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.

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.

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.

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.

Differential inhibition of equine neutrophil function by phosphodiesterase inhibitors.

Neutrophils are recruited to the lungs of horses with chronic obstructive pulmonary disease (COPD) and exhibit increased activity after antigen challenge, which may contribute to inflammation and lung damage. Inhibition of phosphodiesterase isoenzymes (PDEs) has been shown to attenuate human neutrophil functions including superoxide production, leukotriene (LT)B4 biosynthesis, enzyme and chemokine release. As equine neutrophils contain predominantly the isoenzyme, PDE4, the present study was undertaken to investigate the effects of rolipram, a PDE4 inhibitor, on equine neutrophil function. For comparison, the effects of the nonselective PDE inhibitor, theophylline, were examined. Cells from both normal horses and COPD horses in remission were used. Superoxide production was significantly inhibited by both rolipram [32.2 +/- 2.6 vs. 10.1 +/- 1.1 nmol/10(6) cells and 49.8 +/- 6.8 vs. 22.7 +/- 2.2 nmol/10(6) cells for normal and COPD susceptible horses, respectively, in response to 10(-7) M human recombinant (hr) C5a] and theophylline (19.0 +/- 0.6 vs. 10.2 +/- 0.6 nmol/10(6) cells and 24.3 +/- 2.1 vs. 10.7 +/- 0.9 nmol/10(6) cells for normal and COPD susceptible horses, respectively, in response to 10(-7) M C5a). However, superoxide production induced by serum treated zymosan was inhibited only by theophylline (10(-3) M). Neither hrC5a- nor platelet activating factor (PAF)-induced neutrophil adherence to fibronectin coated plastic was reduced by rolipram (10(-5) M). These results demonstrate that the effects of PDE inhibitors on equine neutrophils are both stimulus and function dependent. The PDE4 inhibitors may reduce neutrophil activation in vivo in horses with COPD.

Phosphodiesterase activity in neutrophils from horses with chronic obstructive pulmonary disease.

Neutrophils are recruited to the lungs of horses with chronic obstructive pulmonary disease (COPD) and exhibit increased activity after antigen challenge. Phosphodiesterase type4 (PDE4) inhibitors have been shown to attenuate human neutrophil activation. The aim of this study was to establish the PDE isoenzyme profile of equine neutrophils using isoenzyme selective inhibitors to determine if these compounds should be evaluated in horses with COPD. Total cAMP and cGMP dependent PDE activity was no different in neutrophils from normal (156.2+/-7.1 and 6.8+/-0.6 pmol/min/mg for cAMP and cGMP, respectively) and COPD susceptible horses (146.0+/-10.2 and 5.5+/-0.6 pmol/min/mg for cAMP and cGMP, respectively). The PDE4 inhibitors, CDP840 and rolipram, caused significant, concentration related and almost complete inhibition of PDE activity (IC(50) values=8.8+/-0.1 x 10(-9) and 7.3+/-0.2 x 10(-9)M for CDP840; 1.2+/-0.1 x 10(-6) and 1.1+/-0.1 x 10(-6)M for rolipram in normal and COPD susceptible horses, respectively). The inhibitory effects of the mixed PDE3/ PDE4 inhibitor, zardaverine were of similar magnitude and potency to rolipram. However, the limited inhibitory effects of the PDE3 inhibitor, siguazodan, suggest that zardaverine is acting primarily via PDE4 inhibition. These results indicate that PDE4 is the predominant isoenzyme present in the equine neutrophil and inhibition of PDE activity using selective PDE4 inhibitors may, therefore, modulate equine neutrophil activation in horses with COPD.