Cytokine gene polymorphisms influence mucosal cytokine expression, gastric inflammation, and host specific colonisation during <em>Helicobacter pylori</em> infection

R Rad

A Dossumbekova

B Neu

R Lang

^{Department of Internal Medicine II and Gastroenterology, Technical University of Munich, Munich, Germany}

^{Institute of Medical Microbiology and Immunology, Technical University of Munich, Munich, Germany}

Correspondence to:
Professor C Prinz
Klinikum Rechts der Isar der Technischen Universität München, II Medizinische Klinik, Ismaningerstraβe 22, 81675 München, Germany; ed.mut.zrl@znirp.naitsirhc

Accepted 2003 Dec 23.

Abstract

Background and aims: Recent studies linked cytokine gene polymorphisms to H pylori related gastric cancer development. The current study evaluated the role of cytokine gene polymorphisms for mucosal cytokine expression, the gastric inflammatory response, and bacterial colonisation during H pylori infection.

Patients and methods: In 207 H pylori infected patients with chronic gastritis, polymorphisms at different loci of the interleukin (IL)-10, IL-1B, IL-1 receptor antagonist (IL-1RN), tumour necrosis factor (TNF)-A, and interferon (IFN)-G genes were genotyped by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis, and allelic discriminating TaqMan PCR. Mucosal cytokine mRNA copy numbers were determined by real time quantitative PCR. Presence of bacterial virulence factors was investigated by cagA, vacAs1/2, and babA2 PCR. Biopsies were assessed with regard to the degrees of granulocytic/lymphocytic infiltration and the presence of intestinal metaplasia (IM) and atrophic gastritis (AG).

Results: Proinflammatory IL-1 polymorphisms (IL-1RN*2^{/IL-1B−511T/−31C}⁺) were associated with increased IL-1β expression, more severe degrees of inflammation, and an increased prevalence of IM and AG. Carriers of the IL-10−1082G/−819C/−592C alleles (GCC haplotype) had higher mucosal IL-10 mRNA levels than ATA haplotype carriers and were associated with colonisation by more virulent cagA^{, vacAs1}^{, and babA2}^{H pylori strains. The}TNF-A−307(G/A) and IFN-G+874(A/T) polymorphisms did not influence mucosal cytokine expression or the inflammatory response to H pylori.

Conclusions: Cytokine gene polymorphisms influence mucosal cytokine expression, gastric inflammation, and the long term development of precancerous lesions in H pylori infection. Host polymorphisms are associated with certain bacterial strain types, suggesting host specific colonisation or adaptation. These findings contribute to the understanding of the complex interplay between host and bacterial factors involved in the development of gastric pathology.

Keywords: Helicobacter pylori, cytokines, polymorphisms, cagA, vacA, babA

Abstract

Infection with Helicobacter pylori leads to persistent colonisation and chronic inflammation of the gastric mucosa, thereby increasing the risk of developing peptic ulceration, distal gastric adenocarcinoma, and gastric lymphoma.^1,² There are high interindividual differences in the extent of gastric inflammation among H pylori infected patients, and clinical consequences develop in only a small subgroup. Bacterial virulence factors, such as the cag pathogenicity island (cagPAI), the vacuolating cytotoxin (VacA), and the blood group antigen binding adhesin (BabA) are associated with enhanced inflammation and cancer development.^3–⁹ However, despite the well defined role of virulence factors, it is unclear why a considerable proportion of patients infected with the cagA⁺, vacAs1⁺, or babA2^{H pylori} strains do not develop severe pathologies throughout life.

In addition to bacterial factors, mostly unknown host factors seem to influence the inflammatory response and the development of a more severe pathology. H pylori induced inflammation is implicated in the development of mucosal damage and is characterised by strong granulocytic and lymphocytic infiltration.^10,¹¹ The T helper cell response towards H pylori is generally considered to be of the Th1 phenotype,^10,¹¹ leading to a cell mediated immune response. There is increasing evidence that the H pylori induced Th1 response contributes to cancer development.¹² Downregulation of the Th1 response in mice by concurrent enteric helminth infection or p53 mutation was shown to protect against the development of atrophy, intestinal metaplasia, and invasive gastric carcinoma.^13,¹⁴ However, factors influencing the extent of the H pylori induced Th1 response are currently unknown. Important cytokines characterising Th1 mediated immune responses are interferon γ (IFN-γ), tumour necrosis factor α (TNF-α), and interleukin-1β (IL-1β), all being upregulated during chronic H pylori infection.^15–¹⁷ IL-10, which is also highly expressed in the H pylori infected stomach,¹⁸ is one of the most important regulatory cytokines, inhibiting cell mediated immune responses.

