Culture at high density improves the ability of human macrophages to control mycobacterial growth

The mechanisms through which granuloma formation helps control mycobacteria l infection are poorly understood, but it is possible that the accumulation of macrophages at high density at sites of infection promotes the differen tiation of macrophages into cells with improved mycobactericidal activity. To test this possibility, varying numbers of monocytes were cultured in 96- well plates for 3 days, infected with Mycobacterium bovis bacillus Calmette -Guerin, and mycobacterial number was assessed 7 days after infection based on the measurement of luciferase activity expressed by a mycobacterial rep orter strain or by counting CFU. Mycobacterial growth was optimal in cultur es containing 5 x 10(4) cells/well, but increasing the number of cells to 2 x 10(5) cells/well resulted in complete inhibition of mycobacterial growth . This effect could not be explained by differences in mycobacterial uptake , multiplicity of infection, acidification of the extracellular medium in h igh density cultures, enhanced NO production, or paracrine stimulation resu lting from secretion of cytokines or other proteins. The morphology of cell s cultured at high. density was strikingly different from that of monocytes cultured at 5 x 104 cells/well, including the appearance of numerous giant cells. The bacteriostatic activity of monocyte-derived macrophages was als o dependent on cell number, but fewer of these more mature cells were requi red to control mycobacterial growth. Thus, the ability of human macrophages to control mycobacterial infection in vitro is influenced by the density o f cells present, findings that may help explain why the formation of granul omas in vivo appears to be a key event in the control of mycobacterial infe ctions.