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Title: EVALUATION OF ASSAYS FOR THE MEASUREMENT OF BOVINE NEUTROPHIL REACTIVE OXYGEN SPECIES.

Author
item RINALDI, MANUELA - MILAN, ITALY
item MORONI, PAOLO - MILAN, ITALY
item Paape, Max
item Bannerman, Douglas

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/21/2006
Publication Date: 1/15/2007
Citation: Rinaldi, M., Moroni, P., Paape, M.J., Bannerman, D.D. 2007. Evaluation of assays for the measurement of bovine neutrophil reactive oxygen species. Vet.Immunol. Immunopathol. 115(1-2):107-125.

Interpretive Summary: Neutrophil bactericidal activity is a critical component of the innate immune defense against pathogenic bacteria responsible for an array of cattle diseases, including mastitis. The bactericidal activity of neutrophils is mediated by the generation of toxic molecules known as reactive oxygen species (ROS). It is well known that deficient generation of ROS enhances susceptibility to uncontrollable infection. The current study establishes and validates methods for assessing bovine neutrophil generation of ROS. The impact of this research is the development of assays which can identify defects in critical neutrophil functions that may enhance susceptibility to infection. It is expected that the methods validated for use with bovine neutrophils in the current study will be applied to investigations into the pathogenesis of a wide-array of bacterial-mediated diseases of cattle.

Technical Abstract: 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. Seven different assays were evaluated, including: luminol, isoluminol, and methyl cypridina luciferin analog (MCLA) chemiluminescence assays; Amplex Red, dihydroethidium (DHE), and CM-H2DCFDA fluorescence assays; and the cytochrome c reduction 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 ester (PMA) and 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 study to compare this many ROS assays under similar experimental conditions.