Equine platelets inhibit E. coli growth and can be activated by bacterial lipopolysaccharide and lipoteichoic acid although superoxide anion production does not occur and platelet activation is not associated with enhanced production by neutrophils.

Activated platelets can contribute to host defense through release of products with bactericidal actions such as antimicrobial peptides and reactive oxygen species (ROS), as well as by forming heterotypic aggregates with neutrophils and enhancing their antimicrobial properties. Whilst release of vasoactive mediators from equine platelets in response to stimuli including bacterial lipopolysaccharide (LPS) has been documented, neither ROS production, nor the effects of activated platelets on equine neutrophil ROS production, have been reported. This study first sought evidence that activated equine platelets inhibit bacterial growth. Platelet superoxide production in response to stimuli including Escherichia coli-derived LPS and lipoteichoic acid (LTA) from Staphylococcus aureus was then determined. The ability of LPS and LTA to up-regulate platelet P-selectin expression and induce platelet-neutrophil aggregate formation was investigated and the effect of co-incubating activated platelets with neutrophils on superoxide production measured. Growth of E. coli was inhibited in a time-dependent manner, and to a similar extent, by addition of platelet rich plasma (PRP) or platelet poor plasma (PPP) obtained by centrifugation of PRP. Activation of platelets in PRP by addition of thrombin led to a significant increase in the inhibitory action between 0.5 and 2h. Although phorbol myristate acetate (PMA) caused superoxide production by equine platelets in a protein kinase C-dependent manner, thrombin, platelet activating factor (PAF), LPS, LTA and formyl-methionyl-leucyl phenylalanine (FMLP) were without effect. LPS and LTA did induce platelet activation, measured as an increase in P-selectin expression (% positive cells: 17±3 (un-stimulated); 63±6 (1µg/ml LPS); 64±6 (1µg/ml LTA); n=5) but not platelet superoxide production or heterotypic aggregate formation. Co-incubation of activated platelets with neutrophils did not increase neutrophil superoxide production. This study has demonstrated for the first time that when activated, equine platelets, like those of other species, are capable of releasing ROS that could assist in bacterial killing. However, the findings suggest that neither superoxide production by platelets nor enhancement of production by neutrophils is likely to play a significant role. Nevertheless, as has been reported in man, equine PPP and PRP did inhibit E. coli growth in vitro, and addition of thrombin significantly increased the inhibitory effect of PRP. This suggests that products released from activated platelets could contribute to antimicrobial activity in the horse. The factors in equine plasma and released by activated platelets that are responsible for inhibiting bacterial growth have yet to be determined.

Stimulus-dependent release of tissue-regenerating factors by equine platelets.

REASONS FOR PERFORMING THE STUDY: Platelet-rich plasma (PRP) is increasingly used for treatment of orthopaedic injuries. However, the effects of different stimuli on the release pattern of regenerative and proinflammatory factors from equine platelets are largely unknown and an optimal treatment protocol remains to be established. OBJECTIVES: The aim of this study was to identify a stimulus that enhanced release of histopromotive factors (platelet-derived growth factor BB [PDGF] and transforming growth factor 1ß[TGF]) without causing concurrent release of a proinflammatory mediator (CCL5). METHODS: Washed platelets were prepared from 6 healthy ponies and release of growth factors and CCL5 measured using commercially available ELISAs for human proteins following incubation with or without thrombin, chitosan or equine recombinant tumour necrosis factor (erTNF) over 24 h and subsequently over 96 h. Additionally, noncoagulated samples were analysed. RESULTS: Regardless of whether a stimulus was present or what stimulus was used, PDGF and TGF release was maximal by 0.5-1 h when clot formation took place and very little release was observed after 24 h. Growth factor release was minimal in noncoagulated samples. In contrast, CCL5 release was not associated with coagulation and appeared to persist for much longer. High concentrations of erTNF caused significantly greater release of CCL5 at 6 h than any other stimulus tested. CONCLUSIONS: Growth factor release from equine platelets is dependent on coagulation but independent of the initiating stimulus, and is accompanied by more sustained release of proinflammatory mediators. POTENTIAL RELEVANCE: Supernatants collected from coagulated platelets could be an alternative treatment to PRP.

