. pylori infection of gastric epithelial cells has been shown to involve MAPK, NF-κB and AP-1 pathways (Sharma et al., 1998; Keates et al., 1999; Yamaoka et al., 2004). However, these data come from studies performed in pH-neutral environments. The influence of acidic conditions on H. pylori-mediated IL-8 expression from gastric epithelial cells has not been examined in detail. This study therefore investigated whether an acidic environment augmented H. pylori-induced gastric inflammation.

Results

AGS cell viability in acidic pH medium

To determine whether acidity of the medium affected cell viability, we cultured human gastric epithelial cell line AGS in acidic pH medium ranging from pH 6.8 to pH 3.0. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay showed that cell viability did not decrease at pH 6.8 or pH 6.0 compared with that at pH 7.4 and was only slightly (e.g. approximately 15%) decreased at pH 5.5 and pH 5.0. When H. pylori TN2GF4 was co-cultured at a multiplicity of infection (moi) of 100, cell viability was unchanged compared with uninfected cells at the same pH (data not shown). Therefore, the pH range of 7.4–5.0 was used in subsequent experiments. We used H. pylori TN2GF4 in this study as this strain has been reported to induce gastric cancer in Mongolian gerbils (Watanabe et al., 1998) and has been thought to the typical virulent strains.

Effect of acidic pH and H. pylori infection on IL-8 induction

Helicobacter pylori stimulated IL-8 protein production in a time-dependent manner with maximal levels at 24 h irrespective of the pH (data not shown). Increased production of H. pylori-induced IL-8 was observed as the pH of the culture medium decreased (P < 0.01 compared with pH 7.4) with maximal levels at pH 5.5 (IL-8 levels at 24 h are shown in Fig. 1A). In contrast, IL-8 levels in uninfected AGS cells were much lower than infected samples at each pH tested (P < 0.01).

Fig. 1. IL-8 protein and mRNA levels at specified pH.

Fig. 1

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A. IL-8 protein levels secreted into the AGS cell culture supernatant at 24 h of co-culture were determined by an ELISA.

B. IL-8 mRNA levels in AGS cells at 3 h of co-culture were measured by real-time RT-PCR.

Three independent co-cultures were performed and each was measured in duplicate. Data are expressed as mean ± SE. IL-8 protein levels and IL-8 mRNA levels were significantly higher in infected samples than in uninfected samples at each pH (P < 0.01).

*: P < 0.05 and **: P < 0.01 compared with infected samples at pH 7.4. †: P < 0.05 and ††: P < 0.01 compared with uninfected samples at pH 7.4. We confirmed that the density of PCR product for β-actin was unchanged irrespective of pH (data not shown).

Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) showed that IL-8 mRNA levels reached maximum levels at 3 h irrespective of the pH of the medium (data not shown). In parallel to IL-8 protein levels, IL-8 mRNA levels in infected cells increased as the pH declined (IL-8 mRNA levels at 3 h are shown in Fig. 1B). Levels of IL-8 mRNA in uninfected AGS cells were much lower than in infected samples at each pH (P < 0.01).

Heat-killed H. pylori as well as live H. pylori separated from AGS cells by a permeable membrane were unable to induce IL-8 irrespective of the pH of the medium (data not shown), suggesting that contact with live H. pylori in AGS cells is required to induce IL-8.

Effect of acidic pH and H. pylori infection on activation of the IL-8 promoter

To determine which region of the IL-8 promoter was involved in regulating gene transcription in response to H. pylori infection at acidic pH, AGS cells were transiently transfected with plasmids containing a fragment of the IL-8 promoter (−162/+44). In agreement with previous studies (Yamaoka et al., 2004), luciferase activity was significantly induced by H. pylori at pH 7.4 (Fig. 2A). The luciferase activity induced by H. pylori infection was further increased at acidic pH (e.g. activity normalized to Renilla luciferase vector = 57.9 ± 4.8 and 87.4 ± 6.9; fold increase of uninfected control = 4.8 ± 0.2 and 6.9 ± 0.6 at pH 7.4 and pH 6.4, respectively; P < 0.05 compared with pH 7.4) (Fig. 2A). Significantly more luciferase activity was also observed in infected samples than in uninfected samples at each pH examined (P < 0.01). In contrast to IL-8 protein/mRNA induction, luciferase activity decreased at pH 5.5, possibly due to decreased cell viability (cell viability was reduced approximately 25% after 9 h of co-culture with H. pylori). As cell viability and the ability to transfect luciferase plasmids was markedly less at pH 5.0 (our unpubl. data), we excluded the data at pH 5.0.

Fig. 2. IL-8 promoter activation following H. pylori infection after exposure to acidic pH.

Fig. 2

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A. AGS cells were transfected with luciferase reporter plasmids containing a −162/+44 fragment of the IL-8 promoter and infected with H. pylori at various pH for 9 h.

