Gambero Alessandra, Landucci Elen C T, Toyama Marcos H, Marangoni Sergio, Giglio Jose R, Nader Helena B, Dietrich Carl P, De Nucci Gilberto, Antunes Edson
Department of Pharmacology, Faculty of Medical Sciences, UNICAMP, PO Box 6111, 13081-970, Campinas, SP, Brazil.
Biochem Pharmacol. 2002 Jan 1;63(1):65-72. doi: 10.1016/s0006-2952(01)00841-3.
The purpose of this study was to examine the ability of type I- (porcine pancreas and Naja mocambique mocambique venom), type II- (bothropstoxin-I, bothropstoxin-II, and piratoxin-I), and type III- (Apis mellifera venom) secretory phospholipases A2 (sPLA2s) to induce human neutrophil chemotaxis, and the role of the cell surface proteoglycans, leukotriene B4 (LTB4), and platelet-activating factor (PAF), in mediating this migration. The neutrophil chemotaxis assays were performed by using a 48-well microchemotaxis chamber. Piratoxin-I, bothropstoxin-I, N. m. mocambique venom PLA2 (10-1000 microg/mL each), bothropstoxin-II (30-1000 microg/mL), porcine pancreas PLA2 (0.3-30 microg/mL), and A. mellifera venom PLA2 (30-300 microg/mL) caused concentration-dependent neutrophil chemotaxis. Heparin (10-300 U/mL) concentration-dependently inhibited the neutrophil migration induced by piratoxin-I, bothropstoxin-II, and N. m. mocambique and A. mellifera venom PLA2s (100 microg/mL each), but failed to affect the migration induced by porcine pancreas PLA2. Heparan sulfate (300 and 1000 microg/mL) inhibited neutrophil migration induced by piratoxin-I, whereas dermatan sulfate and chondroitin sulfate (30-1000 microg/mL each) had no effect. Heparitinase I and heparinase (300 mU/mL each) inhibited by 41.5 and 47%, respectively, piratoxin-I-induced chemotaxis, whereas heparitinase II and chondroitinase AC failed to affect the chemotaxis. The PAF receptor antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thienol-[3,2-f] -triazolo-[4,3-a] -diazepine-2-yl]-1-(4-morpholynil)-1-propionate) (0.1-10 microM) and the LTB4 synthesis inhibitor AA-861 [2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone] (0.1-10 microM) significantly inhibited the piratoxin-I-induced chemotaxis. Piratoxin-I (30-300 microg/mL) caused a concentration-dependent release of LTB4. Our results suggest that neutrophil migration in response to sPLA2s is independent of PLA activity, and involves an interaction of sPLA2s with cell surface heparin/heparan binding sites triggering the release of LTB4 and PAF.
本研究的目的是检测I型（猪胰和莫桑比克射毒眼镜蛇毒）、II型（矛头蝮毒素-I、矛头蝮毒素-II和海盗毒素-I）和III型（蜜蜂毒）分泌型磷脂酶A2（sPLA2s）诱导人中性粒细胞趋化性的能力，以及细胞表面蛋白聚糖、白三烯B4（LTB4）和血小板活化因子（PAF）在介导这种迁移中的作用。中性粒细胞趋化性测定采用48孔微量趋化性小室进行。海盗毒素-I、矛头蝮毒素-I、莫桑比克射毒眼镜蛇毒PLA2（各10 - 1000μg/mL）、矛头蝮毒素-II（30 - 1000μg/mL）、猪胰PLA2（0.3 - 30μg/mL）和蜜蜂毒PLA2（30 - 300μg/mL）引起浓度依赖性的中性粒细胞趋化性。肝素（10 - 300 U/mL）浓度依赖性地抑制海盗毒素-I、矛头蝮毒素-II以及莫桑比克射毒眼镜蛇毒和蜜蜂毒PLA2（各100μg/mL）诱导的中性粒细胞迁移，但不影响猪胰PLA2诱导的迁移。硫酸乙酰肝素（300和1000μg/mL）抑制海盗毒素-I诱导的中性粒细胞迁移，而硫酸皮肤素和硫酸软骨素（各30 - 1000μg/mL）无作用。I型类肝素酶和肝素酶（各300 mU/mL）分别抑制海盗毒素-I诱导的趋化性41.5%和47%，而II型类肝素酶和软骨素酶AC不影响趋化性。PAF受体拮抗剂WEB 2086（3 - [4 - (2 - 氯苯基)-9 - 甲基 - 6H - 噻吩并[3,2 - f] - 三唑并[4,3 - a] - 二氮杂卓 - 2 - 基]-1 - (4 - 吗啉基)-1 - 丙酸酯）（0.1 - 10μM）和LTB4合成抑制剂AA - 861 [2 - (12 - 羟基十二碳 - 5,10 - 二炔基)-3,5,6 - 三甲基 - 1,4 - 苯醌]（0.1 - 10μM）显著抑制海盗毒素-I诱导的趋化性。海盗毒素-I（30 - 300μg/mL）引起浓度依赖性的LTB4释放。我们的结果表明，中性粒细胞对sPLA2s的迁移不依赖于PLA活性，并且涉及sPLA2s与细胞表面肝素/硫酸乙酰肝素结合位点的相互作用，从而触发LTB4和PAF的释放。
