title: The use of selective pharmacological inhibitors to delineate signal transduction pathways activated during complement receptor-mediated degranulation in chicken heterophils.
abstract: Complement receptors (CRs), along with Fc receptors, play a primary role in the removal of bacterial pathogens in poultry. The binding of serum-opsonized bacteria to CR results in the secretion of both toxic oxygen metabolites and antibacterial granules. We have previously shown that the stimulation of chicken heterophils with serum-opsonized Salmonella enteritidis induced tyrosine kinase-dependent phosphorylation regulated degranulation. In the present studies, we used selective pharmacological inhibitors to investigate the roles of protein tyrosine kinases, phospholipases C and D (PLC and PLD), phosphatidylinositol 3'-kinase (PI3-K), and the super family of mitogen-activated protein kinases (MAPKs) on CR-mediated heterophil degranulation. Inhibitors of receptor-linked tyrosine kinases (the tryphostins AG1478 and AG1296) had no attenuating effects on CR-mediated degranulation. However, PP2, a selective inhibitor of the src family of protein tyrosine kinases, and piceatannol, an inhibitor of Syk tyrosine kinases, both significantly attenuated the CR-mediated degranulation. Additionally, the specific inhibitors of PLC, U73122, and PI3-K, LY294002, significantly decreased CR-mediated heterophil degranulation. Two inhibitors of PLD-mediated signaling, 2,3-diphosphoglycerate (2,3-DPG) and 1-butanol, hindered degranulation. Addition of purified PLD restored control levels of degranulation in heterophils in which PLD was inhibited. Lastly, SP600125, a selective inhibitor of c-Jun N-terminal kinase (JNK), inhibited degranulation; whereas neither PD98059, the inhibitor of p38 MAPK, nor SB203580, the inhibitor of extracellular signal-regulated kinase, had any effect on CR-mediated heterophil degranulation. These studies demonstrate that CRs on chicken heterophils lack intrinsic tyrosine kinase activity, but that binding of serum-opsonized bacteria activates both proximal tyrosine kinases (src and Syk kinases), but differentially activates downstream tyrosine kinases (JNK, but not p38 nor ERK). Activation of src and Syk kinases plays a significant role in signal transduction of heterophil degranulation probably by stimulating downstream phosphorylation of PLC, PLD, and PI3-K. PI3-K has also been recently shown to be an upstream mediator of JNK activation, suggesting that this enzyme can induce signaling as both a lipid kinase and protein kinase. Engaging CRs on chicken heterophils activates a proximal tyrosine kinase (src and Syk kinases)-->PLC (PLD)-->PI3-K-->JNK signal transduction pathway that induces degranulation.
authors.affiliation: USDA-ARS, Southern Plains Agricultural Research Center, 2881 F&B Road, College Station, TX 77845, USA. kogut@ffsru.tamu.edu
authors.forename: Michael H
authors.initials: MH
authors.lastname: Kogut
authors.forename: Virginia K
authors.initials: VK
authors.lastname: Lowry
authors.forename: Morgan
authors.initials: M
authors.lastname: Farnell
mesh_terms: D000818:Animals; D015550:Cell Degranulation / Q000187:drug effects*; D002469:Cell Separation; D002645:Chickens; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D010586:Phagocytes / Q000187:drug effects*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D011951:Receptors, Complement / Q000187:drug effects*; D011961:Receptors, Fc / Q000187:drug effects; D012477:Salmonella enteritidis / Q000276:immunology; D015398:Signal Transduction / Q000187:drug effects*; D000072377:Syk Kinase; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D002867:Chromones; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D004963:Estrenes; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines; D011760:Pyrrolidinones; D011951:Receptors, Complement; D011961:Receptors, Fc; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases