It is generally recognized that Helicobacter pylori(H. pylori) infection is a major cause of chronic gastritisand peptic ulcers. Although it is suspected thatcytokines contribute to immune response in the devel-opment of gastritis, their mechanisms have not beenclearly defined. The gastric mucosal levels of theproinflammatory cytokines such as interleukin (IL)-1β,IL-6, IL-8, and tumor necrosis factor (TNF)-α havebeen reported to be increased in H. pylori-infected sub-jects (4, 10, 19, 24). We previously investigatedcytokine expression patterns of human gastric mucosalbiopsy specimens by reverse-transcription polymerasechain reaction (RT-PCR) method (35). Our studyshowed that the expression of IL-8 mRNA was signifi-cantly higher in H. pylori-positive gastritis than that inH. pylori-negative controls and that there was a closecorrelation between the degree of the expression of IL-8

mRNA and the severity of gastritis, which suggestedthat IL-8 could play some important roles in mucosalinflammation (23).

In addition, we examined the relationship betweenthe expression of cytokine mRNA and the histologicalfindings in the gastric mucosa of H. pylori-positivepatients (35). The level of TNF-α mRNA expressionwas significantly higher in specimens with moderate tosevere infiltration of mononuclear cells (MNC) andpolymorphonuclear cells (PMN) than in those with nor-mal to mild infiltration in the antrum (12).

TNF-α is a key mediator in a host defense againstbacterial infections and in the septic shock syndromeinduced either by lipopolysaccharide (LPS) or bacterialsuper-antigens. In an experiment using TNF-α gene-

Role of Tumor Necrosis Factor-Alpha and Interferon-Gamma in Helicobacter pylori Infection

Toshiro Yamamoto*, 1, Masakazu Kita1, Tomoyuki Ohno2, Yoichiro Iwakura3, Kenji Sekikawa4,and Jiro Imanishi1

1Department of Microbiology and 2Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto,Kyoto 602–8566, Japan, 3Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Minato-ku,Tokyo 108–8639, Japan, and 4Department of Immunology, National Institute of Animal Health, Tsukuba, Ibaraki 305–0856,Japan

Received April 9, 2004; in revised form, June 11, 2004. Accepted June 25, 2004

Abstract: Immune responses to Helicobacter pylori infection play important roles in gastroduodenal dis-eases. The contributions of tumor necrosis factor-� (TNF-�) and interferon-� (IFN-�) to the induction ofgastric inflammation and to the protection from H. pylori infection were investigated using TNF-� gene-knockout (TNF-��/�) mice and IFN-� gene-knockout (IFN-��/�) mice. We first examined the colonizingability of H. pylori strain CPY2052 in the stomach of C57BL/6 wild-type and knockout mice. The numberof H. pylori colonized in the stomach of IFN-��/� and TNF-��/� mice was higher than that of wild-typemice. These findings suggest that TNF-� and IFN-� may play a protective role in H. pylori infection. Fur-thermore, we examined the contribution of TNF-� and IFN-� to gastric inflammation. The CPY2052-infected TNF-��/� mice showed a moderate infiltration of mononuclear cells in the lamina propria and ero-sions in the gastric epithelium as did wild-type mice, whereas the CPY2052-infected IFN-��/� mice showedno inflammatory findings even 6 months after infection. These results demonstrate that IFN-� may play animportant role in gastric inflammation induced by H. pylori infection, whereas TNF-� may not participatein the development of inflammatory response.

Key words: Helicobacter pylori, TNF-�, IFN-�, Inflammation

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Microbiol. Immunol., 48(9), 647–654, 2004

Abbreviations: cagA, cytotoxin-associated gene A; HSBHI,Helicobacter-selective brain heart infusion; IFN, interferon; IL,interleukin; LPS, lipopolysaccharide; MNC, mononuclear cells;PBMCs, peripheral blood mononuclear cells; PMN, polymor-phonuclear cells; RT-PCR, reverse-transcription polymerasechain reaction; TNF, tumor necrosis factor; VacA, vacuolatingcytotoxin A.

