title: Tumor necrosis factor-alpha promotes survival in methotrexate-exposed macrophages by an NF-kappaB-dependent pathway.
abstract: BACKGROUND
  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-α) treatments. This implies that TNF-α promotes macrophage survival and suggests that TNF-α 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 RAW264.7 macrophage cell line that had been exposed to clinically-relevant concentrations of MTX and TNF-α.
  
  
  RESULTS
  MTX induces apoptosis in primary murine macrophages at concentrations as low as 100 nM in vitro. TNF-α, which has a context-dependent ability to increase or to suppress apoptosis, efficiently suppresses MTX-induced macrophage apoptosis. This depends on NF-κB signaling, initiated through TNF Receptor Type 1 ligation. Macrophage colony stimulating factor, the primary macrophage survival and differentiation factor, does not activate NF-κB or protect macrophages from MTX-induced apoptosis. A weak NF-κB activator, Receptor Activator of NF-κB Ligand (RANKL) is likewise ineffective. Blocking NF-κB in TNF-α-exposed macrophages allowed pro-apoptotic actions of TNF-α to dominate, even in the absence of MTX. MTX itself does not promote apoptosis through interference with NF-κB signaling.
  
  
  CONCLUSIONS
  These findings provide another mechanism by which TNF-α sustains macrophage numbers in inflamed tissue and identify a further point of clinical complementarity between MTX and anti-TNF-α treatments for rheumatoid arthritis.
authors.affiliation: Pharmacology Unit, School of Medicine and Pharmacology, University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia. szylo40@gmail.com
authors.forename: Susan Z Y
authors.initials: SZ
authors.lastname: Lo
authors.forename: James H
authors.initials: JH
authors.lastname: Steer
authors.forename: David A
authors.initials: DA
authors.lastname: Joyce
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D002469:Cell Separation; D002470:Cell Survival; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D008727:Methotrexate / Q000494:pharmacology*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D007166:Immunosuppressive Agents; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D008727:Methotrexate