TNF-a antagonises MTX-induced apoptosis in macrophages, independent of M-CSF action. A. Exposure of primary BMDM to 10 mM MTX for 24 hr in the presence of 30 ng/mL M-CSF resulted in loss of approximately 25% of cells. This could be entirely prevented by introducing TNF-a three hours before MTX. Cell viability was assessed by the MTT reduction assay. Shown are mean ± SEM of normalised data from three independent experiments. B and D, TNF-a suppresses caspase-3 activation (left axis) and annexin-V binding (right axis) in MTX-exposed primary BMDM (B) and RAW264.7 cells (D). MTX was added three hours after TNF-a treatment, and caspase-3 activity or annexin-V binding was measured after 24 hr for BMDM and 6 hr for RAW264.7 cells. Data are mean ± SEM of at least four independent experiments in each case. C and E, MTX dose-dependently increases apoptosis in BMDM (C) and RAW264.7 cells (E). * = P < 0.05; ** = P < 0.01.