Synergistic differentiation-promoting activity of interferon γ and tumor necrosis factor-α: Role of receptor regulation on human neuroblasts

Background: Interferon γ (IFN-γ) and tumor necrosis factor-α (TNF) synergize in inducing human neuroblastoma cells to differentiate terminally in vitro into mature nonproliferating neurons. The mechanisms by which this synergistic activity takes place are still obscure. Purpose: To understand the basis of IFN-γ-TNF synergism, we investigated the constitutive equipment of receptors to IFN-γ and TNF in two human neuroblastoma cell lines (i.e., LAN-5 and GI-LI-N) and their quantitative and functional variations following treatment with IFN-γ or TNF. Methods: IFN-γ receptors and TNF receptors were assessed and functionally characterized by radioreceptor-binding assay before and after treatment of the cells with IFN-γ or TNF. The TNF receptor subtypes were identified by the reverse transcriptase-polymerase chain reaction, chemical cross-linking of receptors to iodinated TNF, and inhibition of TNF binding by type-specific anti-TNF receptor monoclonal antibodies. The effects of cytokines on cell differentiation were assessed by thymidine incorporation inhibition and morphologic maturation. Results: No quantitative or functional modification of IFN-γ receptors was observed in TNF-treated cells. However, after treatment with IFN-γ, TNF receptor numbers were enhanced to a different extent in both cell lines. The two neuroblastoma cell lines expressed, both constitutively and after IFN-γ induction, only one species of TNF receptor, i.e., the p80 form in LAN-5 and the p60 form in GI-LI-N. Sequential treatment with IFN-γ followed by TNF, but not in the opposite order, could reproduce the early effects of differentiation in neuroblastoma cells, supporting a role for TNF receptor up-regulation as a basis for the cooperation between the two cytokines. Conclusion: The results strongly suggest that receptor regulation can be at least one mechanism by which IFN-γ and TNF exert their synergistic effects. Moreover, it appears that the two TNF receptor types are redundant in signaling neuroblastoma cell differentiation. Implications: Our findings can provide a guideline for a rational design of experimental differentiation-based therapeutic protocols in patients with neuroblastoma.