Phenotypic expression of Bcg gene in macrophages : regulation of MHC class II expression and nitric oxide production

In the mouse, the trait of innate resistance or susceptibility to infection with several species of Mycobacteria, including M. bovis BCG, M. lepraemurium, M. intracellulare and M. smegmatis, is controlled by the expression of the single gene on chromosome 1, designated Bcg.In this thesis, macrophage cell lines derived from the bone marrow of B10.A (B10S macrophages) and B10A.$Bcg sp{r}$ (B10R macrophages) were used to study functional parameters associated with Bcg gene activity. We have discovered that there is a significant difference in the production of NO$ sb2 sp-$ of B10S as compared with B10R macrophages in response to IFN-$ gamma.$ The bacteriostatic activity against M. bovis BCG was higher in B10R macrophages compared to B10S macrophages. The bacteriostatic activity of B10R and B10S macrophages correlated with the amount of nitric oxide produced by the macrophages. The antimycobacterial activity was inhibited by N$ rm sp{g}$MMLA, a specific inhibitor of nitrite and nitrate synthesis from L-arginine. Addition of L-arginine to IFN-$ gamma$-stimulated macrophages in the presence of N$ rm sp{g}$MMLA restored nitrite production and bacteriostatic activity of macrophages. Northern blot analysis of macrophage nitric oxide synthase (iNOS) revealed that the difference in NO$ sb2 sp-$ production by IFN-$ gamma$ treated B10S and B10R lines was reflective of the difference in iNOS mRNA expression.The Bcg gene differentially affects the ability of BCG-resistant and -susceptible strains of mice to express important macrophage genes including Major Histocompatibility Complex (MHC) class II genes. We have found that differences at the level of I-A$ sb beta$ gene transcription, and I-A$ sb beta$ and I-A$ sb alpha$ mRNA stability may be responsible for observed differences between steady-state levels of I-A$ sb beta$ mRNA in B10R and B10S macrophages and consequently in the Ia surface protein expression. Differences observed in protein binding to the X box may explain the difference in transcription activation of the I-A$ sb beta$ gene. In addition, we found that B10R macrophages transfected with an Nramp-1 antisense cDNA containing a ribozyme construct expressed lower amounts of Ia antigen compared to mock-transfected macrophages in response to IFN-$ gamma.$ Overall, these findings strongly suggest involvement of the Nramp-1/Bcg gene in the control of Ia antigen expression in macrophages.Finally, I have developed a new PCR-based method that allow an assessment of the mycobacterial content of infected macrophages. This method allows an assessment of the level of M. bovis BCG infection from a variety of sources, including peritoneal macrophages and macrophage lines, within a few hours, making it an assay of choice for rapid determination of the level of mycobacterial growth in infected cells, in experimental models of mycobacterial infection. (Abstract shortened by UMI.