BACKGROUND
neutrophils comprise one of the major cellular components of the innate immune system.
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  such as superoxide anion   <cit> .
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  oxidase which then catalyses the univalent reduction of molecular oxygen  to o2-  <cit> .
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  and its most severe form, acute respiratory distress syndrome , as well as the multiple organ failure characteristic of sepsis.
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.
ovine models have been extensively used in the study of ali/ards  <cit>  and sepsis  <cit> , however they are limited by a lack of understanding of the ovine neutrophil which has been less well studied than its human counterpart.
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.
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  <cit> , some of which have been adapted to study ovine neutrophil respiratory burst function  <cit> .
photometric assays based upon the superoxide dismutase-  inhibitable reduction of cytochrome c have been well standardised for the measurement of human neutrophil respiratory burst function.
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  and n-formylmethionyl-leucyl-phenylalanine , and the maximum rate of o2- release  as the standard measure  <cit> .
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  <cit> , however differences in assay methodology, agonists, and in the units measured have hindered comparisons between the studies.
the relevance of results from these studies has also been hampered by the technical limitations of the assay protocols used.
stopping the cytochrome c reduction by cold-fixing the cells  <cit>  or simply by not taking any further readings  <cit>  means that these assays do not measure the reaction reaching its natural completion, and may bias the assay towards faster reacting agonists.
the technique of measuring sod-inhibitable reduction of cytochrome c has not been utilised to characterise the ovine neutrophil respiratory burst response to biologically relevant agonists such as lipopolysaccharides , the purified endotoxin from escherichia coli which is used in sheep models of sepsis  <cit> , and the inflammatory cytokine, tumour necrosis factor-alpha , which is released from monocytes and tissue macrophages in response to lps, paf and other agonists  <cit> .
priming, a process in which the response of neutrophils to an activating agonist is enhanced by prior exposure to a priming agonist, has been well-characterised in human neutrophils  <cit> , but with the exception of buchta's paper  <cit> , there has been little investigation into the effects that combinations of agonists have on ovine neutrophil respiratory burst function.
this paper aimed to take a standard human neutrophil respiratory burst assay  <cit>  and adapt it for the measurement of ovine neutrophil respiratory burst function.
this assay would also be adapted to measure not just the maximum rate of the reaction , but also the total amount of o2- generated during the reaction, the length of the reaction and the time taken to reach vmax thus allowing us to better characterise neutrophil respiratory burst function.
this modified assay could then be used to define changes in ovine neutrophil respiratory burst function in response to a range of agonists, and by using these agonists both individually and in combination, it was possible to determine if sheep neutrophils demonstrate a response analogous to the priming and activation demonstrated in human neutrophils.
RESULTS
neutrophil isolation
whole blood collected from sheep had an average neutrophil count of  <dig>  ±  <dig>  ×  <dig> cells/l .
the average neutrophil yield of the isolations was  <dig>  ±  <dig> % of starting numbers of neutrophils, with an average purity of  <dig>  ±  <dig> % neutrophils in the total leucocyte population, an average erythrocyte contamination of  <dig>  ±  <dig>  ×  <dig> cells/l, and an average platelet contamination of  <dig>  ±  <dig>  ×  <dig> cells/l.
assessment of respiratory burst stimulation by a single agonist
the respiratory burst function of isolated ovine neutrophils was assessed with either a priming or an activating agonist or with a control  in which buffer only was used, and these results are shown in table  <dig>  maximum rate  results are reported as nmoles o2- per  <dig> ×  <dig> neutrophils per minute, and results for total o2- are reported as nmoles o2- per  <dig> ×  <dig> neutrophils.
unstimulated neutrophils had a low level of respiratory burst function, with an average vmax of  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils/min  and an average total o2- of  <dig>  ±  <dig> .
this result indicates that the cells were not activated during isolation or testing, and that resting ovine neutrophils display low levels of respiratory burst function.
paf
tnf-α
lps
pma
fmlp
ovine neutrophils  were incubated either with buffer only  or one of a range of priming  and activating agents .
total assay length was  <dig> minutes.
parameters were measured as described in methods.
