Natural History of Gastric Mucosal Cytokine Expression in Helicobacter pylori Gastritis in Mongolian Gerbils

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1 INFECTION AND IMMUNITY, Apr. 2005, p Vol. 73, No /05/$ doi: /iai Copyright 2005, American Society for Microbiology. All Rights Reserved. Natural History of Gastric Mucosal Cytokine Expression in Helicobacter pylori Gastritis in Mongolian Gerbils Yoshio Yamaoka, 1 * Kazuyoshi Yamauchi, 2 Hiroyoshi Ota, 3 Atsushi Sugiyama, 4 Satoshi Ishizone, 4 David Y. Graham, 1 Fukuto Maruta, 4 Maki Murakami, 4 and Tsutomu Katsuyama 5 Department of Medicine/Gastroenterology, Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Houston, Texas, 1 and Shinshu University Hospital, 2 Department of Biomedical Laboratory Sciences, School of Health Sciences, 3 and Department of Surgery 4 and Department of Laboratory Medicine, 5 Shinshu University School of Medicine, Matsumoto, Nagano, Japan Received 10 October 2004/Returned for modification 16 November 2004/Accepted 21 December 2004 Data regarding the chronological changes in gastric mucosal cytokines in the different phases of Helicobacter pylori infection are unavailable. We examined Mongolian gerbils for up to 52 weeks after H. pylori (ATCC 43504) inoculation. Levels of mrnas of mucosal cytokines (interleukin-1 [IL-1 ], gamma interferon [IFN- ], IL-4, IL-6, and IL-10) were assessed using real-time reverse transcription-pcr. Starting 26 weeks after H. pylori inoculation, two clinicohistologic patterns appeared: gastric ulcers in 32% and hyperplastic polyps in 68% of gerbils. High levels of mucosal IL-1 mrna were observed early in the infection, reaching maximum at 4 weeks and then rapidly declining. Mucosal IFN- mrna also reached maximal levels at 4 weeks but remained high thereafter. Both IL-1 and IFN- mrna levels were consistently higher in the pyloric mucosa than in the fundic mucosa. In contrast, IL-4, IL-6, and IL-10 mrna levels peaked at 8 to 26 weeks and levels were similar in the pyloric mucosa and the fundic mucosa. IFN- mrna levels were significantly higher in gerbils with ulcers than in those with hyperplastic polyps (median IFN- /glyceraldehyde-3-phosphate dehydrogenase ratio 100, versus 338, respectively [antrum], and 172 versus 40, respectively [corpus]) (P < 0.05). We propose that the different outcomes (e.g., ulcers or hyperplastic polyps) might relate to imbalances among cytokines. Helicobacter pylori infection of the gastric mucosa is characterized by the infiltration of neutrophils, lymphocytes, monocytes, and plasma cells. The initial migration of inflammatory cells into the gastric mucosa and their activation are believed to depend on the production of proinflammatory cytokines (2, 8, 17, 30 32). The inflammatory products from the polymorphonuclear cells (PMNs) and mononuclear cells (MNCs) are also thought to damage the epithelial layer and play a role in disease pathogenesis. T-helper (Th) cells are also found in the gastric lamina propria in H. pylori infection. The cytokine response in the gastric mucosa of patients chronically infected with H. pylori is thought to be predominantly of the Th1 type (1, 2, 5, 7, 8, 10, 11, 15, 17, 18, 22, 24 26). This determination was based in part on the presence of increased numbers of gamma interferon (IFN- )-secreting T cells in H. pylori-infected gastric mucosa compared to normal mucosa (2, 5, 11, 15, 17, 25). H. pylori-infected IFN- -knockout mice developed minimal pathological changes (1, 22, 24), consistent with the response to H. pylori infection being primarily a Th1-type response. However, the