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

Interleukin 4 (IL-4) diminishes cytokine activation of human macrophag e. IL-4 binding to monocyte IL-4R is associated with protein kinase C (PKC) translocation to a nuclear fraction. The cleavage of diacylglyce rol (DAG), an activator of PKC, from membrane phospholipids was invest igated 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 phosphatidy linositol 4,5-bisphosphate (PIP2) hydrolysis, since neither cytosolic calcium flux nor liberation of inositol phosphates was detected in res ponse to IL-4. Experiments were performed using [C-14-methyl]choline-l abeled U937 cells and monocytes to determine whether IL-4R activated p hospholipase C (PLC), PLD, or PLA(2) to use membrane phosphatidylcholi ne (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 cho line (chol) or lyse-PC and glycerolphosphocholine, the respective prod ucts of PC cleavage by PLD or PLA(2), were not detected in IL-4-treate d cells. In contrast, exogenous PLD induced an increase in chol and co ncomitant 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-C-14]palmitoyl, PLD but not IL-4, increased the production of phosphatidic acid (PA) and phosphatidyl-ethanol when pretreated with e thanol. Propanolol, an inhibitor of phosphatidate phosphohydrolase, an d calyculin A, a phosphatase 1 and 2A inhibitor, blocked DAG productio n 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 amou nt of DAG. Evidence that PLA(2) is not coupled to IL-4R is the detecti on of arachidonate production in response to FMLP but not to IL-4. Fur thermore, IL-4R is not coupled to sphingomyelinase (SMase) since IL-4, unlike exogenous SMase, did not generate ceramide but induced the hyd rolysis of PC to pchol that was comparable to exogenous PLC. In summar y, IL-4R signaling in monocytes and U937 cells involves PLC and not PL D, PLA(2), or SMase, and it uses PC and not PIP2 to form DAG.