Batista Camila F, Souza Fernando N, Santos Kamila R, Ramos Sanchez Eduardo M, Reis Luiza Campos, Bertagnon Heloisa G, Blagitz Maiara G, Gomes Renata C, Lage Andrey P, Heinemann Marcos B, Della Libera Alice M M P
Veterinary Clinical Immunology Research Group, Departamento de Clínica Médica, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, 05508-270, São Paulo, São Paulo, Brazil.
Veterinary Clinical Immunology Research Group, Departamento de Clínica Médica, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, 05508-270, São Paulo, São Paulo, Brazil.
Vet Immunol Immunopathol. 2018 Feb;196:53-59. doi: 10.1016/j.vetimm.2017.12.004. Epub 2017 Dec 9.
The present study aimed to validate the use of R-phycoerythrin (R-PE)-labeled Mannheimia haemolytica to simultaneously stimulate phagocytosis and intracellular production of reactive oxygen species (ROS) by blood phagocytes in bronchoalveolar lavage (BAL) fluid. Initially, R-PE-labeled M. haemolytica was inactivated using a water bath at 60 °C for 60 min. Afterwards, R-PE labelling of bacteria was confirmed by flow cytometry. The geometric mean fluorescence intensity of R-PE-labeled bacteria (FL2 detector, 585 ± 42 nm) was analyzed by flow cytometry and was 41.5-fold higher than the respective unlabeled controls, confirming the success of bacterial conjugation to R-PE. Phagocytosis and intracellular production of ROS by blood neutrophils and monocytes, and by BAL CD14 macrophages, in 12 healthy 6-month-old male calves were then performed using R-PE-labeled bacteria and 2',7'-dichlorofluorescein diacetate (DCFH-DA) as probes. Confocal microscopy was used to confirm phagocytosis of R-PE-labeled M. haemolytica by phagocytes and to exclude erroneous measurements of bacteria adhering to the leukocyte membrane. The present study showed that there is no difference in the ROS production without stimulus and in the presence of M. haemolytica by peripheral blood neutrophils and monocytes, in contrast to the increased ROS production by local alveolar macrophages upon stimulation by M. haemolytica. This emphasizes the importance of alveolar macrophages in the maintenance of homeostasis and health of the respiratory system, which can be supported during the inflammatory process by the rapid recruitment of neutrophils with high microbicidal and phagocytic capacity. The method described here provides an easy and feasible tool to measure phagocytosis and intracellular ROS production by phagocytes, especially when commonly used probes for intracellular ROS production were used, such as DCFH-DA and dihydrorhodamine 123.
本研究旨在验证使用R-藻红蛋白（R-PE）标记的溶血曼氏杆菌来同时刺激支气管肺泡灌洗（BAL）液中血液吞噬细胞的吞噬作用和细胞内活性氧（ROS）的产生。首先，使用60℃水浴60分钟使R-PE标记的溶血曼氏杆菌失活。之后，通过流式细胞术确认细菌的R-PE标记。通过流式细胞术分析R-PE标记细菌的几何平均荧光强度（FL2检测器，585±42nm），其比相应的未标记对照高41.5倍，证实了细菌与R-PE结合成功。然后，使用R-PE标记的细菌和2',7'-二氯荧光素二乙酸酯（DCFH-DA）作为探针，对12头健康的6月龄雄性犊牛的血液中性粒细胞和单核细胞以及BAL CD14巨噬细胞进行吞噬作用和细胞内ROS产生的检测。共聚焦显微镜用于确认吞噬细胞对R-PE标记的溶血曼氏杆菌的吞噬作用，并排除白细胞膜上附着细菌的错误测量。本研究表明，外周血中性粒细胞和单核细胞在无刺激和存在溶血曼氏杆菌的情况下ROS产生没有差异，与之形成对比的是，局部肺泡巨噬细胞在溶血曼氏杆菌刺激下ROS产生增加。这强调了肺泡巨噬细胞在维持呼吸系统稳态和健康中的重要性，在炎症过程中，具有高杀菌和吞噬能力的中性粒细胞的快速募集可对此提供支持。这里描述的方法提供了一种简单可行的工具来测量吞噬细胞的吞噬作用和细胞内ROS产生，特别是当使用常用的细胞内ROS产生探针时，如DCFH-DA和二氢罗丹明123。
