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Title:   Author  item
                                            He, Louisitem
                                            Genovese, Kenneth

                                                         - *Ken*item
                                            Nisbet, Daviditem
                                            Kogut, Michael

                                                         - *Mike*
  Submitted to: International Immunopharmacology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 7/13/2006
                                    Publication  Date: 12/5/2006
                                    Citation: He, H., Genovese, K.J., Nisbet, D.J., Kogut, M.H. 2006. Involvement of phosphatidylinositol-phospholipase C in immune response to Salmonella lipopolysacharide in chicken macrophage cells (HD11). International Immunopharmacology. 6:1780-1787.Interpretive Summary: Lipopolysaccharide is a chemical component of bacteria.  Macrophage cells are one type of the white blood cells found in chickens.  The macrophage cells can produce nitric oxide, a chemical that can kill bacteria.  The nitric oxide is a very important bacteria-killing chemical that protects chickens from infection by pathogenic bacteria such as Salmonella.  We have performed experiments to see if and how the macrophage cells produce bacteria-killing nitric oxide when they are exposed to the lipopolysaccharide.  We found that the chicken macrophage can produce nitric oxide after contact with lipopolysaccharide.  We also found that one very important protein called phospholipase C controls the chemical processes for the macrophage cells to produce the nitric oxide.  This information is important to the pharmaceutical and poultry industries in the United States because it shows us that we can possibly manipulate the chicken macrophage cells to produce nitric oxide and to protect chickens from bacterial infection.
                                Technical Abstract: The activation of phospholipases is one of the earliest key events in receptor-mediated cellular responses to a number of extracellular signaling molecules.  Lipopolysaccharide (LPS) is a principle component of the outer membrane of Gram-negative bacteria and a prime target for recognition by the innate immune system.  Recognition of LPS by immune cells is mediated by Toll-like receptor 4 (TLR4) in conjunction with CD14.  It is not clear whether phospholipase activities are involved in TLR4-mediated LPS signaling in chicken macrophages.  In the present study, we evaluated the role of specific phospholipase in the activation of a chicken macrophage cell line HD11 by LPS.  Activation of HD11 cells by LPS results in induction of nitric oxide (NO).  Using selective inhibitors, we have identified that phosphatidylinositol (PI)-phospholipase C (PI-PLC), but not phosphatidylcholine (PC)-phospholipase C (PC-PLC) nor PC-phospholipase D (PC-PLD), was required for LPS-induced NO production.  Preincubation with PI-PLC selective inhibitors (U-73122 and ET-18-OCH3) abrogated LPS-induced NO production in HD11 cells, whereas PC-PLC inhibitor (D609), PAP inhibitor (propranolol), and PC-PLD inhibitor (n-butanol) had no inhibitory effects.  We also showed that inhibition of protein kinase C (PKC) by selective inhibitors Ro 31-8220 and calphostin C and chelating intracellular Ca2+ by BAPTA-AM significantly reduced NO production in LPS-stimulated HD11 cells.  Our results demonstrate that PI-PLC plays a critical role, most likely through activation of PKC pathway, in TLR4 mediated immune responses of avian macrophage cells to LPS.


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