Tumor necrosis factor-alpha promotes survival in methotrexate-exposed macrophages by an NF-kappa B-dependent pathway

Introduction: Methotrexate (MTX) induces macrophage apoptosis in vitro, but there is not much evidence for increased synovial macrophage apoptosis in MTX-treated patients. Macrophage apoptosis is reported, however, during clinical response to anti-tumor necrosis factor-alpha (TNF-alpha) treatments. This implies that TNF-alpha promotes macrophage survival and suggests that TNF-alpha may protect against MTX-induced apoptosis. We, therefore, investigated this proposal and the macrophage signaling pathways underlying it. Methods: Caspase-3 activity, annexin-V binding/7-aminoactinomycin D (7-AAD) exclusion and cell-cycle analysis were used to measure steps in apoptosis of primary murine macrophages and cells of the RAW(264.7) macrophage cell line that had been exposed to clinically-relevant concentrations of MTX and TNF-alpha. Results: MTX induces apoptosis in primary murine macrophages at concentrations as low as 100 nM in vitro. TNF-alpha, which has a context-dependent ability to increase or to suppress apoptosis, efficiently suppresses MTX-induced macrophage apoptosis. This depends on NF-kappa B signaling, initiated through TNF Receptor Type 1 ligation. Macrophage colony stimulating factor, the primary macrophage survival and differentiation factor, does not activate NF-kappa B or protect macrophages from MTX-induced apoptosis. A weak NF-kappa B activator, Receptor Activator of NF-kappa B Ligand (RANKL) is likewise ineffective. Blocking NF-kappa B in TNF-alpha-exposed macrophages allowed pro-apoptotic actions of TNF-alpha to dominate, even in the absence of MTX. MTX itself does not promote apoptosis through interference with NF-kappa B signaling. Conclusions: These findings provide another mechanism by which TNF-alpha sustains macrophage numbers in inflamed tissue and identify a further point of clinical complementarity between MTX and anti-TNF-alpha treatments for rheumatoid arthritis.