*Address correspondence to Dr. Toshiro Yamamoto, Depart-ment of Microbiology, Kyoto Prefectural University of Medi-cine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, Kyoto602–8566, Japan. Fax: �81–75–251–5331. E-mail: yamamoto@koto.kpu-m.ac.jp

knockout (TNF-α�/�) and wild-type mice, for example,Kaneko et al. (15) reported that TNF-α plays an impor-tant role in protective immunity against virulentmycobacterial infections and an indirect role in granulo-ma formation. Taniguchi et al. (33) indicated an impor-tant role for TNF-α in germinal center formation and inthe sepsis-induced hepatocyte apoptosis that precededliver failure. Thus, TNF-α is a cytokine, producedmainly by activated macrophages, with pleiotropicfunctions (21). Although we have demonstrated thatinterferon-γ (IFN-γ) plays an important role in theinduction of gastric inflammation caused by H. pyloriinfection using IFN-γ gene-knockout (IFN-γ�/�) mice(27), the role of TNF-α in H. pylori infection in vivo hasnot been clarified.

The aim of the present study is to investigate theroles of TNF-α in protection against H. pylori infectionand in the development of gastric inflammation in themouse models by using TNF-α�/� mice, compared toIFN-γ�/� mice.

Materials and Methods

Animals. IFN-γ�/�, TNF-α�/� and wild-type C57BL/6mice used in this study were specific pathogen-free 6-week-old males. The wild-type C57BL/6 mice werepurchased from Charles River Japan (Atsugi, Japan).

The IFN-γ�/� were generated by a targeted mutationof the IFN-γ gene as previously described (31). Briefly,the targeting vector containing the disrupted IFN-γ genewas transfected into A3-1 embryonic stem cells. Thesecells were injected into the blastocysts from superovu-lated C57BL/6 females, and then the embryos weretransferred to the uteri of pseudopregnant ICR recipi-ents. The male chimeras were crossed with C57BL/6females, and then embryonic stem cell-derived pupswere backcrossed to C57BL/6 mice. The homozygousIFN-γ�/� mice were produced by intercrossing the het-erozygous mice.

TNF-α�/� mice were generated by a targeted mutationof the TNF-α gene as described previously (33).Briefly, the targeting vector containing the disruptedTNF-α gene was introduced by electroporation intoTT2 ES cells. Three clones were injected into 8-cell-stage C57BL/6 embryos, and the embryos were trans-ferred into the uterus of ICR pseudopregnant femalesafter 24 hr in in vitro culture. TNF-α�/� heterozygoteswere backcrossed with C57BL/6 mice, and the F5 gen-eration was used for preparation of TNF-α�/� homozy-gotes.

The mice were all maintained in a barriered animalfacility under pathogen-free conditions at the KyotoPrefectural University of Medicine. All equipment and

supplies including cages, water bottles, wooden chipsand food pellets were sterilized. Under these condi-tions, the IFN-γ�/� mice, TNF-α�/� mice and wild-typemice grew normally and healthily.

Bacteria. The following H. pylori strains were usedin this study. H. pylori CPY2052 was generously sup-plied by Dr. M. Karita (Houfu Onsen Hospital, Japan)(16). KP142 strain was isolated from human gastricbiopsy specimens at the Kyoto Prefectural University ofMedicine. Helicobacter felis CS1 (�ATCC49179) wasobtained from the American Type Culture Collection(Rockville, Md., U.S.A.). All the strains were Gram-negative spiral rods and H. pylori strains were positivefor H. pylori cagA and vacA genes.

Experimental protocol for H. pylori and H. felisinfection. All mice used in this study were challengedonly once at the beginning of the experiment, after 4-hrfasting, with 0.2 ml of the H. pylori or H. felis culturecontaining 2�108 CFU/ml by use of an oral stainlesssteel catheter. The mice were then maintained on thefast for 4 hr and housed as described in the previoussection. The colonization to gastric mucosa wasassessed at specified times. Each experimental groupwas comprised of 7 to 15 mice.

