Characterization of M. Tuberculosis-derived IL-12-inducing material by alveolar macrophages.
We have investigated the substance derived from Mycobacterium tuberculosis (Mtb) that induces interleukin (IL)-12 production by alveolar macrophages (AMs) in vitro. The cytosol fraction of live Mtb H37Rv induced IL-12 production by AMs in a dose-dependent manner. The addition of interferon-gamma (IFN-gamma) augmented IL-12 production. IL-12-inducing activity by AMs (termed as surely active keeping rescue antigen, SAKRA) was purified by gel filtration and ion exchange column chromatography, and the molecular weight of SAKRA was estimated by gel filtration to be more than 700 kDa. SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting of SAKRA using rabbit anti-SAKRA antibody suggested that SAKRA is composed with several low molecular weight proteins. Amino acids sequence analysis of several bands after SDS-PAGE suggested that SAKRA is a part of ribosomes. RT-PCR showed that SAKRA induced not only expression of IL-12 p40 mRNA, but expression of tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS) mRNA at least 6 h after stimulation, suggesting that SAKRA activates the bactericidal activity of macrophages. To investigate the potential use of SAKRA as a vaccine against tuberculosis, SAKRA was administered to BALB/c mouse that had been immunized with BCG for 18 months, and mouse were infected with Mtb H37Rv via a respiratory route. Replication of Mtb in lungs and spleens was examined 6 weeks after infection. Administration of SAKRA to BCG-vaccinated mice significantly reduced the numbers of Mtb in lungs and spleens as compared with BCG-vaccinated control mice. Taken together, these results suggest that SAKRA is one of the Mtb-derived immunomodulatory substances which induce IL-12 production during infection and also increases mycobactericidal activities of macrophages, and that SAKRA may be a promising new vaccine candidate against tuberculosis.
Higuchi
Kazue
K
Department of Basic Research, Immunology Division, The Research Institute of Tuberculosis, 3-1-24 Matsuyama, Kiyose, Tokyo 204-8533, Japan. higuchi@jata.or.jp
Sekiya
Yukie
Y
Harada
Nobuyuki
N
D000276:Adjuvants, Immunologic; D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D015153:Blotting, Western; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology / Q000276:immunology; D002460:Cell Line; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007202:Indicators and Reagents; D018664:Interleukin-12 / Q000096:biosynthesis*; D016676:Macrophages, Alveolar / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000276:immunology; D032581:Tuberculosis Vaccines / Q000276:immunology*
D000276:Adjuvants, Immunologic; D000907:Antibodies, Bacterial; D007202:Indicators and Reagents; D032581:Tuberculosis Vaccines; D018664:Interleukin-12