B–E. AGS cells were transfected with site-mutated plasmid of the IL-8 promoter (B and C), 5′ deletions of the IL-8 promoter (D) or plasmid containing IL-8 NF-κB trimmer (E) and infected with H. pylori at various pH for 9 h.

Three independent transfections, each done in triplicate, were performed. For each plate, luciferase activity was normalized to Renilla luciferase vector DNA. Data are expressed as mean ± SE of normalized luciferase activity. Numbers on the error bar refer to fold increase of luciferase activity in H. pylori-infected cells relative to uninfected cells at each pH. *: P < 0.05 and **: P < 0.01 compared with pH 7.4. At pH 5.5, both firefly and Renilla luciferase activity were below the detection levels when we used a plasmid containing IL-8 NF-κB trimmer (E) and were excluded. The luciferase activity was significantly higher in infected samples than in uninfected samples at each pH (P < 0.01) for (A), (B) and (D). The luciferase activity was also significantly higher in infected samples than in uninfected samples at pH 7.4 (P < 0.05), pH 6.8 (P < 0.01) and pH 6.4 (P < 0.01) for (E).

In agreement with previous studies (Yamaoka et al., 2004), site mutations of the AP-1 site reduced the luciferase activity induced by H. pylori infection (fold increase of uninfected control) at pH 7.4 (3.6 ± 0.3 versus 4.8 ± 0.2; P < 0.05 compared with −162/+44) (Fig. 2B). However, in contrast to the −162/+44 plasmid, the luciferase activity induced by H. pylori infection was pH independent, consistent with activation of the AP-1 site by H. pylori infection also being pH dependent.

Mutation of the NF-κB site resulted in almost complete inhibition of the promoter activity following H. pylori infection irrespective of the pH (Fig. 2C). Mutations of the interferon-stimulated responsive element (ISRE)-like element reduced the luciferase activity induced by H. pylori infection (fold increase of uninfected control) by approximately 20% irrespective of the pH and the luciferase activity induced by H. pylori infection showed similar pH-dependent increases to −162/+44 plasmid (data not shown) indicating that although activation of ISRE-like element played a role in the IL-8 promoter activation by H. pylori, it was independent of the pH of the medium.

To further investigate the role(s) of the NF-κB binding site in H. pylori infection, AGS cells were transiently transfected with plasmids containing 5′ deletions of the IL-8 promoter (−99/+44) linked to the luciferase reporter gene (Fig. 2D). −99/+44 hIL-8/luc contained binding sites for NF-κB with deletion of the AP-1 and ISRE-like element sites. Deletion to −99 bp reduced the luciferase activity induced by H. pylori (fold increase of uninfected control) irrespective of the pH; however, the luciferase activity induced by H. pylori infection was further increased at acidic pH compared with pH 7.4, indicating that acidic pH played a role in the H. pylori-related activation of the NF-κB site. This result was confirmed using luciferase reporter plasmids containing three repeats of the binding sequence for the IL-8 NF-κB, in which the luciferase activity was gradually increased with decreasing pH, with maximal levels at pH 6.4 (Fig. 2E).

Overall, the analyses of the IL-8 promoter using luciferase reporter gene assays were consistent with the notion that NF-κB and AP-1 transcription factors were involved in acid pH-enhanced IL-8 induction.

Effect of acidic pH and H. pylori infection on NF-κB activation

To confirm the results by luciferase reporter gene assays, we performed electrophoretic mobility shift assay (EMSA). In uninfected cells, the NF-κB binding complex C2 was observed irrespective of the pH; whereas complex C1 was not observed at any pH tested (Fig. 3A, lanes 1–6). Complex C1 increased following H. pylori infection at pH 7.4, and both complexes, especially C1, increased further in infected samples as the pH declined (lanes 7–12). Supershift assays revealed that C1 contained p50 and p65, and C2 contained p50 (lanes 13 and 14) showing that acidic pH is involved in activating NF-κB, especially p65, during H. pylori infection. To confirm these results, we performed quantitative analyses for NF-κB binding using p65 enzyme-linked immunosorbent assay (ELISA) assays (Fig. 3B). p65 activation increased following H. pylori infection at each pH tested (P < 0.01 for each pH compared with uninfected samples) with maximal levels at pH 6.0 (P < 0.01 compared with pH 7.4).

Fig. 3. Effect of acidic pH and H. pylori infection on activation of NF-κB.

Fig. 3

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A. Activation of NF-κB using EMSA for NF-κB binding complexes. Lanes 1–12: Nuclear extracts were prepared from uninfected and infected AGS cells for 1.5 h at specified pH and used for EMSA. Lanes 13 and 14: Supershift interference assay. EMSA was performed using nuclear extracts of AGS cells infected with H. pylori for 1.5 h at pH 5.0 and commercial antibodies against p50 and p65. NS, non-specific band.

