Inhibition of mitogen-induced murine lymphocyte proliferation by Helicobacter pylori cell-free extract.
OBJECTIVE
Previous studies have shown that lysates of Helicobacter pylori inhibit mitogen-induced proliferation of human peripheral blood mononuclear cells. The objective of the present study was to determine whether H. pylori cell-free extract (HPCE) has similar effects on murine lymphoid cells and could, therefore, be used to further delineate the mechanisms of alteration of lymphocyte function by H. pylori.


METHODS
The HPCE was prepared from a H. pylori reference strain and from five clinical strains with varying status of cagA and vacA. Mouse splenic and mesenteric lymph node cells were cultured in microwell plates in the presence or absence of varying concentrations of HPCE (0.625-12.5 microg/mL). T cell mitogens were added into the culture 2 h later and the cells were cultured at 37 degrees C in 5% CO2 for a further 72 h. Cell proliferation was determined by a non-radioactive rapid dye assay and the percentage inhibition caused by HPCE was calculated.


RESULTS
Pre-exposure to HPCE significantly inhibited concanavalin A-induced proliferation of murine spleen and mesenteric lymph node cells (up to 100% inhibition; P < or = 0.01). The HPCE also inhibited lymphocyte proliferation stimulated by mitogens phorbol-myristate-acetate and ionomycin and by the anti-CD3epsilon monoclonal antibody (P < or = 0.05). The inhibition was dose-dependent, but independent of the presence of virulence genes cagA or vacA. Treatment of HPCE at 80 degrees C for 30 min, but not at 55 degrees C for 60 min, completely abolished its inhibitory action. The HPCE, pretreated with pronase E, proteinase K, trypsin, acid or alkali also completely lost its inhibitory effect (P < or = 0.01), while in contrast, treatment with carboxypeptidase and leucine aminopeptidase had no effect.


CONCLUSIONS
Helicobacter pylori produces heat-labile proteins or peptides that suppress T cell mitogen-induced proliferation of murine lymphoid cells in a similar manner to that observed with human peripheral blood mononuclear cells. The mouse cell culture system can, therefore, be used as a model to further study the mechanisms of action and antigen specificity of these immunomodulatory factors.
Chen
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Wakefield Gastroenterology Research Institute, Wakefield Hospital, Wellington, New Zealand. w.chen@clear.net.nz
Shu
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Chadwick
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D000818:Animals; D000942:Antigens, Bacterial; D001426:Bacterial Proteins / Q000235:genetics; D002460:Cell Line; D004306:Dose-Response Relationship, Immunologic; D016480:Helicobacter pylori / Q000276:immunology*; D006801:Humans; D007108:Immune Tolerance / Q000502:physiology*; D008198:Lymph Nodes / Q000276:immunology; D008214:Lymphocytes / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008934:Mitogens / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D013154:Spleen / Q000276:immunology
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D008934:Mitogens; C074689:VacA protein, Helicobacter pylori; C080866:cagA protein, Helicobacter pylori