Preparations of bacterial inoculum. Preparations ofbacterial inoculum were previously described (27).Briefly, frozen CPY2052, KP142 and H. felis strains inBHI broth containing 15% (vol/vol) FBS and 15%(vol/vol) glycerol were thawed and streaked on Heli-cobacter-selective brain heart infusion (HSBHI) agarplates (Eiken Chemical Co., Tokyo), and incubated for 5days at 37 C using an Aeropack System which wascharged with a gas mixture consisting of 80% N2, 15%CO2, and 5% O2 (Mitsubishi Gas Chemical Co., Tokyo).

Microbiological evaluation. Mice were sacrificedafter 4-hr fasting and their stomachs were resected.The resected stomachs were divided longitudinally intotwo halves and weighed. One half of the resected stom-ach was added to 1 ml of PBS and homogenized using atissue grinder (Pellet Pestle Mixer, Kimble/Kontes,U.S.A.). A 100 µl portion of the homogenized stomachwas plated on an HSBHI agar plate and incubated for 5days at 37 C using an Aeropack System as described inthe previous section. The colonies on the plate werecounted and expressed as CFU/g tissue.

For the determination of whether the organisms ofthe colonies were H. pylori or H. felis, a PCR was car-ried out with the DNA preparation of the colonies aspreviously described (36). The primer sequences areshown as follows: H. pylori cagA gene, 5'-GATAACAGGCAAGCTTTTGAGG-3' (sense) and 5'-CTGCAAAAGATTGTTTGGCAGA-3' (antisense), H.felis, 5'-TGCGTAGGCGGGGTTGTAAG-3' (sense) and

648 T. YAMAMOTO ET AL

5'-CAGAGTTGTAGTTTCAAATGC-3' (antisense). PCRwas performed for 35 cycles, each cycle consisting of 1min at 95 C for denaturation, 1 min at 50 C for anneal-ing and 1 min at 72 C for extension. Ten µl of eachPCR product was analyzed by electrophoresis on a1.5% agarose gel containing ethidium bromide, and thebands were visualized under UV light.

Histological evaluation. The other half of eachresected stomach was used for the histological evalua-tion. The longitudinal sections from the gastroe-sophageal junction to the gastroduodenal junction werefixed in neutrally buffered 10% formalin, embedded inparaffin, sectioned at 5 µm, and stained with hema-toxylin and eosin. The degrees of inflammation wereassessed by microscopic observation without knowl-edge of the experimental groups and expressed as fol-lows. The normal appearance of scattered mononuclearcells on the lamina propria (the same degree as unin-fected control mice): none�score 0; moderate infiltra-tion of mononuclear cells in the lamina propria and thesubmucosa, and no erosion in the epithelium:mild�score 1; moderate infiltration of mononuclearcells in the lamina propria and the submucosa, and ero-sions in some parts of the epithelium: moderate�score2; and severe infiltration of mononuclear cells in thelamina propria and the submucosa, and erosions in con-siderable parts of the epithelium: severe�score 3.

Statistics. Colonization and inflammation scoreswere expressed as means and standard errors. Samplemeans were compared by the Mann-Whitney U test. AP value of �0.05 was considered to be significant.

Results

Colonization of H. pylori in Wild-Type, IFN-γ�/� andTNF-α�/� Mice

To examine whether IFN-γ and TNF-α are involvedin protection against H. pylori infection, the followingexperiments were carried out using C57BL/6 wild-typeand IFN-γ�/� and TNF-α�/� mice. Mice were orallyinfected with H. pylori CPY2052, and the number ofcolonized bacteria in the stomach was determined at 1,2, 3 and 6 months after infection (Table 1). The number

of H. pylori colonized in the stomach of IFN-γ�/� andTNF-α�/� mice was significantly higher than that ofwild-type mice from 1 to 6 months after infection.However, there was no significant difference in thenumber of colonized bacteria between IFN-γ�/� andTNF-α�/� mice. These results suggest that TNF-α aswell as IFN-γ may play a protective role against H.pylori infection.

