R-Phycoerythrin-labeled Mannheimia haemolytica for the simultaneous measurement of phagocytosis and intracellular reactive oxygen species production in bovine blood and bronchoalveolar lavage cells.

Autor: Batista CF; 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. Electronic address: camilafb@usp.br., Souza FN; 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., Santos KR; 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., Ramos Sanchez EM; Laboratório de Sorologia e Imunobiologia, Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, Av. Prof. Dr. Enéas de Carvalho Aguiar, 470, 05403-000, São Paulo, São Paulo, Brazil., Reis LC; Laboratório de Sorologia e Imunobiologia, Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, Av. Prof. Dr. Enéas de Carvalho Aguiar, 470, 05403-000, São Paulo, São Paulo, Brazil., Bertagnon HG; 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., Blagitz MG; 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., Gomes RC; 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., Lage AP; Departamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais, Av. Presidente Antônio Carlos, 6627, 31270-010, Belo Horizonte, Minas Gerais, Brazil., Heinemann MB; Departamento de Medicina Veterinária Preventiva e Saúde Animal, 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., Della Libera AMMP; 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.
Jazyk: angličtina
Zdroj: Veterinary immunology and immunopathology [Vet Immunol Immunopathol] 2018 Feb; Vol. 196, pp. 53-59. Date of Electronic Publication: 2017 Dec 09.
DOI: 10.1016/j.vetimm.2017.12.004
Abstrakt: 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.
(Copyright © 2017 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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