Effect of stimulation of human macrophages on intracellular survival of Mycobacterium bovis bacillus Calmette-Guerin - Evaluation with a mycobacterial reporter strain

The mechanisms through which immune and inflammatory responses stimulate th e expression of antimycobacterial activity by human macrophages remain poor ly defined. To study this question, we developed a method permitting the ra pid quantification of viable mycobacteria, based on the detection of lucife rase activity expressed by a Mycobacterium bovis Bacillus Calmette-Guerin ( BCG) reporter strain, and used this approach to evaluate mycobacterial surv ival in human monocyte-derived macrophages following stimulation with cytok ines and through crosslinking of costimulatory molecules expressed on the c ell surface. Modest proliferation, followed by persistence of mycobacteria, was observed in unpretreated macrophages as assessed both by measurement o f luciferase activity and by the evaluation of colony forming units. Of the 19 cytokines tested, only granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to improve the mycobactericid al activity of monocyte-derived macrophages. In both cases, this effect was observed only when macrophages were pretreated with the cytokines prior to infection. In contrast, pretreatment of human macrophages with interferon- gamma, either alone or in combination with other mediators (including tumor necrosis factor-alpha and 1,25[OH](2)-vitamin D-3), did not improve mycoba cterial killing. The stimulation of macrophages through several different c ostimulatory molecules known to participate in macrophage-lymphocyte intera ctions (CD4, CD40, CD45, CD86, CD95 [Fas/Apo-1]) also failed to improve myc obactericidal activity. This study shows that GM-CSF and IL-3, cytokines wh ose receptors are known to share a common subunit and to use common second messengers, may contribute to the stimulation of mycobactericidal activity in humans. The ability to rapidly screen the effects of different macrophag e stimuli on mycobacterial survival through the detection of luciferase act ivity should help define additional signals required for optimal antimycoba cterial responses.