Lipopolysaccharide-induced tumor necrosis factor alpha production by humanmonocytes involves the Raf-1/MEK1-MEK2/ERK1-ERK2 pathway

During gram-negative sepsis, human monocytes are triggered to produce large quantities of proinflammatory cytokines such as tumor necrosis factor alph a (TNF-alpha) in response to endotoxin (lipopolysaccharide [LPS]). Several studies have identified signal transduction pathways that are activated by LPS, Including activation of nuclear factor-kappa B (NF-kappa B) and activa tion of mitogen-activated protein kinases (MAPKs), including ERK1 and ERK2, c-Jun N-terminal kinase, and p38. In this study, the relevance of ERK1 and ERK2 activation for LPS-induced TNF-alpha production by primary human mono cytes has been addressed with PD-098059, which specifically blocks activati on of MAPK kinase (MEK) by Raf-1, TNF-alpha levels in the monocyte culture supernatant, induced by 10 ng of LPS/ml, were reduced by PD-098059 (50 mu M ). In addition, PD-098059 also reduced TNF-alpha mRNA expression when cells were stimulated for 1 h with LPS. On the other hand, LPS-induced interleuk in-10 (IL-10) levels in the monocyte supernatant were only slightly inhibit ed by PD-098059. Ro 09-2210, a recently identified MEK inhibitor, completel y abrogated TNF-alpha levels at nanomolar concentrations. IL-10 levels also were strongly reduced. To show the efficacy of PD-098059 and Ro 09-2210, E RK1 and -2 activation was monitored by Western blotting with an antiserum t hat recognizes the phosphorylated (i.e., activated) forms of ERK1 and ERK2. Addition of LPS to human monocytes resulted in activation of both ERK1 and ERK2 in a time- and concentration (50% effective concentration between 1 a nd 10 ng of LPS/ml)-dependent manner. Activation of ERK2 was blocked by PD- 098059 (50 mu M), whereas ERK1 seemed to be less affected. Ro 09-2210 compl etely prevented LPS-induced ERK1 and ERK2 activation. LPS-induced p38 activ ation also was prevented by Ro 09-2210. These data further support the view that the ERK signal transduction pathway is causally involved in the synth esis of TNF-alpha by human monocytes stimulated with LPS.