Preferential induction of transforming growth factor-beta production in gastric epithelial cells and monocytes by Helicobacter pylori soluble proteins

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M A J O R A R T I C L E

Preferential Induction of Transforming Growth

Factor–b Production in Gastric Epithelial Cells

and Monocytes by Helicobacter pylori

Soluble Proteins

Ming-Shiang Wu,1,a_{Jaw-Town Lin,}1,a_{Ping-Ning Hsu,}2_{Ching-Yi Lin,}4_{Yuan-Ting Hsieh,}4_{Yi-Han Chiu,}4_{Po-Ren Hsueh,}3 and Kuang-Wen Liao4

1_{Department of Internal Medicine,}2_{Graduate Institute of Immunology, College of Medicine,}3_{Department of Laboratory Medicine, National Taiwan}

University Hospital, Taipei,4_{Department of Biological Science and Technology, National Chiao-Tung University, Hsin-Chu, Taiwan}

Background. The cytokines induced by Helicobacter pylori, as well as the intricate balance of proinflammatory and anti-inflammatory cytokines, are relevant to the outcomes of H. pylori infection. Transforming growth factor (TGF)–b and interleukin (IL)–10 are 2 vital anti-inflammatory cytokines that regulate mucosal immunity in various inflammatory and infectious diseases.

Methods. To elucidate whether host-bacteria interaction can influence TGF-b and IL-10 production, we in-vestigated the expression of TGF-b and IL-10 in various mammalian cell lines preincubated with H. pylori and other enteric bacteria.

Results. The amount of TGF-b protein, but not IL-10, was significantly increased after stimulation with H. pylori, but other enteric bacteria did not induce TGF-b production. Different H. pylori strains isolated from patients with gastritis, peptic ulcer, gastric cancer and strains with cagA or vacA isogenic mutations showed similar effects on TGF-b induction, indicating that this effect was a constitutional characteristic of H. pylori and independent of cagA and vacA status.

Conclusion. The results imply the presence of a protein factor (termed “TGF-b–inducing protein”) that induces production of TGF-b. In view of the multiple effects of TGF-b, we conclude the TGF-b–inducing protein of H. pylori might mediate the immune response and contribute to the pathogenesis of H. pylori infection.

Helicobacter pylori infects about half of the world’s pop-ulation. The majority of infected patients have asymp-tomatic gastritis, and 10%–15% develop peptic ulcer, gastric carcinoma, or B cell mucosa-associated lym-phoid tissue (MALT) lymphoma [1]. The variable clin-ical outcomes of H. pylori infection are attributed to variations in bacterial virulence factors as well as

dif-Received 18 January 2007; accepted 22 May 2007; electronically published 1 October 2007.

Potential conflicts of interest: none reported.

Financial support: National Science Council (grants NSC92-2314-B002-122 and NSC92-2314-B002-124); Executive Yuan, Taiwan.

a

M.-S.W. and J.-T.L. contributed equally to this work.

Reprints or correspondence: Kuang-Wen Liao, Dept. of Biological Science and Technology, National Chiao-Tung University, Rm. 205, Zhu-Ming Bldg., 75 Bo-Ai St., Hsin-Chu, Taiwan ([email protected]).

The Journal of Infectious Diseases 2007; 196:1386–93

DOI: 10.1086/522520

ferences in host immune responses [2]. In particular, the immune response against H. pylori virulence factors might provide a direct linkage to the development of gastroduodenal diseases [3].

In the early stages of infection, H. pylori induces the production of chemokines, as well as proinflammatory cytokines [4]. The induction of chemokines or pro-inflammatory cytokines attracts neutrophils, mono-cytes, macrophages, or dendritic cells, which then mi-grate to the inflammatory area and activate a number of innate inflammatory responses. Neutrophils partic-ipating in gastric inflammation are related to the clear-ance of H. pylori [5]. Mucosal macrophages and den-dritic cells, the monocyte-derived cells, can capture and digest pathogenic antigens, soon after infection by the pathogen [6]. Furthermore, specific antibodies against H. pylori that are detectable in patients’ serum may help eradicate the bacterial infection in gastric mucus [6, 7].