relative contribution of the different cytokines during the course of the infection is still unknown. It is generally impossible to characterize the natural history of the immune response to H. pylori in humans, but such studies are possible using animal models. Rodents are excellent model * Corresponding author. Mailing address: Michael E. DeBakey Veterans Affairs Medical Center (111D), Rm. 3A-320, 2002 Holcombe Blvd., Houston, TX Phone: (713) Fax: (713) yyamaoka@bcm.tmc.edu. animals in that they can be infected with H. pylori strains that consistently produce severe gastritis. In particular, Mongolian gerbils (Meriones unguiculatus) infected with selected strains of H. pylori develop an antral-predominant gastritis which progresses with time to corpus gastritis and may include gastric ulcers (12 14, 16, 21) and even gastric cancer (20, 29). There are few studies investigating the chronological changes in cytokine profiles during H. pylori infection in gerbils in part because of the paucity of genomic data regarding gerbils cytokines (3). In the present study, we used real-time reverse transcription-pcr (RT-PCR) to investigate various cytokine profiles in acute and chronic phases of H. pylori infection of Mongolian gerbils. MATERIALS AND METHODS Animals. Specific-pathogen-free 7-week-old male Mongolian gerbils (MGS/ Sea; Seac Yoshitomi, Fukuoka, Japan) were housed in an air-conditioned biohazard room with a 12-h-light 12-h-dark cycle designed for infectious animals. They were provided rodent diet and water ad libitum. All experimental protocols were approved by the Animal Experiment Committee of Shinshu University School of Medicine, Matsumoto, Japan. Bacterial strains and inoculation. We used H. pylori strain ATCC (American Type Culture Collection, Manassas, Va.), which has been shown to colonize gerbils consistently for at least 1 year and to cause reproducible mucosal damage (14, 16, 23). The genotype and/or phenotype with regard to putative virulence factors is cag pathogenicity island positivity, vaca s1-m1 (production of the vacuolating cytotoxin), and functional BabA and OipA. H. pylori was grown in brucella broth (Becton Dickinson, Cockeysville, Md.) supplemented with 10% (vol/vol) horse serum for 40 h at 37 C under microaerobic conditions (15% CO 2 ) and saturated humidity, with shaking at 150 rpm. After fasting for 24 h, each animal was orogastrically inoculated with an 0.8-ml inoculum preparation of H. pylori (10 9 CFU/ml) or sterile brucella broth (as uninfected controls) by using gastric intubation needles. 2205

2 2206 YAMAOKA ET AL. INFECT. IMMUN. Time course and euthanasia. Infected gerbils were euthanized and necropsied at 1, 2, 4, 8, 12, 26, 40, and 52 weeks after H. pylori inoculation. Ten or 11 gerbils were used for each time point. Uninfected control gerbils were euthanized at 7 weeks of age (when the other gerbils were inoculated with H. pylori) (n 10) or at 33 weeks of age (to serve as controls for the infected animals 26 weeks after H. pylori inoculation) (n 5). At necropsy, stomachs were opened along the greater curvature and were divided longitudinally into two parts. One half was fixed in 20% phosphatebuffered formalin for histological examination. The other half was further divided into the pyloric gland mucosa (antrum) and the fundic gland mucosa (corpus) and stored at 80 C. The gastric mucosa was separated as much as possible from the underlying muscle by sharp dissection. In addition, a 1-mm 2 piece of gastric mucosa from the pyloric part of the stomach was taken for culture of H. pylori. H. pylori cultures. The fragments