Figure 5
Pre-incubation with pro-inflammatory cytokines potentiates Poly(I:C)-induced fibronectin aggregation by grey matter astrocytes. (a) Schematic representation of astrocyte treatment and analysis. Primary rat grey matter (gm) and white matter (wm) astrocytes are pre-treated for 24 hours with a mixture of IFNγ (500 units/mL), IL1β (10 ng/mL) and TNFα (10 ng/mL), followed by Poly(I:C) treatment (50 μg/mL) for 48 hours. (b,c) Western blot analysis of fibronectin (Fn) in DOC-soluble (sol, fibronectin dimers) and DOC-insoluble (ins, fibronectin aggregates) extracellular deposits. Representative blots of 3–4 independent experiments are shown in (b), quantification of DOC-insoluble aggregated fibronectin in c. Equal amounts of protein (12 μg) are subjected to DOC-(in)solubility analysis. Note that pre-incubation with cytokines potentiates fibronectin aggregation by gm (p = 0.004), but not wm astrocytes. (d,e) Adhesion assay of astrocytes to fibronectin (n = 3). Astrocytes were treated for 1 hour with a mixture of IFNγ (500 units/mL), IL1β (10 ng/mL) and TNFα (10 ng/mL), followed by adhesion for 2 hours in the absence or presence of Poly(I:C) (50 μg/mL) and/or the absence (d) or presence (e) of Mn2+ (1 mM), which is known to increase integrin affinity. Note that pre-incubation with cytokines potentiates the decreased effect of Poly(I:C) on adhesion (wm + cytokines + Poly(I:C) p = 0.008; gm + cytokines + Poly(I:C) p = 0.022), which cannot be overcome by Mn2+ (wm + cytokines + Poly(I:C) p = 0.048; gm + cytokines + Poly(I:C) p = 0.002). Bars represent mean relative to wm astrocyte control (wm ctrl, c) or untreated control (ctrl, d,e), which was set at 1 for each independent experiment (horizontal line). Error bars show the standard error of the mean. Statistical analyses were performed using a one-sample t-test to test for differences with wm astrocyte control (ctrl, c) or their respective untreated control (d,e) (*p < 0.05, **p < 0.01, ***p < 0.001). A one way ANOVA with a Dunnett multiple comparison post-test was used to compare treatment groups with untreated gm control (c) (###p < 0.001) and a one way ANOVA with a Šidák multiple comparisons post-test was used to test for differences between treatment groups (d,e) (##p < 0.01).
