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Phosphodiesterase activity in neutrophils from horses with chronic obstructive pulmonary disease
Veterinary Immunology and Immunopathology 76 (2000) 319±330
Short communication
Phosphodiesterase activity in neutrophils from horses with chronic obstructive pulmonary disease K.J. Rickardsa,*, C.P. Pagea,b, P. Leesa, F.M. Cunninghama a
Department of Veterinary Basic Sciences, The Royal Veterinary College, Hawkshead Campus, North Mymms, Hertfordshire AL9 7TA, UK b Sackler Institute of Pulmonary Pharmacology, Division of Pharmacology and Therapeutics, GKT School of Biomedical Sciences, King's College London, Guy's Campus, London Bridge, London SE1 9RT, UK Received 1 November 1999; received in revised form 7 June 2000; accepted 20 July 2000
Abstract 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 pro®le 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 signi®cant, concentration related and almost complete inhibition of PDE activity (IC50 values 8:8 0:1 10ÿ9 and 7:3 0:2 10ÿ9 M for CDP840; 1:2 0:1 10ÿ6 and 1:1 0:1 10ÿ6 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. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Horse; COPD; Equine neutrophils; Phosphodiesterase isoenzymes; PDE4 inhibitors
*
Corresponding author. Tel.: 44-1707-666333, ext: 2458; fax: 44-1707-652090 E-mail address: [email protected] (K.J. Rickards). 0165-2427/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 0 ) 0 0 2 2 0 - 8
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1. Introduction Equine chronic obstructive pulmonary disease (COPD) is an allergic respiratory condition characterised by an acute, reversible, bronchoconstriction, airway hyperresponsiveness and neutrophil recruitment to the lungs (Robinson et al., 1996). During acute episodes of COPD there is evidence that the activation of both circulating neutrophils and those recovered from bronchoalveolar lavage ¯uid (BALF) increases (Olszewski and Laber, 1993; Marr et al., 1997). Release of proteases, oxygen radicals and mediators from activated cells could contribute to the lung damage that occurs in horses with this disease. Inhibition of neutrophil activation may therefore be bene®cial in COPD. Leucocyte activation can be reduced by raising intracellular cyclic 30 50 -adenosine monophosphate (cAMP). This can be achieved either by activation of adenylate cyclase or by inhibition of phosphodiesterase enzymes (PDEs). Currently 11 PDE isoenzymes have been identi®ed with PDE4 predominating in human in¯ammatory cells (Torphy, 1998; Fawcett et al., 2000). Compounds that selectively inhibit PDE4 have been shown to reduce a range of human neutrophil functions both in vitro and in vivo (Neilson et al., 1990; Schudt et al., 1991; Griswold et al., 1993). Moreover, the PDE4 selective inhibitor, SB 207499, improved pulmonary function in human patients with moderate COPD after 6 weeks. This disease, like equine COPD, is characterised by neutrophilic airway in¯ammation (Torphy, 1999). Methylxanthines, including theophylline, which are non-selective PDE inhibitors have been used in the treatment of equine COPD, primarily as bronchodilators, but have a narrow therapeutic index (Errecalde et al., 1985). PDE4 selective inhibitors may, in addition to reducing airway in¯ammation, decrease bronchoconstriction, as PDE4 is present in human airway wall smooth muscle (Torphy, 1998) and are likely to cause fewer unwanted effects than the methylxanthines. The principal side effects which have been noted are nausea and emesis but these have only been reported in species with an emetic re¯ex (Maignel et al., 1999, poster communication at the William Harvey Research Conference, PDE Inhibitors: Drugs with an expanding range of therapeutic uses, Nice, France). The PDE isoenzymes present in equine cells and tissues have not been documented previously. The aim of the present study was to compare PDE activity in neutrophils from horses with COPD with that in cells from normal horses and characterise the PDE isoenzymes present. As the sensitivity of PDE to inhibition has been reported to differ in cells from allergic human subjects (Chan and Hani®n, 1993; Landells et al., 1997), the effects of isoenzyme selective inhibitors and the non-selective inhibitor, theophylline were compared in neutrophils from both the COPD susceptible and normal horses. 2. Materials and methods 2.1. Animals Five mixed breed horses with clinical histories of COPD (COPDs) and ®ve New Forest ponies with no history of respiratory disease (normals) were used. The mean (S.D.) age of the horses with COPD was 18:4 4:9 years and that of the normal horses was
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10:2 2:4 years. Lung function changes and radiolabelled neutrophil accumulation in the lungs had previously been shown to occur during a 7-h hay challenge in each of the horses with COPD but not in the normal horses. All horses were free of clinical signs at the start of each study, having been kept in an allergen free environment for at least 4 weeks. In the horses with COPD, the pleural pressure (DPplmax), measured indirectly using an oesophageal balloon catheter (Fairbairn et al., 1993), was 9.0 cm H2O. This is below the average value for normal horses reported by Klein and Deegen (1987). Moreover, the pleural pressure change during expiration was monophasic rather than biphasic, as observed after antigen challenge. All horses were routinely vaccinated against tetanus and regularly dewormed. 2.2. Measurement of neutrophil phosphodiesterase activity Neutrophils were isolated from 100 ml venous blood samples collected into 0.4 M ethylenediaminetetraacetic acid (EDTA) (1:49) by Percoll density gradient centrifugation as previously described (Fairbairn et al., 1993). The purity of the isolated neutrophil population was >98%. Following washing in Hank's balanced salt solution (HBSS) without Ca2 or Mg2, the puri®ed neutrophils were re-suspended in HBSS with Ca2 and Mg2 and counted. Approximately 5 107 neutrophils were lysed in 1 ml modi®ed homogenisation buffer (leupeptin 10 mg/ml, Tris±HCl 20 mM, MgCl2 2 mM, dithiothreitol 1 mM, EDTA 5 mM, benzamidine 1.3 mM, sucrose 0.25 M, Na-p-tosyl-L-lysine chloromethyl ketone 20 mM, triton 1%) and vortexed to expose intracellular PDE. The neutrophil homogenates were frozen at ÿ808C until required. PDE activity was measured using a two-step radioactive assay modi®ed from that of Thompson and Appleman (1971). Brie¯y, 1 mCi [3H]-cAMP or [3H]-cGMP was added to a reaction mixture containing 50 ml neutrophil homogenate or homogenisation buffer, assay buffer (pH8) and the PDE inhibitor/activator under investigation or vehicle (DMSO). The mixture was incubated at 308C for 25 min after which 0.1 mg/ml snake venom was added. Following a 10 min incubation at 308C samples were eluted through Dowex and the radioactivity of the elute was counted. Each sample was assayed in duplicate, and for each assay, tubes containing tritiated substrate alone were included (Tc). The protein content of each sample was determined using the colorimetric method of Lowry et al. (1951). PDE activity was then calculated as follows: PDE activity pmol=min=mg