from the pyloric part of the stomach were minced with brucella broth and placed on commercially available H. pyloriselective agar plates (Eiken Chemical Co., Tokyo, Japan). Cultures were incubated under microaerobic conditions and high humidity at 37 C for 7 days. Gram-negative and oxidase-, catalase-, and urease-positive spiral curved rods were identified as H. pylori. Histology. Histological examination was performed as previously described (14). Briefly, tissue sections were stained with hematoxylin and eosin for morphological observations and immunostained for H. pylori (rabbit anti-h. pylori polyclonal antibody at 1:20; DAKO, Glostrup, Denmark). The degree of inflammation was graded according to the updated Sydney system (6). All histological examination was performed blindly by one pathologist (H.O.). Serology. Before euthanasia, blood samples were obtained from the orbital plexus by using hematocrit tubes. Sera were used to measure the titer of anti-h. pylori immunoglobulin G (IgG) antibody as previously described (14, 16). The titers of antibody were expressed as an arbitrary index as previously described (14). An arbitrary index value of 1.5 indicated the presence of H. pylori antibodies. Sequence analysis of Mongolian gerbil cytokine cdna. Total RNA was extracted from the gastric mucosa by using an RNA extraction kit (Isogen; Nippon Gene, Tokyo, Japan). After DNase treatment, a 5- g portion of total RNA solution was subjected to reverse transcription with 200 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Gaithersburg, Md.) and 1 mol of oligo(dt) 16 primers/liter. To identify gerbil-specific cytokine cdna clones, we used a cross-species RT-PCR technique. We selected parts of the sequence conserved among murine, rat, and human cdna in the biologically active portion of interleukin-1 (IL-1 ) and IL-6. We chose a forward primer (IL1up, 5 -CTG AAA GCT CTC CAC CTC AAT GG-3 ) and a reverse primer (IL1down, 5 -AGG TGC TGA TGT ACC AGT TG-3 ) for PCR and amplified a segment of 325 bp from the cdna in the gastric mucosa of gerbils. Using the same methods, we identified IL-6 cdna clones by using a forward primer (IL6up, 5 -CAG AAA ACA AYC TGA AAC TTC C-3 ; Y is C or T) and reverse primer (IL6down, 5 -GTT CTT CRT AGA GAA CAA CA-3 ; RisAorG)and amplified a segment of 460 bp. The PCR products were cloned into plasmid pt7blue (Novagen, Madison, Wis.), and the nucleotide sequence of the insert was confirmed by the dideoxy chain termination procedure. We used gerbilspecific IL-4, IL-10, and IFN- cdna sequences which have been deposited in GenBank (accession numbers L37779, L37781, and L37782, respectively) for real-time RT-PCR. We normalized the cytokine mrna expression levels by using previously identified clones of cdna of the housekeeping gene gerbilspecific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (21). Analysis of cytokines by real-time quantitative PCR. The quantification of cytokine mrna and GAPDH mrna levels was performed using an ABI Prism 7700 sequence detection system (Perkin-Elmer Applied Biosystems, Foster City, Calif.). Specific primers and TaqMan probes were designed with the aid of the Primer Express program (Perkin-Elmer Applied Biosystems) (Table 1). A standard curve was constructed by using 10-fold serial dilutions of each cdna, which was subcloned into plasmid pcr2.1 (Invitrogen). We used forward and reverse primers described in Table 1 for subcloning into pcr2.1. Reaction mixtures for PCR (50 l) were prepared by mixing 5 l of synthesized cdna solution with 2 TaqMan Universal PCR Master Mix (Perkin-Elmer Applied Biosystems), 