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

Abstract

Summary Synthetic lipopeptides based on bacterial lipoprotein are efficient activators for monocytes/macrophages inducing the release of interleukin (IL)‐1, IL‐6, tumour necrosis factor‐α (TNF‐α), reactive oxygen/nitrogen intermediates, and the translocation of nuclear factor κB (NFκB). In this report we investigate the signal transduction pathways involved in leucocyte activation by the synthetic lipopeptide N ‐palmitoyl‐ S ‐[2,3‐bis(palmitoyloxy)‐(2R, S )‐propyl]‐(R)‐cysteinyl‐seryl‐(lysyl)3‐lysine (P 3 CSK 4 ). We show that P 3 CSK 4 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 differences between the P 3 CSK 4 and lipopolysaccharide (LPS)‐induced phosphorylation 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 and LPS responder mice (C57BL/10ScSn) or LPS non‐responder mice (C57BL/10ScCr). The lipopeptide activated the MAPK‐signalling cascade in both LPS responder and non‐responder macrophages, whereas LPS induced the MAPK signalling pathway only in macrophages derived from LPS responder mice. An approximately 70% decrease of lipopeptide induced NFκB 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 ERK1/2 after stimulation with P 3 CSK 4 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.