asterisks represent level of significance compared to unstimulated cells ; * p <  <dig> ; ** p <  <dig> ; *** p <  <dig> .
when incubated individually with ovine neutrophils each of the priming agonists: paf, tnf-α, and lps displayed low levels of respiratory burst function with vmax values of  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils/min,  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils/min, and  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils/min respectively; and with total o2- values of  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils,  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils, and  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils respectively.
this did not represent a significant increase in respiratory burst function compared to unstimulated ovine neutrophils for any of the agonists .
of the activating agonists phorbol 12-myristate 13-acetate  produced the strongest response with an average vmax of  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils/min and an average total o2- of  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils.
this represented a significant increase in respiratory burst function compared to unstimulated ovine neutrophils .
analyses of the time taken to reach vmax  and the length of the reaction  showed that the pma-induced respiratory burst was a slow reaction with a long duration.
the other activating agonist, fmlp, produced a weaker response with an average vmax of  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils/min and an average total o2- generated of  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils.
this did not represent a significant increase in respiratory burst function compared to unstimulated ovine neutrophils .
combinations of agonists
in order to investigate the ability of combinations of agonists to enhance the respiratory burst function of ovine neutrophils, cells were incubated firstly with a priming agonist before the subsequent addition of an activating agonist.
pma was added following a  <dig> minute incubation with either paf or tnf-α, or following a  <dig> minute incubation with lps , and fmlp was added after a  <dig> minute incubation with either paf or tnf-α, or after a  <dig> minute incubation with lps .
compared to ovine neutrophils that were incubated with pma alone , there was a significant increase in vmax  when the cells were first incubated with paf for  <dig> minutes prior to the subsequent addition of pma .
there was, however no significant change in total o2- generated .
analyses of the time taken to reach vmax  and the duration of the reaction  showed no significant change  when compared to pma alone.
the pma-induced respiratory burst response of ovine neutrophils showed no significant changes in either vmax or total o2- generated  when the cells were incubated with tnf-α for  <dig> minutes  or with lps for  <dig> minutes  relative to cells that received pma only.
there were also no significant changes  in the time taken to reach vmax or in the duration of the reaction relative to cells stimulated with pma alone .
the fmlp-induced respiratory burst response of ovine neutrophils showed no significant changes in either vmax or total o2- generated  when the cells were incubated with paf for  <dig> minutes , tnf-α for  <dig> minutes  or with lps for  <dig> minutes  relative to cells that received fmlp only .
there were also no significant changes  in the time taken to reach vmax or in the duration of the reaction relative to cells stimulated with fmlp alone .
enhanced respiratory burst function?
in order to investigate whether incubation of ovine neutrophils with paf causes an enhanced respiratory function following subsequent pma stimulation, the duration of incubation was varied and the order of addition was reversed  and results are shown in figure  <dig>
when ovine neutrophils were incubated with paf for  <dig> minutes prior to the addition of pma there was an average vmax of  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils/min  and an average total o2- generated of  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils .
when ovine neutrophils were incubated with both paf and pma simultaneously  there was an average vmax of  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils/min  and an average total o2- generated of  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils .
when the order of addition was reversed, and ovine neutrophils were incubated with pma for  <dig> minutes prior to the addition of paf there was an average vmax of  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils/min  and an average total o2- generated of  <dig>  ±  <dig>  nmol o2-/ <dig> ×  <dig> neutrophils .
discussion
the ability of neutrophils to destroy bacteria or other pathogens is dependant upon their ability to mount an effective respiratory burst response.
the respiratory burst function is commonly investigated by assays based upon the sod-inhibitable reduction of cytochrome c by o2-.
changes to the respiratory burst function of isolated human neutrophils induced by a range of priming and activating agonists has been well characterised, however the respiratory burst function of ovine neutrophils has been less well characterised.
there have been fewer reports of ovine neutrophil respiratory function, and these studies are limited due to either: the methodology used, the small number of agonists investigated, or the lack of investigation into combinations of agonists.
this study aimed to address these limitations by adapting a standardised method of investigating human neutrophil respiratory burst function to investigate the changes in ovine neutrophil respiratory burst function due to incubation with a range of agonists, both individually and in combination.