To confirm that TNF-α plays a protective role ininfection against another strain of H. pylori, wild-typeand TNF-α�/� mice were orally infected with CPY2052 and KP142, and the colonization number in thestomach was determined 6 months after infection (Fig.1). The number of KP142 as well as CPY2052 thatcolonized the stomach of TNF-α�/�mice was signifi-cantly higher than that of wild-type mice.

Histology of the Stomachs of Wild-Type, IFN-γ�/� andTNF-α�/� Mice after Infection

To investigate the role of IFN-γ and TNF-α in gastric

649TNF-α AND IFN-γ IN H. PYLORI INFECTION

Table 1. Colonization of H. pylori CPY2052 strain in wild-type, IFN-γ�/� andTNF-α�/� mice

MiceMonths after infection

1 2 3 6

Wild-type 3.8�0.1 3.5�0.2 4.1�0.1 4.0�0.2IFN-γ�/� 4.8�0.2** 4.5�0.2** 4.5�0.1* 4.6�0.1**TNF-α�/� 4.3�0.1** 4.0�0.1* 4.5�0.1* 4.9�0.1**

*: P�0.05, **: P�0.005.Data are expressed as means�S.E. (log of CFU/g tissue) (n�7–10).

Fig. 1. Number of H. pylori (CPY2052 and KP142) colonized inthe stomach of wild-type ( ) and TNF-α�/� ( ) mice 6months after infection. Data are expressed as means�S.E.(n�7). *: P�0.005.

650 T. YAMAMOTO ET AL

Fig. 2. Histology of the gastric tissue of wild-type (a, c, e), TNF-α�/� (b, d) and IFN-γ�/� (f) mice 2 (a, b)or 6 (c, d, e, f) months after infection with H. pylori CPY2052. (Hematoxylin-Eosin stain: original mag-nification �100).

inflammation induced by H. pylori infection, the histol-ogy of stomachs of wild-type, IFN-γ�/� and TNF-α�/�

mice was examined after infection with H. pyloriCPY2052. In wild-type and TNF-α�/�mice, a moderateinfiltration of mononuclear cells was observed in thesubmucosa and the lamina propria 2 months after infec-tion. Erosions were also recognized in some parts of theepithelium (Fig. 2, a and b). Six months after infection,the erosions of the epithelium became severer and a

moderate infiltration of mononuclear cells or lymphoidfollicles appeared in the submucosa and the lamina pro-pria (Fig. 2, c and d). In contrast, in the CPY2052-infected IFN-γ�/� mice, no inflammatory findings wereobserved even 6 months after infection (Fig. 2, e and f,Table 2).

To confirm the role of TNF-α in the process ofinflammation caused by H. pylori infection, we carriedout an experiment of long-term infection with H. felis

651TNF-α AND IFN-γ IN H. PYLORI INFECTION

Fig. 3. Histology of the gastric tissue of wild-type (a, c), and TNF-α�/� (b, d) mice 6 months after infection with KP142 (a, b) and H.felis (c, d). (Hematoxylin-Eosin stain: original magnification �100).

Table 2. Histopathological inflammation score in wild-type, IFN-γ�/� andTNF-α�/� mice after infection with H. pylori CPY2052

MiceMonths after infection

1 2 3 6

Wild-type 0�0 0.1�0.1 0.8�0.1 1.3�0.3IFN-γ�/� 0�0 0�0 0�0 0�0*TNF-α�/� 0�0 0.1�0.1 1.0�0.1 1.4�0.2

*: P�0.005.Data are expressed as means�S.E. (n�7–10).

and H. pylori KP142, and compared the results to thoseobtained with CPY2052. Wild-type and TNF-α�/� micewere orally infected with H. felis or H. pylori KP142,and the histopathological inflammation scores werecompared at 6 months after infection. KP142-infectedmice developed more severe infiltration of mononuclearcells in the lamina propria and erosions in the gastricepithelium than did CPY2052-infected mice. Therewere no significant differences in histopathologicalinflammation scores between wild-type and TNF-α�/�

mice infected with KP142 and H. felis (Figs. 3 and 4).