B. Transcription factor ELISA for NF-κB p65. Three independent co-cultures were performed and each was measured in duplicate. Data are expressed as fold increase of OD for uninfected samples at pH 7.4. Data are expressed as mean ± SE. *: P < 0.05 and **: P < 0.01 compared with pH 7.4. The NF-κB p65 activation was significantly higher in infected samples than in uninfected samples at each pH (P < 0.01).

C. Immunoblot analyses for IκB, phospho-IκB and β-actin using the cytoplasmic protein extracted after 1.5 h of H. pylori stimulation or without H. pylori infection at various pH.

IκB phosphorylation and subsequent degradation in the cytoplasm are required for the translocation of NF-κB to the nucleus. Thus, immunoblot analysis of IκB was performed on the cytoplasmic extract after 1.5 h of H. pylori stimulation (Fig. 3C). Levels of total and phosphorylated IκB did not change at acidic pH in uninfected cells indicating that acidic pH alone does not trigger IκB phosphorylation and subsequent degradation. However, when AGS cells were co-cultured with H. pylori, IκB phosphorylation and degradation of total IκB were observed as pH decreased and IκB phosphorylation reached maximal levels at pH 6.4–6.0.

Effect of acidic pH and H. pylori infection on activation of AP-1

EMSA showed that constitutive AP-1 binding was observed in extracts from uninfected AGS cells at each pH tested (Fig. 4A, lanes 1–6). In H. pylori-infected samples, more AP-1 binding was observed than in uninfected cells at each pH (lanes 7–12). Supershift assays showed c-Jun binding to the AP-1 site (lane 13). Treatment with anti-c-Fos antibody reduced the binding complexes, although clear supershifted bands were not observed, indicating that c-Fos is also a component of the AP-1 complex induced by H. pylori infection (lane 14). The AP-1 binding complex appeared to be greater at acidic pHs than at pH 7.4 in infected cells. To confirm the data, quantitative analyses of AP-1 binding was performed using ELISA assays for the Fos (c-Fos, FosB, Fra-1 and Fra-2) and Jun (c-Jun, JunB and JunD) proto-oncogene families. Following H. pylori infection c-Jun activation was greater at each pH (P < 0.01 for each compared with uninfected samples) with maximal levels at pH 6.4 (P < 0.01 compared with pH 7.4) (Fig. 4B). c-Jun activation was unchanged in uninfected cells irrespective of the pH. Similarly, H. pylori-induced c-Fos activation reached maximal levels at pH 6.8–6.4 (Fig. 4C).

Fig. 4. Effect of acidic pH and H. pylori infection on AP-1 activation.

Fig. 4

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A. Activation of AP-1 using EMSA for AP-1 binding complex. Lanes 1–12: Nuclear extracts were prepared from uninfected and infected AGS cells for 1.5 h at specified pH and used for EMSA. Lanes 13 and 14: Supershift interference assay. EMSA was performed using nuclear extracts of AGS cells infected with H. pylori for 1.5 h at pH 5.0 and commercial antibodies against c-Jun and c-Fos.

B and C. Transcription factor ELISA for c-Jun (B) and c-Fos (C).

Three independent co-cultures were performed and each was measured in duplicate. Data are expressed as fold increase of OD for uninfected samples at pH 7.4. Data are expressed as mean ± SE. †: P < 0.05 and ††: P < 0.01 compared with uninfected samples at pH 7.4. *: P < 0.05 and **: P < 0.01 compared with infected samples at pH 7.4. The c-Jun activation was significantly higher in infected samples than in uninfected samples at each pH (P < 0.01). The c-Fos activation was significantly higher in infected samples than in uninfected samples at pH 7.4 to pH 6.4 (P < 0.01) and at pH 6.0 to pH 5.5 (P < 0.05).

Interestingly, c-Fos activation was inversely correlated with decreasing pH, even in uninfected cells. At pH 5.0, c-Fos activation was similar in uninfected and infected cells. These results suggest that increased c-Fos activation might induce small amounts of IL-8 mRNA in uninfected cells at acidic pH as shown in Fig. 2B. Activation of other AP-1 family members such as FosB, Fra-1, Fra-2, JunB and JunD were not significantly affected by H. pylori and/or by pH stimulation (data not shown).

Effect of acidic pH and H. pylori infection on phosphorylation of MAPKs

Phosphorylated and total MAPKs were evaluated with or without H. pylori infection (for 90 min). Extracellular signal-regulated kinase (ERK) phosphorylation was faint or invisible except at pH 5.0 in uninfected cells (Fig. 5A), indicating that acidic pH medium (pH 5.0) triggered ERK phosphorylation even in uninfected cells. In contrast, H. pylori infection markedly induced ERK phosphorylation as the pH declined to pH 5.0. ERK phosphorylation at pH 5.5 was much more prominent than at pH 7.4 during the 4 h after H. pylori infection (Fig. 5B). H. pylori induced phosphorylation of p38 and c-jun N-terminal kinase (JNK); however, the phosphorylation was pH independent (data not shown).