in previous studies of neutrophil respiratory burst function the maximum reaction rate or vmax has been the principal measure  <cit> .
the current study collected data on vmax, but has additionally collected data on the time taken to reach vmax, the total amount of o2- released during the reaction and the duration of the reaction.
this additional data allowed a more complete characterisation of the ovine neutrophil respiratory burst function.
the assay used in this study was run for a two hour period as compared to the  <dig> –  <dig> minute time period that previous studies were run for  <cit> .
this extra time proved to be important because it allowed the reactions to run to completion, and allowed sufficient time for the priming periods previously shown to be optimal for these agonists  <cit> .
the reaction duration and time taken to reach vmax data obtained in this study for pma-induced ovine neutrophil respiratory burst function  indicates that in buchta's study  <cit>  when these reactions had been stopped at  <dig> minutes, the reaction would not have been completed, and would not have reached vmax.
this would indicate that buchta's study may have underestimated the pma-induced ovine neutrophil respiratory burst function.
since the reactions in this study were allowed to run to completion, with regular readings taken throughout the assay, the data presented here is likely to be more accurate.
in this study the agonists considered to be priming agonists for human neutrophils  did not induce a significant change in ovine neutrophil respiratory burst function when added individually.
this result is consistent with those from studies of human neutrophil respiratory burst function showing that only some priming agonists are capable of inducing a significant response of their own, and even then this is only when they are added in high concentrations that may not be physiologically relevant  <cit> .
of the two agonists considered to be activating agonists for human neutrophils , when added individually only pma induced any significant change in ovine neutrophil respiratory burst function relative to unstimulated cells, with titrations showing that this response was dose-dependent .
the pathways by which pma and fmlp activate neutrophil respiratory burst function are different.
studies of human neutrophils have shown that pma stimulates the signalling element, protein kinase c  directly whereas fmlp does so indirectly by binding to g-protein linked receptors on the surface of the neutrophil that subsequently dissociate and activate multiple signalling pathways including mitogen -activated protein kinase  and lipid kinases, as well as phospholipases which produce the second messengers responsible for activation of pkc  <cit> .
in humans it has been shown that while some priming agonists cause an increase in fmlp receptor expression, the receptor-generated response is generally not the rate-limiting step in the priming and activation response and so this does not cause any increased response  <cit> .
previous reports have shown that fmlp has a negligible effect on ovine neutrophil respiratory burst function, and have hypothesised that this is due to either the low expression or the absence of receptors for this peptide on the surface of the cell  <cit> , so it is possible that this may be a reaction-limiting step in ovine neutrophils.
this study therefore investigated the possibility that incubation of ovine neutrophils with a priming agonist prior to the subsequent addition of fmlp would increase the expression of fmlp receptors to a level at which ovine neutrophil respiratory burst function to fmlp could be measured.
the incubation periods and the concentrations used in this study demonstrated that neither paf, tnf-α, or lps was capable of enhancing ovine neutrophil respiratory burst function in response to fmlp.
this provides evidence supporting the hypothesis that fmlp receptors are not expressed on the surface of ovine neutrophils, however to prove this hypothesis conclusively, ovine neutrophil surface expression studies using anti-fmlp receptor antibodies would be required.
studies into human neutrophil respiratory burst function have demonstrated that priming agonists act to increase the response to pma although the exact mechanisms by which they act remains uncertain and are thought to vary for different agonists.
paf is thought to act by enhancing pkc activity via a calcium-dependent mechanism  <cit>  whereas tnf-α priming has been demonstrated to be independent of extracellular calcium  <cit>  and priming of pma-induced respiratory burst activity by lps has not yet been demonstrated.
based upon these differences in priming mechanisms for the priming agonists used in this study, the finding that only paf appeared to affect the pma-induced respiratory burst function of ovine neutrophils was not unexpected nor was it novel.
this study demonstrated that there was a significant increase in vmax for ovine neutrophils incubated with paf followed by pma or when they were added simultaneously but not when incubated with pma followed by paf, however this did not correlate with any significant change in the total amount of o2- released during the reactions.
