CpG-ODN-induced nitric oxide production is mediated through clathrin-dependent endocytosis, endosomal maturation, and activation of PKC, MEK1/2 and p38 MAPK, and NF-κB pathways in avian macrophage cells (HD11)

Abstract

We have characterized the nitric oxide (NO) induction by CpG oligodeoxydinucleotide (CpG-ODN) and lipopolysaccharide (LPS) in an avian macrophage cell line (HD11) and evaluated signal transduction pathways by using selective inhibitors. Our results indicate that while CpG-ODN and LPS both stimulate inducible NO synthase (iNOS) to produce NO through common signalling pathways involving activation of protein kinase C (PKC), mitogen-activated protein kinases (p38 MAPK and MEK1/2) and transcription factor NF-κB; CpG-ODN inducing NO production distinctively requires a clathrin-dependent endocytosis and subsequent endosomal maturation. Inhibitors of clathrin-dependent endocytosis such as monodansylcadaverine and hyperosmolar sucrose completely abolished CpG-ODN stimulated NO production by HD11 cells, but have no or less effect on LPS-induced NO production. The endosomal maturation is also critical for stimulation of NO induction by CpG-ODN, but not by LPS. Our findings are the first to demonstrate cellular signalling pathways that mediate CpG-ODN immunostimulatory activity in cells from non-mammalian species.