sample count ÿ blank count Tc ÿ blank count 1 1 1 400 25 0:05 protein
where 400 is the number of moles of substrate (pmol), 25 the incubation time (min), 0.05 the homogenate volume (ml), and protein (mg). 2.3. Study design In part 1 of the study the effects of isoenzyme selective inhibitors for PDE3, 4 and 5, and a combined PDE3/4 inhibitor were examined as these isoenzymes have been
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identi®ed in human in¯ammatory cells (Torphy, 1998). PDE1 and 2 have also been identi®ed in small amounts from some in¯ammatory cells (Torphy, 1998). As no selective inhibitors are available, compounds that activate these isoenzymes were added. All compounds were added at a high single concentration, known in other species to be greater than the concentration that inhibits or activates PDE activity for the relevant isoenzyme by 50% (IC50 and EC50, respectively), whilst still conferring selectivity (Nicholson and Shahid, 1993). Total PDE activity and PDE activity in the presence of each inhibitor or activator was calculated. In view of the data obtained in part 1 of the study, the sensitivity to inhibition of PDE activity in neutrophils from normal and COPD susceptible horses was compared by constructing concentration response curves to PDE3, PDE4 and PDE3/4 selective inhibitors. The effects of the non-selective PDE inhibitor, theophylline, were also examined in this study. Finally, the effect of antigen challenge on neutrophil PDE activity was determined in the two groups of horses. Venous blood samples were taken for measurement of leucocyte counts and neutrophil PDE activity and lung function measurements (DPplmax and respiratory rate) were recorded before the start of a 6-h hay and straw challenge (Fairbairn et al., 1993). Further blood samples were taken at 6 h for measurement of neutrophil PDE activity and at 24 h for measurement of PDE activity and leucocyte counts. Respiratory rate was measured at 6 h and DPplmax at 24 h after the start of the challenge. These time points were chosen for measurement of PDE activity as neutrophils begin to accumulate in the lungs (Fairbairn et al., 1993) and appear in BALF (McGorum et al., 1993) at 4±5 h and changes in circulating neutrophil function have been observed at 24 h (Marr et al., 1997). In the three studies described above, neutrophils were isolated from ®ve COPD susceptible and ®ve normal horses with cells from one normal and one COPD susceptible horse being used in each PDE assay. In the studies described above, the horses in the two groups could not be age-matched because of the limited availability of COPD susceptible animals for such work. Preuss and Goldie (1999) have reported age-related differences in PDE activity in both the rat and the guinea pig. Therefore, as a separate study, both total PDE activity and the potency of inhibition of PDE4 activity by rolipram were compared in the three oldest (mean age 17 0:6 years) and three youngest (mean age 5 0:6 years) of the available clinically normal horses and in three of the horses with COPD that could be age-matched with three normal animals (mean age 14 2:1 versus 15 1:5 for normal and COPD susceptible horses, respectively). 2.4. Data analysis Values are presented as means standard error of the mean. Data from the isoenzyme pro®le studies were analysed by repeated measures two-way analysis of variance followed by Tukey's multiple comparison test using Graph Pad Prism (Graphpad Software, San Diego, CA). Values for the concentration response curves were converted to percentage of total PDE activity and then analysed by analysis of covariance on the linear portion of the graph using Minitab (Minitab, PA). IC50 values were calculated from log concentration response curves ®tted by a modi®ed Marquat procedure using Multi®t
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(Day Computing, Cambridge, UK). The IC50 values for the normal and COPD susceptible horses were compared by Student's unpaired t-test using Multistat Biosoft (Day Computing, Cambridge, UK). The total PDE activities and IC50 values in neutrophils from young versus older horses and that in neutrophils from age-matched normal and COPD susceptible horses were also compared using Student's unpaired t-test. Leucocyte and lung function measurements at either 6 or 24 h following antigen were compared with pre-challenge values by Student's paired t-test using Multistat Biosoft (Day Computing, Cambridge, UK). Total PDE activity in the antigen challenge study was compared at the three time points by repeated measures two-way analysis of variance using Minitab. The null hypothesis was rejected when P < 0:05. 2.5. Materials [3H]-cAMP and [3H]-cGMP were from Amersham International, Bucks, UK. Rolipram (ZK 62711) was from Schering AG, Berlin, Germany. CDP840 was from Celltech Chiroscience, Berks, UK. Siguazodan was from SmithKline Beecham Pharmaceuticals, King of Prussia, PA. Zardaverine (B 842-90) was from Forschungslaboratorien Byk Gulden, Konstanz, Germany. Zaprinast (M&B 22 948) was from Rhone-Poulenc Rorer, Dagenham, UK. HBSS with Ca2 and Mg2 was from GibcoBRL, Life Technologies, Paisley, UK. Methanol was from Merck, Leics, UK. Optiphase Hisafe was from Fisher Scienti®c, Leics, UK. All other chemicals were from Sigma Diagnostics, Dorset, UK. 3. Results 3.1. PDE isoenzyme pro®le As shown in Fig. 1a there was no difference in the total PDE activity in neutrophils from normal and COPD susceptible horses. Furthermore, there was no signi®cant difference in total neutrophil PDE activity between either young and older horses (39:7 3:6 versus 36:2 0:9 pmol=min=mg for young and older horses, respectively) or age-matched COPD and normal horses (34:4 1:0 versus 27:5 4:3 pmol=min=mg for normal and COPD horses, respectively). Addition of either a PDE4 or a mixed PDE3/4 inhibitor signi®cantly inhibited neutrophil PDE activity in both groups of horses. In contrast neither a PDE3 inhibitor nor a PDE2 stimulant/PDE3 inhibitor had a signi®cant effect. Surprisingly a PDE1 stimulant signi®cantly reduced PDE activity in neutrophils from both normal and COPD affected horses (Fig. 1a). As shown in Fig. 1b, total cGMP PDE activity was also similar in neutrophils from normal and COPD susceptible horses. However, the activity was considerably lower than that of cAMP PDE. No signi®cant inhibition of cGMP PDE activity in neutrophils from either group of horses was seen after addition of a PDE5 inhibitor or a PDE1 stimulant. 3.2. Concentration Ð response relationships for PDE isoenzyme inhibitors CDP840 and rolipram, a second PDE4 inhibitor, both caused concentration dependent inhibition of cAMP PDE activity in neutrophils from normal and COPD
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Fig. 1. PDE activity in neutrophils from normal horses (®lled columns) and COPD horses in remission (open columns). Columns represent mean values and lines represent standard error of the mean (n 5 for both groups). : P < 0:05 when compared to total cAMP PDE activity (cA) or total cGMP PDE activity (cG) (repeated measures two-way analysis of variance followed by Tukey's multiple comparison test).