Genes encoding cytokines and related molecules harbour polymorphic regions, which are considered to alter gene transcription and thereby influence inflammatory processes in response to infectious diseases.^19,²⁰ Polymorphisms in the human IL-10, IL-1B, TNF-A, IFN-G, and IL-1 receptor antagonist (IL-1RN) genes have been reported to influence cytokine expression. In the IL-10 promoter, single nucleotide polymorphisms (SNPs) at positions −1082 (G/A), −819 (C/T), and −592 (C/A) from the transcription start site have been reported to produce mainly three haplotypes: GCC, ACC, and ATA.²¹ Although there are stimulus or cell type dependent variations, haplotype GCC seems to be associated with high and ATA with low IL-10 production capacity from in vitro stimulated peripheral blood mononuclear cells (PBMC).^22,²³ Several polymorphisms have been reported in the TNF-A promoter, most of which are functionally silent.²⁴ The majority of studies focused on the G/A polymorphism at position −307, which was originally misnumbered and is therefore better known as TNF-A−308.²⁴ While in some in vitro studies allele A of this SNP was associated with heightened TNF-α secretion, other studies did not find such a correlation.^24,²⁵ In the IFN-G gene, a T/A polymorphism at position +874 has been reported.²⁶ Allele T of this SNP shows an absolute correlation with allele 2 of a penta-allelic CA microsatellite polymorphism, which has been associated with high IFN-γ production from PBMC in one study.²⁷ In the promoter region of the IL-1B gene, IL-1B−511T, which is in complete linkage disequilibrium with IL-1B−31C,²⁸ was previously associated with slightly, but not significantly, increased IL-1β secretion from stimulated PBMC.²⁰ The IL-1RN gene has a penta-allelic 86 bp variable number of tandem repeat region (VNTR) in intron 2, of which allele 2 (IL-1RN*2) was previously associated with enhanced IL-1β secretion.^20,^29,³⁰

In the context of H pylori infection, the genetic basis for the high interindividual differences in cytokine responses is poorly understood. However, recent reports linked cytokine gene polymorphisms to gastric cancer.^28,^31,³² In the current study, we investigated a large H pylori infected patient group with chronic gastritis and determined host cytokine gene polymorphisms, corresponding mucosal cytokine expression, histopathological features of gastritis, and the infecting bacterial strain type. The aim of the study was to define the influence of different polymorphisms on the H pylori induced cytokine response and to investigate the resulting consequences for gastric inflammation and bacterial colonisation.

Acknowledgments

The authors wish to thank Dr Stefan Wagenpfeil for statistical advice, Dr Vera Pravica for kindly providing us with control DNA, and Dr El-Omar for providing us with primer and probe sequences for IL-1B polymorphisms. Part of this work was performed by Roland Rad as an MD thesis at the TUM. Sponsored by DFG Pr 411/7-1 (Heisenberg Program to CP); a grant from Astra Hässle, Wedel, Germany; and Kuratorium für Klinische Forschung der TUM 8733153 and 8733156. Roland Rad is a recipient of a grant from the “Bund der Freunde der TU München”. Sponsored by Dr Else Kröner Fresenius Stiftung, Germany

Acknowledgments

Abbreviations

cagPAI, cag pathogenicity island
VacA, vacuolating cytotoxin
BabA, blood group antigen binding adhesin
IFN-γ, interferon γ
TNF-α, tumour necrosis factor α
IL, interleukin
IL-1RN, IL-1 receptor antagonist
SNP, single nucleotide polymorphism
PBMC, peripheral blood mononuclear cells
VNTR, variable number of tandem repeat region
IM, intestinal metaplasia
AG, atrophic gastritis
PCR, polymerase chain reaction
RFLP, restriction fragment length polymorphism
OR, odds ratio
GAPDH, glyceraldehyde-3-phosphate dehydrogenase