in other words, the total amount of o2- released in response to pma stimulation remained constant regardless of paf co-stimulation.
it would make sense that if the total amount of o2- released during the reaction remains constant while the vmax increases, then the duration of the reaction should decrease, however this was found to be the case only when paf and pma were added simultaneously and not when there pma was added after a  <dig> or  <dig> minute incubation with paf.
a possible explanation for this is that the magnitude of paf-priming of the pma-induced respiratory burst function of ovine neutrophils is at its maximum either immediately or shortly after paf addition, and then decreases rapidly over the  <dig> minute time period tested here.
this study has provided evidence that paf is capable of having an enhanced effect upon the subsequent pma-induced respiratory burst function of ovine neutrophils.
firstly the vmax values observed for ovine neutrophils stimulated by both paf and pma was not just greater than that of either agonist alone but also was greater than the combined vmax values obtained by simply adding together the effects of each agonist alone.
additionally, the vmax data presented here indicates that paf is enhancing pma-induced respiratory burst function rather than pma enhancing paf-induced respiratory burst function.
further evidence, such as that from blocking studies using pkc inhibitors or paf antagonists, would be required to better elucidate the relationship between paf and pma and to ascertain whether this is analogous to the priming and activation described for human neutrophils.
these experiments have demonstrated that lps and tnf-α did not enhance either fmlp- or pma-induced ovine neutrophil respiratory burst function.
this is not unexpected because even for human neutrophils the pma-induced respiratory burst function has not been demonstrated to be primed by lps, and the evidence from this and other studies  <cit>  indicates that there is no significant fmlp-induced ovine neutrophil respiratory burst function.
so while this study has not been able to demonstrate priming by either lps or tnf-α, further studies using alternative activating agonists may demonstrate whether lps and tnf-α are capable of priming ovine neutrophil respiratory burst function.
alternatively the investigation of specific surface molecule markers of neutrophil priming and activation would provide a measure of ovine neutrophil priming and activation independent of respiratory burst function, and this different technique may also demonstrate whether lps and tnf-α are capable of priming ovine neutrophils.
studies to define the role of endogenous paf in ovine biomedical models have trialled paf-receptor antagonists with mixed results  <cit> , so an alternative method of assessing this would be beneficial.
while pma is not a biologically relevant agonist, its characterisation here and in other studies  <cit>  has demonstrated that it is useful as an in-vitro activating agonist for the ovine neutrophil respiratory burst function.
the possibility that paf may be capable of enhancing or priming the ovine neutrophil respiratory burst function for subsequent activation by pma is an important finding because it means that it may be possible to investigate endogenous paf priming of ovine neutrophils by isolating them and stimulating them with pma.
it has been demonstrated in humans that neutrophils isolated from injured or sick patients display a reduced respiratory burst function in response to priming and activating agonists as compared to healthy controls  <cit> , so by using the assay described in this study with the agonists paf and pma, it may be possible to investigate whether neutrophils isolated from sick or injured sheep display a similar reduced respiratory burst function.
this would greatly enhance the relevance of ovine biomedical models of human pathologies such as ali/ards and sepsis.
another way to enhance these models would be to directly compare the expression of key markers linked to neutrophil priming and activation on the surfaces of both human and ovine neutrophils.
CONCLUSIONS
this study has described an assay based upon the sod-inhibitable reduction of cytochrome c to measure several aspects of ovine neutrophil respiratory burst function.
the actions of several well-characterised human neutrophil priming and activating agonists were investigated both individually and in combination.
it was shown that the priming agonists paf, tnf-α and lps did not on their own induce the ovine neutrophil respiratory burst.
it was demonstrated that fmlp, both on its own, and after priming by either paf, tnf-α and lps, did not induce the ovine neutrophil respiratory burst.
pma, however was capable of inducing the ovine neutrophil respiratory burst by itself, and this response was significantly enhanced by pre-incubation with paf, with an increase in vmax and a decrease in the duration of the reaction.
these changes indicate that paf in some way, possibly analogous to the priming described for human neutrophils, enhances the pma-induced ovine neutrophil respiratory burst.
the findings of this study advance our knowledge and understanding of ovine neutrophils, and improve the validity of ovine biomedical models of human pathologies such as ali/ards and sepsis in which neutrophils have been implicated.

