Nitric oxide and TNF-alpha production by murine peritoneal macrophages activated with a novel 20-kDa protein isolated from Bacillus thuringiensis var. thuringiensis parasporal bodies.

The effects of a novel 20-kDa protein isolated from Bacillus thuringiensis var. thuringiensis (BTp20) parasporal bodies on macrophage activation were investigated and compared with the activity of LPS. Murine resident or IFN-gamma-primed peritoneal macrophages (50 U/ml of IFN-gamma for 16 h) were treated with 50 microg/ml of BTp20, alone or in combination with LPS (1 to 100 ng/ml). BTp20 was not toxic for macrophages as determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction assay, however, 16% reduction of viability was observed when macrophages were treated with a combination of IFN-gamma, BTp20, and LPS at 100 ng/ml. BTp20 was able to stimulate significant cellular adherence and spreading, and significant production of nitrite and TNF-alpha by resident macrophages after 1.5 h of treatment; nitrite release, however, was induced with only 10 min of macrophage exposure to BTp20. BTp20 activities were significantly potentiated by IFN-gamma pretreatment. It was also observed that nitrite release by IFN-gamma-primed macrophages activated with LPS (1-100 ng/ml) was 20 times lower than that induced by IFN-gamma + BTp20. BTp20 and LPS independently stimulated significant TNF-alpha production by IFN-gamma-primed macrophages, the effect of BTp20 + LPS (1 and 10 ng/ml) combination was additive. In summary, this study demonstrated that BTp20 activates macrophage functions in the absence of IFN-gamma or LPS, and that IFN-gamma enhances BTp20 activities.