Figure 4.

IFNγ selectively kills motoneurons in a LIGHT-dependent manner. (a–c) Isolated Hb9∷GFP motoneurons were immunostained 24 h after seeding with antibodies directed against IFNγR1 (a), IFNγR2 (b) or with hamster (c) or mouse (not shown) irrelevant IgG as control. Scale bar, 10 μm. (d) Motoneurons were cultured for 24 h and incubated with increasing concentrations of soluble mouse recombinant IFNγ from two different sources. The percentage of surviving motoneurons was determined 48 h later. The distance between the x-axis values is arbitrary. (e) Motoneuron survival was determined 48 h following treatment or not with LT-βR-Fc (100 ng/ml), HVEM-Fc (10 ng/ml), Fas-Fc (1 μg/ml) or TNFR1-Fc (100 ng/ml) in combination or not with 250 ng/ml of IFNγ. The number of surviving motoneurons is expressed as a percentage of the number of motoneurons in the control condition (none). (f) Motoneurons were isolated from E12.5 embryos of indicated genotype and cultured for 24 h before being treated with 250 ng/ml of IFNγ. Motoneuron survival was determined 48 h later and expressed relative to the non-treated condition for each genotype. (g) Immunoblot analysis of cortical, hippocampal, sensory, striatal and motoneurons proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Actin was used as a loading control. (h) Cell survival assay was performed as in (d), with neuronal cells being treated with 250 ng/ml of IFNγ. (i and j) Motoneurons were cultured for 24 h and incubated with 250 ng/ml of IFNγ. Eight hours later, cells were lysed and expression levels of LIGHT (i) and LT-βR (j) were determined by western blotting with indicated antibodies. Fold-increase of LIGHT and LT-βR over non-treated conditions was determined by densitometric analysis of immunoreactive bands, normalized to their respective actin signals (n=3, means±S.D.). Results shown in (d, e, f and h) are the mean values±S.D. of three independent experiments performed in triplicate