Abbreviations

REFERENCES

References

1. Suerbaum S , Michetti PHelicobacter pylori infection. N Engl J Med 2002;347:1175–86. [[PubMed][Google Scholar]
2. Covacci A , Telford JL, Del GG, et al. Helicobacter pylori virulence and genetic geography. Science 1999;284:1328–33. [[PubMed]
3. Blaser MJ, Berg DEHelicobacter pylori genetic diversity and risk of human disease. J Clin Invest 2001;107:767–73. [Google Scholar]
4. Prinz C , Schoniger M, Rad R, et al. Key importance of the Helicobacter pylori adherence factor blood group antigen binding adhesin during chronic gastric inflammation. Cancer Res 2001;61:1903–9. [[PubMed]
5. Gerhard M , Lehn N, Neumayer N, et al. Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin. Proc Natl Acad Sci U S A 1999;96:12778–83.
6. Blaser MJ, Perez PG, Kleanthous H, et al. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res 1995;55:2111–15. [[PubMed]
7. Atherton JC, Cao P, Peek-RM J, et al. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration. J Biol Chem 1995;270:17771–7. [[PubMed]
8. Censini S , Lange C, Xiang Z, et al. cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc Natl Acad Sci U S A 1996;93:14648–53.
9. Crabtree JE, Taylor JD, Wyatt JI, et al. Mucosal IgA recognition of Helicobacter pylori 120 kDa protein, peptic ulceration, and gastric pathology. Lancet 1991;338:332–5. [[PubMed]
10. Ernst PB, Gold BDThe disease spectrum of Helicobacter pylori: the immunopathogenesis of gastroduodenal ulcer and gastric cancer. Annu Rev Microbiol 2000;54:615–40. [[PubMed][Google Scholar]
11. Shimoyama T , Crabtree JEBacterial factors and immune pathogenesis in Helicobacter pylori infection. Gut 1998;43 (suppl 1) :S2–5. [Google Scholar]
12. Ernst P Review article: the role of inflammation in the pathogenesis of gastric cancer. Aliment Pharmacol Ther 1999;13 (suppl 1) :13–18. [[PubMed][Google Scholar]
13. Fox JG, Beck P, Dangler CA, et al. Concurrent enteric helminth infection modulates inflammation and gastric immune responses and reduces Helicobacter-induced gastric atrophy. Nat Med 2000;6:536–42. [[PubMed]
14. Fox JG, Sheppard BJ, Dangler CA, et al. Germ-line p53-targeted disruption inhibits Helicobacter-induced premalignant lesions and invasive gastric carcinoma through down-regulation of Th1 proinflammatory responses. Cancer Res 2002;62:696–702. [[PubMed]
15. Bodger K , Crabtree JEHelicobacter pylori and gastric inflammation. Br Med Bull 1998;54:139–50. [[PubMed][Google Scholar]
16. Yamaoka Y , Kita M, Kodama T, et al. Induction of various cytokines and development of severe mucosal inflammation by cagA gene positive Helicobacter pylori strains. Gut 1997;41:442–51.
17. Rad R , Gerhard M, Lang R, et al. The Helicobacter pylori blood group antigen-binding adhesin facilitates bacterial colonization and augments a nonspecific immune response. J Immunol 2002;168:3033–41. [[PubMed]
18. Karttunen RA, Karttunen TJ, Yousfi MM, et al. Expression of mRNA for interferon-gamma, interleukin-10, and interleukin-12 (p40) in normal gastric mucosa and in mucosa infected with Helicobacter pylori. Scand J Gastroenterol 1997;32:22–7. [[PubMed]
19. Bidwell J , Keen L, Gallagher G, et al. Cytokine gene polymorphism in human disease: on-line databases. Genes Immun 1999;1:3–19. [[PubMed]
20. Hurme M , Lahdenpohja N, Santtila SGene polymorphisms of interleukins 1 and 10 in infectious and autoimmune diseases. Ann Med 1998;30:469–73. [[PubMed][Google Scholar]
21. Eskdale J , Keijsers V, Huizinga T, et al. Microsatellite alleles and single nucleotide polymorphisms (SNP) combine to form four major haplotype families at the human interleukin-10 (IL-10) locus. Genes Immun 1999;1:151–5. [[PubMed]
22. Turner DM, Williams DM, Sankaran D, et al. An investigation of polymorphism in the interleukin-10 gene promoter. Eur J Immunogenet 1997;24:1–8. [[PubMed]