Fig. 5. Effect of acidic pH and H. pylori infection on ERK phosphorylation by immunoblot.

Fig. 5

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A. Phosphorylated and total ERK in AGS cells with or without H. pylori infection for 90 min at various pH.

B. Phosphorylated and total ERK in AGS cells with H. pylori infection at pH 7.4 and pH 5.5 at various time points.

Inhibition of MAPK pathways and NF-κB pathways in acidic pH-induced IL-8 induction

Inhibition of all three MAPKs and NF-κB with chemical inhibitors significantly decreased IL-8 production induced by H. pylori infection at pH 7.4 (Fig. 6A). The acidic pH-dependent enhancement of IL-8 production was almost abrogated by inhibiting the ERK pathways (U0126) and the NF-κB pathways (MG-132) suggesting that these are the main pathways for enhanced H. pylori-induced IL-8 production from AGS cells. As MG-132, the proteasome inhibitor, is not a specific NF-κB inhibitor, we also used the small interfering RNA (siRNA) technique to knock-down NF-κB expression. The results for siRNA for NF-κB p65 were identical to those with MG-132 (data not shown). Pre-treatment with the p38 inhibitor (SB203580) diminished IL-8 production at both neutral and acid pH. Despite p38 inhibition, IL-8 levels were significantly increased at pH 5.5, suggesting that p38 was not involved in the pH-dependent enhancement of IL-8 production. JNK inhibition did not reduce IL-8 at pH 5.5.

Fig. 6. Inhibition of MAPK pathways and NF-κB pathways in acidic pH-induced IL-8 induction.

Fig. 6

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A. After AGS cells were incubated with each inhibitor at pH 7.4 for 1 h, the medium was maintained at pH 7.4 or changed to acidic pH. H. pylori was added 1 h later. MAPK inhibitor (U; U0126, SB; SB203580, SP; SP600125) (10 μM) and MG; MG-132 (5 μM) were used. IL-8 protein levels secreted into the AGS cell culture supernatant after 24 h of co-culture were determined by ELISA in triplicate. Data are expressed as mean ± SE. *: P < 0.05 and **: P < 0.01 compared with samples without inhibitors. ††: P < 0.01 compared with samples at pH 7.4 at corresponding samples. B and C. EMSA for NF-κB (B) and for AP-1 (C) using the nuclear proteins extracted 90 min after H. pylori stimulation at pH 5.5 with or without inhibitors.

D–F. Transcription factor ELISA for NF-κB p65 (D), c-Jun (E) and c-Fos (F) using the same nuclear extracts as used for EMSA.

Three independent co-cultures were performed and each was measured in duplicate. Data are expressed as mean ± SE. **: P < 0.01 compared with infected samples without inhibitors.

We next used EMSA (Fig. 6B and C) and ELISA (Fig. 6D–F) to determine whether MAPK pathways were involved in NF-κB and AP-1 activation at pH 5.5. Inhibition of MAPKs did not reduce NF-κB activation, consistent with ERK not being in the direct upstream pathway leading to enhanced NF-κB binding activity. EMSA at pH 5.5 showed that inhibition of MEK and JNK, but not p38, reduced AP-1 binding activity, consistent with previous reports at neutral pH (Naumann et al., 1999; Meyer-ter-Vehn et al., 2000). Inhibition of MEK inhibited the activation of both c-Jun and c-Fos; whereas inhibition of JNK only inhibited the activation of c-Jun.

Effects of H. pylori virulence factors on IL-8 induction at acidic pH

As H. pylori virulence factors, especially the cag pathogenicity island (PAI) and OipA, are known to affect IL-8 production from gastric epithelial cells as well as in gastric mucosa in vivo (Yamaoka et al., 1996; 2000; 2002; 2004; Sharma et al., 1998; Keates et al., 1999), we examined isogenic mutants for their ability to induce IL-8 at acidic pH. As expected, both cag PAI and oipA mutants induced smaller amounts of IL-8 induction than parental H. pylori at pH 7.4 (P < 0.01) (Fig. 7A and B). Exposure to pH 5.5 enhanced IL-8 production compared with pH 7.4 (P < 0.01), showing that the effects of the cag PAI and OipA on IL-8 induction are pH independent. Of interest, IL-8 mRNA levels induced by oipA mutants at pH 5.5 were even greater than those produced by wild-type H. pylori at pH 7.4. cag PAI mutants at pH 5.5, and wild-type H. pylori at pH 7.4, induced similar levels of IL-8 mRNA.

Fig. 7. Effects of H. pylori virulence factors on IL-8 induction at acidic pH.

Fig. 7

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A and B. IL-8 protein levels secreted into the AGS cell culture supernatant at 24 h of co-culture were determined by an ELISA (A) and IL-8 mRNA levels in AGS cells at 3 h of co-culture were measured by real-time RT-PCR (B).

