Ho J L, Zhu B, He S, Du B, Rothman R
Department of Medicine, Cornell University Medical College, New York 10021.
J Exp Med. 1994 Oct 1;180(4):1457-69. doi: 10.1084/jem.180.4.1457.
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.
白细胞介素4（IL-4）可减少人巨噬细胞的细胞因子激活。IL-4与单核细胞IL-4R结合与蛋白激酶C（PKC）转位至细胞核部分有关。研究了从膜磷脂中裂解二酰基甘油（DAG）（PKC的激活剂）以确定IL-4R信号传导的近端事件。IL-4诱导了具有统计学意义的DAG的时间和剂量依赖性生成。IL-4触发的DAG产生并非源自磷脂酰肌醇4,5-二磷酸（PIP2）水解，因为在对IL-4的反应中未检测到胞质钙通量或肌醇磷酸的释放。使用[14C-甲基]胆碱标记的U937细胞和单核细胞进行实验，以确定IL-4R是否激活磷脂酶C（PLC）、磷脂酶D（PLD）或磷脂酶A2（PLA2）以利用膜磷脂酰胆碱（PC）形成DAG。IL-4诱导了磷酸胆碱（pchol）的时间和剂量依赖性增加，同时膜PC降解（与对照相比p<0.05）。PC的峰值降低与pchol的峰值产生相当这一发现表明IL-4R信号传导涉及PC特异性PLC的激活。在IL-4处理的细胞中未检测到胆碱（chol）或溶血磷脂酰胆碱以及甘油磷酸胆碱（分别是PLD或PLA2对PC裂解的产物）的变化。相反，外源性PLD诱导了胆碱增加以及膜PC的相应损失。进一步的研究表明IL-4R信号传导不涉及PLD。在用L-溶血-3-PC 1-[1-14C]棕榈酰标记的细胞中，PLD而非IL-4在用乙醇预处理后增加了磷脂酸（PA）和磷脂酰乙醇的产生。普萘洛尔（一种磷脂酸磷酸水解酶抑制剂）和花萼海绵诱癌素A（一种磷酸酶1和2A抑制剂）可阻断对FMLP而非IL-4的反应中的DAG产生。在普萘洛尔预处理的细胞中，佛波醇酯（PMA）而非IL-4触发了PA的产生并降低了DAG的量。PLA2不与IL-4R偶联的证据是检测到对FMLP而非IL-4的反应中有花生四烯酸产生。此外，IL-4R不与鞘磷脂酶（SMase）偶联，因为与外源性SMase不同，IL-4不会产生神经酰胺，而是诱导PC水解为pchol，这与外源性PLC相当。总之，单核细胞和U937细胞中的IL-4R信号传导涉及PLC而非PLD、PLA2或SMase，并且它利用PC而非PIP2形成DAG。
