Available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/dci Phospholipase C, phosphatidylinositol 3-kinase, and intracellular [Ca 2+ ] mediate the activation of chicken HD11 macrophage cells by CpG oligodeoxynucleotide Haiqi He à , Kenneth J. Genovese, David J. Nisbet, Michael H. Kogut Southern Plain Agricultural Research Center, USDA-ARS, 2881 F&B Road, College Station, TX 77845, USA Received 30 November 2007; received in revised form 25 February 2008; accepted 27 February 2008 Available online 24 March 2008 KEYWORDS CpG ODN; Nitric oxide; Phospholipase C; Phosphatidylinositol 3-kinase; Intracellular [Ca 2+ ]; Protein kinase C; Chicken macrophage cells Summary 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 phosphatidy- linositol (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. Published by Elsevier Ltd. Introduction Microbial DNA is a powerful stimulus of the innate immune system of vertebrates. Unmethylated CpG dinucleotides (CpG motifs) that are relatively abundant in bacterial and viral genomic DNAs but are rare in vertebrate genomes are responsible for the immune stimulatory activities [1]. ARTICLE IN PRESS 0145-305X/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.dci.2008.02.009 à Corresponding author. Tel.: +1979 2603771; fax: +1 979 260 9332. E-mail address: he@ffsru.usda.gov (H. He). Developmental and Comparative Immunology (2008) 32, 1111–1118