500 nm (each) primer, and 250 nm TaqMan probe. These prepared samples were placed in the analyzer, and PCR was carried out at 50 C for 2 min and 95 C for 10 min, followed by 50 cycles of 95 C for 15 s and 60 C for 60 s according to the manufacturer s instructions. The expression levels of cytokine mrna were expressed as the ratio of cytokine mrna to GAPDH mrna (cytokine mrna [units per microliter]/gapdh mrna [units per microliter] ratio 100,000). Each assay was performed in triplicate. Each cytokine assay was performed TABLE 1. Primers and probes used in this study Probe or Sequence primer type a GAPDH...F: 5 -CATGGCCTTCCGAGTTCCT-3 R: 5 -TTCTGCAGTCGGCATGTCA-3 Probe: 5 -VIC-CCCCCAACGTGTCTGTCGTGGA- IL-1...F: 5 -GGTGACACAAGCAGCAACAAA-3 R: 5 -CATCACACAGGACAGGTACAGATTCT-3 Probe: 5 -FAM-TACCGGTGGCCTTGGGCCTCA- IFN-...F: 5 -TTGGGCCCTCTGACTTCGT-3 R: 5 -CAGTGTGTAGCGTTCATGGTCTCT-3 Probe: 5 -FAM-AGGAGCCGGACTTGCCCTGCC- IL-4...F: 5 -CAGGGTGCTCCGCAAATTT-3 R: 5 -GACCCCGGAGTTGTTCTTCA-3 Probe: 5 -FAM- ACTTCCCACGAGAGGTGACTCCATGC- IL-6...F: 5 -AGGATCCAGGTCAAATAGTCTTTCC-3 R: 5 -TTCCGTCTGTGACTCCAGTTTCT-3 Probe: 5 -FAM- CCCAACTTCCGAGGCGCTCCTACT- IL-10...F: 5 -CAAGGCAGCCTTGCAGAAG-3 R: 5 -TCCAGCCAGTAAGATTAGGCAATA-3 Probe: 5 -FAM-AGCTCCATCATGCCCAGCTCG G- a Abbreviations: F, forward; R, reverse; FAM, 6-carboxyfluorescein; TAMURA, 6-carboxy-N,N,N,N -tetramethylrhodamine. during the same time period and after all mrna samples to be analyzed had been obtained. Statistical analyses. Statistical analyses were performed using SigmaStat version 3.0 (SPSS, Chicago, Ill.). Results are presented as medians when the data were not distributed normally and means and standard errors when they were distributed normally. Statistical analyses used Student s t test or the Mann- Whitney rank sum test depending on whether the data were normally distributed. P values of 0.05 were considered to be significant. The Spearman rank order correlation test was performed for the correlation between inflammation and cytokine mrna levels or among cytokine mrna levels. RESULTS Macroscopic and histological changes in H. pylori-inoculated gerbils. Macroscopic and histological changes in H. pylori-inoculated gerbils were similar to those observed in our previous studies (14, 16). In brief, there were no visible changes in the gastric mucosa of any uninfected gerbils or gerbils at 2 weeks after inoculation. At 8 and 12 weeks after inoculation, histological changes reached their maximum levels in the pyloric mucosa (Fig. 1). The area of the fundic mucosa decreased significantly in parallel with an expansion of the pyloric mucosa. In the pyloric mucosa (antrum), PMN and MNC infiltration scores reached their maximum levels at 8 ( ) and 4 ( ) weeks after H. pylori inoculation, respectively, and gradually decreased (e.g., PMNs at 52 weeks [P compared with 8 weeks] and MNC at 52 weeks [P compared with 4 weeks]) (Fig. 1). In the fundic

3 VOL. 73, 2005 CYTOKINES AND H. PYLORI IN MONGOLIAN GERBILS 2207 FIG. 1. Cellular inflammation in the gastric mucosa of Mongolian gerbils infected with H. pylori. PMN and MNC infiltration scores are presented as means standard errors. *, P 0.05; **, P 0.01; ***, P compared with week 0. #, P 0.05; ##, P 0.01; ###, P compared with weeks at peak scores. This comparison was performed only between weeks at peak scores and weeks after peak scores., P 0.05;, P 0.01;, P 0.001, comparing scores in the pyloric mucosa with those in the fundic mucosa. Downloaded from mucosa (corpus), histological changes reached their maximum levels at approximately 12 weeks after H. pylori inoculation and plateaued