Accepted for publication in ALTEX:
Characterization of the mouse astrocyte cell line IMA 2.1 as alternative model system for primary astrocyte cultures
Stefan Schildknecht 1, Susanne Kirner 1, Anja Henn 1, Karlo Gasparic 1, Regina Pape 1, Liudmila Efremova 1, Olaf Maier 2, Roman Fischer 2, Marcel Leist 1
1Doerenkamp-Zbinden Chair for In vitro Toxicology and Biomedicine, University of Konstanz, 78457 Konstanz, Germany
2Institute for Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
Short title: IMA 2.1 astrocyte cell line
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 alternative to the currently used primary astrocyte cultures. IMA 2.1 were directly compared with primary mouse astrocytes with respect to their response to proinflammatory stimuli, the expression of typical astrocyte markers, as well as to the cell line´s capacity to metabolize the experimental parkinsonian drug MPTP to its toxic metabolite MPP+. Under inflammatory conditions, mimicked by the addition of a cytokine mix, IMA 2.1 responded similar to primary astrocytes by mRNA upregulation, expression of iNOS and COX-2, as well as by 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 fast as in primary cells. For all endpoints investigated, the cell line delivered reproducible results over a period of several years and allowed upscaling of experiments due to its easy handling compared with primary cells.
Accepted for publication in J Immunol:
Role of caspases in cytokine-induced barrier breakdown in human brain endothelial cells
M. Alejandro Lopez-Ramirez a, Roman Fischer b, Claudia C. Torres-Badillo a, Heather A. Davies a, Karen Logan a, Klaus Pfizenmaier b, David K. Male a, Basil Sharrack c and Ignacio A. Romero a,*
a Department of Life, Health & Chemical Sciences, The Open University, Milton Keynes MK7 6AA, UK.
b Institute of Cell Biology and Immunology, University Stuttgart, Stuttgart, Germany.
c Department of Neurology, Sheffield Teaching Hospitals NHS Trust, University of Sheffield, Sheffield, UK.
During neuroinflammation, cytokines such as TNFa and IFNg secreted by activated leukocytes and/or central nervous system (CNS) resident cells have been shown to alter the phenotype and function of brain endothelial cells (BECs) leading to blood-brain barrier (BBB) breakdown. Here, we show that the human brain endothelial cell line, hCMEC/D3, expresses the receptors for TNFa, TNFR1 and TNFR2, and for IFNg. BEC activation with TNFa alone or in combination with IFNg induced endothelial leakage of paracellular tracers. At high cytokine concentrations (10 and 100 ng/ml), this effect was associated with caspase-3/7 activation and apoptotic cell death as determined by Annexin V binding or DNA fragmentation by TUNEL assays. In addition, inhibition of JNK and PKC activation at these doses partially prevented activation of caspase-3/7 although only JNK inhibition was able to partially prevent the increase in BEC paracellular permeability induced by cytokines. By contrast, lower cytokine concentrations (1 ng/ml) also led to effector caspase activation, increased paracellular flux and redistribution of ZO-1 and VE-cadherin but not to apoptosis. Specific caspase-3 and 9 but not caspase-8 inhibitors partially reversed cytokine-induced disruption of tight and adherens junctions and increased BEC paracellular permeability at low cytokine concentrations. Our results suggest that the concentration of cytokines in the CNS endothelial microenvironment determine the extent of caspase-mediated barrier permeability changes which may be generalised as a result of apoptosis or more subtle as a result of alterations in the organization of junctional complex molecules.