 neutrophils comprise one of the major cellular components of the innate immune system. 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 such as superoxide anion. 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 oxidase which then catalyses the univalent reduction of molecular oxygen to o2-. 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 and its most severe form, acute respiratory distress syndrome, as well as the multiple organ failure characteristic of sepsis. 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. 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. 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. 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. photometric assays based upon the superoxide dismutase- inhibitable reduction of cytochrome c have been well standardised for the measurement of human neutrophil respiratory burst function. 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 and n-formylmethionyl-leucyl-phenylalanine, and the maximum rate of o2- release as the standard measure. 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. the relevance of results from these studies has also been hampered by the technical limitations of the assay protocols used. stopping the cytochrome c reduction by cold-fixing the cells or simply by not taking any further readings means that these assays do not measure the reaction reaching its natural completion, and may bias the assay towards faster reacting agonists. the technique of measuring sod-inhibitable reduction of cytochrome c has not been utilised to characterise the ovine neutrophil respiratory burst response to biologically relevant agonists such as lipopolysaccharides, the purified endotoxin from escherichia coli which is used in sheep models of sepsis, and the inflammatory cytokine, tumour necrosis factor-alpha, which is released from monocytes and tissue macrophages in response to lps, paf and other agonists. priming, a process in which the response of neutrophils to an activating agonist is enhanced by prior exposure to a priming agonist, has been well-characterised in human neutrophils, but with the exception of buchta's paper, there has been little investigation into the effects that combinations of agonists have on ovine neutrophil respiratory burst function. this paper aimed to take a standard human neutrophil respiratory burst assay and adapt it for the measurement of ovine neutrophil respiratory burst function. this assay would also be adapted to measure not just the maximum rate of the reaction, but also the total amount of o2- generated during the reaction, the length of the reaction and the time taken to reach vmax thus allowing us to better characterise neutrophil respiratory burst function. this modified assay could then be used to define changes in ovine neutrophil respiratory burst function in response to a range of agonists, and by using these agonists both individually and in combination, it was possible to determine if sheep neutrophils demonstrate a response analogous to the priming and activation demonstrated in human neutrophils.  neutrophil isolation whole blood collected from sheep had an average neutrophil count of ± × cells/l. the average neutrophil yield of the isolations was ± % of starting numbers of neutrophils, with an average purity of ± % neutrophils in the total leucocyte population, an average erythrocyte contamination of ± × cells/l, and an average platelet contamination of ± × cells/l. assessment of respiratory burst stimulation by a single agonist the respiratory burst function of isolated ovine neutrophils was assessed with either a priming or an activating agonist or with a control in which buffer only was used, and these results are shown in table maximum rate results are reported as nmoles o2- per × neutrophils per minute, and results for total o2- are reported as nmoles o2- per × neutrophils. unstimulated neutrophils had a low level of respiratory burst function, with an average vmax of ± nmol o2-/ × neutrophils/min and an average total o2- of ±. this result indicates that the cells were not activated during isolation or testing, and that resting ovine neutrophils display low levels of respiratory burst function. paf tnf-α lps pma fmlp ovine neutrophils were incubated either with buffer only or one of a range of priming and activating agents. total assay length was minutes. parameters were measured as described in methods. asterisks represent level of significance compared to unstimulated cells; * p <; ** p <; *** p <. when incubated individually with ovine neutrophils each of the priming agonists: paf, tnf-α, and lps displayed low levels of respiratory burst function with vmax values of ± nmol o2-/ × neutrophils/min, ± nmol o2-/ × neutrophils/min, and ± nmol o2-/ × neutrophils/min respectively; and with total o2- values of ± nmol o2-/ × neutrophils, ± nmol o2-/ × neutrophils, and ± nmol o2-/ × neutrophils respectively. this did not represent a significant increase in respiratory burst function compared to unstimulated ovine neutrophils for any of the agonists. of the activating agonists phorbol 12-myristate 13-acetate produced the strongest response with an average vmax of ± nmol o2-/ × neutrophils/min and an average total o2- of ± nmol o2-/ × neutrophils. this represented a significant increase in respiratory burst function compared to