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Table 1 IC50 values for inhibition of PDE activity in neutrophils from normal and COPD susceptible horsesa IC50 (M) Normals CDP840 Rolipram Zardaverine Siguazodan Theophylline a
COPDs ÿ9
8:8 0:1 10 1:2 0:1 10ÿ6 8:2 0:3 10ÿ7 2:2 0:1 10ÿ3 6:8 0:4 10ÿ3
7:3 0:2 10ÿ9 1:1 0:1 10ÿ6 8:6 0:9 10ÿ7 1:3 0:5 10ÿ3 5:6 0:2 10ÿ3
Results are expressed as mean standard error of the mean IC50; n 5 horses for both groups.
susceptible horses, although rolipram was considerably less potent. IC50 values for the PDE isoenzyme selective inhibitors are shown in Table 1. Zardaverine, also caused concentration related inhibition and was of similar potency to rolipram. There was also some inhibition of PDE activity in neutrophils from both groups of horses when siguazodan, was added, but this was only observed at high concentrations. Finally, the non-speci®c PDE inhibitor, theophylline, caused concentration dependent inhibition of cAMP PDE activity in neutrophils from both groups of animals but was at least 1000-fold less potent than either the PDE4 or PDE3/4 selective compounds (Table 1). There was no signi®cant difference in potency of any of the PDE inhibitors when IC50 values were compared in neutrophils from normal and COPD susceptible horses. Additionally, no difference in potency was found in cells from either young or older horses (IC50 values 4:5 1:3 10ÿ7 and 4:8 1:2 10ÿ7 M for young and older horses, respectively) or age-matched COPD versus normal horses (IC50 values 5:5 1:2 10ÿ7 and 3:6 1:8 10ÿ7 M for normal and COPD susceptible horses, respectively). 3.3. Effect of antigen challenge on PDE activity Antigen challenge caused a statistically signi®cant increase in the total blood leucocyte and neutrophil counts at 24 h post-challenge in the COPD susceptible horses but not the normal horses. DPplmax and/or respiratory rate increased in all the COPD affected horses. Additionally the expiratory pattern in the horses with COPD was biphasic post-challenge. However, the mean changes were not statistically signi®cant due to the variability of responses in individual horses. There were no changes in leucocyte counts or lung function in the normal horses (Table 2). Antigen challenge had no signi®cant effect on total PDE activity in neutrophils from either the normal or COPD susceptible horses. Moreover, there was no signi®cant difference in the cAMP PDE inhibitory potency of rolipram at 6 or 24 h when compared to pre-challenge values (Table 3).
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Normals COPDs a *
Respiratory rate (minÿ1)
Total leucocyte count 109 =l
Neutrophil count 109 =l
DPplmax (cm H2O)
0h
24 h
0h
24 h
0h
24 h
0h
6h
9:3 0:9 10:4 0:8
10:0 0:7 12:6 1:2*
4:9 0:4 5:6 0:4
5:6 0:4 7:9 0:8*
6:1 0:7 7:0 1:7
5:2 1:0 11:2 2:3
18:2 6:1 20:6 5:4
20:6 5:2 25:6 6:3
Results are expressed as mean standard error of the mean; n 5 horses for both groups. P < 0:05 compared with 0 h using Student's paired t-test.
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Table 2 Leucocyte counts and lung function changes after antigen challenge of normal and COPD susceptible horsesa
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Table 3 Neutrophil PDE activity and potency of inhibition by rolipram before and after antigen challenge of normal and COPD susceptible horsesa Time after challenge (h)
0 6 24
PDE activity (pmol/min/mg)
IC50 (M)
Normals
Normals
82:3 7:5 82:2 8:4 82:2 8:4 a
COPDs 71:7 9:9 66:1 4:5 63:9 9:4
COPDs ÿ6
1:0 0:5 10 9:1 0:6 10ÿ7 8:5 1:1 10ÿ7
1:1 1:9 10ÿ6 1:1 1:7 10ÿ6 9:7 2:4 10ÿ7
Results are expressed as mean standard error of the mean; n 5 horses for both groups.
4. Discussion This study has shown that total cAMP and cGMP PDE activities are similar in neutrophils from COPD susceptible and normal horses. This ®nding is in agreement with that reported by Goldberg et al. (1994) in neutrophils from normal and asthmatic human subjects. There was considerably more cAMP than cGMP PDE activity in equine neutrophils, which also agrees with data obtained using human neutrophils from normal subjects (Grady and Thomas, 1986). However, it should be noted that although the amount of cGMP PDE was similar, high background counts are obtained in the assay because of the poor adherence of unreacted substrate to the Dowex columns. This may mean that the amount of cGMP PDE present in the equine neutrophil has been underestimated. COPD is a disease that affects older animals and the mean age was higher in the COPD susceptible group. However no difference was found between total PDE activity in neutrophils from three age-matched normal and COPD susceptible horses. Similarly, age did not seem to affect PDE activity when neutrophils from a small group of young and older horses were used. Moreover, the potency of inhibition of PDE activity by the PDE4 selective inhibitor, rolipram, was also similar in neutrophils from both the age-matched and young versus older horses. Thus although Preuss and Goldie (1999) have reported differences in PDE activity in old versus young rats and guinea pigs, the limited study of neutrophils from old versus young horses and age-matched COPD and normal horses provides no evidence of such a difference in this species over the age range used. The isoenzyme pro®le analysis of equine neutrophil PDE has demonstrated that PDE4 is the predominant isoenzyme present, which agrees with the isoenzyme pro®le of human neutrophils (Neilson et al., 1990). Whilst both CDP840 and rolipram produced concentration related, and almost complete, inhibition of PDE activity, CDP840, a second-generation PDE4 inhibitor, was over 100-fold more potent than the archetypal PDE4 inhibitor, rolipram. In human neutrophils CDP840 is approximately 25 times more potent than rolipram. In contrast in guinea-pig neutrophils the two compounds are equipotent (Cooper et al., 1999). This demonstrates that species variability exists in the ability of these two PDE4 inhibitors to suppress neutrophil PDE4 activity. No signi®cant difference in potency of PDE4 inhibition was demonstrated in neutrophils from COPD susceptible horses when compared to that obtained in neutrophils from normal horses.