during the observation periods (Fig. 1). Starting 26 weeks after H. pylori inoculation, the animals could be grouped into two categories in agreement with our previous studies (14, 16). One group (32%) developed gastric ulcers, located close to the transitional zone between fundic and pyloric mucosa (ulcer group), and the other group (68%) developed many sessile hyperplastic polyps (hyperplastic group). Ulcers were seen in 5 of 11 (45%) gerbils at 26 weeks, 2 of 10 (20%) at 40 weeks, and 3 of 10 (30%) at 52 weeks after inoculation. PMN and MNC scores were significantly greater in the ulcer group than in the hyperplastic group. The PMN score was (ulcer group) versus (hyperplastic group) (P 0.002) in the pyloric mucosa and (ulcer group) versus (hyperplastic group) (P 0.001) in the fundic mucosa. The MNC score was (ulcer group) versus (hyperplastic group) in the pyloric mucosa and (ulcer group) versus (hyperplastic group) in the fundic mucosa (P 0.05 for both). Intestinal metaplasia was present in 100% of infected gerbils starting at 26 weeks after inoculation. Establishment of H. pylori infection. Bacteriological, histological, and serological examination showed no detectable H. pylori in control gerbils. H. pylori was cultured from the pyloric mucosa of 3 of 10 inoculated gerbils at 1 week, 8 of 10 gerbils at 2 weeks, all gerbils at 4 to 26 weeks, and 5 of 10 gerbils at 40 and 52 weeks. H. pylori density reached its maximal levels at 4 weeks (median, CFU/mg of tissue) and then decreased (e.g., CFU at 26 weeks). All gerbils in the ulcer group were positive by culture compared to 48% (10 of 21) in the hyperplastic group. Serological examination showed that the titer of anti-h. pylori IgG antibody increased consistently after inoculation: 100% of gerbils had positive levels at 8 weeks or later after inoculation. The titer in the ulcer group was significantly higher than that in the hyperplastic group ( versus , respectively) (P 0.005) in agreement with our previous study (14). Histological examination confirmed successful infection in all except two gerbils at 1 week (n 82). These two gerbils were also negative for culture and serology and were excluded from further studies of cytokine mrna levels. As expected, H. pylori was not present on metaplastic glands. Expression of cytokine mrnas in gastric mucosa of Mongolian gerbils. Partial gerbil-specific IL-1 and IL-6 cdna sequences were successfully cloned (GenBank accession numbers AB and AB164706, respectively). A high degree of homology was observed for IL-1 sequences among the rodents and humans: the 298-bp and 99-amino-acid sequences of gerbil IL-1 were 91.3 and 90.9% identical to rat, 90.6 and 90.9% identical to murine, and 85.2 and 80.8% identical to human sequences, respectively. A high degree of homology was also observed for IL-6 sequences among the rodents but not between rodents and humans: the 483-nucleotide and 127- amino-acid sequences for gerbil IL-6 were 84.1 and 75.6% on December 10, 2018 by guest

4 2208 YAMAOKA ET AL. INFECT. IMMUN. FIG. 2. Cytokine mrna levels in the Mongolian gerbil gastric mucosa. (A) IL-1 ; (B) IFN- ; (C) IL-4; (D) IL-6; (E) IL-10. Cytokine mrna levels (cytokine mrna/gapdh mrna ratio 100,000) are presented as means and standard errors. *, P 0.05; **, P 0.01; ***, P compared with week 0. #, P 0.05; ##, P 0.01; ###, P compared with weeks at peak cytokine mrna levels. This comparison was performed only between weeks at peak levels and weeks after peak levels., P 0.05;, P 0.01;, P 0.001, comparing cytokine mrna levels in the pyloric mucosa with those in the fundic mucosa.