unstimulated ovine neutrophils. analyses of the time taken to reach vmax and the length of the reaction showed that the pma-induced respiratory burst was a slow reaction with a long duration. the other activating agonist, fmlp, produced a weaker response with an average vmax of ± nmol o2-/ × neutrophils/min and an average total o2- generated of ± nmol o2-/ × neutrophils. this did not represent a significant increase in respiratory burst function compared to unstimulated ovine neutrophils. combinations of agonists in order to investigate the ability of combinations of agonists to enhance the respiratory burst function of ovine neutrophils, cells were incubated firstly with a priming agonist before the subsequent addition of an activating agonist. pma was added following a minute incubation with either paf or tnf-α, or following a minute incubation with lps, and fmlp was added after a minute incubation with either paf or tnf-α, or after a minute incubation with lps. compared to ovine neutrophils that were incubated with pma alone, there was a significant increase in vmax when the cells were first incubated with paf for minutes prior to the subsequent addition of pma. there was, however no significant change in total o2- generated. analyses of the time taken to reach vmax and the duration of the reaction showed no significant change when compared to pma alone. the pma-induced respiratory burst response of ovine neutrophils showed no significant changes in either vmax or total o2- generated when the cells were incubated with tnf-α for minutes or with lps for minutes relative to cells that received pma only. there were also no significant changes in the time taken to reach vmax or in the duration of the reaction relative to cells stimulated with pma alone. the fmlp-induced respiratory burst response of ovine neutrophils showed no significant changes in either vmax or total o2- generated when the cells were incubated with paf for minutes, tnf-α for minutes or with lps for minutes relative to cells that received fmlp only. there were also no significant changes in the time taken to reach vmax or in the duration of the reaction relative to cells stimulated with fmlp alone. enhanced respiratory burst function? in order to investigate whether incubation of ovine neutrophils with paf causes an enhanced respiratory function following subsequent pma stimulation, the duration of incubation was varied and the order of addition was reversed and results are shown in figure when ovine neutrophils were incubated with paf for minutes prior to the addition of pma there was an average vmax of ± nmol o2-/ × neutrophils/min and an average total o2- generated of ± nmol o2-/ × neutrophils. when ovine neutrophils were incubated with both paf and pma simultaneously there was an average vmax of ± nmol o2-/ × neutrophils/min and an average total o2- generated of ± nmol o2-/ × neutrophils. when the order of addition was reversed, and ovine neutrophils were incubated with pma for minutes prior to the addition of paf there was an average vmax of ± nmol o2-/ × neutrophils/min and an average total o2- generated of ± nmol o2-/ × neutrophils. discussion the ability of neutrophils to destroy bacteria or other pathogens is dependant upon their ability to mount an effective respiratory burst response. the respiratory burst function is commonly investigated by assays based upon the sod-inhibitable reduction of cytochrome c by o2-. changes to the respiratory burst function of isolated human neutrophils induced by a range of priming and activating agonists has been well characterised, however the respiratory burst function of ovine neutrophils has been less well characterised. there have been fewer reports of ovine neutrophil respiratory function, and these studies are limited due to either: the methodology used, the small number of agonists investigated, or the lack of investigation into combinations of agonists. this study aimed to address these limitations by adapting a standardised method of investigating human neutrophil respiratory burst function to investigate the changes in ovine neutrophil respiratory burst function due to incubation with a range of agonists, both individually and in combination. in previous studies of neutrophil respiratory burst function the maximum reaction rate or vmax has been the principal measure. the current study collected data on vmax, but has additionally collected data on the time taken to reach vmax, the total amount of o2- released during the reaction and the duration of the reaction. this additional data allowed a more complete characterisation of the ovine neutrophil respiratory burst function. the assay used in this study was run for a two hour period as compared to the – minute time period that previous studies were run for. this extra time proved to be important because it allowed the reactions to run to completion, and allowed sufficient time for the priming periods previously shown to be optimal for these agonists. the reaction duration and time taken to reach vmax data obtained in this study for pma-induced ovine neutrophil respiratory burst function indicates that in buchta's study when these reactions had been stopped at minutes, the reaction would not have been completed, and would not have reached vmax. this would indicate that buchta's study may have underestimated the