Regulation of the release of tumour necrosis factor (TNF)α and soluble TNF Receptor by γ irradiation and interferon γ in Ewing's sarcoma/peripheral primitive neuroectodermal tumour cells

This study analyses the production of tumour necrosis factor (TNF)α and soluble TNF receptor (sTNF-R) before and after exposure to γ irradiation and interferon γ (IFNγ) in 12 cell lines derived from Ewing's sarcoma (ES)/peripheral primitive neuroectodermal tumours (pPNET). Supernatants from ES/pPNET cell cultures were tested in a TNFα-specific amplified enzyme-linked immunosorbent assay (ELISA), a bioassay, and sTNF-Rp55 and sTNF-Rp75 ELISA. The tumour cell lines released minimal amounts of TNFα, prominent amounts of sTNF-Rp55 (7/12 cell lines) and no sTNF-Rp75. Exposure to γ irradiation (5 Gy) either induced (3/12) cell lines) or up-regulated (3/12 cell lines) TNFα release without changing sTNF-Rp55 and sTNF-Rp75 levels. Priming of cultures with recombinant human IFNγ (rhIFNγ) markedly enhanced TNFα secretion in the radiation-responsive cell lines and had no influence on sTNF-Rp55 and sTNF-Rp75 levels. rhIFNγ affected the magnitude rather than the sensitivity of the radiation response. The TNFα secreted was bioactive, as shown by its cytotoxic effect of WEHI-164 cells, and neutralization of its activity by anti-TNFα monoclonal antibody. Herbimycin A (a tyrosine-specific protein kinase inhibitor) but not calphostin C (a protein kinase C inhibitor), H89 (a protein kinase A inhibitor), AACOCF3 (a specific inhibitor of phospholipase A2) and MK-886 (a specific inhibitor of 5-lipoxygenase) abrogated γ-irradiation-stimulated TNFα release. The antioxidantsN-acetylcysteine, nordihydroguaiaretic acid and mepacrine dose-dependently inhibited γ-irradiation-mediated TNFα production. Collectively our findings indicate that IFNγ priming potentiates the secretion of bioactive TNFα by ES/pPNET cells in response to γ irradiation without affecting sTNF-R release. The data suggest a requirement for protein tyrosine kinase activity and a role for reactive oxygen species in the γ-irradiation-mediated intracellular signalling pathway leading to TNFα production.