PKC isoenzymes in equine platelets and stimulus induced activation.

Protein kinase C (PKC) is an important regulator of platelet activation and different isoenzymes can play positive and negative regulatory roles. The PKC isoenzymes expressed in equine platelets have not been documented but pharmacological inhibition has suggested a role for PKC delta (δ) in modulating responsiveness to platelet activating factor (PAF) (Brooks et al., 2009). Here the PKC isoenzyme profile in equine platelets has been characterised and PKCδ activation by PAF investigated. Platelet lysates were probed by Western blotting using a panel of antibodies against individual PKC isoenzymes. PKCδ and eight other isoenzymes were identified, namely classical PKCs alpha (α), beta (ß), (both ßI and ßII) and gamma (γ), the novel PKCs epsilon (ɛ), eta (η) and theta (θ) and atypical PKC zeta (ζ). Having shown PKCδ to be present, a method was developed to measure PAF-induced isoenzyme translocation by preparing cytosolic and membrane fractions from digitonin permeabilised platelets. Phorbol 12-myristate 13-acetate (PMA) was shown to cause translocation of PKCδ to the membrane within 5s. PAF also caused PKCδ translocation although the response occurred more slowly; a significant, 7.6 ± 1.2 fold, increase in band density compared to unstimulated platelets was observed at 15 min; p=0.036, n=3. These data support a role for PKCδ in regulating PAF-induced functional responses in equine platelets.

Endotoxin-induced HIF-1alpha stabilisation in equine endothelial cells: synergistic action with hypoxia.

OBJECTIVE AND DESIGN: Hypoxia may enhance the deleterious effects of lipopolysaccharide (LPS) in the endotoxaemic horse. This study has examined some of the actions of LPS and hypoxia, alone and in combination, on cultured equine digital vein endothelial cells (EDVEC) and the signalling molecules involved. METHODS: EDVEC were exposed to LPS, 5% O(2) and LPS then 5% O(2) for up to 24 h. HIF-1alpha stabilisation, neutrophil adhesion and EDVEC permeability were assessed by immunoblotting, measurement of myeloperoxidase and movement of FITC-dextran, respectively. Pharmacological inhibitors were used to assess the roles of p38 MAPK and HIF-1alpha. RESULTS: LPS and hypoxia significantly increased HIF-1alpha stabilisation, neutrophil adhesion and EDVEC permeability and the effects of the two stimuli in combination on HIF-1alpha stabilisation and neutrophil adhesion were more than additive. The effect of LPS, but not 5% O(2), on neutrophil adherence required activation of p38 MAPK, whereas EDVEC permeability in response to both stimuli was dependent on p38 MAPK and HIF-1alpha. CONCLUSIONS: Exposure of EDVEC to LPS prior to induction of hypoxia up-regulates responses that may enhance LPS-induced tissue damage in the endotoxaemic horse. Inhibitors of p38 MAPK or HIF-1alpha could reduce such unwanted effects.

Neutrophil and platelet activation in equine recurrent airway obstruction is associated with increased neutrophil CD13 expression, but not platelet CD41/61 and CD62P or neutrophil-platelet aggregate formation.

Recurrent airway obstruction (RAO) in mature horses is characterized by reversible airway obstruction and neutrophilic inflammation; there is also functional activation of circulating platelets and neutrophils. This study was undertaken to determine if changes in activation marker expression and heterotypic aggregate formation can be used as an indicator of this increased functional responsiveness. In vitro conditions for flow cytometric measurement of CD13, CD41/61 and CD62P expression on activated cells and heterotypic aggregate formation were established. Values were then compared before and after antigen challenge of RAO and healthy horses. Platelet adhesion to serum-coated plastic was measured as a functional marker of platelet activation. In vitro activation resulted in increased expression of neutrophil CD13 and platelet CD41/61 and CD62P. Activation of both cell types caused a significant increase in neutrophil-platelet aggregates. In horses with RAO, but not controls, there was a significant increase in the percentage of CD13 positive neutrophils at 10h and 24h and in the mean fluorescence intensity at 10h. This was accompanied at 24h by an increased mean platelet side scatter and thrombin-stimulated platelet adhesion. In conclusion, CD13 expression can be used as an indicator of equine neutrophil activation both in vitro and in vivo. Equine platelet activation in vitro can be detected by measuring CD41/61 or CD62P expression, and PAF-activated platelets and neutrophils form aggregates. However, despite evidence of circulating platelet activation, neither a change in expression of platelet activation markers, nor heterotypic aggregate aggregate formation could be detected.