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PDE4 inhibition has not been compared in neutrophils from normal subjects and those with respiratory disease. In man PDE4 in monocytes from asthmatic subjects is less sensitive to inhibition by rolipram (Landells et al., 1997). Whether differences exist in the potency of inhibition of PDE4 activity in mononuclear cells from horses with COPD remains to be established. A signi®cant reduction in PDE activity was also obtained with zardaverine, which inhibits both PDE4 and PDE3. However, in the concentration response studies, the PDE3 selective inhibitor, siguazodan, only produced marked inhibition of neutrophil PDE activity at concentrations of 10ÿ4 and 10ÿ3 M (28 2% and 28 3% at 10ÿ4 M; 73 2% and 72 2% at 10ÿ3 M for normal and COPD susceptible horses, respectively). It is unlikely that, at these concentrations, the inhibitor is selective for PDE3 (Nicholson and Shahid, 1993). Hence, it seems likely that zardaverine is producing its effects primarily via inhibition of PDE4. Moreover, as PDE4 hydrolyses both cGMP and cAMP the small reduction in neutrophil PDE activity caused by addition of the PDE5 inhibitor, zaprinast (Fig. 1b), could be due to an effect on PDE4 (IC50 for inhibition of PDE4 by zaprinast 2:5 10ÿ5 M; Nicholson and Shahid, 1993). Theophylline only caused inhibition of PDE activity in equine neutrophils at relatively high concentrations (10ÿ3 M). This also agrees with data obtained in human neutrophils, for which the IC50 for theophylline is reported to be 2 10ÿ4 M (Neilson et al., 1990). Interestingly CaCAM, which stimulates PDE1, produced no increase in PDE activity but instead caused inhibition. The explanation for this effect is not apparent, although a similar observation has been made using human mononuclear cells (Landells and Page, personal communication). As there was no signi®cant difference in the PDE isoenzyme pro®le or inhibitory potency of PDE inhibitors in COPD susceptible horses in remission, when compared to normal horses, it was of interest to determine whether neutrophil activation during antigen challenge would alter either the total PDE activity or the inhibitory potency of the PDE4 inhibitor, rolipram. However, following antigen challenge, there was no difference between the two groups of horses with respect to either total cAMP PDE activity or the sensitivity to inhibition of PDE4, suggesting neutrophil activation in COPD susceptible horses does not affect cAMP PDE activity. Although human neutrophils have not been studied, these results do agree with data from human monocytes where there was no change in either total PDE activity or sensitivity to inhibition of PDE4 activity by rolipram following antigen challenge (Landells et al., 1999). The values for total cAMP PDE activity varied in the different studies reported here. This may be a natural phenomenon as discussed by Schudt et al. (1995) as variation in technique, reagents and storage of samples can be excluded. Although PDE activity and sensitivity to inhibition is no different in horses with COPD when compared with normal horses, these data suggest that PDE4 inhibitors, which reduce neutrophil function in other species, may be bene®cial in equine COPD by inhibiting neutrophil activation. Additional symptomatic bene®t may be achieved by inhibiting PDE4 in other in¯ammatory cells, as well as in airway wall smooth muscle. Thus, inhibition of PDE4 may cause bronchodilation directly, as well as inhibiting the release of mediators that contribute to increased cholinergic tone and in¯ammation.
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Acknowledgements The authors wish to thank the owners who kindly allowed their COPD susceptible animals to be used in this study and the Home of Rest for Horses for the award of a Welfare Grant that provided ®nancial support. References Chan, S.C., Hani®n, J.M., 1993. Differential inhibitor effects on cyclic adenosine monophosphate± phosphodiesterase isoforms in atopic and normal leukocytes. J. Lab. Clin. Med. 121, 44±51. Cooper, N., Teixeira, M.M., Warneck, J., Miotla, J.M., Wills, R.E., Macari, D.M.T., Gristwood, R.W., Hellewell, P.G., 1999. A comparison of the inhibitory activity of PDE4 inhibitors on leukocyte PDE4 activity in vitro and eosinophil traf®cking in vivo. Br. J. Pharmacol. 126, 1863±1871. Errecalde, J.O., Button, C., Mulders, M.S.G., 1985. Some dynamic and toxic effects of theophylline in horses. J. Vet. Pharmacol. Therap. 8, 320±327. Fairbairn, S.M., Page, C.P., Lees, P., Cunningham, F.M., 1993. Early neutrophil but not eosinophil or platelet recruitment to the lungs of allergic horses following antigen exposure. Clin. Exp. Allergy 23, 821±828. Fawcett, L., Baxendale, R., Stacey, P., McGrouther, C., Harrow, I., Soderling, S., Hetman, J., Beavo, J.A., Phillips, S.C., 2000. Molecular cloning and characterization of a distinct human phosphodiesterase gene family: PDE11A. Proc. Natl. Acad. Sci. USA 97, 3702±3707. Goldberg, M.D., Lad, P.M., Ghekiere, L., 1994. Phosphodiesterase activity and superoxide production in normal and asthmatic subjects neutrophils. J. Allergy Clin. Immunol. 93, 166. Grady, P.G., Thomas, L.L., 1986. Characterization of cyclic-nucleotide phospho-diesterase activities in resting and N-formylmethionylleucyl±phenylalanine-stimulated human neutrophils. Biochim. Biophys. Acta 885, 282±293. Griswold, D.E., Webb, E.F., Breton, J., White, J.R., Marshall, P.J., Torphy, T.J., 1993. Effect of selective phosphodiesterase type IV inhibitor, rolipram, on ¯uid and cellular phases of in¯ammatory responses. In¯ammation 17, 333±344. Klein, H.J., Deegen, E., 1987. The measurement of interpleural pressure Ð a method to assess lung mechanics in the horse. Pferdeheilkunde Z. f. Wissenschaft und Praxis 3, 141±147. Landells, L.J., Spina, D., Souness, J.E., O'Connor, B.J., Page, C.P., 1997. cAMP phosphodiesterase activity in monocytes from healthy and asthmatic subjects. Eur. Resp. J. 10, 443S. Landells, L.J., Jensen, M.W., Orr, L.M., Spina, D., O'Connor, B.J., Page, CP., 1999. Cyclic AMP phosphodiesterase activity in monocytes from asthmatic subjects: effect of allergen challenge. Am. J. Resp. Crit. Care Med. 159, A624. