Effects of TNFα on immature and mature oligodendrocytes and their progenitors in vitro

Abstract

Tumor necrosis factor α (TNFα) appears to take part in the pathogenesis of multiple sclerosis and to contribute to the degeneration of oligodendrocytes as well as neurons. TNFα is produced by microglia and astrocytes, which also produce hormones and cytokines that influence its biological activity. Thus, in mixed cultures the effects of exogenous TNFα might be modified by products of astrocytes and microglia. The effects of TNFα in oligodendrocyte-enriched cultures are reported below. We prepared the cultures by shaking oligodendrocytes off primary mixed glial-cell cultures from brains of 2-day-old rats at 7 days in vitro and plating them (0 days post-shake, DPS). Platelet-derived growth factor and fibroblast growth factor were included in the media at 1–5 DPS in order to encourage proliferation. At 2 DPS media were added with no TNFα (controls) or 1000, 2000 or 5000 U/ml of TNFα, and at 5 DPS media were replaced with fresh serum-free media. Cultures were fixed with 4% paraformaldehyde at 5, 7, 9 and 12 DPS and immunostained. Oligodendrocyte progenitors were not reduced in numbers immediately after the incubation with TNFα (i.e. at 5 DPS). However, after an additional 4 days in culture fewer progenitors remained in the cultures that had been treated with TNFα than in the untreated cultures. In the absence of the growth factors there were fewer progenitors, but their numbers also were reduced by TNFα. Maturation to the myelin basic protein (MBP)-positive stage was inhibited by about 36% at 9 DPS by 1000–2000 U/ml of TNFα, while numbers of O4+/MBP− precursors were unaffected. It is interesting that the steady-state number of O4-positive precursors was unchanged by TNFα at 9 DPS, when there were reductions in the numbers of A2B5-positive progenitors and MBP-positive mature oligodendrocytes. That observation suggests that the rates of proliferation, death and maturation are controlled by multiple factors, with a particularly vulnerable time at the maturation to the MBP-positive stage. At 5000 U/ml TNFα the specific effect on maturation was overtaken cytotoxicity. These data and a summary of the literature suggest that inhibition of MBP expression is sensitive to lower TNFα concentrations and incubation times than is cell survival. Specific effects on numbers of MBP-positive cells, morphology and MBP expression occur at 1000–2000 U/ml for 48–72 h or at up to 10 000 U/ml for≤24 h, and the deficits remain after removal of the TNFα.