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 (IF...

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Bibliographic Details
Published in:Vaccine 2004-01, Vol.22 (5-6), p.724-734
Main Authors: Higuchi, Kazue, Sekiya, Yukie, Harada, Nobuyuki
Format: Article
Language:English
Subjects:
Adjuvants, Immunologic
Alveolar macrophages
Amino Acid Sequence
Animals
Antibodies, Bacterial - biosynthesis
Applied microbiology
Bacterial diseases
Biological and medical sciences
Blotting, Western
Bronchoalveolar Lavage Fluid - cytology
Bronchoalveolar Lavage Fluid - immunology
Cell Line
Cells, Cultured
Chromatography, Gel
Chromatography, Ion Exchange
Cytosol - immunology
Enzyme-Linked Immunosorbent Assay
Female
Fundamental and applied biological sciences. Psychology
Human bacterial diseases
Humans
IL-12
Indicators and Reagents
Infectious diseases
Interleukin-12 - biosynthesis
M. tuberculosis
Macrophages, Alveolar - immunology
Medical sciences
Mice
Mice, Inbred BALB C
Microbiology
Molecular Sequence Data
Mycobacterium tuberculosis
Mycobacterium tuberculosis - chemistry
Mycobacterium tuberculosis - immunology
Reverse Transcriptase Polymerase Chain Reaction
Subcellular Fractions - immunology
Tuberculosis and atypical mycobacterial infections
Tuberculosis Vaccines - immunology
Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies (general aspects)
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Summary: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-γ) 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 700kDa. 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)-α and inducible nitric oxide synthase (iNOS) mRNA at least 6h 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.
ISSN:0264-410X
1873-2518
DOI:10.1016/j.vaccine.2003.08.018