Jurkat T cells were treated for 18 h with 400 uM of H2O2 neverless and apoptosis was confirmed by an annexin Inhibitors,Modulators,Libraries V assay. Apoptotic Jurkat T cells were then added to a culture of BV 2 cells treated under different conditions with a ratio of Jurkat to BV 2 cells of 8,1. After 2 h incu bation, the co culture was analyzed by flow cytometry to quantify cell uptake. As shown in Figure 7A, we observed very little phagocytosis under control condi tions where BV 2 cells were resting. However Jurkat en gulfment increased significantly when BV 2 cells were pre treated for 24 h with 1 ug ml of sPLA2 IIA or 100 UI ml of IFN��, as increasing number of microglia cells showed FL3 fluorescence positive signals. In a separate experiment, the cells were also stained with DAPI and studied using a confocal microscope to visually confirm the ingestion of apoptotic cells.

The orthog onal reconstruction images showed the spatial relation of ingested cells to the BV 2 cell nucleus and confirm that Jurkat cells were not merely bound to the cell surface. In subsequent experiments, we examined whether transactivation of EGFR is also a key step for controlling sPLA2 IIA mediated efferocytosis. Consistent with the Inhibitors,Modulators,Libraries signaling mechanism recruited by the secreted phospho lipase to promote proliferation of BV 2, we found that the presence of the selective inhibitors GM6001, CMK and TAPI 1 also abolished the phagocytic response trig gered by the sPLA2 IIA on microglial cells, as it previously did on sPLA2 IIA enhanced cell growth.

sPLA2 Inhibitors,Modulators,Libraries IIA promotes synthesis and secretion of inflammatory mediators in BV 2 cells Finally, we examined whether Inhibitors,Modulators,Libraries sPLA2 IIA could affect the expression levels of pro inflammatory mediators in BV 2 microglia cells. Then, BV 2 cells were treated with the optimal concentration of 1 ug ml of sPLA2 IIA or 100 UI ml of IFN�� for 4 and 8 h, and the expression of COX 2 was examined in the cell lysate by western blot. Our results revealed that both treatments markedly induced the expression of the pro inflammatory protein COX 2. We also measured the production Inhibitors,Modulators,Libraries of the cytokine TNF using a commercial ELISA assay. We observed that in the supernatant of cells treated with sPLA2 IIA or IFN�� for 24 h, the levels of TNF were significantly enhanced, compared with untreated cells which did not produce TNF spontaneously.

In contrast, the release or accumulation of anti inflammatory mediators, such as IL 10 was not detected in any of our culture conditions. Lastly, we further examined whether blockage of EGFR signaling at different levels, as demonstrated in previous sections, affects the expression of these inflammatory sellckchem proteins induced by sPLA2 IIA. Figure 8C and D show that sPLA2 IIA induced up regulation of COX 2 and secretion of TNF was significantly inhibited by the presence of the inhibitors AG1478, GM6001, TAPI 1 and CMK, as well as by the polyclonal anti HB EGF antibody.