Discussion

H. pylori infection is always associated with inflam-mation; however, ulcerative diseases and gastric carci-noma occur only in a subset of patients. There are twoproposed explanations for such diverse clinical out-comes of chronic H. pylori infection. The first is agenetic diversity of virulence factors and antigenic pro-files of various H. pylori strains, including enhancedmotility, production of urease, catalase, and phospholi-pase C, specific adherence to gastric epithelial cells,production of a vacuolating cytotoxin A (VacA), andthe presence of a bacterial gene cluster (a pathogenicityisland) with cagA (cytotoxin-associated gene A). Thesecond is a genetic difference in the individual immuneresponses to the pathogen, which may result in failure toeradicate the infection and lead to chronic mucosaldamage (9); for example, IL-1 gene cluster polymor-phisms may be associated with an increased risk of H.pylori-induced hypochlorhydria and gastric cancer (8).

H. pylori-activated PBMCs produce IL-1, IL-2, IL-6,

IL-8, TNF-α, IFN-γ, and surface Fas L (2–4, 7, 17, 37).Increased TNF-α secretion is associated with anincreased level of apoptosis (18), independent ofexpression of the vacuolating cytotoxin (32) or thecagA status (25) of H. pylori. Apoptosis has been recog-nized as a factor in H. pylori-related mucosal injury(11, 13, 23, 28, 38). We therefore hypothesized thatthere was a reduced mucosal inflammatory responsedue to the loss of TNF-α in TNF-α-deficient mice. Theresults of our study, however, demonstrate that therewas no effect on the grade of gastritis in TNF-α-defi-cient mice compared with isogenic wild-type controls.Gastric inflammation in H. pylori infection seems to beindependent of the function of the TNF-α. Thalmaier etal. (34) reported that there was no detectable differencein the grade or activity of gastritis in TNF-R1-deficientmice when they were compared with wild-type mice.Our results are in agreement with their results.

In contrast to TNF-α, IFN-γ plays an important rolein the induction of gastric inflammation caused by H.pylori infection. Our results showed that IFN-γ�/�micewere colonized but did not show any inflammatorysigns after H. pylori infection (14, 27, 30). Th1cytokines such as IFN-γ, released by lymphocytes andneutrophils that infiltrate gastric mucosa are associatedwith immune activation and tissue injury. It was report-ed that Fas antigen expression was enhanced by IFN-γ,and this elevated expression of Fas antigen causes apo-ptosis (26). These data suggest that IFN-γ, viaenhancement of apoptosis, may contribute to anincrease in the pathogenicity of H. pylori infection.

On the other hand, IL-4 does not seem to be criticalin H. pylori infection (1, 5, 20), whereas it plays animportant role in H. felis infection of mice (22), andSmythies et al. (29) found stronger gastric inflamma-tion in IL-4-deficient mice. Chen et al. reported signifi-cantly reduced colonization of the gastric mucosa in IL-10-deficient mice so that IL-10 seemed to be aninhibitor of the protective immune response to H. pyloriinfection (6). Thus, a role of Th1 type response may beimportant in the pathogenesis of H. pylori-associateddisease.

In conclusion, our results showed significantlyenhanced colonization of the gastric mucosa in TNF-α-deficient mice so that TNF-α seemed to be a protectivefactor in H. pylori infection, whereas TNF-α does notappear to play a role in gastric inflammation induced byH. pylori infection. However, further investigations arerequired to clarify the importance of TNF-α in patho-genesis and modulation of inflammatory processes fol-lowing H. pylori infection leading to H. pylori-associat-ed gastroduodenal diseases.

652 T. YAMAMOTO ET AL

Fig. 4. Histopathological inflammation score in wild-type ( )and TNF-α�/� ( ) mice 6 months after infection with H. pylori(CPY2052 and KP142) and H. felis. Data are expressed asmeans�S.E. Each group consisted of 7 mice.

We are grateful to Dr. M. Karita, Houfu Onsen Hospital,Japan for the generous supply of H. pylori CPY2052.

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