5 VOL. 73, 2005 CYTOKINES AND H. PYLORI IN MONGOLIAN GERBILS 2209 FIG. 2 Continued. identical to rat, 81.8 and 70.1% to identical to murine, and 60.2 and 44.1% identical to human sequences, respectively. IL-1 mrna was not induced in uninfected gerbils in either the pyloric or the fundic mucosa at 7 weeks of age (when the other gerbils were inoculated with H. pylori) or at 33 weeks of age (controls for the infected animals 26 weeks after H. pylori inoculation) (median IL-1 /GAPDH ratio 100,000 0 for each). In contrast, IL-1 mrna was strongly induced 4 weeks after H. pylori inoculation ( in the pyloric mucosa and in the fundic mucosa) (P compared with week 0) and then rapidly declined in both the pyloric and the fundic mucosa (Fig. 2A). In the fundic mucosa IL-1 mrna levels returned to the baseline at 8 weeks after inoculation. IL-1 mrna levels were consistently higher in the pyloric than in the fundic mucosa from 4 to 40 weeks (Fig. 2A). IFN- mrna was not induced in uninfected gerbils in either the pyloric or fundic mucosa at 7 or 33 weeks of age (median IFN- /GAPDH ratio 100,000 0 for each). IFN- mrna was strongly induced 4 weeks after H. pylori inoculation ( in the pyloric mucosa and in the fundic mucosa) (P compared with week 0) and remained elevated throughout the observation period (Fig. 2B). Similar to the results with IL-1 mrna, IFN- mrna levels were consistently higher in the pyloric than in the fundic mucosa from 4 to 52 weeks (Fig. 2B). IL-4 mrna levels were very low in uninfected gerbils at 7 weeks of age (median IL-4/GAPDH ratio 100,000 0inthe pyloric mucosa and 0.7 in the fundic mucosa) and at 33 weeks of age (1.4 in the pyloric mucosa and 1.0 in the fundic mucosa). IL-4 mrna was induced at 8 weeks ( in the fundic mucosa) after H. pylori inoculation and reached its maximal levels at 12 weeks in the pyloric mucosa (66.7) (P compared with that of week 0) and at 8 weeks in the fundic mucosa ( )(P 0.01 compared with that of week 0) (Fig. 2C). IL-4 mrna levels remained high throughout the observation period (Fig. 2C). In contrast to IL-1 and IFN- mrna, IL-4 mrna levels were similar in the pyloric and fundic mucosa throughout the observation period (Fig. 2C). IL-6 mrna levels (median IL-6/GAPDH ratio 100,000) in uninfected gerbils at 7 weeks of age were 14.7 in the pyloric mucosa and 11.5 in the fundic mucosa and at 33 weeks of age were 20.1 in the pyloric mucosa and 23.0 in the fundic mucosa. In infected gerbils, IL-6 mrna reached its maximal levels 12 weeks after inoculation (85.4 in the pyloric mucosa and 90 in the fundic mucosa) (Fig. 2D). IL-6 mrna levels remained high until 40 weeks (Fig. 2D). IL-6 mrna levels were similar in the pyloric and fundic mucosa throughout the observation period (Fig. 2D). IL-10 mrna levels (median IL-10/GAPDH ratio 100,000) in uninfected gerbils at 7 weeks of age were 16.5 in the pyloric mucosa and 14.1 in the fundic mucosa and at 33 weeks of age were 21.2 in the pyloric mucosa and 19.5 in the fundic mucosa. IL-10 mrna was induced rapidly, reaching its maximal levels at 26 weeks (93.8 in the pyloric mucosa and in the fundic mucosa) (P 0.01 compared with week 0), and remained high until sacrifice at 52 weeks (Fig. 2E). Similar to IL-4 and IL-6, IL-10 mrna levels were similar in the pyloric and fundic mucosa during the observation period (Fig. 2E). In infected gerbils, IL-1 mrna levels correlated with IFN- mrna levels in both the pyloric (r 0.75) (P 0.001) and fundic (r 0.26) (P 0.05) mucosa. There were also significant correlations between IFN- mrna levels and IL-4, IL-6, or IL-10 mrnas in the fundic mucosa: r 0.25, P 0.05, for IL-4; r 0.37, P 0.001, for IL-6; and r 0.61, P 0.001, for