23. Crawley E , Kay R, Sillibourne J, et al. Polymorphic haplotypes of the interleukin-10 5′ flanking region determine variable interleukin-10 transcription and are associated with particular phenotypes of juvenile rheumatoid arthritis. Arthritis Rheum 1999;42:1101–8. [[PubMed]
24. Allen RDPolymorphism of the human TNF-alpha promoter—random variation or functional diversity? Mol Immunol 1999;36:1017–27. [[PubMed][Google Scholar]
25. de Jong BA, Westendorp RG, Bakker AM, et al. Polymorphisms in or near tumour necrosis factor (TNF)-gene do not determine levels of endotoxin-induced TNF production. Genes Immun 2002;3:25–9. [[PubMed]
26. Pravica V , Perrey C, Stevens A, et al. A single nucleotide polymorphism in the first intron of the human IFN-gamma gene: absolute correlation with a polymorphic CA microsatellite marker of high IFN-gamma production. Hum Immunol 2000;61:863–6. [[PubMed]
27. Pravica V , Asderakis A, Perrey C, et al. In vitro production of IFN-gamma correlates with CA repeat polymorphism in the human IFN-gamma gene. Eur J Immunogenet 1999;26:1–3. [[PubMed]
28. El Omar EM, Carrington M, Chow WH, et al. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature 2000;404:398–402. [[PubMed]
29. Santtila S , Savinainen K, Hurme MPresence of the IL-1RA allele 2 (IL1RN*2) is associated with enhanced IL-1beta production in vitro. Scand J Immunol 1998;47:195–8. [[PubMed][Google Scholar]
30. Hwang IR, Kodama T, Kikuchi S, et al. Effect of interleukin 1 polymorphisms on gastric mucosal interleukin 1beta production in Helicobacter pylori infection. Gastroenterology 2002;123:1793–803. [[PubMed]
31. Wu MS, Wu CY, Chen CJ, et al. Interleukin-10 genotypes associate with the risk of gastric carcinoma in Taiwanese Chinese. Int J Cancer 2003;104:617–23. [[PubMed]
32. El Omar EM, Rabkin CS, Gammon MD, et al. Increased risk of noncardia gastric cancer associated with proinflammatory cytokine gene polymorphisms. Gastroenterology 2003;124:1193–201. [[PubMed]
33. Rad R , Prinz C, Neu B, et al. Synergistic effect of Helicobacter pylori virulence factors and interleukin-1 polymorphisms for the development of severe histological changes in the gastric mucosa. J Infect Dis 2003;188:272–81. [[PubMed]
34. Covacci A , Rappuoli RHelicobacter pylori: molecular evolution of a bacterial quasi-species. Curr Opin Microbiol 1998;1:96–102. [[PubMed][Google Scholar]
35. Kirschner DE, Blaser MJThe dynamics of Helicobacter pylori infection of the human stomach. J Theor Biol 1995;176:281–90. [[PubMed][Google Scholar]
36. Dubois A , Berg DE, Incecik ET, et al. Host specificity of Helicobacter pylori strains and host responses in experimentally challenged nonhuman primates. Gastroenterology 1999;116:90–6. [[PubMed]
37. Philpott DJ, Belaid D, Troubadour P, et al. Reduced activation of inflammatory responses in host cells by mouse-adapted Helicobacter pylory isolates. Cell Microbiol 2002;4:285–96. [[PubMed]
38. Sutton P , Kolesnikow T, Danon S, et al. Dominant nonresponsiveness to Helicobacter pylori infection is associated with production of interleukin 10 but not gamma interferon. Infect Immun 2000;68:4802–4.
39. Kuipers EJ, Israel DA, Kusters JG, et al. Quasispecies development of Helicobacter pylori observed in paired isolates obtained years apart from the same host. J Infect Dis 2000;181:273–82.
40. Israel DA, Salama N, Krishna U, et al. Helicobacter pylori genetic diversity within the gastric niche of a single human host. Proc Natl Acad Sci U S A 2001;98:14625–30.
41. Webb GF, Blaser MJDynamics of bacterial phenotype selection in a colonized host. Proc Natl Acad Sci U S A 2002;99:3135–40. [Google Scholar]
42. Kersulyte D , Chalkauskas H, Berg DEEmergence of recombinant strains of Helicobacter pylori during human infection. Mol Microbiol 1999;31:31–43. [[PubMed][Google Scholar]