at National Chiao Tung University Library on April 25, 2014

http://jid.oxfordjournals.org/

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With respect to cell-mediated immunity, evidence has shown

that CD4+_{T cells play an important role in protective immunity}

against H. pylori [8, 9]. However, H. pylori infection is not eliminated in patients with detectable Th1 and antibody re-sponses. It is presumed that the pathogen has evolved a number of strategies to circumvent protective immune responses. In this regard, studies have shown that H. pylori can induce ap-optosis of various types of cells, including gastric epithelial cells, macrophages, and T cells [10–12]. Moreover, the local cytokine milieu, particularly the intricate balance between proinflam-matory and anti-inflamproinflam-matory cytokines, can influence T cell development, the efficacy of immune responses, and gastric pathology [13, 14]. An early and persistent Th1-dominated CD4 response appears to be critical in the prevention of chronic infection but may lead to more severe gastric inflammation [15]. In contrast, the development of chronic infection is linked to a weak or absent H. pylori–specific Th1 response and to the presence of Th2-type cytokines; but only minimal gastritis was found under such conditions [16]. Among known Th2 cyto-kines, interleukin (IL)–4 and IL-5 were virtually absent from gastric lymphocytes from patients infected with H. pylori [17– 19].

Recently, transforming growth factor (TGF)–b and IL-10 have been reported to exert potent anti-proliferative and anergy-inducing effects on CD4 cells [20]. Such pathogen-stim-ulated IL-10 or TGF-b production might play a vital role in the prevention of infection-induced immunopathology or pro-longation of persistence, by suppressing Th1 responses [21, 22]. However, only few studies have investigated the effects of H. pylori on TGF-b and IL-10 production and have reported con-troversial results [23–31]. We investigated whether H. pylori infection could modulate the production of TGF-b and IL-10 in various mammalian epithelial cells. We show that H. pylori may secrete some soluble protein(s) to induce TGF-b produc-tion in gastric epithelial cells and monocytes and that this ability is a constitutional characteristic of H. pylori independent of cagA status and disease status. We assumed that H. pylori might use this capability to escape from or interfere with T cell func-tions and inflammatory responses.

MATERIALS AND METHODS

Bacterial strains. The H. pylori strains were obtained from ATCC (ATCC 43504) or freshly isolated from gastric biopsy specimens at National Taiwan University Hospital. Clinical iso-lates were cultured on blood agar piso-lates under microaerobic

conditions (5% O2, 10% CO2, and 85% N2) [32]. After isolation,

a single colony was subcultured. The strains were then

pre-served at⫺70C in Brucella broth (Difco Laboratories)

sup-plemented with 15% glycerol (vol/vol). H. pylori strains isolated from 15 patients with chronic gastritis, 15 patients with gastric ulcer, 14 patients with duodenal ulcer, and 15 patients with

gastric cancer were randomly selected from more than 300 clinical isolates for further studies. All these strains were de-termined to be cagA-positive by PCR. The cagA- or vacA-neg-ative isogenic mutants were kindly provided by Professor Jin-Town Wang [32]. Before the infection of epithelial cells, pure cultures of H. pylori were recovered from stocks cultured on blood agar plates under microaerophilic conditions at 37C for 3 days. The culture plate was washed with 2 mL PBS, and the amount of bacteria suspended in PBS was determined by mea-suring the optical density at 600 nm.

Bacterial strains including Escherichia coli, Klebsiella pneu-moniae, Enterobacter cloacae, Pseudomonas aeruginosa, Shigella flexneri, and group B Salmonella were obtained from National Taiwan University Hospital and were identified by standard microbiological techniques.

Epithelial cell cultures. Different epithelial cell lines were obtained from the ATCC, including gastric cancer cells (human stomach adenocarninoma [AGS] cells, N87, SUN-1, SUN-16, Hs578T, T-47D, L48, and TSGSH), colonic cancer cells (colo 320), breast cancer cells (NIC-H157), and hepatoma cells (HepG2). The cell lines were maintained in Dulbecco’s mod-ified Eagle’s medium (DMEM; Sigma) supplemented with 10% fetal calf serum and 50 mg/mL penicillin-streptomycin.