Regulation of platelet activating factor-induced equine platelet activation by intracellular kinases.

Lipopolysaccharide (LPS) can activate equine platelets directly or indirectly, via leukocyte-derived platelet activating factor (PAF). Thromboxane (Tx) production by LPS-stimulated equine platelets requires p38 MAPK and this kinase has been suggested as a therapeutic target in endotoxaemia. The present study has utilised selective inhibitors to investigate the role of p38 MAPK and two other kinases, phosphatidylinositol-3 kinase (PI3K) and protein kinase C (PKC), in regulating PAF-induced Tx production, aggregation and 5-HT release in equine platelets, and the modification of these responses by LPS. LPS enhanced PAF-induced 5-HT release, an effect that was reduced by the p38 MAPK inhibitor, SB203580 (60 +/- 8% reduction; n = 6). SB203580 did not affect responses to PAF alone; whereas inhibition of PKC reduced PAF-induced 5-HT release, Tx production and aggregation (maximal inhibition by the PKCdelta inhibitor, rottlerin: 69 +/- 13%, 63 +/- 14% and 97 +/- 1%, respectively; n = 6). Wortmannin and LY249002, which inhibit PI3K, also caused significant inhibition of PAF-induced aggregation (maximal inhibition 78 +/- 3% and 88 +/- 2%, respectively; n = 6). These data suggest that inhibition of platelet p38 MAPK may be of benefit in equine endotoxaemia by counteracting some of the effects of LPS. However, detrimental effects of platelet activation mediated by PAF and not enhanced by LPS are unlikely to be markedly affected.

Role of intracellular kinases in the regulation of equine eosinophil migration and actin polymerization.

Inappropriately activated eosinophils can contribute to disease pathogenesis and intracellular signalling pathways that regulate functional responses may represent a therapeutic target. Little is known about intracellular signalling in equine eosinophils and this study examined the role of phospholipase C (PLC) and a range of protein kinases on responses to histamine and CCL11. Histamine (10(-4) M) or CCL11 (5.6 x 10(-9) M)-induced actin polymerization, migration and superoxide production by eosinophils from healthy horses were compared in the presence and absence of selective kinase inhibitors. Inhibition of phosphatidylinositol-3 kinase (PI3K) significantly reduced the response in each assay. In contrast, whilst inhibition of PLC decreased actin polymerization and superoxide production, an increase in migration was observed; the latter effect was also seen when protein kinase C (PKC) was inhibited. With the exception of histamine-induced migration, which was significantly reduced by blocking extracellular regulated kinase (ERK)1/2, activation of ERK1/2, p38 MAPK and tyrosine kinase did not appear to play an important role in the responses studied. These results suggest that equine eosinophil activation by histamine and CCL11 is mediated through PI3K. Whilst PLC activation is required for actin polymerization and superoxide production, migration may be negatively regulated by PLC and PKC. These kinases represent potential targets for modulating eosinophil activation by multiple stimuli.

Platelet activation in ponies with airway inflammation.