C–E. Activation of NF-κB p65 (C), c-Jun (D) and c-Fos (E) in AGS cells at 90 min of co-culture were determined by transcription factor ELISA.

F. Phosphorylated and total ERK in AGS cells with or without H. pylori infection for 90 min were determined by immunoblot. In each assay, three independent co-cultures were performed and each was measured in duplicate.

Data are expressed as fold increase of OD for uninfected samples at pH 7.4 (C–E). Data are expressed as mean ± SE (A–E). *: P < 0.05 and **: P < 0.01 compared with uninfected samples.

Quantitative analyses showed that significantly less NF-κB p65, c-Jun and c-Fos activation was observed with both mutants than with wild-type strains at both pH 7.4 and pH 5.5 (P < 0.01) (Fig. 7C–E). Importantly, oipA mutants at pH 5.5, and wild-type H. pylori at pH 7.4, induced similar levels of NF-κB p65, c-Jun and c-Fos activation. In addition, cag PAI mutants at pH 5.5, and wild-type H. pylori at pH 7.4, also induced similar levels of c-Fos activation. Further, immunoblots for ERK showed that both cag PAI and oipA mutants induced more phospho-ERK at pH 5.5 than at pH 7.4 and both mutants induced more phospho-ERK at pH 5.5 than did wild-type strains at pH 7.4 (Fig. 7F). Overall, the less virulent strains were able to induce high IL-8 mRNA/protein at acidic pHs, consistent with their ability to induce severe gastric inflammation.

Effect of short-term acidic pH exposure on H. pylori-induced IL-8 induction

In the above experiments, we co-cultured H. pylori with AGS cells in acidic pH medium during the observation periods. We therefore examined the effects of short-term acidic pH stimulation of the gastric cells. Exponentially growing AGS cells were exposed to acidic pH medium for 1–60 min following which the medium was changed to pH 7.4 medium for 1 h, then H. pylori were added to AGS cells for the indicated periods of time. Therefore, AGS cells were exposed to acidic pH but the H. pylori were not. IL-8 protein and mRNA levels were both significantly increased by exposure to pH 5.5 medium as short as 1 min (P < 0.01 compared with pH 7.4) (Fig. 8A and B), emphasizing the importance of host factors in generating H. pylori-associated enhanced IL-8 production at acidic pH.

Fig. 8. Effects of short-term acidic pH exposure on H. pylori-induced IL-8 induction.

Fig. 8

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A and B. AGS cells were cultured at pH 5.5 for the designated time and then the medium was changed to pH 7.4. One hour later H. pylori was co-cultured and IL-8 protein levels secreted into the AGS cell culture supernatant after 24 h of co-culture were determined by an ELISA (A) and IL-8 mRNA levels in AGS cells at 3 h of co-culture were measured by real-time RT-PCR (B). Continuous stimulation at pH 7.4 or 5.5 were used as controls. Three independent co-cultures were performed and each was measured in duplicate. Data are expressed as mean ± SE. IL-8 protein levels and IL-8 mRNA levels were significantly higher in infected samples than in uninfected samples at each pH with each time point (P < 0.01). **: P < 0.01 compared with infected samples at pH 7.4. †: P < 0.05 compared with uninfected samples at pH 7.4.

C. Inhibition of MAPK pathways and NF-κB pathways in short-term acidic pH-induced IL-8 induction. After AGS cells were incubated with each inhibitor at pH 7.4 for 1 h, the medium was maintained at pH 7.4 or changed to acidic pH for designated time, and then the medium was changed to pH 7.4. One hour later H. pylori was co-cultured and IL-8 protein levels secreted into the AGS cell culture supernatant at 24 h of co-culture were determined by an ELISA in triplicate. Each MAPK inhibitor (10 μM) and MG-132 (5 μM) were used. Data are expressed as mean ± SE. **: P < 0.01 compared with infected samples without inhibitors.

D–F. Transcription factor ELISA for NF-κB p65 (D), c-Jun (E) and c-Fos (F). AGS cells were cultured in pH 5.5 medium for 1 min, the medium was changed to pH 7.4, and 1 h later H. pylori was co-cultured for 90 min and extracted nuclear proteins were used for ELISA. Continuous stimulation at pH 7.4 or 5.5 were used as controls. Three independent co-cultures were performed and each was measured in duplicate. Data are expressed as fold increase of OD for uninfected samples at pH 7.4. Data are expressed as mean ± SE. †: P < 0.05 and ††: P < 0.01 compared with uninfected samples at pH 7.4. *: P < 0.05 and **: P < 0.01 compared with infected samples at pH 7.4. The NF-κB p65, c-Jun and c-Fos activation was significantly higher in infected samples than in uninfected samples at short-term exposure to pH 5.5 (P < 0.01).

