We exposed the cells to CS medium which has been treated with polymixin beads. Macrophages stimulated with polymyxin bead treated CS medium did not show a significant decrease in the amounts of IL 8 generation. The amount of IL 8 release in response was studied. The amounts of IL 8 release after stimulation with PMA ionomycin was monitored. PMA stimulates selleck chemical Imatinib Mesylate PKC and ionomycin increases intracellular cal cium. The cytokine production by human monocyte derived macrophages is modulated by PKC. We pre treated the cells with anti human TLR4 antibody before CS medium exposure and examined the amounts of IL 8 generation. We demonstrated that macrophages produce IL 8 in response to PMA ionomycin and the amount of IL 8 release is not affected by TLR4 neutralizing antibody.

Indeed, the same levels of IL 8 production by macrophages following PMA ionomycin stimulation in the presence or absence of neutralizing antibody suggest that Anti TLR4 inhibition of CS induced IL 8 release is not due to cellular damage but blockade of TLR4. Then, TLR4 and its downstream pathways were studied. TLR4 ligation leads to NF B activation and signals via IRAK and TRAF. Our observations show that the signaling cascade of TLR4 ligation by CS medium involves IRAK 1 phos phorylation. Additionally, we found that TRAF6 degradation is also involved in the signaling path ways. Ligation of TLR4 by LPS activates NF B and induces production of cytokines in human myeloid cells. Moreover, induced transcriptional activity of NF B leads to maximal amount of IL 8 generation.

We demonstrated increases in phosphorylated I B lev els after CS medium stimulation of macrophages. The proteosome inhibitor MG 132 blocks the degra dation of I B. As shown in Figure 5A, an increase in the phospho I B level was detected in the CS treated samples. In contrast, the samples pretreated with MG 132 did not show such levels of phospho I B upon CS stimulation. Moreover, the degradation of I B is blocked when the cells were exposed to MG 132. The natural product curcumin is a known inhibitor of activation of NF B. Involvement of NF B in CS induced IL 8 production was demonstrated when macrophages were treated with NF B inhibitor curcumin prior to CS medium exposure. Cur cumin completely blocked the CS induced IL 8 produc tion.

The anti inflammatory properties of curcumin and its ability to inhibit the immune response upon exposure to a variety of external stimuli may, at least in part, result from inhibition of the activation of NF B by these GSK-3 external signals, since many of the genes that are implicated in the immune inflammatory response are up regulated by NF B. For example, curcumin inhibits the LPS induced production of IL 1 and TNF which NF B is implicated in these signaling pathways. SB 203580 is an inhibitor of p38 MAP kinase.