Am. J. Respir. Crit. Care Med., Volume 159, Number 5, May 1999, 1629-1637

Effect of Stimulation of Human Macrophages on Intracellular Survival of Mycobacterium bovis Bacillus Calmette-Guerin
Evaluation with a Mycobacterial Reporter Strain

MARCEL BONAY, FRANCINE BOUCHONNET, VLADIMIR PELICIC, BEATRICE LAGIER, MARTINE GRANDSAIGNE, DENISE LECOSSIER, ALAIN GRODET, MARTIN VOKURKA, BRIGITTE GICQUEL, and ALLAN J. HANCE

INSERM U82, Faculté de Médecine Xavier Bichat, Paris; and Unité de Génétique Mycobactérienne, Institut Pasteur, Paris, France

The mechanisms through which immune and inflammatory responses stimulate the expression of antimycobacterial activity by human macrophages remain poorly defined. To study this question, we developed a method permitting the rapid quantification of viable mycobacteria, based on the detection of luciferase activity expressed by a Mycobacterium bovis Bacillus Calmette-Guerin (BCG) reporter strain, and used this approach to evaluate mycobacterial survival in human monocyte-derived macrophages following stimulation with cytokines and through crosslinking of costimulatory molecules expressed on the cell surface. Modest proliferation, followed by persistence of mycobacteria, was observed in unpretreated macrophages as assessed both by measurement of 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 mycobactericidal 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-, either alone or in combination with other mediators (including tumor necrosis factor- and 1,25[OH]₂-vitamin D₃), did not improve mycobacterial killing. The stimulation of macrophages through several different costimulatory molecules known to participate in macrophage-lymphocyte interactions (CD4, CD40, CD45, CD86, CD95 [Fas/Apo-1]) also failed to improve mycobactericidal activity. This study shows that GM-CSF and IL-3, cytokines whose 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 macrophage stimuli on mycobacterial survival through the detection of luciferase activity should help define additional signals required for optimal antimycobacterial responses.

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