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265±275. Marr, K.A., Foster, A.P., Lees, P., Cunningham, F.M., Page, C.P., 1997. Effect of antigen challenge on the activation of peripheral blood neutrophils from horses with chronic obstructive pulmonary disease. Res. Vet. Sci. 62, 253±260. McGorum, B.C., Dixon, P.M., Halliwell, R.E.W., 1993. Responses of horses affected with chronic obstructive pulmonary disease to inhalation challenges with mould antigens. Equine Vet. J. 25, 261±267. Neilson, C.P., Vestal, R.E., Sturm, R.J., Heaslip, R., 1990. Effects of selective phosphodiesterase inhibitors on the polymorphonuclear leukocyte respiratory burst. J. Allergy Clin. Immunol. 86, 801±808. Nicholson, C.D., Shahid, M., 1993. Inhibitors of cyclic nucleotide phosphodiesterase isoenzymes Ð their potential utility in the therapy of asthma. Pulm. Pharmacol. 6, 101±116. Olszewski, M., Laber, G., 1993. Production of free oxygen radicals by phagocytes from respiratory tract lavage as well as from peripheral blood of horses with chronic obstructive pulmonary disease (COPD) in comparison to healthy animals. Wiener Tierartzliche Monatsschrift 80, 332±337. Preuss, J.M.H., Goldie, R.G., 1999. Age-related changes in airway responsiveness to phosphodiesterase inhibitors and activators of adenylyl cyclase and guanylyl cyclase. Pulm. Pharmacol. Ther. 12, 237±243.
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Short communication
Phosphodiesterase activity in neutrophils from horses with chronic obstructive pulmonary disease K.J. Rickardsa,*, C.P. Pagea,b, P. Leesa, F.M. Cunninghama a
Department of Veterinary Basic Sciences, The Royal Veterinary College, Hawkshead Campus, North Mymms, Hertfordshire AL9 7TA, UK b Sackler Institute of Pulmonary Pharmacology, Division of Pharmacology and Therapeutics, GKT School of Biomedical Sciences, King's College London, Guy's Campus, London Bridge, London SE1 9RT, UK Received 1 November 1999; received in revised form 7 June 2000; accepted 20 July 2000
Abstract 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 pro®le 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 signi®cant, concentration related and almost complete inhibition of PDE activity (IC50 values 8:8 0:1 10ÿ9 and 7:3 0:2 10ÿ9 M for CDP840; 1:2 0:1 10ÿ6 and 1:1 0:1 10ÿ6 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. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Horse; COPD; Equine neutrophils; Phosphodiesterase isoenzymes; PDE4 inhibitors
*
Corresponding author. Tel.: 44-1707-666333, ext: 2458; fax: 44-1707-652090 E-mail address: [email protected] (K.J. Rickards). 0165-2427/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 0 ) 0 0 2 2 0 - 8
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1. Introduction Equine chronic obstructive pulmonary disease (COPD) is an allergic respiratory condition characterised by an acute, reversible, bronchoconstriction, airway hyperresponsiveness and neutrophil recruitment to the lungs (Robinson et al., 1996). During acute episodes of COPD there is evidence that the activation of both circulating neutrophils and those recovered from bronchoalveolar lavage ¯uid (BALF) increases (Olszewski and Laber, 1993; Marr et al., 1997). Release of proteases, oxygen radicals and mediators from activated cells could contribute to the lung damage that occurs in horses with this disease. Inhibition of neutrophil activation may therefore be bene®cial in COPD. Leucocyte activation can be reduced by raising intracellular cyclic 30 50 -adenosine monophosphate (cAMP). This can be achieved either by activation of adenylate cyclase or by inhibition of phosphodiesterase enzymes (PDEs). Currently 11 PDE isoenzymes have been identi®ed with PDE4 predominating in human in¯ammatory cells (Torphy, 1998; Fawcett et al., 2000). Compounds that selectively inhibit PDE4 have been shown to reduce a range of human neutrophil functions both in vitro and in vivo (Neilson et al., 1990; Schudt et al., 1991; Griswold et al., 1993). Moreover, the PDE4 selective inhibitor, SB 207499, improved pulmonary function in human patients with moderate COPD after 6 weeks. This disease, like equine COPD, is characterised by neutrophilic airway in¯ammation (Torphy, 1999). Methylxanthines, including theophylline, which are non-selective PDE inhibitors have been used in the treatment of equine COPD, primarily as bronchodilators, but have a narrow therapeutic index (Errecalde et al., 1985). PDE4 selective inhibitors may, in addition to reducing airway in¯ammation, decrease bronchoconstriction, as PDE4 is present in human airway wall smooth muscle (Torphy, 1998) and are likely to cause fewer unwanted effects than the methylxanthines. The principal side effects which have been noted are nausea and emesis but these have only been reported in species with an emetic re¯ex (Maignel et al., 1999, poster communication at the William Harvey Research Conference, PDE Inhibitors: Drugs with an expanding range of therapeutic uses, Nice, France). The PDE isoenzymes present in equine cells and tissues have not been documented previously. The aim of the present study was to compare PDE activity in neutrophils from horses with COPD with that in cells from normal horses and characterise the PDE isoenzymes present. As the sensitivity of PDE to inhibition has been reported to differ in cells from allergic human subjects (Chan and Hani®n, 1993; Landells et al., 1997), the effects of isoenzyme selective inhibitors and the non-selective inhibitor, theophylline were compared in neutrophils from both the COPD susceptible and normal horses. 2. Materials and methods 2.1. Animals Five mixed breed horses with clinical histories of COPD (COPDs) and ®ve New Forest ponies with no history of respiratory disease (normals) were used. The mean (S.D.) age of the horses with COPD was 18:4 4:9 years and that of the normal horses was
