TLR2 hypersensitivity of astrocytes as functional consequence of previous inflammatory episodes.

Precedent inflammatory episodes may drastically modify the function and reactivity of cells. We investigated whether priming of astrocytes by microglia-derived cytokines alters their subsequent reaction to pathogen-associated danger signals not recognized in the quiescent state. Resting primary murine astrocytes expressed little TLR2, and neither the TLR2/6 ligand fibroblast-stimulating lipopeptide-1 (FSL1) nor the TLR1/2 ligand Pam(3)CysSK(4) (P3C) triggered NF-κB translocation or IL-6 release. We made use of single-cell detection of NF-κB translocation as easily detectable and sharply regulated upstream indicator of an inflammatory response or of c-Jun phosphorylation to measure restimulation events in astrocytes under varying conditions. Cells prestimulated with IL-1ß, with a TLR3 ligand, with a complete cytokine mix consisting of TNF-α, IL-1ß, and IFN-γ, or with media conditioned by activated microglia responded strongly to FSL1 or P3C stimulation, whereas the sensitivity of the NF-κB response to other pattern recognition receptors was unchanged. This sensitization to TLR2 ligands was associated with an initial upregulation of TLR2, displayed a "memory" window of several days, and was largely independent of the length of prestimulation. The altered signaling led to altered function, as FSL1 or P3C triggered the release of IL-6, CCL-20, and CXCL-2 in primed cells, but not in resting astrocytes. These data confirmed the hypothesis that astrocytes exposed to activated microglia assume a different functional phenotype involving longer term TLR2 responsiveness, even after the initial stimulation by inflammatory mediators has ended.

Characterization of mouse cell line IMA 2.1 as a potential model system to study astrocyte functions.

Astrocytes are activated in most chronic neurodegenerative diseases associated with inflammatory events such as Parkinson's disease or Alzheimer's disease, but also in stroke. Due to an aging population worldwide, research efforts in these areas are likely to expand in the future. This will entail an increased demand for appropriate experimental models. We introduce here the new immortalized mouse astrocyte cell line IMA 2.1 as an alternative to currently used primary astrocyte cultures. IMA 2.1 were directly compared with primary mouse astrocytes with respect to their response to proinflammatory stimuli, expression of typical astrocyte markers, and to the cell line's capacity to metabolize the parkinsonian toxin MPTP to its toxic metabolite MPP+. Under inflammatory conditions, mimicked with the addition of a cytokine mix, IMA 2.1 responded similarly to primary astrocytes with mRNA upregulation, expression of iNOS and COX-2, and the release of various inflammatory mediators. Analysis of astrocytic markers indicated that IMA 2.1 represent a relatively early, GFAP-negative stage of astrocyte development. Moreover, conversion of MPTP by monoamine oxidase-B proceeded in IMA at least as quickly as in primary cells. For all endpoints investigated, the cell line IMA 2.1, derived from a single clone, delivered reproducible results over a period of several years and allowed upscaling of experiments due to its easy handling compared with primary cells.