REASON FOR PERFORMING STUDY: Platelet activation occurs in human obstructive airway diseases and in laboratory animal models. However, there is limited evidence that platelets may be involved in equine recurrent airway obstruction (RAO) and other inflammatory diseases. This study investigated whether platelet activation also occurred in RAO. HYPOTHESIS: Platelet function is altered in ponies with active RAO. This alteration can be detected ex vivo by measuring platelet adhesion. METHODS: An in vitro platelet adhesion assay measuring acid phosphatase (AcP) activity colorimetrically was adapted for use with equine platelets and responses to selected agonists were established. Platelet adhesion and aggregation was evaluated in vitro on platelets isolated from 6 ponies with RAO before, during and after a 7 h natural antigen challenge. Three ponies with no history of airway disease were also studied. RESULTS: Adhesion of equine platelets to serum coated plastic was detected at concentrations of 10-100 radicaló 10(9)/l. Adhesion increased in response to stimulation with platelet activating factor and thrombin, but not equine interleukin 8. Prior to the antigen challenge, adhesion of nonstimulated platelets was low and increased significantly (P<0.05) 24 h after initiation of the challenge in RAOs, but not in the normal animals. No changes in platelet aggregation were noted in either group. CONCLUSIONS: The described assay offers an alternative method to evaluate platelet function in healthy and diseased horses and can detect changes not observed using a classic aggregation assay. Circulating platelets are activated 24 h after antigen challenge of ponies with RAO and may play a role in pulmonary inflammation and/or the pathophysiology of RAO. POTENTIAL RELEVANCE: Investigating platelet function in RAO and airway inflammation may reveal new aspects of the pathogenesis of inflammatory lung disease in the horse.

Endotoxin-induced activation of equine platelets: evidence for direct activation of p38 MAPK pathways and vasoactive mediator production.

OBJECTIVE AND DESIGN: The aim of this study was to determine the effects of endotoxin on p38 MAPK activation in equine platelets and leukocytes in vivo and in vitro and its role in thromboxane (Tx) production with reference to equine endotoxaemia. METHODS: Six adult Thoroughbred horses were used for in vivo infusion studies and separate in vitro studies. For in vivo studies, following collection of a pre-infusion sample, horses were infused with E. Coli O55:B5 LPS (30 ng/kg; 30 min) during and after which platelets were harvested. For in vitro studies isolated platelets and leukocytes were exposed to LPS (10 pg/ml-1 microg/ml). p38 MAPK activity was assessed by SDS-PAGE followed by immunoblotting. TxA2 release was measured by radioimmunoassay. RESULTS: LPS infusion caused increased phospho-p38 MAPK in equine platelets and leukocytes (1492 +/- 486 % and 83 +/- 45 above basal, respectively) from 10 min after the start of the infusion, which returned to basal by 60 min. In vitro, platelets were 1,000 times more sensitive to LPS than leukocytes in terms of both TxA2 production (EC50 66 pg/ml versus 110 ng/ml, respectively) and p38 MAPK phosphorylation (EC50 11.1 +/- 2 pg/ml versus 14.8 +/- 4 ng/ml, respectively). p38 MAPK inhibitors SB203580 and PD169316 attenuated LPS-induced TxA2 release in platelets, but not leukocytes. CONCLUSIONS: In vivo, LPS stimulates TxA2 production and p38 MAPK phosphorylation in equine platelets and leukocytes at a concentration within a similar range to those reported in clinical endotoxaemia. These data suggest that LPS-induced eicosanoid production in the early phase of clinical endotoxaemia may involve direct effects of LPS upon platelets, mediated via activation of p38 MAPK.

Platelets in equine recurrent airway obstruction.

Platelets contribute to the pathogenesis of human allergic airway disease. The aim of this study was to compare platelet activating factor (PAF)-induced platelet aggregation and thromboxane (Tx) production, plasma Tx and 5-hydroxytryptamine (5-HT) in ponies with recurrent airway obstruction (RAO), an hypersensitivity to inhaled antigens, and normal ponies, before and after antigen exposure. Plasma 5-HT was significantly higher in ponies with RAO but was not further increased by antigen challenge. There was no difference between PAF-induced platelet aggregation or Tx production, or in plasma Tx before or after challenge. These data suggest there may be a difference between platelet 5-HT uptake in RAO and normal ponies but do not provide evidence of platelet activation following antigen exposure.

Equine CCL11 induces eosinophil cytoskeletal reorganization and activation.

