Locati M, Lamorte G, Luini W, Introna M, Bernasconi S, Mantovani A, Sozzani S
Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
J Biol Chem. 1996 Mar 15;271(11):6010-6. doi: 10.1074/jbc.271.11.6010.
Monocyte chemotactic protein (MCP)-1, a member of the C-C (or beta) branch of the chemokine superfamily, at chemotactic concentrations, induced a rapid release of [3H]arachidonic acid but not of [14C]oleic acid from prelabeled human monocytes. This effect was associated with an increase in the intensity of the immunoreactive band corresponding to the phosphorylated form of cytosolic phospholipase A2, (cPLA2). To address the role of cPLA2 in the induction of monocyte chemotaxis, cells were treated with a specific antisense oligonucleotide. Monocytes cultured in the presence of 10 microM antisense oligonucleotide for 48 h showed a marked decrease (57 +/- 5%; n = 4) of cPLA2 expression, as evaluated by Western blot analysis and a nearly complete inhibition (81.8 +/- 4.2%; n = 3) of [3H]arachidonic acid release in MCP-1-stimulated cells. Monocyte chemotaxis in response to MCP-l also was inhibited in a concentration-dependent manner by cPLA2 antisense oligonucleotide (IC50 = 1.9 +/- 1.1 microM; n = 3), with complete inhibition observed between 3 and 10 microM. No inhibition of chemotactic response was observed in monocytes treated with a control oligonucleotide. Monocyte migration in response to MCP-3, RANTES (regulated on activation normal T cells expressed and secreted), and MIP-1 alpha/LD78 also was inhibited (>70%) in antisense oligonucleotide-treated cells. On the contrary, the chemotactic response elicited by formyl-methionyl-leucyl-phenylalanine and C5a, two "classical" chemotactic agonists, was minimally affected (<20%) by antisense oligonucleotide treatment. These data show that cPLA2 plays a major role in [3H]arachidonic acid release by MCP-1 in human monocytes and provide direct evidence for the involvement of cPLA2 in C-C chemokine-induced monocyte chemotaxis.
单核细胞趋化蛋白（MCP）-1是趋化因子超家族C-C（或β）分支的成员，在趋化浓度下，可诱导预先标记的人单核细胞快速释放[3H]花生四烯酸，但不释放[14C]油酸。这种效应与对应于胞质磷脂酶A2（cPLA2）磷酸化形式的免疫反应条带强度增加有关。为了探讨cPLA2在单核细胞趋化诱导中的作用，用特异性反义寡核苷酸处理细胞。通过蛋白质印迹分析评估，在10μM反义寡核苷酸存在下培养48小时的单核细胞显示cPLA2表达显著降低（57±5%；n = 4），并且在MCP-1刺激的细胞中[3H]花生四烯酸释放几乎完全受到抑制（81.8±4.2%；n = 3）。cPLA2反义寡核苷酸也以浓度依赖性方式抑制单核细胞对MCP-1的趋化作用（IC50 = 1.9±1.1μM；n = 3），在3至10μM之间观察到完全抑制。在用对照寡核苷酸处理的单核细胞中未观察到趋化反应的抑制。在反义寡核苷酸处理的细胞中，单核细胞对MCP-3、调节激活正常T细胞表达和分泌的趋化因子（RANTES）以及巨噬细胞炎性蛋白-1α/LD78的迁移也受到抑制（>70%）。相反，甲酰甲硫氨酰亮氨酰苯丙氨酸和C5a这两种“经典”趋化激动剂引发的趋化反应受反义寡核苷酸处理的影响最小（<20%）。这些数据表明，cPLA2在MCP-1诱导人单核细胞释放[3H]花生四烯酸中起主要作用，并为cPLA2参与C-C趋化因子诱导的单核细胞趋化提供了直接证据。
