Rinaldi Manuela, Moroni Paolo, Paape Max J, Bannerman Douglas D
Department of Veterinary Pathology, Hygiene and Public Health, University of Milan, Milan 20133, Italy.
Vet Immunol Immunopathol. 2007 Jan 15;115(1-2):107-25. doi: 10.1016/j.vetimm.2006.09.009. Epub 2006 Sep 26.
During mastitis and other bacterial-mediated diseases of cattle, neutrophils play a critical role in the host innate immune response to infection. Neutrophils are among the earliest leukocytes recruited to the site of infection and contribute to host innate immune defenses through their ability to phagocytose and kill bacteria. The bactericidal activity of neutrophils is mediated, in part, through the generation of reactive oxygen species (ROS). Extracellular release of ROS can induce injury to host tissue as well, and aberrant release of ROS has been implicated in the pathogenesis of certain inflammatory-mediated diseases. Due to their essential role in bacterial clearance and implicated involvement in the pathogenesis of other diseases, there is much interest in the study of neutrophil-generated ROS. Several assays have been developed to measure ROS production, however, many of these have not been evaluated with bovine neutrophils. The objectives of the current study were to evaluate different assays capable of measuring bovine neutrophil ROS, and to compare the results of assays never previously tested with bovine neutrophils to those obtained from more well-established assays frequently used with these cells. Eight different assays were evaluated, including: luminol, isoluminol, and methyl cypridina luciferin analog (MCLA) chemiluminescence assays; Amplex Red, dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), and dihydrorhodamine 123 fluorescence assays; and the cytochrome c absorbance assay. The assays were evaluated in the context of their abilities to detect ROS produced in response to two agonists commonly used to induce neutrophil activation, phorbol 12-myristate, 13-acetate (PMA) and opsonized zymosan. Diphenyleneiodonium chloride, a NADPH oxidase inhibitor, was used to assess the specificity of the assays to detect ROS. The ability of these assays to discriminate between intra- and extracellular ROS and to specifically detect distinct ROS was evaluated using superoxide dismutase and catalase, which scavenge extracellular superoxide and hydrogen peroxide, respectively. With the exception of the DHE assay, all assays detected bovine neutrophil ROS generation elicited by PMA and zymosan. PMA, but not zymosan, was able to stimulate neutrophil generation of ROS at levels that were detectable with DHE. The MCLA chemiluminescence assay was the only assay that detected ROS produced in response to each of the lowest concentrations of PMA and zymosan tested. To our knowledge, this is the first study to evaluate DHE-, MCLA-, Amplex Red-, and isoluminol-based assays for the measurement of bovine neutrophil ROS, and the most comprehensive comparative study of ROS assays under similar experimental conditions.
在奶牛乳腺炎及其他细菌介导的疾病中，中性粒细胞在宿主对感染的固有免疫反应中发挥关键作用。中性粒细胞是最早被招募到感染部位的白细胞之一，通过其吞噬和杀灭细菌的能力，为宿主的固有免疫防御做出贡献。中性粒细胞的杀菌活性部分是通过活性氧（ROS）的产生来介导的。ROS的细胞外释放也可诱导宿主组织损伤，并且ROS的异常释放与某些炎症介导疾病的发病机制有关。由于它们在细菌清除中的重要作用以及与其他疾病发病机制的潜在关联，对中性粒细胞产生的ROS的研究备受关注。已经开发了几种测定ROS产生的方法，然而，其中许多方法尚未在牛中性粒细胞中进行评估。本研究的目的是评估能够测量牛中性粒细胞ROS的不同方法，并将以前从未用牛中性粒细胞测试过的方法的结果与从这些细胞常用的更成熟方法获得的结果进行比较。评估了八种不同的方法，包括：鲁米诺、异鲁米诺和甲基环丙啶荧光素类似物（MCLA）化学发光法；Amplex Red、二氢乙锭（DHE）、二氯二氢荧光素二乙酸酯（CM-H₂DCFDA）和二氢罗丹明123荧光法；以及细胞色素c吸光度法。在检测由两种常用于诱导中性粒细胞活化的激动剂佛波醇12-肉豆蔻酸酯、13-乙酸酯（PMA）和调理酵母聚糖诱导产生的ROS的能力背景下评估这些方法。使用NADPH氧化酶抑制剂氯化二苯碘鎓来评估这些方法检测ROS的特异性。使用超氧化物歧化酶和过氧化氢酶分别清除细胞外超氧化物和过氧化氢，评估这些方法区分细胞内和细胞外ROS以及特异性检测不同ROS的能力。除DHE法外，所有方法均检测到PMA和酵母聚糖诱导的牛中性粒细胞ROS生成。PMA能够刺激中性粒细胞产生可被DHE检测到水平的ROS，而酵母聚糖则不能。MCLA化学发光法是唯一检测到对测试的每种最低浓度PMA和酵母聚糖产生的ROS的方法。据我们所知，这是第一项评估基于DHE、MCLA、Amplex Red和异鲁米诺的方法用于测量牛中性粒细胞ROS的研究，也是在相似实验条件下对ROS测定方法最全面的比较研究。