Infection procedure. Epithelial cell lines were seeded in 24-well culture plates in a volume of 1 mL per 24-well and grown at

37C in a 5% CO2 atmosphere to reach confluence. Prior to

infection, each well was washed twice in 1 mL of antibiotic-free cell culture medium. Bacterial cells were harvested, washed with PBS and then resuspended in the same medium. The bacteria were added to the cultured cells at an MOI of 400.

After incubation for 16 h at 37C in a 5% CO2 atmosphere,

the concentrations of TGF-b or IL-10 in the supernatant were measured at indicated time points. In addition, an uninfected control was included in each experiment.

Preparation of soluble extract of H. pylori. H. pylori was

harvested, washed and resuspended in PBS (₄⫻ 107cfu/mL).

Soluble extract was prepared in PBS by sonication, centrifuged at 13,400g for 10 min and passed through a 0.20-mm filter. The protein concentration of soluble extract was determined by the bicinchoninic acid method with bovine serum albumin as a standard.

Preparation of peripheral blood mononuclear cells (PBMCs) and primary gastric epithelial cell lines. Monocytes were iso-lated from buffy coat prepared from a H. pylori–negative vol-unteer from our laboratory staff. The plasma of blood samples was carefully removed, PBMCs were prepared with the use of Ficoll-Hypaque density gradient centrifugation and resus-pended in RPMI 1640 with 5% inactivated fetal calf serum. Cells were incubated in Petri dishes at 37C for 1 h. Nonadher-ent cells were removed by several washes with PBS. The

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Figure 1. Transforming growth factor (TGF)–b production of human gastric epithelial cells induced by Helicobacter pylori (Hp). Results of 3 experiments are shown, expressed as mean values SEM. A, TGF-b production in human gastric cancer cell lines cultured with H. pylori. Black

bars, H. pylori soluble extract; white bars, supernatants of cells treated

with PBS; striped bars, supernatants of cells without any treatment. B, Time-dependent TGF-b production, as measured by ELISA, in human stom-ach adenocarninoma (AGS) cells exposed to clinically isolated H. pylori strains from patients with gastritis (HC-9), duodenal ulcer (HD-27), and gastric cancer (HS-22). C, Relationship between TGF-b production in AGS cells with different MOI values for H. pylori.

herent cells were cultured in RPMI 1640 with 10% fetal calf

serum at 37 in an atmosphere with 5% CO2and 95% humidity.

Primary cultures of human gastric epithelial cells were es-tablished from gastric biopsies taken at gastroscopic exami-nations, as detailed elsewhere [33]. In the experiments to de-termine TGF-b induction, the concentrations of both primary

gastric epithelial cell lines and PBMCs were adjusted to 105

cells/mL.

Size exclusion assays. The supernatants from H. pylori were poured into a centrifugal filter device (Amicon Ultra-15; Mil-lipore) with 100-kDa ultrafiltration membranes. The device was centrifuged at 3300g for 30 min, and the supernatant in the upper layer or lower layer was separately collected. The su-pernatant in the lower layer was later centrifuged again at 1200g for 30 min with a 50 kDa Vivaspin concentrator device (Viva-Science) with a 50-kDa ultrafiltration membrane. The protein concentrations of supernatants were measured with a BCA pro-tein assay kit (Pierce), and their TGF-b–inducing activities were determined.

Gel filtration and SDS-PAGE. Fractionation to collect the protein with TGF-b–inducing activity (∼50–100 kDa) was ap-plied on a Sephadex G200 column (Amersham Biosciences). The protein was monitored by Coomassie Plus Bradford assay with absorbance set at 595 nm. Gel filtration was carried out at room temperature with the buffer containing 1x PBS (pH, 7.4). Every 5 fractionations (5 mL/fraction) collected by gel filtration were mixed, and the TGF-b–inducing ability was ex-amined in AGS cells. The TGF-b–producing fraction was then subjected to 10% SDS-PAGE electrophoresis. The gels were lat-er stained with Coomassie blue and their molecular equivalent masses were determined by comparing them with standard markers.