OBJECTIVES: To assess the biological effects of purified recombinant equine CCL11 on equine eosinophil function. METHODS: Following stimulation of eosinophils from normal horses, the polymerised form of actin was measured by flow cytometry using fluorescently labelled phalloidin. Migration was determined in a 96 well plate chemotaxis assay using 8 microm pore membranes, and adherence of eosinophils to serum-coated plastic was assessed using a colorimetric assay for eosinophil peroxidase. Superoxide generation was measured by the reduction of cytochrome C in a colorimetric assay. RESULTS: Equine CCL11 induced significant (p < 0.001), concentration-dependent actin polymerisation and migration of equine eosinophils. Stimulation with CCL11 did not induce significant adherence to serum coated plastic, or superoxide production. CONCLUSIONS: Equine CCL11 stimulates cytoskeletal reorganization and migration of equine eosinophils, suggesting that it may be involved in the regulation of eosinophil trafficking in horses.

The role of protein kinase C in regulating equine eosinophil adherence and superoxide production.

OBJECTIVE: To determine if protein kinase C (PKC) regulates equine eosinophil function. MATERIAL OR SUBJECTS: Blood eosinophils were obtained from healthy ponies. METHODS: IL-5- and histamine-induced adherence to serum-coated plastic was measured as the eosinophil peroxidase content of adherent cells and serum treated zymosan (STZ)-and IL-5-induced superoxide production by the reduction of cytochrome C. Eosinophil PKC activity was quantitated as the rate of transfer of (32)P from ATP to substrate. The effects of Ro31-8220 (isotype non-selective PKC inhibitor), Go6976 (conventional PKC inhibitor), and rottlerin (PKCdelta inhibitor) were determined by ANOVA and Bonferroni's or Dunnett's test. RESULTS: Ro31-8220 and Go6976 reduced superoxide production whereas only Go6976 inhibited adherence. Rottlerin inhibited histamine-induced adherence and increased STZ-induced superoxide production. Ro31-8220 and Go6976, but not rottlerin, inhibited PKC activity. CONCLUSIONS: PKC is involved in regulating equine eosinophil adherence and superoxide production. The role of PKCdelta appears to depend upon the stimulus used and response measured.

Principles of pharmacodynamics and their applications in veterinary pharmacology.

Pharmacodynamics (PDs) is the science of drug action on the body or on microorganisms and other parasites within or on the body. It may be studied at many organizational levels--sub-molecular, molecular, cellular, tissue/organ and whole body--using in vivo, ex vivo and in vitro methods and utilizing a wide range of techniques. A few drugs owe their PD properties to some physico-chemical property or action and, in such cases, detailed molecular drug structure plays little or no role in the response elicited. For the great majority of drugs, however, action on the body is crucially dependent on chemical structure, so that a very small change, e.g. substitution of a proton by a methyl group, can markedly alter the potency of the drug, even to the point of loss of activity. In the late 19th century and first half of the 20th century recognition of these facts by Langley, Ehrlich, Dale, Clarke and others provided the foundation for the receptor site hypothesis of drug action. According to these early ideas the drug, in order to elicit its effect, had to first combine with a specific 'target molecule' on either the cell surface or an intracellular organelle. It was soon realized that the 'right' chemical structure was required for drug-target site interaction (and the subsequent pharmacological response). In addition, from this requirement, for specificity of chemical structure requirement, developed not only the modern science of pharmacology but also that of toxicology. In relation to drug actions on microbes and parasites, for example, the early work of Ehrlich led to the introduction of molecules selectively toxic for them and relatively safe for the animal host. In the whole animal drugs may act on many target molecules in many tissues. These actions may lead to primary responses which, in turn, may induce secondary responses, that may either enhance or diminish the primary response. Therefore, it is common to investigate drug pharmacodynamics (PDs) in the first instance at molecular, cellular and tissue levels in vitro, so that the primary effects can be better understood without interference from the complexities involved in whole animal studies. When a drug, hormone or neurotransmitter combines with a target molecule, it is described as a ligand. Ligands are classified into two groups, agonists (which initiate a chain of reactions leading, usually via the release or formation of secondary messengers, to the response) and antagonists (which fail to initiate the transduction pathways but nevertheless compete with agonists for occupancy of receptor sites and thereby inhibit their actions). The parameters which characterize drug receptor interaction are affinity, efficacy, potency and sensitivity, each of which can be elucidated quantitatively for a particular drug acting on a particular receptor in a particular tissue. The most fundamental objective of PDs is to use the derived numerical values for these parameters to classify and sub-classify receptors and to compare and classify drugs on the basis of their affinity, efficacy, potency and sensitivity. This review introduces and summarizes the principles of PDs and illustrates them with examples drawn from both basic and veterinary pharmacology. Drugs acting on adrenoceptors and cardiovascular, non-steroidal anti-inflammatory and antimicrobial drugs are considered briefly to provide a foundation for subsequent reviews in this issue which deal with pharmacokinetic (PK)-PD modelling and integration of these drug classes. Drug action on receptors has many features in common with enzyme kinetics and gas adsorption onto surfaces, as defined by Michaelis-Menten and Langmuir absorption equations, respectively. These and other derived equations are outlined in this review. There is, however, no single theory which adequately explains all aspects of drug-receptor interaction. The early 'occupation' and 'rate' theories each explain some, but not all, experimental observations. From these basic theories the operational model and the two-state theory have been developed. For a discussion of more advanced theories see Kenakin (1997).

