Differential modulation of interleukin‐6 expression by interleukin‐1β in neuronal and glial cultures

Pictures

Figure 1. Immunostaining of neuronal and glial cultures. Neuronal cultures double-marked with NF and GFAP antibodies. A and B show the same microscopic field with different UV filters. Scale bar: 100 µm.
C, immunofluorescence detection of GFAP in glial cultures. D, the same microscopic field of C, stained with Hoechst nuclear fluorochrome. Scale bar: 50 µm.

Figure 2. A. Upper side: representative histogram of IL-1β mRNA densitometric analysis in neuronal and astrocyte cultures measured under each set of conditions; N = 10 for both kinds of cell cultures. *** means p < 0.005 of hypoxic cultures versus normoxic and reoxygenation conditions (Wilcoxon's test for paired data); A, lower side: an example of multiplex RT-PCR for IL-1β mRNA and the 18S rRNA internal standard respectively, for neuronal and glial cultures. Molecular weight markers are indicated by “MM”. B, IL-1β release measured by ELISA in culture medium under the same conditions. * p = 0.05 versus normoxic and p = 0.02 versus reoxygenated neuronal cultures. ** means p < 0.05 versus normoxic cultures of astrocytes (Wilcoxon's test).

Figure 3.  Neuronal cultures. A, upper panel: graphical representation of data from densitometric analysis of IL-6 RT – PCR amplifications. N = 12. *** and °°° under hypoxic state means p < 0.005 versus normoxic and reoxygenated levels as determined by one-way ANOVA followed by post-hoc, multiple comparison analysis with Scheffè's test. For each condition, data from treated cultures were significantly different from untreated (p < 0.005, Wilcoxon's test, not shown). Lower panel: results of multiplex RT-PCR amplification showing one representative experiment for each conditions. “MM” means molecular weight marker, B: Protein release, measured in the media of the same hippocampal neuronal cultures, parallels transcript expression. Significant differences (*** and °°° = p < 0.005) were evident after exposure to mild hypoxia (ANOVA followed by Sheffe's test), and between treated and untreated neuronal cultures for each condition (### = p < 0.005, Wilcoxon's test).

Figure 4. IL-6 expression as a function of IL-1β expression in untreated hippocampal neurones. A, B and C: linear regression analysis of IL-6 and IL-1β mRNAs under normoxic conditions, after mild hypoxia and after three hours of reoxygenation respectively. D, E, and F: linear regression analysis of IL-6 and IL-1β release measured in the culture media of the same cultures, parallels transcript expression. Each point represents a single experiment. The lines are computer-generated least-squares linear regression plots (Pearson's correlation).

Figure 5. Comparative histogram showing the effects of IL-1β immunoneutralization on IL-6 levels in cultured astrocytes. A, upper side: the densitometric analysis of mRNA amplifications showed a significant difference between IL-1β-immunoneutralised and untreated cultures of astrocytes during normal condition and after mild hypoxia (### means p < 0.005, Wilcoxon's test). After recovery, no significant differences were observed between treated and untreated cultures. *** means p < 0.005 under hypoxic-untreated cultures versus normoxic- and reoxygenated-untreated cultures (ANOVA followed by Sheffe's test). The hypoxic immunoneutralised cultures of astrocytes showed significant differences when compared with normoxic and reoxygenated cultures (°°°p < 0.005, ANOVA followed by Sheffe's test) N = 12 for each condition. A, lower side: RT – PCR products amplification. One representative experiment showing each condition. “MM” indicates molecular weight marker. B: shows protein histogram of IL-6 protein measured by ELISA. In all conditions, significant differences were observed between treated and untreated cultures (## means p < 0.05, ### means p < 0.005, Paired data were analysed by Wilcoxon's rank test. °°° p < 0.005 by ANOVA, followed by Sheffe's test.