There have been several studies that have used photometric methods to investigate the respiratory burst function of neutrophils isolated from the blood of healthy sheep, however differences in assay methodology, agonists, and in the units measured have hindered comparisons between the studies. Aspects such as the agonists used and their concentrations and incubation times, as well as which parts of the human neutrophil respiratory burst function that the assays measure are of importance, with the standard agonists being Platelet Activating Factor (PAF) and N-formylmethionyl-leucyl-phenylalanine (fMLP), and the maximum rate of O 2 - release ( V max) as the standard measure. Photometric assays based upon the superoxide dismutase- (SOD) inhibitable reduction of cytochrome c have been well standardised for the measurement of human neutrophil respiratory burst function. Respiratory burst function provides a key measure of neutrophil function, and studies investigating this have used different techniques such as luminometry, fluorometry, precipitation reactions and photometry, some of which have been adapted to study ovine neutrophil respiratory burst function. Due to the neutrophil's central role in innate immunity and its postulated role in pathologies such as ALI/ARDS and sepsis, a better understanding of ovine neutrophils is crucial to improving the validity of these ovine biomedical models as well as improving the understanding of immunology and pathology in sheep.
Ovine models have been extensively used in the study of ALI/ARDS and sepsis, however they are limited by a lack of understanding of the ovine neutrophil which has been less well studied than its human counterpart. As the scope for investigating these pathologies in humans is limited, the development of relevant in-vivo animal models is crucial in attempting to elucidate the mechanisms by which they develop. In healthy individuals this process is closely regulated as the inappropriate release of ROS by neutrophils can cause damage to the surrounding tissue and is thought to be a key factor in the development of pathologies such as acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), as well as the multiple organ failure characteristic of sepsis. ROS are generated by a variety of intracellular mechanisms, although the predominant mechanism, referred to as respiratory burst, is based upon the assembly and activation of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase which then catalyses the univalent reduction of molecular oxygen (O 2) to O 2. They are rapidly recruited to sites of infection and their response includes phagocytosis of the injurious element, the release of pre-formed granular enzymes and proteins and the de novo production of a range of potentially damaging but ephemeral, reactive oxygen species (ROS) such as superoxide anion (O 2 -). Neutrophils comprise one of the major cellular components of the innate immune system. By varying the length of pre-incubation with PAF it was demonstrated that this effect decreased as the duration of pre-incubation with PAF increased, and that PAF was enhancing PMA's effects rather than PMA enhancing PAF's effects. The PMA-induced respiratory burst function was further enhanced by pre-incubation with PAF, but not with TNF-α or LPS. In the absence of priming or activating agonists, ovine neutrophils displayed a low level of respiratory burst function which was not enhanced by either PAF, TNF-α, LPS or fMLP, but was significantly enhanced by PMA.
As well as for unstimulated neutrophils, these aspects were also characterised after incubation with a priming agonist (platelet activating factor, tumour necrosis factor alpha and lipopolysaccharides ) activating agonists (N-formylmethionyl-leucyl-phenylalanine and phorbol 12-myristate 13-acetate ) or a combination of a priming and an activating agonist. Aspects of ovine neutrophil respiratory burst function to be characterised were: i) the maximum rate of O 2 - generated ( V max) ii) the time taken to reach V max iii) the total amount of O 2 - generated during the reaction and iv) the duration of the reaction.