Allergen challenge alters lymphocyte phosphodiesterase activity in horses with heaves.

Heaves is an allergic airway disease in horses characterised by reversible airway obstruction, bronchial hyperresponsiveness and airway inflammation associated with a Th(2) response. Cyclic nucleotide-dependent signalling pathways can regulate lymphocyte function. In this study, we examined lymphocyte PDE activity comparing horses with heaves to healthy control animals. Total PDE activity and the effects of isoenzyme selective inhibitors were measured before, 5 and 24 h after the start of a 7 h allergen challenge. Allergen challenge had no effect on either total cAMP PDE activity or its inhibition by the PDE4 selective inhibitor, rolipram, and the non-selective PDE inhibitor, theophylline. In contrast, the PDE3 selective inhibitor, quazinone, caused significantly greater inhibition of cAMP PDE activity before challenge in the heaves susceptible group. Additionally, total cGMP PDE activity was significantly lower 24 h after the start of challenge in the heaves affected group (11+/-2 and 21+/-3 pmol/min/mg for heaves and control animals, respectively) and the PDE5 selective inhibitor, zaprinast, caused significantly less inhibition in the heaves group at this time point. The functional significance of these findings was explored by examining the effect of PDE3, PDE4 and PDE5 selective inhibitors on mitogen-induced mononuclear cell proliferation before and 24 h after the start of allergen challenge. Proliferation decreased after challenge in the heaves group (stimulation index=328+/-110 and 200+/-72 before and after challenge, respectively) whilst remaining constant in the control group (stimulation index=161+/-13 and 183+/-45 before and after challenge, respectively). However, all three PDE inhibitors caused a similar amount of inhibition at each time point and the effect of a combination of a PDE3 and a PDE5 inhibitor was simply additive in both groups. These results suggest differences in the control of lymphocyte PDE activity in horses with heaves.

Biochemical and functional assessment of equine lymphocyte phosphodiesterases and protein kinase C.

Lymphocytes play an important role in allergic inflammation and have been implicated in the pathogenesis of equine allergic skin and respiratory disease. Targeting intracellular signalling pathways in human lymphocytes has demonstrated a role for both phosphodiesterase and protein kinase C in cell activation. The aim of this study was to measure total cyclic nucleotide hydrolysing phosphodiesterase activity and to identify the phosphodiesterase and protein kinase C isoenzymes present in equine lymphocytes. The functional significance of these isoenzymes was then investigated by examining their role in peripheral blood mononuclear cell proliferation using isoenzyme selective inhibitors. Total cyclic adenosine monophosphate hydrolysing phosphodiesterase activity was double that of cyclic guanosine monophosphate (30+/-2 pmol/min mg versus 16+/-3 pmol/min mg for cyclic adenosine and cyclic guanosine monophosphate phosphodiesterase activity, respectively). Evidence for the presence of PDE1, 3, 4 and 5 was obtained and PKCalpha, beta, delta, eta, iota, theta and zeta were identified. Selective inhibitors of PDE4, PKCdelta and conventional PKCs alpha and beta caused significant inhibition of mitogen-induced peripheral blood mononuclear cell proliferation. This study demonstrates a functional role for specific signalling isoenzymes and suggests that, in the context of allergic inflammation, targeting inflammatory cells involved in disease pathogenesis with relevant isoenzyme inhibitors may have therapeutic potential.

