SYNERGISTIC DIFFERENTIATION-PROMOTING ACTIVITY OF INTERFERON-GAMMA AND TUMOR-NECROSIS-FACTOR-ALPHA - ROLE OF RECEPTOR REGULATION ON HUMAN NEUROBLASTS

Background: Interferon gamma (IFN-gamma) and tumor necrosis factor-alp ha (TNF) synergize in inducing human neuroblastoma cells to differenti ate terminally in vitro into mature nonproliferating neurons. The mech anisms by which this synergistic activity takes place are still obscur e, Purpose: To understand the basis of IFN-gamma-TNF synergism, we inv estigated the constitutive equipment of receptors to IFN-gamma and TNF in two human neuroblastoma cell lines (i.e., LAN-5 and GI-LI-N) and t heir quantitative and functional variations following treatment with I FN-gamma or TNF. Methods: IFN-gamma receptors and TNF receptors were a ssessed and functionally characterized by radioreceptor-binding assay before and after treatment of the cells with IFN-gamma or TNF. The TNF receptor subtypes were identified by the reverse transcriptase-polyme rase 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 differentiatio n were assessed by thymidine incorporation inhibition and morphologic maturation. Results: No quantitative or functional modification of IFN -gamma receptors was observed in TNF-treated cells. However, after tre atment with IFN-gamma, TNF receptor numbers were enhanced to a differe nt extent in both cell lines. The two neuroblastoma cell lines express ed, both constitutively and after IFN-gamma induction, only one specie s of TNF receptor, i.e., the p80 form in LAN-5 and the p60 form in CI- LI-N. Sequential treatment with IFN-gamma followed by TNF, but not in the opposite order, could reproduce the early effects of differentiati on in neuroblastoma cells, supporting a role for TNF receptor up-regul ation as a basis for the cooperation between the two cytokines. Conclu sion: The results strongly suggest that receptor regulation can be at least one mechanism by which IFN-gamma and TNF exert their synergistic effects. Moreover, it appears that the two TNF receptor types are red undant in signaling neuroblastoma cell differentiation. Implications: Our findings can provide a guideline for a rational design of experime ntal differentiation-based therapeutic protocols in patients with neur oblastoma.