Inhibition of either the NF-κB or MAPK pathways significantly suppressed IL-8 production induced by H. pylori infection following 1 min exposure to pH 5.5 (Fig. 8C). In contrast to continuous pH 5.5 exposure, during short-term exposure to pH 5.5, JNK inhibition reduced IL-8 induction. This result suggests that JNK-related IL-8 induction is involved in the response to short-term acidic exposure or at the neutral pH environment. We used ELISA to quantify NF-κB p65, c-Jun and c-Fos binding during short-term exposure to acidic pH (Fig. 8D–F) and the results were similar to those seen with continuous exposure to acidic pH.

Discussion

This study examined one important aspect of the host–bacterium interaction: the interaction between an acidic environment and IL-8 production from H. pylori-infected gastric epithelial cells. While prior reports (Allan et al., 2001; Ang et al., 2001; Wen et al., 2003) focused on H. pylori gene expression in acidic environments, this study explored changes in gastric epithelial cells at increasingly acidic pH. In contrast to many other types of cells (Shi et al., 1999; 2000; Xu and Fidler, 2000; Karashima et al., 2003), and consistent with the fact that the normal niche of gastric cells is in the acidic stomach, acid exposure alone did not result in IL-8 production from gastric epithelial cells. Rather, IL-8 production required the presence of H. pylori and the effect of H. pylori was further enhanced by exposure of the epithelial cells to an acid pH. This effect was not specific for cag PAI-positive, OipA-positive H. pylori, but was also seen following exposure to H. pylori lacking these pro-inflammatory virulence factors. Acid exposure-enhanced IL-8 production occurred with low-virulence H. pylori which is consistent with the clinical experience that both peptic ulcer and gastric cancer associated with H. pylori without these putative virulence factors. Moreover, we found that gastric epithelial cells exposed to acid and then infected with H. pylori in a neutral environment also exhibited a greater enhancement of IL-8 production than gastric epithelial cells that were not exposed to an acidic pH. These results indicate acid appears to prime gastric epithelial cells for enhanced IL-8 secretion and act as another step in addition to acid exposure-related changes in gene expression in H. pylori. These results are consistent with the results of clinical studies in which IL-8 levels in the antral gastric mucosa of patients with duodenal ulcer were shown to be greater than in patients with simple H. pylori gastritis (Yamaoka et al., 1999). Patients with duodenal ulcer typically have high levels of inflammation and a high H. pylori density in the antrum and lower levels of H. pylori density in the corpus (Yamaoka et al., 1999). Our results therefore seem to mirror the H. pylori–host interaction in the antrum. The interaction in the corpus is clearly more complicated. For example, events that reduce gastric acid secretion such as the use of an antisecretory drug or a highly selective vagotomy lead to a very rapid increase in both the severity and depth of corpus inflammation and in IL-8 production (Graham et al., 2004). In the antrum, the pH in environment of the gastric pits reflects that of the lumen whereas in the corpus, the pits are the conduit for the transit of 160 mM HCl from the parietal cell to the lumen. It remains unclear if or how much our in vitro studies reflect bacterial host interactions in the corpus of duodenal ulcer patients.

Few tissues or cells in the body are regularly exposed to acidic conditions (e.g. pH < 6) and therefore it is not surprising that exposure to acid might lead to changes in intracellular signalling. For example, acidic pH has been shown to activate NF-κB and/or AP-1 (Bellocq et al., 1998; Shi et al., 2000; Xu and Fidler, 2000; Karashima et al., 2003; Martinez et al., 2006) as well as ERK phosphorylation in a variety of cell types (Xue and Lucocq, 1997; Xu et al., 2002). Gastric epithelial cells are normally exposed to acid pH and we found that acidic pH alone (pH 6.0 to pH 5.0) did not induce measurable amounts of IL-8 protein. These results differ from those of O’Toole et al. (2005), who reported that acidic pH alone activated NF-κB with NF-κB binding activity with maximal levels at pH 6.8 and falling to negligible levels at pH 6.4 in AGS cells. This phenomenon occurred both with and without H. pylori infection. In contrast, we found that acidic pH alone did not activate NF-κB binding and that H. pylori-induced NF-κB binding activity reached maximal levels at pH 6.4–6.0. O’Toole et al. (2005) also suggested that cell viability was substantially compromised at pHs less than pH 6.6. However, in our experiments greater than 85% cell viability was maintained after 4 h culture at pH 5.5. The reasons for these discrepant results are unknown. Importantly, our finding of the absence of acid exposure-stimulated NF-κB activation and IL-8 secretion is consistent with clinical observation that normal (uninfected) gastric epithelium is typically devoid of inflammatory cells and that in normal gastric mucosa the levels of pro-inflammatory cytokines are low to absent.