Protein kinase C (PKC) isotype profile in eosinophils from ponies with sweet itch and role in histamine-induced eosinophil activation.

Eosinophils have been implicated in the pathogenesis of the seasonal equine allergic skin disease, sweet itch. Protein kinase C (PKC) is involved in regulating eosinophil function and antigen challenge has been reported to alter PKC isotype expression in blood eosinophils from allergic human subjects. Here we have compared the pattern of PKC isotype expression in eosinophils from sweet itch ponies with that in cells from normal ponies both during the active and inactive phases of the disease. A role for PKC in histamine-induced eosinophil activation was also investigated. Conventional PKCs alpha and beta, novel PKCs delta and epsilon and atypical PKCs iota and zeta were identified in eosinophils pooled from four allergic ponies during the inactive phase, when no clinical signs were evident. The PKC isotypes, like those in eosinophils from normal ponies, were located primarily in the particulate fraction of the cell. Isotype expression in cells from normal and allergic animals did not appear to be different. In contrast, during the active phase of the disease, when the sweet itch ponies had clinical signs, the expression of PKCs beta, epsilon and iota in eosinophils from these animals appeared to be increased relative to that in cells from normal ponies. When PKC expression in eosinophils from five individual normal and sweet itch ponies was compared, small, but statistically significant, increases in PKC epsilon and PKCdelta expression were evident in eosinophils from the sweet itch ponies during the active and inactive phases, respectively. The non-selective PKC inhibitors, staurosporine and Ro31-8220, significantly reduced histamine-induced superoxide production. Use of Gö6976, an inhibitor of conventional PKCs, suggested that PKCalpha and/or beta were involved and that there was significantly greater inhibition of the response in eosinophils obtained from sweet itch ponies during the active phase. There was no significant difference in histamine-induced superoxide production by eosinophils from allergic and normal ponies and the functional significance of the increased PKC isotype expression in eosinophils from sweet itch ponies relative to that in cells from healthy animals remains to be established.

Cloning, expression and biological activity of equine interleukin (IL)-5.

The cytokine, interleukin (IL)-5 stimulates eosinophil differentiation, activation and survival and can prime these cells, increasing the response to other mediators. In view of its many effects on eosinophils, IL-5 has been implicated in the pathogenesis of allergic disease in man. Here we report the cloning of equine IL-5 and expression of the recombinant protein by transfection of Chinese hamster ovary (CHO) cells. The cloned cDNA sequence consisted of 405 nucleotides and encoded a protein of 135 amino acids. There is >85% identity with feline, bovine, ovine, canine, and human IL-5 sequences at the nucleotide and protein level. Supernatants containing equine IL-5 were also examined for biological activity. CHO supernatant containing equine recombinant (eqr) IL-5, like the human ortholog (hrIL-5), induced concentration dependent equine eosinophil adherence to autologous serum-coated plastic (9.7+/-1.5% with a 1:100 dilution of eqrIL-5 and 9.1+/-1.6% adherence with 1 nM hrIL-5; n = 4). The eqr protein also caused concentration dependent superoxide production (11.9+/-2.4 nmol (reduced cytochrome (cyt) C)/10(6) cells at a 1:50 dilution, n = 4). In contrast, hrIL-5 only caused significant superoxide production when diluted in conditioned CHO medium, an effect that was inhibited by the anti-human mAb, TRFK5 (4.4+/-0.3 versus 0.3+/-0.4 nmol/10(6) cells for 0.5 nM hrIL-5 in the presence of the isotype matched IgG1 control (10 microM) and TRFK5 (10 microM), respectively). TRFK5 also significantly inhibited hrIL-5 induced adherence at concentrations of 0.3 microg/ml and above but had no significant inhibitory effect on either superoxide or adherence caused by eqrIL-5. These results demonstrate that equine IL-5 expressed by CHO cells stimulates equine eosinophils, suggesting that this cytokine could play a role in eosinophil recruitment and activation in equine allergic disease. The anti-human and murine moAb TRFK5 does not appear to recognise the equine protein.