Quantitation of TGF-b and IL-10. TGF-b and IL-10 pro-teins in the supernatant were measured with ELISA kits pur-chased from Promega and used according to the manufacturer’s instructions.

Statistical analysis. Triplicate results of 3 experiments (n p 9) are expressed as mean values SEM. Statistical dif-ferences were determined with the software program School-stat (White Ant Occasional Publishing) using overall analysis of variance and the independent t test.

RESULTS

Induction of TGF-b production in human gastric epithelial cell lines exposed to H. pylori. To examine the effects of H. pylori on TGF-b production by gastric epithelial cells, H. pylori were cultured with different human gastric cancer cell lines in the initial experiments. As shown in figure 1A, quantification of TGF-b in supernatants after incubation for 16 h demon-strated a significant increase in TGF-b, compared with cells

unexposed to H. pylori (P!_.05). Three clinical isolates of H.

pylori isolated from patients with gastritis, duodenal ulcer, or gastric cancer were tested in AGS cells to determine the re-sponse time for TGF-b production. The expression of TGF-b increased 5 min after H. pylori infection, reached its maximal

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Figure 2. Influence of disease status and virulence factors cagA and vacA on induction of transforming growth factor (TGF)–b production by

Helicobacter pylori. Results of 3 experiments are shown, expressed as mean values SEM. A, Variations in the ability to induce TGF-b production

for cell lines exposed to H. pylori (MOI, 400). PBS alone was the negative control for each cell line. B, TGF-b concentrations in supernatants obtained after incubation of human stomach adenocarninoma (AGS) cells exposed to H. pylori and other enteric bacteria (Escherichia coli, Klebsiella pneumoniae,

Enterobacter cloacae, Pseudomonas aeruginosa, Shigella flexneri, and group B Salmonella; MOI, 400). C, TGF-b production in supernatants of cultures

of AGS cells and clinical isolates of H. pylori from patients with gastritis (HC), duodenal ulcer (HS), gastric ulcer (HU), and gastric cancer (HD). D, TGF-b production in supernatants of cultures of AGS cells with wild-type H. pylori and 5 knockout (KO) strains of H. pylori.

level at 90 min, and remained at this high level till the end of the experiment (16 h after infection) (figure 1B). Production of TGF-b in AGS cells was noted to depend on the size of the inoculum; the plateau was found to be at an MOI value of 400 (figure 1C). On the basis of these results, the following analyses of H. pylori–induced TGF-b production were performed using an MOI value of 400 and an incubation period of 16 h, unless otherwise stated.

Induction of TGF-b production by H. pylori, according to

cagA, vacA and the disease status. H. pylori could

signifi-cantly increase the production of TGF-b in various mammalian

nongastric cell lines (P!_.05) (figure 2A). In contrast, there was

no alteration in the IL-10 level after culture of H. pylori with various mammalian cell lines (data not shown). The induction of TGF-b in epithelial cells was specific to H. pylori, because other enteric bacteria (E. coli, K. pneumoniae, E. cloacae, P. aeruginosa, S. flexneri, and group B Salmonella) did not induce TGF-b production (figure 2B). There was no strain variability with respect to TGF-b production: cultures of AGS cells with various H. pylori strains isolated from patients with gastritis, duodenal ulcer, gastric ulcer, or gastric cancer showed no sig-nificant difference in the mean level of TGF-b (figure 2C).

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Figure 3. Influence of Helicobacter pylori–derived soluble proteins on the production of transforming growth factor (TGF)–b from the gastric cancer cell lines. Human stomach adenocarninoma (AGS) cells were stim-ulated with live and dead H. pylori, boiled and unboiled soluble fractions (supernatants), and boiled and unboiled insoluble fractions (H. pylori cell pellets). Results of 3 experiments are shown, expressed as mean values SEM.

Figure 4. Induction of transforming growth factor (TGF)–b by

Helico-bacter pylori (Hp)–derived soluble proteins in exposed to Escherichia coli

or H. pylori or their soluble proteins. A, Induction in primary gastric epithelial cells. B, Induction in monocytes. Results of 3 experiments are shown, expressed as mean values SEM.