IL-10. Significant correlations between IFN- mrna levels and IL-4, IL-6, or IL-10 mrnas were not present in the pyloric mucosa (data not shown). There was no correlation between IL-1 mrna levels and IL-4, IL-6, or IL-10 mrna levels in either the pyloric or the fundic mucosa (data not shown). The induction of IL-4, IL-6, or IL-10 mrnas correlated with the induction of any other of those cytokines in the pyloric mucosa (r 0.80 for IL-6 versus IL-10, r 0.63 for IL-6 versus IL-4, and r 0.49 for IL-4 versus IL-10) (P for each) and the fundic mucosa (r 0.54 for IL-6 versus IL-10, r 0.62 for IL-6 versus IL-4, and r 0.70 for IL-4 versus IL-10) (P for each). Expression of cytokine mrnas, cellular infiltration, and H. pylori density in infected gastric mucosa of gerbils. In the pyloric mucosa, both PMN and MNC scores strongly correlated with IFN- mrna levels (r 0.51 and 0.60, respectively) (P for both) (Fig. 3). These scores also correlated with other cytokine mrna levels in the pyloric mucosa; however, the correlation was weaker than with IFN- mrna (r 0.25 to 0.46). In the fundic mucosa, both PMN and MNC scores corre-

6 2210 YAMAOKA ET AL. INFECT. IMMUN. FIG. 3. Correlation between IFN- mrna levels and cellular infiltration scores in the pyloric mucosa. IFN- mrna levels are presented as IFN- mrna/gapdh mrna ratio 100,000. lated with IL-4, IL-6, or IL-10 mrna levels: r 0.44 and 0.41 for IL-10 (P for both), r 0.29 and 0.31 for IL-6 (P 0.01 for both), and r 0.35 and 0.36 for IL-4 (P for both). In contrast, these scores were independent of IL-1 mrna and IFN- mrna levels in the fundic mucosa. H. pylori was successfully cultured from all gerbils at 4 to 26 weeks. During 4 to 26 weeks, H. pylori density as defined by cultures of the pyloric mucosa correlated with IL-1 mrna levels (r 0.52) (P 0.001). H. pylori density did not correlate with any other cytokine mrna levels. Expression of cytokine mrnas and the titer of anti-h. pylori IgG antibody. The titer of anti-h. pylori IgG antibody correlated with IL-4, IL-6, or IL-10 mrna levels in the fundic mucosa (r 0.40 for IL-4, r 0.46 for IL-6, and r 0.40 for IL-10) (P for each). The titer was also related to IFN- mrna levels in the fundic mucosa (r 0.35) (P 0.001). In contrast, the titer was not related to IL-1 mrna levels in the fundic mucosa and was not related to any cytokine mrna levels in the pyloric mucosa. Expression of IFN- mrna and gastric ulcer. IFN- mrna levels were significantly higher in the ulcer group than in the hyperplastic group in both the antrum (median IFN- / GAPDH ratio 100, versus 338, respectively) (P 0.02) and the corpus (172 versus 40.4, respectively) (P 0.01) (Fig. 4). There were no relationships between the other cytokine mrna levels and clinical presentation (data not shown). mrna levels by real-time RT-PCR. This is the first report to our knowledge that employs real-time RT-PCR for detection of various cytokine messages in H. pylori-infected gerbils. As in prior experiments, Mongolian gerbils infected with DISCUSSION The fact that genomic sequences of Mongolian gerbils differ from those of other rodents precluded use of commercial enzyme-linked immunosorbent assays to determine tissue cytokine levels. We therefore cloned partial sequences of the gerbil IL-1 and IL-6 mrnas, which allowed us to measure cytokine FIG. 4. IFN- mrna levels in the Mongolian gerbil gastric mucosa in ulcer group and hyperplastic group. IFN- mrna levels are presented as IFN- mrna/gapdh mrna ratio 100,000. The ends of the bars indicate the 25th and 75th percentiles. The median is indicated with a solid line in the box, and the 10th and 90th percentiles are indicated with error bars.

