Reversible inhibitory effects of interferon-gamma and tumour necrosis factor-alpha on oligodendroglial lineage cell proliferation and differentiation in vitro

We have investigated the effects of the two prominent inflammatory cytokines, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), on oligodendroglial lineage cell development and survival. Purified oligodendrocytes and oligodendrocyte precursors obtained from neonatal rat brain primary cultures were subcultured in a defined, serum-free medium and exposed to IFN-gamma (1-100 U/ml), TNF-alpha (25-100 ng/ml) or both (100 U/ml and 50 ng/ml respectively) from day 1 to day 3 or from day 3 to day 6. While cell survival was not affected in any of the conditions tested, IFN-gamma dose-dependently inhibited [H-3]thymidine or bromodeoxyuridine incorporation (by up to 50%) and the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; by up to 33%). TNF-alpha synergized with IFN-gamma but was ineffective by itself. Moreover, IFN-gamma totally antagonized the induction by basic fibroblast growth factor and platelet-derived growth factor of the proliferation of the oligodendroglial lineage cell population under study. IFN-gamma also blocked the differentiation of oligodendrocyte precursors, as evidenced by cell morphology, immunostaining for early and late differentiation markers (galactocerebroside and myelin basic protein respectively) and activity of ceramide galactosyl transferase. Again, the effect of IFN-gamma was potentiated by TNF-alpha, which was ineffective when tested alone. The inhibitory activity of IFN-gamma was rapidly reversible: 3 days after removal of the cytokine, administered from day 1 to day 3, complete recovery of cell proliferation and differentiation could be documented. The cytokine-induced arrest in the expression of differentiation antigens was accompanied by perturbations in the expression of the corresponding mRNAs, revealed by a semiquantitative reverse transcription-polymerase chain reaction method. In particular, the message for myelin basic protein (and, in the case of treatment from days 3 to 6, also that for myelin associated glycoprotein) was decreased in cultures exposed to IFN-gamma, and further depressed in cultures treated with IFN-gamma and TNF-alpha, while TNF-alpha alone was ineffective. The above observations may help explain the role of IFN-gamma and TNF-alpha in the pathogenesis of inflammatory demyelinating diseases, in which increases in the levels of these substances have been described. In particular, in the case of multiple sclerosis, our results may bear on the problem of defective remyelination and are consistent with the frequent relapsing-remitting course of the disease.