Furthermore, TGF-b production was similar between cagA-negative isogenic mutants and cagA-positive strains (figure 2D). Induction of TGF-b by soluble proteins of H. pylori. Stim-ulation of AGS cells with soluble and insoluble fractions of live and dead H. pylori showed that only the insoluble fraction could not induce TGF-b production (figure 3). To further determine whether the modulatory factors present in H. pylori prepara-tions were protein or nonprotein factors, the soluble lysate was pretreated with boiling. After boiling, the capability of induc-tion of TGF-b was significantly decreased (figure 3). Further-more, digestion of the supernatant with proteinase K com-pletely stopped TGF-b production, indicating that soluble proteins account for the inducing effect (data not shown).

Induction of TGF-b by the soluble proteins of H. pylori in PBMCs and gastric epithelial cells. Although epithelial cells from cancer cell lines can be stimulated to express TGF-b, whether the normal gastric epithelial cells and PBMCs can re-spond to H. pylori is unknown. Therefore, the human primary gastric epithelial cells were incubated with H. pylori or the soluble supernatant to monitor the secretion of TGF-b into medium. The results revealed that H. pylori and its soluble proteins could induce TGF-b secretion in the gastric epithelial cells and in PBMCs, but E. coli could not (figure 4A and 4B). Partial characterization of the TGF-b inducing fac-tor(s). TGF-b inducing activity was detectable in the retentate after concentration with a 50–100 kDa–cutoff concentrator (results not shown). After eluates that passed through the 100-kDa filter were reseparated with the gel filtration column, and the TGF-b inducing activities in the fractions were determined (figure 5A), SDS-PAGE analysis of fractions with peak TGF-b inducing activity revealed 2 major bands with a molecular mass

of∼70 kDa (figure 5B).

DISCUSSION

The mechanisms by which H. pylori causes chronic infection and interacts with immune systems remain unclear. Proinflam-matory cytokines, particularly Th1 cytokines, have long been considered as the main mediators of the immune response to H. pylori infection [34]. In view of the recent findings on the critical role of anti-inflammatory cytokines in diverse pathogens [20], this study focuses on the anti-inflammatory cytokines TGF-b and IL-10. We first demonstrated that TGF-b, rather

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Figure 5. Partial characterization of factors inducting transforming growth factor (TGF)–b by gel filtration and SDS-PAGE. A, Gel filtration profile of

Helicobacter pylori supernatants, showing the fractionation of TGF-b–inducing factors and their ability to induce TGF-b production. Results of 3

experiments are shown, expressed as mean values SEM. B, SDS-PAGE of gel filtration exclusion chromatography eluted fractions. The molecular weight marker position is shown at left. Lane 1, pooled fractions 1–5; lane 2, pooled fractions 6–10.

than IL-10, was elicited in vitro in gastric epithelial cells and monocytes by secreted soluble proteins of H. pylori. Our results indicate a new interaction between H. pylori and epithelial and PBMCs through the induction of TGF-b.

Several studies in which H. pylori and epithelial cell lines have been cultured together yielded contradictory findings: both increased IL-10 production and no change of IL-10 pro-duction have been found [23–26]. This discrepancy may be the result of different experimental conditions, such as differences in bacteria strains, cell lines, host-bacteria interaction, and methods of quantitation (eg, use of reverse-transcription PCR or ELISA). In a recent publication, Nakachi et al. [30] found

that H. pylori infection did not increase the levels of TGF-b mRNA in human gastric epithelial cells. However, Takagi et al. [28] have demonstrated that TGF-b was produced by gastric cancer cells after exposure to H. pylori. In agreement with the latter report, our results show that H. pylori induced a rapid accumulation of TGF-b in various cell lines. It is important to note that H. pylori also stimulated TGF-b production in pri-mary gastric epithelial cells and PBMCs. Because PBMCs com-prise a major part of the cellular inflammatory response to H. pylori infection, our finding for PBMCs strengthens the im-portance of the TGF-b in vitro model. Further evidence for the involvement of TGF-b in H. pylori infection was found by

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immunohistochemical staining of gastric biopsy specimens. Compared with cells from uninfected subjects, cells from H. pylori–infected patients have significantly higher TGF-b levels [35]. Moreover, Messa et al. [29] have demonstrated that TGF-b expression dropped remarkaTGF-bly after successful eradication of H. pylori. Confirming these observations, our data suggest that H. pylori infection can induce TGF-b production in epi-thelial cells and PBMCs.