7 VOL. 73, 2005 CYTOKINES AND H. PYLORI IN MONGOLIAN GERBILS 2211 H. pylori ATCC developed an antral-predominant gastritis which progressed to corpus gastritis. This pattern is typical of what is seen in humans living in regions of high gastric cancer incidence, where the area of the fundic mucosa decreases over time as the atrophic border advances from the antrum into the corpus. Accordingly, maximal pyloric inflammation appeared relatively early in the natural history of H. pylori gastroduodenal disease and dropped rather dramatically by 52 weeks. In contrast, fundic inflammation plateaued after reaching maximum levels. These inflammation patterns (Fig. 1) tended to parallel the patterns of cytokine expression, especially in relationship to IFN- (Fig. 2). The different profiles in gastric inflammation and cytokine expression at the different anatomic sites (e.g., pyloric, fundic, or both) show that understanding the chronological changes requires examination of both the pyloric and fundic mucosa. The proinflammatory cytokine IL-1 mrna increased rapidly after inoculation, reaching its maximum levels at 4 weeks, followed by a rapid decline in both the pyloric and fundic mucosa (Fig. 2A). Although IL-1 is also a potent inhibitor of gastric acid secretion (27, 28), this early finding suggests that the induction of acute inflammation without inhibition of gastric acid secretion is probably a main role of IL-1 in gerbils. Two patterns of disease appeared in the gerbils irrespective of identical experimental conditions and diet, one ulcerative and the other associated with hyperplastic polyps. Interestingly, two patterns of disease were able to be identified at 26 weeks, which coincided with the time when most of the cytokine levels were beginning to decrease in those with polyps, as did the gastric inflammation and H. pylori density, possibly because H. pylori was absent from the metaplastic glands. In contrast, H. pylori density and gastric inflammation remained severe in animals that developed ulcers. IFN- mrna levels were also significantly greater in the animals in the ulcer group than in those in the hyperplastic group. The critical factors responsible for these different outcomes and for the different IFN- mrna induction levels remain unknown. The present experiments do not allow us to determine whether the increased IFN- mrna levels were related to the H. pylori density or whether they were directly related to the development of ulceration. However, the fact that, among the cytokines examined, only the IFN- mrna levels were increased suggests a possible relationship to ulcer development. These findings are in agreement with prior reports suggesting that IFN- plays an important role in development of gastric ulceration (4, 5). The early phase of the infection of gerbils (e.g., 4 weeks) was associated with a strong IFN- response and a very low IL-4 response. This result is consistent with previous studies of humans, mice, monkeys, and cats showing that the cytokine response in the infected gastric mucosa is predominantly of the Th1 type (1, 2, 5, 7, 8, 10, 11, 15, 17, 18, 22, 24 26). In contrast, well-known Th2 marker IL-4 mrna levels were elevated in the infected gerbil mucosa in the chronic phase of the infection. IL-4 mrna levels were also closely correlated with the cellular infiltration, especially in the fundic mucosa. In contrast, previous investigations have reported that the expression of Th2-type cytokines was either unchanged or slightly elevated in animals infected with different Helicobacter species (1, 7, 9, 10, 18, 19, 22, 24, 26). Further studies will be necessary to confirm whether Th2-type responses play important roles in gastric inflammation in the chronic phase of the infection in gerbils, especially in the corpus. However, the fact that IL-4 mrna levels were closely related to mrna levels for other cytokines (IFN-, IL-6, and IL-10 in the pyloric mucosa and IL-6 and IL-10 in the fundic mucosa) is consistent with the notion that the regulation of proinflammatory cytokines, regulatory cytokines, and Th1-Th2 cytokines in infection is part of a closely linked network, with Th2 cytokines possibly playing a role in maintenance and regulation of chronic inflammation. From these data we hypothesize that IL-1 and IFN- play important roles in the acute phase of pyloric inflammation and that IFN- as well as IL-4, IL-6, and IL-10 is important in maintenance and regulation of the severity of gastric inflammation in gerbils. If these hypotheses are correct, the underlying effect may be related to an imbalance that predisposes to a specific clinical outcome (e.g., strong IFN- production induces ulceration). ACKNOWLEDGMENTS This study was supported by Grant-in-Aid for Scientific Research C (to H.O.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; by a grant from the Hokuto Foundation for Bioscience (to H.O.); and by National Institutes of Health grants R01 DK62813 (to Y.Y.). D.Y.G. and Y.Y. are supported in part by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs. We thank James G. 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