Cytokine mRNA and protein levels were ana lyzed using a two way ANOVA. Phos phorylation of STAT3 levels were analyzed using a two way ANOVA. Post hoc Stu dents t test of least square means was used to deter mine if Gefitinib EGFR inhibitor treatment means were significantly different from one another. All data are presented as mean SEM. Results IL 6 and LPS induce STAT3 phosphorylation in microglia and neurons To verify the presence of the subunits involved in IL 6 and LPS signaling, the cell surface expression of IL Inhibitors,Modulators,Libraries 6R, gp130, and TLR 4 on BV. 2 and Neuro. 2A cells was examined. Figure 1A shows more than 50% of the microglial BV. 2 cells expressed gp130 while nearly 90% expressed IL 6R, approximately 50% of the BV. 2 cells expressed both IL 6R and gp130. In contrast, about 90% of the Neuro.

2A cells expressed gp130, 3% expressed IL 6R, and 3% co expressed IL 6R and gp130. Approxi mately 80% of the BV. 2 cells expressed TLR 4 compared to 30% of the Neuro. 2A cells. Although IL 6 can activate multiple transcription factors, in CNS cells activation of the IL 6 recep tor upregulates STAT3 phosphorylation. Thus, the capacity of IL 6 to induce Inhibitors,Modulators,Libraries the phosphorylation of STAT3 in BV. 2 and Neuro. 2A cell cultures was examined. Figure 2 shows that IL 6 at a higher concen tration increased phosphorylated STAT3 similarly in microglia and neurons. However, at a lower concentration, IL 6 only increased STAT3 phosphorylation in microglia, which is consistent with the greater proportion of these cells that expressed IL 6R.

These findings sug gest that classic and trans signaling can occur on both neurons and Inhibitors,Modulators,Libraries microglia, although neurons may be more readily regulated through the mechanism of trans sig naling since gp130 is highly expressed on this cell type. IL 6 trans signaling in microglia and neurons Previous studies have shown that gp130 is expressed con stitutively on all cell types and this expression facil itates trans signaling in the presence of Inhibitors,Modulators,Libraries IL 6 and sIL 6R. Figure 3A shows that pretreatment of microglia and neurons with sIL 6R increased IL 6 induced STAT3 phosphorylation and, respectively. Consistent with the increase in STAT3 phosphorylation, a sIL 6R �� LPS inter action was evident whereby sIL 6R upregulated LPS induced IL 6 production in microglia, and neurons. Although not statistically significant, there was some constitutive STAT3 phosphorylation and IL 6 expression in samples pretreated with sIL 6R.

sgp130 attenuated IL 6R activation in microglia and Inhibitors,Modulators,Libraries neurons We next investigated the ability of sgp130 to alter phos phorylation of STAT3 and expression of IL 6. A sgp130 �� LPS interaction revealed that pretreatment of BV. 2 microglial and Neuro. 2A neuronal cells with sgp130 decreased small molecule LPS induced activation of STAT3 and, respec tively and inhibited LPS induced IL 6 production in both BV. 2 and Neuro. 2A cells. These data demonstrate that sgp130 inhi bits LPS induced IL 6 production in microglia and neu rons.