The phenomenon of TGF-b induction by H. pylori is quite unique, and different from the findings for other commonly isolated enteric pathogens. Only H. pylori can influence the release of TGF-b by the gastric epithelial cells. Intriguingly, the target of the TGF-b–inducing protein exists in not only gastric epithelial cells but also other types of cell. It implies that the ability to induce the secretion of TGF-b is specific to H. pylori, but the receptor for the modulating factor may be expressed on different cells.

Previous studies have demonstrated that cytokine production is dependent on direct contact between epithelial cells and bac-teria [23–28] and is influenced by bacbac-terial strains with an intact cag pathogenicity island [36]. The results of our study suggest that the capability to induce TGF-b production can be regarded as a constitutional characteristic of H. pylori, because all strains from different disease status show this activity. There was no relation between the inducing activity and the cagA or vacA status of the strain.

The induction of TGF-b by H. pylori probably has biological significance, in view of the central role of TGF-b in immune regulation [37]. As a regulator of site-specific T cell inflam-matory response [21], TGF-b is relevant in gastric pathology during H. pylori infection. By its immunosuppressive function, TGF-b may protect the gastric mucosa from severe damage caused by gastritis, in one way, and contribute to the persistence of H. pylori infection, in another. The role of TGF-b is also elusive in other infectious and inflammatory bowel diseases [38, 39]. Buzoni-Gatel et al. [37] have shown that the regulation of the ileal inflammatory process in T. gondii infection is de-pendent on TGF-b–producing intraepithelial lymphocytes. In colitis, TGF-b production is an essential mechanism of counter-regulation of Th1 cell-medicated mucosal inflammation [39]. In addition, perturbation of TGF-b signaling in animal models is linked to development of severe mucosal inflammation of gastrointestinal tract [40, 41]. In patients with H. pylori–related duodenal ulcer, TGF-b production in the metaplastic epithe-lium of the duodenum is decreased, compared with production in nonmetaplastic epithelium [31]. Pathogen-stimulated TGF-b production in innate cells might prevent infection-induced immunopathology or prolong pathogen persistence by sup-pressing protective Th1 responses [20].

In accordance with previous reports and our data, we pro-pose a hypothetical explanation of the persistence of H. pylori

infection in the face of strong immune responses. We speculate that H. pylori, through the induction of TGF-b, might construct an immune privileged area in the stomach that mimics the ocular microenvironment, where immune effector responses and inflammation are suppressed [22]. Then the organisms protect themselves in the acidic environment with urease. How-ever, the adhesion would alert the host immune system and induce proinflammatory cytokines and chemokines to trigger the migration of immune cells to the gastric epithelium. To prevent destruction by these immune responses, H. pylori se-cretes TGF-b–inducing protein to induce TGF-b production, and the gastric epithelial cell-derived TGF-b diffuses in the gastric epithelium to protect H. pylori from the immediate immune attack at the early stage of infection. Moreover, the secreted TGF-b–inducing protein will interact with macro-phages to trigger the second burst of TGF-b secretion, which causes more-extensive immunosuppression. The macrophages expressing TGF-b may interact with T cells to induce anergy. As the H. pylori bacteria invade further, they magnify the TGF-b–dependent immune-privileged area. Therefore, although the immune effectors will be attracted by chemokines, immune cells might lose their activity once they enter the TGF-b–in-ducing, protein-induced, immune-privileged area. The signif-icance of TGF-b and TGF-b–inducing protein for the patho-genesis of H. pylori–related diseases remains speculative at present. Nevertheless, it is potent and worth investigating in future studies.

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