Immunostimulation by the synthetic lipopeptide P3CSK4: TLR4-independent activation of the ERK1/2 signal transduction pathway in macrophages

Synthetic lipopeptides based on bacterial lipoprotein are efficient activat ors for monocytes/macrophages inducing the release of interleukin (IL)-1, I L-6, tumour necrosis factor-alpha (TNF-alpha), reactive oxygen/nitrogen int ermediates, and the translocation of nuclear factor kappaB (NF kappaB). In this report we investigate the signal transduction pathways involved in leu cocyte activation by the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmi toyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine (P3CSK4). We sh ow that P3CSK4 activates mitogen-activated protein (MAP)-kinases ERK1/2 and MAP kinase (MAPK)-kinases MEK1/2 in bone-marrow-derived macrophages (BMDM) and in the macrophage cell line RAW 264.7. Additionally, we could detect d ifferences between the P3CSK4 and lipopolysaccharide (LPS)-induced phosphor ylation of MAP kinases: Different levels in phosphorylation were found both in kinetics and dose-response using RAW 264.7 cells or BMDM from BALB/c an d LPS responder mice (C57BL/10ScSn) or LPS nonresponder mice (C57BL/10ScCr) . The lipopeptide activated the MAPK-signalling cascade in both LPS respond er and non-responder macrophages, whereas LPS induced the MAPK signalling p athway only in macrophages derived from LPS responder mice. An approximatel y 70% decrease of lipopeptide induced NF kappaB translocation and an about 50% reduction of nitric oxide (NO) release was observed in the presence of anti-CD14. These data correspond to the reduction of phosphorylation of ERK 1/2 after stimulation with P3CSK4 in the presence of anti-CD14 antibodies. Inhibition of MEK1/2 by PD98059 completely reduced the lipopeptide-induced phosphorylation of ERK1/2 indicating that MEK1/2 are solely responsible for the phosphorylation of the downstream-located MAP kinases ERK1/2.