Sequential activation of alveolar macrophages by IFN-gamma and LPS is required for enhanced growth inhibition of virulent Mycobacterium bovis but not M. bovis BCG.

Alveolar macrophages (AM) form the first line of defence against most respiratory pathogens and, unlike tissue macrophages, are constantly exposed to a wide variety of antigenic stimuli. In this study we investigated the in vitro effects of IFN-gamma and LPS on growth of virulent Mycobacterium bovis and M. bovis bacille Calmette-Guerin (BCG) in bovine AM. Bovine AM were purified from bronchial lavage fluid and cultured in serum-free medium. Pretreatment of bovine AM with IFN-gamma resulted in growth inhibition of M. bovis BCG but only partially inhibited growth of virulent M.bovis. Enhanced inhibition of virulent M.bovis by bovine AM required sequential stimulation with IFN-gamma and LPS and was associated with increased induction of nitric oxide (NO) and IL-12 mRNa. Growth inhibition of M. bovis was not affected by treatment of macrophages with the L-arginine analogue, NG-monomethyl-L-arginine although this treatment decreased NO production. These results suggest that a second activation signal in the form of TNF-alpha or LPS may be required to induce bacteriostasis of virulent M. bovis by bovine AM in vivo. The ability of bovine AM to respond to activation stimuli in vitro suggests that these cells may play an important role in preventing establishment of intracellular bacterial infections in the lung.