Indeed, it has been reported that sPLA2 IIA influences survival of some cellular types within the CNS including oligodendrocytes and neurons, independently of its catalytic activity. In this study, we provide data demonstrating research use the func tional consequences of microglial cell exposure to the activating agent sPLA2 IIA. We have measured prolifera tive responses, phagocytic capabilities and synthesis and release of several molecules with pro inflammatory activities, Inhibitors,Modulators,Libraries for example, tumor necrosis factor and cycloxigenase 2, as indices of microglial activation. In addition, we have characterized several of the potential molecular mechanisms involved in these events. Methods Reagents A C127 mouse fibroblast cell line, stably transfected with the coding sequence of sPLA2 IIA from human placenta, was kindly provided by Dr Olivier and used as a source of human recombinant enzyme in some experiments to ascertain specificity.

sPLA2 IIA was obtained and purified as described previously. The absence of lipo polysaccharide in the preparation Inhibitors,Modulators,Libraries was confirmed by the limulus amebocyte lysate assay test in the batches used for the experiments. Moreover, experiments are conducted in the absence of fetal calf serum, which ensures that the effect is observed in the absence of LPS binding protein, necessary for the action of low concentrations of LPS. Bee venom sPLA2 III and human recombinant sPLA2 V were from Cayman. Rapamycin, pyrazole pyrimidine type 2, porcine sPLA2 IB, LPS, both anti rabbit and anti mouse fluorescein isothiocyanate secondary antibodies, FITC dextran Inhibitors,Modulators,Libraries and other chemicals were from Sigma Chemical Co.

PD98059 and AG1478 inhibitors were from Tocris Biosciece. Policlonal Inhibitors,Modulators,Libraries anti heparin binding epidermal growth factor neutralizing antibody and the inhibitors GM6001, chloromethylke tone and TNF proteinase inhibitor 1 were from Calbiochem. Rabbit anti mitogen activated protein kinase was from Zymed Laboratories. Rabbit antibody phosphorylated ERK1 2, phospho S6 ribosomal protein and phospho P70S6 kinase were from Cell Signaling Technology, Inc. The Rabbit phosphor Src, phospho EGF, phospho EGF, anti actin, and COX 2 anti bodies were from Santa Cruz Biotechnology Inc. Hybond P membrane was from Amersham Biosciences. DMEM and the cell culture supple ments, including FCS, were purchased from Gibco BRL.

Cell culture BV 2 murine microglia cells, a generous gift from Dr JR Bethea, were cultured at 37 C in a humidified atmosphere Inhibitors,Modulators,Libraries of 5% CO2 in high sucrose DMEM, supple mented with 100U ml penicillin, 100 ug ml strepto mycin, 50 ug ml gentamicin, 2 mM glutamine, and 10% heat inactivated fetal calf serum. Primary microglia enriched cultures were obtained from primary mixed glial cultures from 2 to 4 day old neonatal C57BL 6 mice. To obtain mixed glial cultures, cerebral cortices were dissected, carefully stripped of their meninges, and digested with 0. 25% trypsin EDTA solution JQ1 purchase for 25 minutes at 37 C.