Our study also provides new insights into the regulation of gastric mucosal IL-8 secretion. We showed that the NF-κB pathways, ERK→c-Fos/c-Jun→AP-1 pathways, JNK→c-Jun→AP-1 pathways and p38 pathways all are involved in H. pylori-induced IL-8 induction in neutral pH environments. The underlying mechanism leading to H. pylori-induced augmentation of IL-8 at acidic pH was through the NF-κB pathways and ERK→c-Fos→AP-1 pathways which were markedly upregulated at acidic pH. In contrast, activation of JNK→c-Jun→AP-1 pathways and p38 pathways were pH independent. These results agree with previous studies that JNK and p38 phosphorylation are pH independent in human glioblastoma cells (Xu et al., 2002) and human neutrophils (Martinez et al., 2006) and are consistent with activation of c-Jun at acidic pH being mainly regulated by the phosphorylation of ERK. Interestingly, ERK and c-Fos were activated by acid alone especially at pH 5.0 (Figs 4C and 5A) suggesting that the ERK→c-Fos→AP-1 pathways are also activated in uninfected cells at acidic pH. However, activation of these pathways alone is unable to induce measurable amounts of IL-8 suggesting that synergistic activations of NF-κB and AP-1 are required for IL-8 induction.

We found that H. pylori-induced augmentation of IL-8 at acidic pH involved the NF-κB pathways and ERK→c-Fos→AP-1 pathways. Both NF-κB and ERK pathways have been reported to be involved in H. pylori-induced IL-8 secretion at neutral pH (Sharma et al., 1998; Keates et al., 1999). We and others have previously reported that inhibition of the ERK pathway did not reduce NF-κB binding activity in AGS, MKN28 or MKN45 gastric epithelial cells (Keates et al., 1999; Kudo et al., 2005; Lu et al., 2005) and that inhibition of ERK impeded NF-κB transactivation, but not DNA binding in H. pylori-infected MKN28 cells (Lu et al., 2005). This same phenomenon was observed in infected AGS cells (our unpubl. data). In this study we confirmed that ERK inhibition did not reduce NF-κB binding activity consistent with the notion that ERK is not the direct upstream kinase of NF-κB at either acidic or neutral pH. As above, we were unable to confirm O’Toole et al. (2005) report that ERK inhibition resulted in reduced NF-κB binding activity in uninfected AGS cells.

We found that within the pH range tested, H. pylori-induced IL-8 levels were maximal at pH 5.5, NF-κB levels were maximal at pH 6.4–6.0 and AP-1 at pH 6.8–6.4. Maximal levels of ERK, measured by immunoblot, and c-Fos, measured by ELISA, were at pH 5.0. IL-8 transcription, measured by luciferase reporter gene assay, was maximal at pH 6.8–6.0, which differed from maximal IL-8 mRNA/protein levels. Reasons for the slight differences in results are unclear and could relate to difference in the methods used or the timing of individual experiments. In addition, outcome (e.g. maximal IL-8 production) of the complicated pathways may not involve maximal stimulation. Most importantly, we found that a wide range of acidity (pH 6.8–5.5) resulted in activation of NF-κB, AP-1 and ERK, followed by induction of IL-8 in H. pylori-infected AGS cells.

Experimental procedures

Helicobacter pylori and epithelial cells co-culture

Helicobacter pylori TN2GF4, its isogenic oipA mutant (Yamaoka et al., 2000) and its cag PAI-deleted mutants (Kudo et al., 2005) were used.

The human gastric epithelial cell line AGS (American Type Culture Collection, Manassas, VA) was cultured according to standard procedures. One hour before H. pylori treatment, the culture medium was changed to a defined pH medium whose pH had been adjusted with HCl immediately before use. In all experiments, the pH of the culture medium was measured at the start of the experiments and at the end of the incubation to ensure that pH remained unaltered. H. pylori were suspended in PBS and added to exponentially growing AGS cells at an moi of 100 for the indicated periods of time. To avoid the influence of serum, epithelial cells were serum starved for 16 h before experiments. In the case of short-term pH stimulation experiments, exponentially growing AGS cells were exposed to acidic pH medium for the indicated periods of time, the medium was changed to pH 7.4 medium for 1 h, and H. pylori were added to AGS cells for the indicated periods of time.

In some experiments, heat-killed H. pylori were used at the same moi or the same concentration of live bacteria were added to the upper well of a transwell plate (Falcon, Lincoln Park, NJ); the lower well contained subconfluent epithelial cells. In some experiments, gastric cells were pre-treated with U0126 (a specific inhibitor of MAPK/ERK1/2), SB203580 (a specific inhibitor of p38), SP600125 (a specific inhibitor of JNK), or the proteasome inhibitor MG-132, which inhibits NF-κB activation. All inhibitors were purchased from Calbiochem (San Diego, CA). We used 10 μM for each MAPK inhibitor and 5 μM for MG-132 according to our previous analyses of optimal concentration (Lu et al., 2005). After incubating AGS cells with each inhibitor at pH 7.4 for 1 h, the medium was maintained at pH 7.4 or changed to an acidic pH medium, and H. pylori were added 1 h later.

