Phospholipase C, phosphatidylinositol 3-kinase, and intracellular [Ca(2+)] mediate the activation of chicken HD11 macrophage cells by CpG oligodeoxynucleotide

Abstract

The activation of phospholipases is one of the earliest key events in receptor-mediated cellular responses to a number of extracellular signaling molecules. Oligodeoxynucleotides containing CpG motifs (CpG ODN) mimic microbial DNA and are immunostimulatory to most vertebrate species. In the present study, we used the production of nitric oxide (NO) as an indicator to evaluate the involvement of the signaling cascades of phospholipases and phosphatidylinositol 3-kinase (PI3K) in the activation of chicken HD11 macrophage cells by CpG ODN. Using selective inhibitors, we have identified the involvement of phosphatidylinositol (PI)-phospholipase C (PI-PLC), but not phosphatidylcholine (PC)-phospholipase C (PC-PLC) and PC-phospholipase D (PC-PLD), in CpG ODN-induced NO production in HD11 cells. Preincubation with PI-PLC selective inhibitors (U-73122) completely abrogated CpG ODN-induced NO production in HD11 cells, whereas PC-PLC inhibitor (D609) and PC-PLD inhibitor (n-butanol) had no inhibitory effects. Additionally, inhibition of PI3K and protein kinase C (PKC) with selective inhibitors and chelation of intracellular [Ca(2+)] also significantly attenuated NO production in CpG ODN-activated HD11 cells. Our results demonstrate that PI-PLC, PI3 K, PKC, and intracellular [Ca(2+)] are important components of the CpG ODN-induced signaling pathway that leads to the production of NO in avian macrophage cells.