pma-induced ovine neutrophil respiratory burst function. since the reactions in this study were allowed to run to completion, with regular readings taken throughout the assay, the data presented here is likely to be more accurate. in this study the agonists considered to be priming agonists for human neutrophils did not induce a significant change in ovine neutrophil respiratory burst function when added individually. this result is consistent with those from studies of human neutrophil respiratory burst function showing that only some priming agonists are capable of inducing a significant response of their own, and even then this is only when they are added in high concentrations that may not be physiologically relevant. of the two agonists considered to be activating agonists for human neutrophils, when added individually only pma induced any significant change in ovine neutrophil respiratory burst function relative to unstimulated cells, with titrations showing that this response was dose-dependent. the pathways by which pma and fmlp activate neutrophil respiratory burst function are different. studies of human neutrophils have shown that pma stimulates the signalling element, protein kinase c directly whereas fmlp does so indirectly by binding to g-protein linked receptors on the surface of the neutrophil that subsequently dissociate and activate multiple signalling pathways including mitogen -activated protein kinase and lipid kinases, as well as phospholipases which produce the second messengers responsible for activation of pkc. in humans it has been shown that while some priming agonists cause an increase in fmlp receptor expression, the receptor-generated response is generally not the rate-limiting step in the priming and activation response and so this does not cause any increased response. previous reports have shown that fmlp has a negligible effect on ovine neutrophil respiratory burst function, and have hypothesised that this is due to either the low expression or the absence of receptors for this peptide on the surface of the cell, so it is possible that this may be a reaction-limiting step in ovine neutrophils. this study therefore investigated the possibility that incubation of ovine neutrophils with a priming agonist prior to the subsequent addition of fmlp would increase the expression of fmlp receptors to a level at which ovine neutrophil respiratory burst function to fmlp could be measured. the incubation periods and the concentrations used in this study demonstrated that neither paf, tnf-α, or lps was capable of enhancing ovine neutrophil respiratory burst function in response to fmlp. this provides evidence supporting the hypothesis that fmlp receptors are not expressed on the surface of ovine neutrophils, however to prove this hypothesis conclusively, ovine neutrophil surface expression studies using anti-fmlp receptor antibodies would be required. studies into human neutrophil respiratory burst function have demonstrated that priming agonists act to increase the response to pma although the exact mechanisms by which they act remains uncertain and are thought to vary for different agonists. paf is thought to act by enhancing pkc activity via a calcium-dependent mechanism whereas tnf-α priming has been demonstrated to be independent of extracellular calcium and priming of pma-induced respiratory burst activity by lps has not yet been demonstrated. based upon these differences in priming mechanisms for the priming agonists used in this study, the finding that only paf appeared to affect the pma-induced respiratory burst function of ovine neutrophils was not unexpected nor was it novel. this study demonstrated that there was a significant increase in vmax for ovine neutrophils incubated with paf followed by pma or when they were added simultaneously but not when incubated with pma followed by paf, however this did not correlate with any significant change in the total amount of o2- released during the reactions. in other words, the total amount of o2- released in response to pma stimulation remained constant regardless of paf co-stimulation. it would make sense that if the total amount of o2- released during the reaction remains constant while the vmax increases, then the duration of the reaction should decrease, however this was found to be the case only when paf and pma were added simultaneously and not when there pma was added after a or minute incubation with paf. a possible explanation for this is that the magnitude of paf-priming of the pma-induced respiratory burst function of ovine neutrophils is at its maximum either immediately or shortly after paf addition, and then decreases rapidly over the minute time period tested here. this study has provided evidence that paf is capable of having an enhanced effect upon the subsequent pma-induced respiratory burst function of ovine neutrophils. firstly the vmax values observed for ovine neutrophils stimulated by both paf and pma was not just greater than that of either agonist alone but also was greater than the combined vmax values obtained by simply adding together the effects of each agonist alone. additionally, the vmax data presented here indicates that paf is enhancing pma-induced respiratory burst function rather than pma enhancing paf-induced respiratory burst function. further evidence, such as that from