Cell viability assay

Viability of AGS cells co-cultured with H. pylori in 96-well plates was measured using an MTT assay (Roche Applied Bioscience, Indianapolis, IN). The spectrophotometrical absorbance at 560 nm was measured, and viability of acidic samples was expressed as percentage of pH 7.4 samples at each time point.

siRNA for knock-down of NF-κB expression

siRNA to interfere with NF-κB p65 mRNA was performed as previously described (Kudo et al., 2005). NF-κB p65 siRNA expression plasmids were obtained from Panomics (Redwood City, CA). For siRNA knock-down, NF-κB p65 siRNA plasmids or empty plasmids were transfected 72 h prior to H. pylori infection using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions.

IL-8 protein levels from AGS cells co-cultured with H. pylori

The amount of IL-8 secreted into the AGS cell culture supernatant was determined using an ELISA(R&D Systems, Minneapolis, MN) in triplicate as previously described (Yamaoka et al., 2004).

IL-8 mRNA levels from AGS cells co-cultured with H. pylori

Total RNA was extracted from infected and untreated AGS cells using the RNeasy Mini Kit (Qiagen, Chatsworth, CA) and converted cDNA was then used for real-time RT-PCR by standard procedures. Primers and probe sets used to amplify IL-8 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Applied Biosystems (ABI, Foster City, CA). PCR products were cloned into the plasmid pCR2.1 TOPO (Invitrogen) and used as a standard. IL-8 mRNA levels were expressed as the ratio of IL-8 mRNA to GAPDH mRNA as previously described (Yamauchi et al., 2003).

Luciferase reporter gene assay

Human IL-8 promoter-firefly luciferase reporter plasmid (−162/+44), plasmids containing site-directed mutations in three sites (ISRE-like element, AP-1 or NF-κB), plasmids containing 5′ deletions of the IL-8 promoter (−99/+44) linked to a luciferase reporter gene, and plasmids containing three copies of the IL-8 NF-κB binding sites, were described previously (Casola et al., 2000; Yamaoka et al., 2004). Nine hours after treatment with H. pylori, lysates were assayed for luciferase activity using the Dual-Luciferase® reporter assay (Promega, Madison, WI) as previously described (Yamaoka et al., 2004). Normalized luciferase activity is presented as firefly luciferase activity/Renilla luciferase activity. This activity is expressed as fold increase of luciferase activity in infected cells relative to uninfected samples at each pH.

Electrophoretic mobility shift assay

Nuclear extracts of infected and uninfected AGS cells (90 min) were prepared using hypotonic/non-ionic detergent lysis (Yamaoka et al., 2004). Equal amounts of nuclear extracts were used for EMSA with duplexed NF-κB and AP-1 binding site oligonucleotides (Promega), as previously described (Yamaoka et al., 2004). We used the same nuclear extracts as used for transcription factor ELISA assays (Active Motif, Carlsbad, CA). In the gel mobility super shift assays, commercial antibodies (anti-p50, anti-p65, anti-c-Fos and anti-c-Jun; Santa Cruz Biotechnology, Santa Cruz, CA) were added to the binding reactions before adding the probes.

Transcription factor assay

Transcription factor activation was also measured quantitatively using commercially available ELISA kits (Active Motif, Carlsbad, CA) that measure DNA binding activity using the same nuclear extracts as used for EMSA. We used the TransAM™ NF-κB kit for p65 or TransAM™ AP-1 family kit for c-Jun, c-Fos, Fra-1, Fra-2, JunB, JunD and FosB.

Immunoblot analyses

Immunoblots were performed by standard methods using cytoplasmic extracts for IκBα and whole-cell lysates for MAPKs. We used phospho-specific antibodies as well as control total antibodies for the IκBα (Ser32), JNK, ERK and p38 (Cell Signaling Technology) as previously described (Yamaoka et al., 2004; Kudo et al., 2005; Lu et al., 2005). Anti-β actin antibody (Santa Cruz Biotechnology) was used to normalize the immunoblot analyses. Primary antibodies were used at a 1:1000 dilution. Horseradish peroxidase-conjugated IgG (1:2000) (Cell Signaling Technology) was used as the secondary antibody. Detection was performed using ECL reagents (Amersham Life Science, Arlington Heights, IL) and exposure to X-ray film.

Statistical analyses

Statistical analysis was performed using the Mann–Whitney Rank Sum test and the paired t-test depending on the data set of concern. Data are presented as mean ± standard error (SE). P < 0.05 was accepted as statistically significant.

Acknowledgments

This report is based on work supported in part by grants from the National Institutes of Health DK62813 (Y.Y.), the Office of Research and Development Medical Research Service Department of Veterans Affairs (D.Y.G.) and a Public Health Service grant (DK56338) which funds the Texas Gulf Coast Digestive Diseases Center.

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