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10:2 2:4 years. Lung function changes and radiolabelled neutrophil accumulation in the lungs had previously been shown to occur during a 7-h hay challenge in each of the horses with COPD but not in the normal horses. All horses were free of clinical signs at the start of each study, having been kept in an allergen free environment for at least 4 weeks. In the horses with COPD, the pleural pressure (DPplmax), measured indirectly using an oesophageal balloon catheter (Fairbairn et al., 1993), was 9.0 cm H2O. This is below the average value for normal horses reported by Klein and Deegen (1987). Moreover, the pleural pressure change during expiration was monophasic rather than biphasic, as observed after antigen challenge. All horses were routinely vaccinated against tetanus and regularly dewormed. 2.2. Measurement of neutrophil phosphodiesterase activity Neutrophils were isolated from 100 ml venous blood samples collected into 0.4 M ethylenediaminetetraacetic acid (EDTA) (1:49) by Percoll density gradient centrifugation as previously described (Fairbairn et al., 1993). The purity of the isolated neutrophil population was >98%. Following washing in Hank's balanced salt solution (HBSS) without Ca2 or Mg2, the puri®ed neutrophils were re-suspended in HBSS with Ca2 and Mg2 and counted. Approximately 5 107 neutrophils were lysed in 1 ml modi®ed homogenisation buffer (leupeptin 10 mg/ml, Tris±HCl 20 mM, MgCl2 2 mM, dithiothreitol 1 mM, EDTA 5 mM, benzamidine 1.3 mM, sucrose 0.25 M, Na-p-tosyl-L-lysine chloromethyl ketone 20 mM, triton 1%) and vortexed to expose intracellular PDE. The neutrophil homogenates were frozen at ÿ808C until required. PDE activity was measured using a two-step radioactive assay modi®ed from that of Thompson and Appleman (1971). Brie¯y, 1 mCi [3H]-cAMP or [3H]-cGMP was added to a reaction mixture containing 50 ml neutrophil homogenate or homogenisation buffer, assay buffer (pH8) and the PDE inhibitor/activator under investigation or vehicle (DMSO). The mixture was incubated at 308C for 25 min after which 0.1 mg/ml snake venom was added. Following a 10 min incubation at 308C samples were eluted through Dowex and the radioactivity of the elute was counted. Each sample was assayed in duplicate, and for each assay, tubes containing tritiated substrate alone were included (Tc). The protein content of each sample was determined using the colorimetric method of Lowry et al. (1951). PDE activity was then calculated as follows: PDE activity pmol=min=mg
sample count ÿ blank count Tc ÿ blank count 1 1 1 400 25 0:05 protein
where 400 is the number of moles of substrate (pmol), 25 the incubation time (min), 0.05 the homogenate volume (ml), and protein (mg). 2.3. Study design In part 1 of the study the effects of isoenzyme selective inhibitors for PDE3, 4 and 5, and a combined PDE3/4 inhibitor were examined as these isoenzymes have been
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identi®ed in human in¯ammatory cells (Torphy, 1998). PDE1 and 2 have also been identi®ed in small amounts from some in¯ammatory cells (Torphy, 1998). As no selective inhibitors are available, compounds that activate these isoenzymes were added. All compounds were added at a high single concentration, known in other species to be greater than the concentration that inhibits or activates PDE activity for the relevant isoenzyme by 50% (IC50 and EC50, respectively), whilst still conferring selectivity (Nicholson and Shahid, 1993). Total PDE activity and PDE activity in the presence of each inhibitor or activator was calculated. In view of the data obtained in part 1 of the study, the sensitivity to inhibition of PDE activity in neutrophils from normal and COPD susceptible horses was compared by constructing concentration response curves to PDE3, PDE4 and PDE3/4 selective inhibitors. The effects of the non-selective PDE inhibitor, theophylline, were also examined in this study. Finally, the effect of antigen challenge on neutrophil PDE activity was determined in the two groups of horses. Venous blood samples were taken for measurement of leucocyte counts and neutrophil PDE activity and lung function measurements (DPplmax and respiratory rate) were recorded before the start of a 6-h hay and straw challenge (Fairbairn et al., 1993). Further blood samples were taken at 6 h for measurement of neutrophil PDE activity and at 24 h for measurement of PDE activity and leucocyte counts. Respiratory rate was measured at 6 h and DPplmax at 24 h after the start of the challenge. These time points were chosen for measurement of PDE activity as neutrophils begin to accumulate in the lungs (Fairbairn et al., 1993) and appear in BALF (McGorum et al., 1993) at 4±5 h and changes in circulating neutrophil function have been observed at 24 h (Marr et al., 1997). In the three studies described above, neutrophils were isolated from ®ve COPD susceptible and ®ve normal horses with cells from one normal and one COPD susceptible horse being used in each PDE assay. In the studies described above, the horses in the two groups could not be age-matched because of the limited availability of COPD susceptible animals for such work. Preuss and Goldie (1999) have reported age-related differences in PDE activity in both the rat and the guinea pig. Therefore, as a separate study, both total PDE activity and the potency of inhibition of PDE4 activity by rolipram were compared in the three oldest (mean age 17 0:6 years) and three youngest (mean age 5 0:6 years) of the available clinically normal horses and in three of the horses with COPD that could be age-matched with three normal animals (mean age 14 2:1 versus 15 1:5 for normal and COPD susceptible horses, respectively). 2.4. Data analysis Values are presented as means standard error of the mean. Data from the isoenzyme pro®le studies were analysed by repeated measures two-way analysis of variance followed by Tukey's multiple comparison test using Graph Pad Prism (Graphpad Software, San Diego, CA). Values for the concentration response curves were converted to percentage of total PDE activity and then analysed by analysis of covariance on the linear portion of the graph using Minitab (Minitab, PA). IC50 values were calculated from log concentration response curves ®tted by a modi®ed Marquat procedure using Multi®t
