Differential Expression of Gamma Interferon mRNA Induced by Attenuated and Virulent Mycobacterium tuberculosis in Guinea Pig Cells after Mycobacterium
Department of Medical Microbiology and Immunology, Texas A&M University System Health Science Center,1 Department of Statistics, Texas A&M University, College Station, Texas 77843,3 Laboratory of Molecular Immunoregulation, National Cancer Institute—Frederick, Frederick, Maryland 217022$2:%{8|, 百拇医药
Received 20 June 2002/ Returned for modification 11 September 2002/ Accepted 18 September 2002$2:%{8|, 百拇医药
ABSTRACT$2:%{8|, 百拇医药
To determine whether Mycobacterium bovis BCG vaccination would alter gamma interferon (IFN-) mRNA expression in guinea pig cells exposed to Mycobacterium tuberculosis, we cloned a cDNA encoding guinea pig IFN- from a spleen cell cDNA library. The cDNA is composed of 1,110 bp, with an open reading frame encoding a 166-amino-acid protein which shows 56 and 41% amino acid sequence homology to human and mouse IFN-, respectively. Spleen or lymph node cells from naïve and BCG-vaccinated guinea pigs were stimulated with purified protein derivative (PPD) or M. tuberculosis H37Ra or H37Rv, and the total RNA was subjected to Northern blot analysis with a 32P-labeled probe derived from the cDNA clone. Compared to the IFN- mRNA expression in cells of naïve animals, that in spleen and lymph node cells exposed to various stimuli was enhanced after BCG vaccination. However, there was a significant reduction in IFN- mRNA levels when cells were stimulated with a multiplicity of infection of greater than 1 virulent M. tuberculosis bacterium per 10 cells. The enhanced IFN- mRNA response in BCG-vaccinated animals was associated with an increase in the proportions of CD4+ T cells in the spleens, as determined by fluorescence-activated cell sorter analysis. Furthermore, the nonadherent population in the spleens enriched either by panning with anti-guinea pig immunoglobulin G-coated plates or by purification on nylon wool columns produced more IFN- mRNA than whole spleen cells following stimulation with concanavalin A or PPD. This indicates that T cells are principally responsible for the upregulation of IFN-{gamma} mRNA expression following BCG vaccination. The mechanism by which virulent mycobacteria suppress IFN- mRNA accumulation is currently under investigation.(Amminikutty Jeevan Teizo Yoshimura Kyeong Eun Lee and David N. McMurray)
Received 20 June 2002/ Returned for modification 11 September 2002/ Accepted 18 September 2002$2:%{8|, 百拇医药
ABSTRACT$2:%{8|, 百拇医药
To determine whether Mycobacterium bovis BCG vaccination would alter gamma interferon (IFN-) mRNA expression in guinea pig cells exposed to Mycobacterium tuberculosis, we cloned a cDNA encoding guinea pig IFN- from a spleen cell cDNA library. The cDNA is composed of 1,110 bp, with an open reading frame encoding a 166-amino-acid protein which shows 56 and 41% amino acid sequence homology to human and mouse IFN-, respectively. Spleen or lymph node cells from naïve and BCG-vaccinated guinea pigs were stimulated with purified protein derivative (PPD) or M. tuberculosis H37Ra or H37Rv, and the total RNA was subjected to Northern blot analysis with a 32P-labeled probe derived from the cDNA clone. Compared to the IFN- mRNA expression in cells of naïve animals, that in spleen and lymph node cells exposed to various stimuli was enhanced after BCG vaccination. However, there was a significant reduction in IFN- mRNA levels when cells were stimulated with a multiplicity of infection of greater than 1 virulent M. tuberculosis bacterium per 10 cells. The enhanced IFN- mRNA response in BCG-vaccinated animals was associated with an increase in the proportions of CD4+ T cells in the spleens, as determined by fluorescence-activated cell sorter analysis. Furthermore, the nonadherent population in the spleens enriched either by panning with anti-guinea pig immunoglobulin G-coated plates or by purification on nylon wool columns produced more IFN- mRNA than whole spleen cells following stimulation with concanavalin A or PPD. This indicates that T cells are principally responsible for the upregulation of IFN-{gamma} mRNA expression following BCG vaccination. The mechanism by which virulent mycobacteria suppress IFN- mRNA accumulation is currently under investigation.(Amminikutty Jeevan Teizo Yoshimura Kyeong Eun Lee and David N. McMurray)