43. Eaton KA, Mefford M, Thevenot TThe role of T cell subsets and cytokines in the pathogenesis of Helicobacter pylori gastritis in mice. J Immunol 2001;166:7456–61. [[PubMed][Google Scholar]
44. Bouma G , Crusius JB, Oudkerk PM, et al. Secretion of tumour necrosis factor alpha and lymphotoxin alpha in relation to polymorphisms in the TNF genes and HLA-DR alleles. Relevance for inflammatory bowel disease. Scand J Immunol 1996;43:456–63. [[PubMed]
45. Louis E , Franchimont D, Piron A, et al. Tumour necrosis factor (TNF) gene polymorphism influences TNF-alpha production in lipopolysaccharide (LPS)-stimulated whole blood cell culture in healthy humans. Clin Exp Immunol 1998;113:401–6.
46. Huizinga TW, Westendorp RG, Bollen EL, et al. TNF-alpha promoter polymorphisms, production and susceptibility to multiple sclerosis in different groups of patients. J Neuroimmunol 1997;72:149–53. [[PubMed]
47. Pociot F , Briant L, Jongeneel CV, et al. Association of tumor necrosis factor (TNF) and class II major histocompatibility complex alleles with the secretion of TNF-alpha and TNF-beta by human mononuclear cells: a possible link to insulin-dependent diabetes mellitus. Eur J Immunol 1993;23:224–31. [[PubMed]
48. Mycko M , Kowalski W, Kwinkowski M, et al. Multiple sclerosis: the frequency of allelic forms of tumor necrosis factor and lymphotoxin-alpha. J Neuroimmunol 1998;84:198–206. [[PubMed]
49. Machado JC, Pharoah P, Sousa S, et al. Interleukin 1B and interleukin 1RN polymorphisms are associated with increased risk of gastric carcinoma. Gastroenterology 2001;121:823–9. [[PubMed]
50. Furuta T , El Omar EM, Xiao F, et al. Interleukin 1beta polymorphisms increase risk of hypochlorhydria and atrophic gastritis and reduce risk of duodenal ulcer recurrence in Japan. Gastroenterology 2002;123:92–105. [[PubMed]
51. Figueiredo C , Machado JC, Pharoah P, et al. Helicobacter pylori and interleukin 1 genotyping: an opportunity to identify high-risk individuals for gastric carcinoma. J Natl Cancer Inst 2002;94:1680–7. [[PubMed]
52. Schepp W , Dehne K, Herrmuth H, et al. Identification and functional importance of IL-1 receptors on rat parietal cells. Am J Physiol 1998;275:G1094–105. [[PubMed]
53. Beales IP, Calam JInterleukin 1 beta and tumour necrosis factor alpha inhibit acid secretion in cultured rabbit parietal cells by multiple pathways. Gut 1998;42:227–34. [Google Scholar]
54. Prinz C , Neumayer N, Mahr S, et al. Functional impairment of rat enterochromaffin-like cells by interleukin 1 beta. Gastroenterology 1997;112:364–75. [[PubMed]
55. Mahr S , Neumayer N, Gerhard M, et al. IL-1beta-induced apoptosis in rat gastric enterochromaffin-like cells is mediated by iNOS, NF-kappaB, and Bax protein. Gastroenterology 2000;118:515–24. [[PubMed]
56. Takashima M , Furuta T, Hanai H, et al. Effects of Helicobacter pylori infection on gastric acid secretion and serum gastrin levels in Mongolian gerbils. Gut 2001;48:765–73.
57. Gillen D , El Omar EM, Wirz AA, et al. The acid response to gastrin distinguishes duodenal ulcer patients from Helicobacter pylori-infected healthy subjects. Gastroenterology 1998;114:50–7. [[PubMed]
58. El Omar EM, Penman ID, Ardill JE, et al. Helicobacter pylori infection and abnormalities of acid secretion in patients with duodenal ulcer disease. Gastroenterology 1995;109:681–91. [[PubMed]
59. El Omar EM, Oien K, El Nujumi A, et al. Helicobacter pylori infection and chronic gastric acid hyposecretion. Gastroenterology 1997;113:15–24. [[PubMed]
60. Sachs G , Weeks DL, Melchers K, et al. The gastric biology of Helicobacter pylori. Annu Rev Physiol 2003;65:349–69. [[PubMed]
61. El Omar EMThe importance of interleukin 1beta in Helicobacter pylori associated disease. Gut 2001;48:743–7. [Google Scholar]
62. Garcia-Gonzalez MA, Lanas A, Santolaria S, et al. The polymorphic IL-1B and IL-1RN genes in the aetiopathogenesis of peptic ulcer. Clin Exp Immunol 2001;125:368–75.