Abstract
BackgroundRespiratory burst function resulting in the release of reactive oxygen species such as superoxide anion (O2-) from neutrophils is one of the key mechanisms of the innate immune system, and maladaptive control of this mechanism is thought to play a pivotal role in the development of pathologies such as acute lung injury and sepsis. Ovine models of these pathologies are limited by the poor understanding of ovine neutrophil respiratory burst function.ResultsAspects of ovine neutrophil respiratory burst function to be characterised were: i) the maximum rate of O2- generated (Vmax); ii) the time taken to reach Vmax; iii) the total amount of O2- generated during the reaction; and iv) the duration of the reaction. As well as for unstimulated neutrophils, these aspects were also characterised after incubation with a priming agonist (platelet activating factor [PAF], tumour necrosis factor alpha [TNF-α] and lipopolysaccharides [LPS]) activating agonists (N-formylmethionyl-leucyl-phenylalanine [fMLP] and phorbol 12-myristate 13-acetate [PMA]) or a combination of a priming and an activating agonist. 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. The PMA-induced respiratory burst function was further enhanced by pre-incubation with PAF, but not with TNF-α or LPS. 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.ConclusionThis study successfully adapted a commonly used method of measuring human neutrophil respiratory burst function to characterise different aspects of ovine neutrophil respiratory burst function. This improved understanding of ovine neutrophils will facilitate the validitation of ovine biomedical models of human pathologies in which neutrophils have been implicated.
Highlights
Respiratory burst function resulting in the release of reactive oxygen species such as superoxide anion (O2-) from neutrophils is one of the key mechanisms of the innate immune system, and maladaptive control of this mechanism is thought to play a pivotal role in the development of pathologies such as acute lung injury and sepsis
The incubation periods and the concentrations used in this study demonstrated that neither Platelet Activating Factor (PAF), TNF-α, or LPS was capable of enhancing ovine neutrophil respiratory burst function in response to fMLP
This study demonstrated that there was a significant increase in Vmax for ovine neutrophils incubated with PAF followed by phorbol 12-myristate 13-acetate (PMA) or when they were added simultaneously but not when incubated with PMA followed by PAF, this did not correlate with any significant change in the total amount of molecular oxygen (O2)- released during the reactions
Summary
Respiratory burst function resulting in the release of reactive oxygen species such as superoxide anion (O2-) from neutrophils is one of the key mechanisms of the innate immune system, and maladaptive control of this mechanism is thought to play a pivotal role in the development of pathologies such as acute lung injury and sepsis. ROS are generated by a variety of intracellular mechanisms, the predominant mechanism, referred to as respiratory burst, is based upon the assembly and activation of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase which catalyses the univalent reduction of molecular oxygen (O2) to O2- [1,2,3,4] 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. 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
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