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(Day Computing, Cambridge, UK). The IC50 values for the normal and COPD susceptible horses were compared by Student's unpaired t-test using Multistat Biosoft (Day Computing, Cambridge, UK). The total PDE activities and IC50 values in neutrophils from young versus older horses and that in neutrophils from age-matched normal and COPD susceptible horses were also compared using Student's unpaired t-test. Leucocyte and lung function measurements at either 6 or 24 h following antigen were compared with pre-challenge values by Student's paired t-test using Multistat Biosoft (Day Computing, Cambridge, UK). Total PDE activity in the antigen challenge study was compared at the three time points by repeated measures two-way analysis of variance using Minitab. The null hypothesis was rejected when P < 0:05. 2.5. Materials [3H]-cAMP and [3H]-cGMP were from Amersham International, Bucks, UK. Rolipram (ZK 62711) was from Schering AG, Berlin, Germany. CDP840 was from Celltech Chiroscience, Berks, UK. Siguazodan was from SmithKline Beecham Pharmaceuticals, King of Prussia, PA. Zardaverine (B 842-90) was from Forschungslaboratorien Byk Gulden, Konstanz, Germany. Zaprinast (M&B 22 948) was from Rhone-Poulenc Rorer, Dagenham, UK. HBSS with Ca2 and Mg2 was from GibcoBRL, Life Technologies, Paisley, UK. Methanol was from Merck, Leics, UK. Optiphase Hisafe was from Fisher Scienti®c, Leics, UK. All other chemicals were from Sigma Diagnostics, Dorset, UK. 3. Results 3.1. PDE isoenzyme pro®le As shown in Fig. 1a there was no difference in the total PDE activity in neutrophils from normal and COPD susceptible horses. Furthermore, there was no signi®cant difference in total neutrophil PDE activity between either young and older horses (39:7 3:6 versus 36:2 0:9 pmol=min=mg for young and older horses, respectively) or age-matched COPD and normal horses (34:4 1:0 versus 27:5 4:3 pmol=min=mg for normal and COPD horses, respectively). Addition of either a PDE4 or a mixed PDE3/4 inhibitor signi®cantly inhibited neutrophil PDE activity in both groups of horses. In contrast neither a PDE3 inhibitor nor a PDE2 stimulant/PDE3 inhibitor had a signi®cant effect. Surprisingly a PDE1 stimulant signi®cantly reduced PDE activity in neutrophils from both normal and COPD affected horses (Fig. 1a). As shown in Fig. 1b, total cGMP PDE activity was also similar in neutrophils from normal and COPD susceptible horses. However, the activity was considerably lower than that of cAMP PDE. No signi®cant inhibition of cGMP PDE activity in neutrophils from either group of horses was seen after addition of a PDE5 inhibitor or a PDE1 stimulant. 3.2. Concentration Ð response relationships for PDE isoenzyme inhibitors CDP840 and rolipram, a second PDE4 inhibitor, both caused concentration dependent inhibition of cAMP PDE activity in neutrophils from normal and COPD
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Fig. 1. PDE activity in neutrophils from normal horses (®lled columns) and COPD horses in remission (open columns). Columns represent mean values and lines represent standard error of the mean (n 5 for both groups). : P < 0:05 when compared to total cAMP PDE activity (cA) or total cGMP PDE activity (cG) (repeated measures two-way analysis of variance followed by Tukey's multiple comparison test).
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Table 1 IC50 values for inhibition of PDE activity in neutrophils from normal and COPD susceptible horsesa IC50 (M) Normals CDP840 Rolipram Zardaverine Siguazodan Theophylline a
COPDs ÿ9
8:8 0:1 10 1:2 0:1 10ÿ6 8:2 0:3 10ÿ7 2:2 0:1 10ÿ3 6:8 0:4 10ÿ3
7:3 0:2 10ÿ9 1:1 0:1 10ÿ6 8:6 0:9 10ÿ7 1:3 0:5 10ÿ3 5:6 0:2 10ÿ3
Results are expressed as mean standard error of the mean IC50; n 5 horses for both groups.
susceptible horses, although rolipram was considerably less potent. IC50 values for the PDE isoenzyme selective inhibitors are shown in Table 1. Zardaverine, also caused concentration related inhibition and was of similar potency to rolipram. There was also some inhibition of PDE activity in neutrophils from both groups of horses when siguazodan, was added, but this was only observed at high concentrations. Finally, the non-speci®c PDE inhibitor, theophylline, caused concentration dependent inhibition of cAMP PDE activity in neutrophils from both groups of animals but was at least 1000-fold less potent than either the PDE4 or PDE3/4 selective compounds (Table 1). There was no signi®cant difference in potency of any of the PDE inhibitors when IC50 values were compared in neutrophils from normal and COPD susceptible horses. Additionally, no difference in potency was found in cells from either young or older horses (IC50 values 4:5 1:3 10ÿ7 and 4:8 1:2 10ÿ7 M for young and older horses, respectively) or age-matched COPD versus normal horses (IC50 values 5:5 1:2 10ÿ7 and 3:6 1:8 10ÿ7 M for normal and COPD susceptible horses, respectively). 3.3. Effect of antigen challenge on PDE activity Antigen challenge caused a statistically signi®cant increase in the total blood leucocyte and neutrophil counts at 24 h post-challenge in the COPD susceptible horses but not the normal horses. DPplmax and/or respiratory rate increased in all the COPD affected horses. Additionally the expiratory pattern in the horses with COPD was biphasic post-challenge. However, the mean changes were not statistically signi®cant due to the variability of responses in individual horses. There were no changes in leucocyte counts or lung function in the normal horses (Table 2). Antigen challenge had no signi®cant effect on total PDE activity in neutrophils from either the normal or COPD susceptible horses. Moreover, there was no signi®cant difference in the cAMP PDE inhibitory potency of rolipram at 6 or 24 h when compared to pre-challenge values (Table 3).
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Normals COPDs a *
Respiratory rate (minÿ1)
Total leucocyte count 109 =l
Neutrophil count 109 =l
DPplmax (cm H2O)
0h
24 h
0h
24 h
0h
24 h
0h
6h
9:3 0:9 10:4 0:8
10:0 0:7 12:6 1:2*
4:9 0:4 5:6 0:4
5:6 0:4 7:9 0:8*
6:1 0:7 7:0 1:7
5:2 1:0 11:2 2:3
18:2 6:1 20:6 5:4
20:6 5:2 25:6 6:3
Results are expressed as mean standard error of the mean; n 5 horses for both groups. P < 0:05 compared with 0 h using Student's paired t-test.
