Analysis of interleukin-1 signal transduction in striatal primary cultures

Characterisation of striatal primary cultures using immunocytochemistry (ICC) and calcium imaging has demonstrated that the cultures consist primarily of neurones (70%), and that the remaining cells display morphology typical of astrocytes. Both cell types were found to express the Type I IL-1 receptor. Treatment of striatal cells in vitro with IL-1 stimulated two mitogen activated protein (MAP) kinase families. Extracellular-regulated kinase (ERK) and p38 MAP kinase were both phosphorylated in a time- and concentration-dependent manner. Phosphorylation was found to correlate with induction of kinase activity, and therefore MAP kinase activation. Further studies utilising ICC revealed that p38 MAP kinase phosphorylation occurred specifically within the astrocyte nucleus. Downstream of the MAP kinases, IL-1 activated the transcription factor c-Jun by phosphorylation. The MAP kinase substrates ATF-2 and CREB were not activated upon IL-1 stimulation. Furthermore, IL-1 stimulated phosphorylation and nuclear translocation of the transcription factor NF-kB, an important mediator of immune responses. NF-kB translocation was observed specifically in astrocytes. Gene expression was studied directly using the TaqMan quantitative real time PCR technique. These studies revealed that IL-1 stimulated transcription of the cytokines tumour necrosis factor alpha (TNF-α), IL-6 and IL-1 itself. Gene expression of other cytokines (including IL-1 receptors, the IL-1 receptor antagonist and IL-18) were not stimulated by IL-1 in striatal cells. ELISA analysis of protein levels of selected cytokines (TNF-α and IL-6) revealed that gene expression did not correlate with protein release. These studies reveal for the first time important aspects of IL-1 signalling in a functionally significant region of the rat brain.