Figure 1

Necrotic neurons induce expression of activation markers by microglia. (A) Microglia were stimulated with necrotic HT22 neurons at a ratio of 1:1 for 24 hours and the expression of several activation markers analysed by FACS. The staining intensity is represented on histogram overlays for the mentioned proteins in non-stimulated microglia (grey filled), and in microglia stimulated either with IFN-γ (100 U/ml) (dashed line) or with necrotic neurons (solid line). (B) These results are shown as fold increases ("fold activation") of the Geo Mean when divided by the average expression of triplicates of untreated microglia. The fold increase in the expression of the different markers was compared to the effect on B7.1 expression. Necrotic neurons were significantly more efficient than apoptotic HT22 cells in inducing CD40 expression by microglia (graph on the right). (C) Histogram overlays for CD40 staining intensity of microglia cultured in medium only (grey filled), stimulated with necrotic HT22 neurons (solid line), or incubated with different necrotic cell types: histogram plot on the left, primary cerebellar granule neurons (CGN) (dashed line), astrocyte-enriched cell population (dotted line); histogram plot on the right, EL-4 (dotted line) and WHEI-164 (dashed line) cell lines. (D) Microglial cells were stimulated for 24 hours with necrotic neurons or with IFN-γ (10 U/ml), and the expression of COX-2, IDO and iNOS was analysed by RT-PCR. (E) Microglial cells were plated in an insert containing a membrane with a cut-off of 8 μm. The number of microglial cells migrated through the membrane was quantified 3 hours after adding necrotic HT22 neurons (50 μg/ml) to the lower chamber. ATP (100 μM) was used as a positive control for chemotaxis. Data are representative of at least two experiments done in triplicate. *** p < 0.001; **p < 0.01 and * p < 0.05.