blocking studies using pkc inhibitors or paf antagonists, would be required to better elucidate the relationship between paf and pma and to ascertain whether this is analogous to the priming and activation described for human neutrophils. these experiments have demonstrated that lps and tnf-α did not enhance either fmlp- or pma-induced ovine neutrophil respiratory burst function. this is not unexpected because even for human neutrophils the pma-induced respiratory burst function has not been demonstrated to be primed by lps, and the evidence from this and other studies indicates that there is no significant fmlp-induced ovine neutrophil respiratory burst function. so while this study has not been able to demonstrate priming by either lps or tnf-α, further studies using alternative activating agonists may demonstrate whether lps and tnf-α are capable of priming ovine neutrophil respiratory burst function. alternatively the investigation of specific surface molecule markers of neutrophil priming and activation would provide a measure of ovine neutrophil priming and activation independent of respiratory burst function, and this different technique may also demonstrate whether lps and tnf-α are capable of priming ovine neutrophils. studies to define the role of endogenous paf in ovine biomedical models have trialled paf-receptor antagonists with mixed results, so an alternative method of assessing this would be beneficial. while pma is not a biologically relevant agonist, its characterisation here and in other studies has demonstrated that it is useful as an in-vitro activating agonist for the ovine neutrophil respiratory burst function. the possibility that paf may be capable of enhancing or priming the ovine neutrophil respiratory burst function for subsequent activation by pma is an important finding because it means that it may be possible to investigate endogenous paf priming of ovine neutrophils by isolating them and stimulating them with pma. it has been demonstrated in humans that neutrophils isolated from injured or sick patients display a reduced respiratory burst function in response to priming and activating agonists as compared to healthy controls, so by using the assay described in this study with the agonists paf and pma, it may be possible to investigate whether neutrophils isolated from sick or injured sheep display a similar reduced respiratory burst function. this would greatly enhance the relevance of ovine biomedical models of human pathologies such as ali/ards and sepsis. another way to enhance these models would be to directly compare the expression of key markers linked to neutrophil priming and activation on the surfaces of both human and ovine neutrophils.  this study has described an assay based upon the sod-inhibitable reduction of cytochrome c to measure several aspects of ovine neutrophil respiratory burst function. the actions of several well-characterised human neutrophil priming and activating agonists were investigated both individually and in combination. it was shown that the priming agonists paf, tnf-α and lps did not on their own induce the ovine neutrophil respiratory burst. it was demonstrated that fmlp, both on its own, and after priming by either paf, tnf-α and lps, did not induce the ovine neutrophil respiratory burst. pma, however was capable of inducing the ovine neutrophil respiratory burst by itself, and this response was significantly enhanced by pre-incubation with paf, with an increase in vmax and a decrease in the duration of the reaction. these changes indicate that paf in some way, possibly analogous to the priming described for human neutrophils, enhances the pma-induced ovine neutrophil respiratory burst. the findings of this study advance our knowledge and understanding of ovine neutrophils, and improve the validity of ovine biomedical models of human pathologies such as ali/ards and sepsis in which neutrophils have been implicated. 
ovine models of these pathologies are limited by the poor understanding of ovine neutrophil respiratory burst function. 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. this improved understanding of ovine neutrophils will facilitate the validitation of ovine biomedical models of human pathologies in which neutrophils have been implicated. respiratory burst function resulting in the release of reactive oxygen species such as superoxide anion 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. aspects of ovine neutrophil respiratory burst function to be characterised were: i) the maximum rate of o2- generated; ii) the time taken to reach vmax; iii) the total amount of o2- generated during the reaction; and iv) the duration of the reaction. this study successfully adapted a commonly used method of measuring human neutrophil respiratory burst function to characterise different aspects of ovine neutrophil respiratory burst function.
 respiratory burst function resulting in the release of reactive oxygen species such as superoxide anion 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.  aspects of ovine neutrophil respiratory burst function to be characterised were: i) the maximum rate of o2- generated; 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 activating agonists 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.  this 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. 