title: Interleukin 4 receptor signaling in human monocytes and U937 cells involves the activation of a phosphatidylcholine-specific phospholipase C: a comparison with chemotactic peptide, FMLP, phospholipase D, and sphingomyelinase.
abstract: Interleukin 4 (IL-4) diminishes cytokine activation of human macrophage. IL-4 binding to monocyte IL-4R is associated with protein kinase C (PKC) translocation to a nuclear fraction. The cleavage of diacyglycerol (DAG), an activator of PKC, from membrane phospholipids was investigated to define the proximal events of IL-4R signaling. IL-4 induced a statistically significant time-and dose-dependent generation of DAG. The IL-4-triggered production of DAG was not derived from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, since neither cytosolic calcium flux nor liberation of inositol phosphates was detected in response to IL-4. Experiments were performed using [14C-methyl]choline-labeled U937 cells and monocytes to determine whether IL-4R activated phospholipase C (PLC), PLD, or PLA2 to use membrane phosphatidylcholine (PC) to form DAG. IL-4 induced a time- and dose-dependent increase of phosphocholine (pchol) with concomitant degradation of membrane PC (p < 0.05 compared with control). The finding that the peak reduction of PC was equivalent to peak production of pchol suggested that IL-4R signaling involved the activation of a PC-specific PLC. Changes in choline (chol) or lyso-PC and glycerolphosphocholine, the respective products of PC cleavage by PLD or PLA2, were not detected in IL-4-treated cells. In contrast, exogenous PLD induced an increase in chol and concomitant loss of membrane PC. Additional investigation suggested that IL-4R signaling does not involve PLD. In cells labeled with L-lyso-3-PC 1-[1-14C]palmitoyl, PLD but not IL-4, increased the production of phosphatidic acid (PA) and phosphatidyl-ethanol when pretreated with ethanol. Propranolol, an inhibitor of phosphatidate phosphohydrolase, and calyculin A, a phosphatase 1 and 2A inhibitor, blocked DAG production in response to FMLP but not to IL-4. In propranolol pretreated cells, PMA but not IL-4 triggered the production of PA and lowered the amount of DAG. Evidence that PLA2 is not coupled to IL-4R is the detection of arachidonate production in response to FMLP but not to IL-4. Furthermore, IL-4R is not coupled to sphingomyelinase (SMase) since IL-4, unlike exogenous SMase, did not generate ceramide but induced the hydrolysis of PC to pchol that was comparable to exogenous PLC. In summary, IL-4R signaling in monocytes and U937 cells involves PLC and not PLD, PLA2, or SMase, and it uses PC and not PIP2 to form DAG.
authors.affiliation: Department of Medicine, Cornell University Medical College, New York 10021.
authors.forename: J L
authors.initials: JL
authors.lastname: Ho
authors.forename: B
authors.initials: B
authors.lastname: Zhu
authors.forename: S
authors.initials: S
authors.lastname: He
authors.forename: B
authors.initials: B
authors.lastname: Du
authors.forename: R
authors.initials: R
authors.lastname: Rothman
mesh_terms: D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D004075:Diglycerides / Q000096:biosynthesis; D004789:Enzyme Activation; D006801:Humans; D015847:Interleukin-4 / Q000378:metabolism / Q000494:pharmacology*; D009000:Monocytes / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D010713:Phosphatidylcholines / Q000378:metabolism; D010739:Phospholipase D / Q000502:physiology*; D018123:Receptors, Interleukin / Q000502:physiology*; D019948:Receptors, Interleukin-4; D013108:Sphingomyelin Phosphodiesterase / Q000502:physiology*; D010738:Type C Phospholipases / Q000502:physiology*
chemical_list: C011439:1,2-diacylglycerol; D004075:Diglycerides; D010713:Phosphatidylcholines; D018123:Receptors, Interleukin; D019948:Receptors, Interleukin-4; D015847:Interleukin-4; D016718:Arachidonic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase; D010739:Phospholipase D; D002118:Calcium