多形核粒细胞在金黄色葡萄球菌杀白细胞素相关肺炎性损伤中的作用

The role of polymorphonuclear leukocyte in Staphylococcus aureus Panton-Valentine leukocidin-induced acute lung inflammation and injury

目的探讨多形核粒细胞（PMN）在金黄色葡萄球菌Panton—Valentine杀白细胞素（PVL）介导的肺炎性损伤中的作用。方法取15只新西兰大白兔均分为3组，构建肺炎性损伤模型。对照组使用PBS灌肺，粒细胞正常兔直接用重组PVL灌肺（rPVL组），粒细胞减少症兔先用长春新碱（VCR）处理，再用rPVL灌肺（VCR＋rPVL组）。造模后9h，计数外周血和支气管肺泡灌洗液（BALF）中PMN，同时检测BALF上清液中乳酸脱氢酶（LDH）含量、肺通透指数（LPI），BALF中PMN凋亡率、坏死率、活性氧自由基（ROs）释放量。取肺组织检测肺湿干比，并进行病理学检查。组间比较采用t检验。结果rPVL组外周血PMN为（2．69±0．34）×10^6／mL，显著低于对照组的（3．63±0．38）×10^6／mL（t=4．12，P〈0．05）。对照组、rPVL组和VCR＋rPVL组BALF中PMN分别为（O．57±0．01）×10^6／mL、（3．01±0．02）×10^6／mL和（O．10±0．02）×10^6／mL，rPVL组显著高于对照组（t=254．39，P％0．05）。rPVL组兔LDH、LPI和肺湿重／干重比均显著高于对照组，但后者与VCR＋rPVL组比较，差异无统计学意义。rPVL组BALF中PMN晚期凋亡率和坏死率分别为（18．98±1．04）％、（63．56±3．53）％，对照组分别为（1．03±0．17）％、（0．95±0．33）％（t=8．24，39．48；均P〈0．05），VCR＋rPVL组凋亡率和坏死率分别为（1．17±0．24）％、（1．13±0．17）％。rPVL组、对照组和VCR＋rPVL组ROS分别为1．56±0．39、0．41±0．03和0．39±0．02，rPVL组明显升高（t=．58，P％0．05）。rPVL组肺组织有弥漫性炎性细胞浸润、出血、水肿，VCR＋rPVL组仅见支气管周围与肺泡间隔极少量炎性细胞浸润。结论rPVL可引起粒细胞正常兔肺炎性损伤，但对粒细胞减少症兔肺损伤极小。PVL引起肺炎性损伤可能是依赖PMN的招募和聚集，继而坏死和（或）激活，释放细胞毒素颗粒内容物和（或）活性氧代谢产物等。

Objective To explore the role of polymorphonuclear leukocyte （PMN） in Panton- Valentine leucocidin （PVL）-induced acute lung inflammation and injury. Methods Fifteen New Zealand rabbits were divided into 3 groups with five rabbits in each group. The controls were treated with phosphate buffer solution （PBS）, the rabbits with normal granulocyte in rPVL group were treated with endotracheal instillation of rPVL, the granulocytopenia rabbits in vincristine （VCR）＋rPVL group were firstly treated with VCR, then with endotracheal instillation of rPVL. Nine hours after injection, the peripheral blood and bronchoalveolar lavage fluid （BALF） were collected forcounting PMN. The lactate dehydrogenase （LDH） activity in BALF, lung permeability index （LPI）, PMN apoptosis and necrosis and the release of reactive oxygen species （ROS） in BALF were measured. After the rabbits sacrificed, the lung tissue samples were collected for determining wet/dry （W/D） ratio and histopathological examination. The comparison among groups was done by t test. Results The PMN count in the peripheral blood was （2.69±0.34） ）〈 10^6/mL in rPVL group, which was significantly lower than control group [（3. 63±0. 38） ）〈10^6/mL] （t=4. 12, P〈0.05）. The PMN counts in BALF in control group, rPVL group and VCR..rPVL group were （0.57±0.01））〈 10^6/mL, （3.01±0.02））〈 10^6/mL and （0. 10±0. 02））〈10^6/mL, respectively; that in rPVL group was significantly higher than those in control group （t=254.39, P〈0.05）. The LDH activity,LPI and W/D ratio in rPVL group were all significantly higher than control group, while those in VCR± rPVL group were not significantly different from control group. The PMN apoptosis rate and necrosis rate in VCR..rPVL group were （1.17±0.24）% and （1.13±0.17）%, respectively. The releases of ROS （meanfluorescence intensity） in rPVL group, control group and VCR..rPVL group were 1.56± 0.39, 0.41 ±0. 03 and 0. 39± 0. 02, respectively, and that in rPVL group was significantly higher （t=6. 58, P〈0. 05）. Histopathological examination of the lung showed the diffuse infiltration of inflammatory cells, hemorrhage and edema in rPVL group, while there was only thimbleful infiltration of inflammatory cells observed in surrounding bronchia and alveolar septum in VCR.. rPVL group. Conclusions rPVL can induce lung inflammation and injury in rabbits with normal granulocyte, but not in neutropenic rabbits. Lung inflammation and injury may be the result of recruitment, aggregation and subsequent lysis and/or activation of PMN, which can damage the lung by releasing the contents of cytotoxic granules and/or reactive oxygen metabolites.