Differential effects of phosphodiesterase inhibitors on platelet activating factor (PAF)- and adenosine diphosphate (ADP)-induced equine platelet aggregation.

Compounds that activate adenylate cyclase, increasing intracellular cyclic adenosine monophosphate (cAMP), inhibit equine platelet aggregation. Cyclic AMP is broken down by phosphodiesterase (PDE) and, in the present study, the effects of theophylline, a nonselective PDE inhibitor, and selective inhibitors of PDE isoenzymes PDE3, PDE4 and PDE5, on equine platelet aggregation in response to platelet activating factor (PAF) and adenosine diphosphate (ADP) have been examined. Theophylline and the PDE3 inhibitors, trequinsin and quazinone, inhibited both PAF and ADP-induced aggregation in a concentration dependent manner. The inhibition of PAF-induced aggregation was, however, significantly greater than that of the response to ADP. The inhibitory effects of theophylline and the PDE3 inhibitors on ADP- but not PAF-, induced aggregation were reversed by addition of the calcium ionophore, A23187. Rolipram and zaprinast, inhibitors of PDE4 and PDE5, respectively, had no effect on either PAF- or ADP-induced aggregation. These results demonstrate that inhibition of aggregation caused by PAF or ADP can be achieved by selective inhibition of PDE3 but suggest that there may be agonist-specific differences in the intracellular signalling pathways that regulate equine platelet aggregation.

In vitro and ex vivo effects of the phosphodiesterase 4 inhibitor, rolipram, on thromboxane production in equine blood.

Phosphodiesterase 4 (PDE4) inhibitors have been shown to inhibit equine neutrophil function in vitro and may be of benefit in recurrent airway obstruction (RAO), an allergy-based respiratory disease characterized by inflammatory cell recruitment and activation within the lungs following exposure of susceptible horses to allergens in mouldy hay. The aim of this study was to evaluate the inhibitory effects of the PDE4 inhibitor, rolipram, in an in vitro assay of thromboxane (Tx) production. The assay was then used to monitor the activity of this compound in vivo in normal and RAO-affected horses. Rolipram and the structurally distinct PDE4 inhibitor, denbufylline, attenuated both lipopolysaccharide (LPS)-induced and unstimulated Tx production in blood from normal horses. Thromboxane production appeared to involve a calcium-dependent interaction between leucocytes and platelets (LPS-induced Tx production = 2.3 +/- 0.4, 4.5 +/- 1.1 and 20.8 +/- 3.6 ng/mL for platelets, leucocytes and blood, respectively) and rolipram-inhibited Tx production via an effect on leucocytes. Inhibition of ex vivo LPS induced Tx production was detected after intravenous administration of rolipram (5 microg/kg) to normal ponies. This dose did not significantly affect either lung function or neutrophil accumulation when administered to three horses with clinical signs of RAO. This study suggests that inhibition of Tx production in equine blood can be used to measure PDE4 activity. However, PDE4 inhibitors with improved therapeutic profiles are required for evaluation in RAO.

Substance P induces activation, adherence and migration of equine eosinophils.

The tachykinin, substance P (SP), affects eosinophil function by direct and indirect mechanisms and has been shown to cause equine eosinophils to adhere to vascular endothelium and to release cytokines that increase cell adherence. The aim of this study was to determine whether SP could act directly on equine eosinophils in vitro. Eosinophil activation was also compared in cells from normal ponies and those with insect hypersensitivity as SP may be released in the skin of hypersensitive animals. SP caused equine eosinophils to adhere, migrate and produce superoxide, although high concentrations were required to produce these effects [10 +/- 2% adherence, 45 +/- 20 cells/0.3 mm2 and 48 +/- 7 nmol (of reduced cytochrome C)/106 cells, respectively, at 3 x 10-4 m]. That the 7-11, but not the 1-7, amino acid fragment of SP caused superoxide production, suggested the effects of SP were receptor mediated. Eosinophils from hypersensitive ponies produced more superoxide in response to SP, but not phorbol myristate acetate or histamine, over the concentration range tested when compared with cells from normal ponies. The data obtained in this study suggest that although SP can directly activate equine eosinophils, in view of the high concentrations required, such actions may be of less relevance physiologically than other SP-mediated effects.