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Table 2 Leucocyte counts and lung function changes after antigen challenge of normal and COPD susceptible horsesa
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Table 3 Neutrophil PDE activity and potency of inhibition by rolipram before and after antigen challenge of normal and COPD susceptible horsesa Time after challenge (h)
0 6 24
PDE activity (pmol/min/mg)
IC50 (M)
Normals
Normals
82:3 7:5 82:2 8:4 82:2 8:4 a
COPDs 71:7 9:9 66:1 4:5 63:9 9:4
COPDs ÿ6
1:0 0:5 10 9:1 0:6 10ÿ7 8:5 1:1 10ÿ7
1:1 1:9 10ÿ6 1:1 1:7 10ÿ6 9:7 2:4 10ÿ7
Results are expressed as mean standard error of the mean; n 5 horses for both groups.
4. Discussion This study has shown that total cAMP and cGMP PDE activities are similar in neutrophils from COPD susceptible and normal horses. This ®nding is in agreement with that reported by Goldberg et al. (1994) in neutrophils from normal and asthmatic human subjects. There was considerably more cAMP than cGMP PDE activity in equine neutrophils, which also agrees with data obtained using human neutrophils from normal subjects (Grady and Thomas, 1986). However, it should be noted that although the amount of cGMP PDE was similar, high background counts are obtained in the assay because of the poor adherence of unreacted substrate to the Dowex columns. This may mean that the amount of cGMP PDE present in the equine neutrophil has been underestimated. COPD is a disease that affects older animals and the mean age was higher in the COPD susceptible group. However no difference was found between total PDE activity in neutrophils from three age-matched normal and COPD susceptible horses. Similarly, age did not seem to affect PDE activity when neutrophils from a small group of young and older horses were used. Moreover, the potency of inhibition of PDE activity by the PDE4 selective inhibitor, rolipram, was also similar in neutrophils from both the age-matched and young versus older horses. Thus although Preuss and Goldie (1999) have reported differences in PDE activity in old versus young rats and guinea pigs, the limited study of neutrophils from old versus young horses and age-matched COPD and normal horses provides no evidence of such a difference in this species over the age range used. The isoenzyme pro®le analysis of equine neutrophil PDE has demonstrated that PDE4 is the predominant isoenzyme present, which agrees with the isoenzyme pro®le of human neutrophils (Neilson et al., 1990). Whilst both CDP840 and rolipram produced concentration related, and almost complete, inhibition of PDE activity, CDP840, a second-generation PDE4 inhibitor, was over 100-fold more potent than the archetypal PDE4 inhibitor, rolipram. In human neutrophils CDP840 is approximately 25 times more potent than rolipram. In contrast in guinea-pig neutrophils the two compounds are equipotent (Cooper et al., 1999). This demonstrates that species variability exists in the ability of these two PDE4 inhibitors to suppress neutrophil PDE4 activity. No signi®cant difference in potency of PDE4 inhibition was demonstrated in neutrophils from COPD susceptible horses when compared to that obtained in neutrophils from normal horses.
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PDE4 inhibition has not been compared in neutrophils from normal subjects and those with respiratory disease. In man PDE4 in monocytes from asthmatic subjects is less sensitive to inhibition by rolipram (Landells et al., 1997). Whether differences exist in the potency of inhibition of PDE4 activity in mononuclear cells from horses with COPD remains to be established. A signi®cant reduction in PDE activity was also obtained with zardaverine, which inhibits both PDE4 and PDE3. However, in the concentration response studies, the PDE3 selective inhibitor, siguazodan, only produced marked inhibition of neutrophil PDE activity at concentrations of 10ÿ4 and 10ÿ3 M (28 2% and 28 3% at 10ÿ4 M; 73 2% and 72 2% at 10ÿ3 M for normal and COPD susceptible horses, respectively). It is unlikely that, at these concentrations, the inhibitor is selective for PDE3 (Nicholson and Shahid, 1993). Hence, it seems likely that zardaverine is producing its effects primarily via inhibition of PDE4. Moreover, as PDE4 hydrolyses both cGMP and cAMP the small reduction in neutrophil PDE activity caused by addition of the PDE5 inhibitor, zaprinast (Fig. 1b), could be due to an effect on PDE4 (IC50 for inhibition of PDE4 by zaprinast 2:5 10ÿ5 M; Nicholson and Shahid, 1993). Theophylline only caused inhibition of PDE activity in equine neutrophils at relatively high concentrations (10ÿ3 M). This also agrees with data obtained in human neutrophils, for which the IC50 for theophylline is reported to be 2 10ÿ4 M (Neilson et al., 1990). Interestingly CaCAM, which stimulates PDE1, produced no increase in PDE activity but instead caused inhibition. The explanation for this effect is not apparent, although a similar observation has been made using human mononuclear cells (Landells and Page, personal communication). As there was no signi®cant difference in the PDE isoenzyme pro®le or inhibitory potency of PDE inhibitors in COPD susceptible horses in remission, when compared to normal horses, it was of interest to determine whether neutrophil activation during antigen challenge would alter either the total PDE activity or the inhibitory potency of the PDE4 inhibitor, rolipram. However, following antigen challenge, there was no difference between the two groups of horses with respect to either total cAMP PDE activity or the sensitivity to inhibition of PDE4, suggesting neutrophil activation in COPD susceptible horses does not affect cAMP PDE activity. Although human neutrophils have not been studied, these results do agree with data from human monocytes where there was no change in either total PDE activity or sensitivity to inhibition of PDE4 activity by rolipram following antigen challenge (Landells et al., 1999). The values for total cAMP PDE activity varied in the different studies reported here. This may be a natural phenomenon as discussed by Schudt et al. (1995) as variation in technique, reagents and storage of samples can be excluded. Although PDE activity and sensitivity to inhibition is no different in horses with COPD when compared with normal horses, these data suggest that PDE4 inhibitors, which reduce neutrophil function in other species, may be bene®cial in equine COPD by inhibiting neutrophil activation. Additional symptomatic bene®t may be achieved by inhibiting PDE4 in other in¯ammatory cells, as well as in airway wall smooth muscle. Thus, inhibition of PDE4 may cause bronchodilation directly, as well as inhibiting the release of mediators that contribute to increased cholinergic tone and in¯ammation.
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