title: [Analysis of Mycobacterium tuberculosis-derived substance which induces interleukin-12 production from macrophages].
abstract: Protection of hosts against tuberculosis depends on expression of cellular immunity. To express cellular immunity, interleukin 12 (IL-12) has been shown to play an important role. Although Mycobacterium tuberculosis is known to induce IL-12 from macrophages (M phi s), the mechanism for the induction is still unclear. To understand the mechanisms of IL-12 induction from M phi s by M. tuberculosis, the IL -12-inducing ability of substances derived from M. tuberculosis was investigated in vitro. Production of IL-12 in culture medium of M phi s was measured by ELISA system using specific antibodies. Live M. tuberculosis H37Rv induced slightly higher IL-12 production than live M. tuberculosis H37Ra upon stimulation of human or mouse alveolar macrophages (hAM phi s or mAM phi s). Heat-killed M. tuberculosis failed to induce IL-12 production of alveolar macrophages (AM phi). The responses of hAM phi s and mAM phi s to M. tuberculosis were remarkably different. mAM phi s produced five times larger amount of IL-12, compared with that from hAM phi s. Human peripheral blood mononuclear cells (PBMC) obtained by the density gradient centrifugation were also used for induction of IL-12 production. Although production levels of IL-12 from PBMC stimulated with M. tuberculosis were below the detectable level, addition of interferon-gamma (IFN-gamma) or neutralizing antibody against IL-10 augmented the production of IL-12 from PBMC, suggesting that IFN-gamma and IL-10 regulate the production of IL-12 from M phi positively and negatively, respectively. To characterize the physicochemical properties of IL-12-inducing molecules, M. tuberculosis H37Rv was disrupted by pressing with 1,000 bar and centrifuged and separated into cytosol and cell wall fraction. The culture filtrate was also examined on IL-12-inducing activity. Among the three subjects examined, cytosol was found to induce the highest production of IL-12 from mAM phi s 1 day after the stimulation. Addition of IFN-gamma to the cytosol fraction markedly increased the production of IL-12 from mAM phi s. The molecular weight of IL-12-inducing substance was shown to be more than 30kDa by fractionating with molecular filters. Treatment of 30kDa-fraction with IL-12-inducing activity by proteinase K completely abolished the activity. Furthermore, approximately 90% of IL-12-inducing activity of 30kDa-fraction was lost by proteinase K treatment even in the presence of IFN-gamma. These results indicate that the major component of IL-12-inducing activity is a protein. The identification of this IL-12-inducing active substance may provide a new therapeutic tool for tuberculosis.
authors.affiliation: Department of Basic Research, Japan Anti-Tuberculosis Association, Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Higuchi
authors.forename: N
authors.initials: N
authors.lastname: Harada
authors.forename: T
authors.initials: T
authors.lastname: Uchiyama
authors.forename: H
authors.initials: H
authors.lastname: Fujiwara
authors.forename: C
authors.initials: C
authors.lastname: Ueda
authors.forename: I
authors.initials: I
authors.lastname: Tsuyuguchi
authors.forename: R M
authors.initials: RM
authors.lastname: Nakamura
authors.forename: K
authors.initials: K
authors.lastname: Kobayashi
authors.forename: M
authors.initials: M
authors.lastname: Aoki
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002478:Cells, Cultured; D019286:Endopeptidase K / Q000494:pharmacology; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D016753:Interleukin-10 / Q000494:pharmacology; D018664:Interleukin-12 / Q000096:biosynthesis*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D016676:Macrophages, Alveolar / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000166:cytology; D013268:Stimulation, Chemical
chemical_list: D001426:Bacterial Proteins; D016753:Interleukin-10; D018664:Interleukin-12; D007371:Interferon-gamma; D019286:Endopeptidase K