Research Paper Mediators of Inflammation, 8, 153–157 (1999)

The aim of the present study was to compare the effects of selective phosphodiesterase (PDE) 3, 4 and 5 inhibitors on antigen-induced airway hyperresponsiveness in sensitized guinea-pigs. When the sensitized guinea-pigs were orally pre-treated with the selective PDE4 inhibitor, Ro 20-1724 (30 mg/kg), and studied 48h after OA, a significant reduction (P<0.01) of the leftward shift of the dose-response curve to ACh was noted, whereas it was ineffective at the lower dose (10 mg/kg). Administration of the selective PDE3 inhibitor, milrinone (30 mg/kg) also elicited a significant reduction (P<0.01) of the airway hyperresponsiveness, whereas the PDE5 inhibitor zaprinast (30 mg/kg) was ineffective. These results show that both PDE3 and PDE4 inhibitors are able to inhibit the antigen-induced airway hyperresponsiveness in sensitized guinea-pigs and support the potential utility of selective PDE inhibitors in the treatment of asthma.


Introduction
Bronchial asthma is a disease characterize d by variable airw ay obstruction and airw ay hype rre sponsivene ss that has be en linked to mucosal inflammation and partic ularly, the influx and activation of e osinophils. 1 One w ay to reduce the development of airw ay hyperresp onsive ness is to inhibit the associate d inflammation by anti-inflammatory drugs. How ever, under certain conditions, airw ay hyperre sponsivene ss has bee n obse rved despite the abse nce of any inflammatory proce ss, such as e osinophil re cruitme nt. We have pre viously de monstrated that substanc e P is able to induce airw ay hyperre sponsivene ss not assoc iate d w ith eosinophil rec ruitment, but w ith an enhanc eme nt of alve olar mac rophage ac tivation. 2 The cyclic nucle otides, cyclic adenosine monophosphate (cyclic AMP) and cyclic guanosine monophosphate (c yclic GMP), are important second messengers of c ellular func tion. Phosphodieste rases (PDEs) are a group of enzymes that regulate the bre akdow n of c yclic nucleotides. Among PDEs, PDE3 and PDE4 appear to be the most important for the regulation of cyclic AMP, w hereas PDE5 is re sponsible for the level of intrac ellular cGMP. As far as antiinflammatory drugs are conce rne d, much attention has been focuse d on the therapeutic pote ntial of se lec tive PDE4 inhibitors (for re view see Refs 3 and 4). It has also been frequently reported that PDE3 inhibitor s do not have potent anti-inflammatory e ffe cts. This has been cle arly demonstrated on e osinophil re c ruitment induced by antige n challenge , PAF ae rosol ex posure or intratra cheal administration of IL-5. 5,6 This is probably due to the fac t that in most inflammatory cells, including eosinophils, the low K m c yclic AMP-specific members of the PDE4 family are the most prominently ex pre ssed. 4,7 How e ver, in mononucle ar cells, PDE3 is also involved in the re gulation of cyclic AMP levels. In mac rophage s, in the presence of the adenylyl cyclase ac tivator, PGE 2 , PDE3 inhibitors are as effec tive as PDE4-selective drugs in inhibiting TNF-a rele ase. 8 We also re c ently demonstrated that PDE3 and PDE4 isoenzymes are present in alveolar mac rophage s from sensitized guinea-pigs and that PDE4, but also PDE3, regulate the re lease of inflammatory me diators. 9 Numerous studie s have reported the ac tual efficacy of PDE inhibitors, name ly selective PDE4 inhibitor s, on the development of airw ay hyperresponsiveness. How ever the effects of PDE3 inhibitors appe ar controversial. 10 -15 The p re se nt study w as undertaken in order to compare the e ffec t of milrinone , a selec tive PDE3 inhibitor, Ro 20-1724, a sele ctive PDE4 inhibitor, and zaprinast, a selec tive PDE5 inhib itor, on the de ve lopme nt of airw ay hyperresp onsive ness in sensitize d and challe nged guine a-p igs.

Sensitization procedure and challenge
Spe cific pathogen-fre e male Hartle y guinea-pigs (300-350 g, Charles Rive r, St. Aubin le s Elboe uf, France ) w e re use d throughout the study. Follow ing purchase, the y w e re house d in our standard animal care facilities. All guinea-pigs w e re fe d standard pe lle t (UAR, Ville moisson-Sur-Orge, Franc e) and given w ater a d libitu m . Guinea-pigs w ere sensitize d and challenge d as pre viously described. 16 Briefly, the y w ere plac ed in a Plex iglas chambe r (30 3 50 3 30 c m) and ex pose d tw ice for 30 min to an ae rosol of ovalbumin (OA) 2 mg/ml in saline (NaCl, 0.9 %), w ith a 48-h inte rval. The aerosol w as generated by a De vilbiss ultrasonic ne bulizer (Ae rodynamic mean mass median particle diameter of 0.5-5 m M, ULTRA-NEB 99, Somme rset, PA, USA). Fiftee n to 20 days afte r the initial se nsitization procedure, the guinea-pigs w e re challenge d by 15-min ex posures to five succ essive solutions of OA of respec tively 10 m g/ml, 100 m g/ml, 1 mg/ml, 5 mg/ml and 10 mg/ml. Control guinea-pigs w ere ex posed to a saline solution for an equivale nt period of time.

Assessment of airway hyperresponsiveness
Bronchopulmonary re ac tivity to ac etylcholine w as asse sse d 48 h after antigen challenge or saline ex posure. Guinea-pigs are anaesthe tize d (urethane, 1.2 g/kg, i.p.) and place d in a dorsal rec umbe nt position. A trache a cannula w as inserte d and the lungs w ere mechanically ventilated w ith a constant tidal volume (1 ml laboratory air/100 g body w eight) w ith a respiratory pump (Ugo Basile, Vare se , Italy, 60 breaths/min). Spontaneous breathing w as abolishe d w ith panc uronium bromide (2 mg/kg), injec te d in the poste rior penis ve in. Airw ay inflation pressure (AIP), an index of intrathoracic airw ay c aliber, w as monitored from a late ral port of the ventilator circ uit using a Ugo basile bronchospasm transduce r acc ording to the previously desc ribed method. 16 After a 10-min equilibration pe riod, thre e succ essive 1-min aerosol administrations of Ac h (50, 100, 200 and 500 m g/ml) w e re performed at 10-min inte rvals w ith c onstant monitoring of the airw ay inflation pressure . The aerosol w as gene rated by a De vilbiss 'Pulmosonic ' ultrasonic nebulizer permane ntly conne cte d in se rie s w ith the affe rent limb of the ventilator circuit. The airw ay inflation p re ssure w as ex pressed as p ercent change over the 100% obtained by clamping the trache al cannula at the end of the ex pe riment.

Protocol
All drugs w ere pre pare d ex te mporaneously in distilled w ater c ontaining 5% Arabic gum. Sensitize d guinea-pigs w ere tre ated orally w ith either Ro 20-1724 (10 or 30 mg/kg) or milrinone (30 mg/kg) or zaprinast (30 mg/kg), 24 and 3 h before the antigen challe nge or saline ex posure .

Data analysis
Results are ex pressed as means± SEM. Statistical differences betw e en the dose -resp onses to Ac h in the groups rec eiving the various treatme nt w ere analyze d by tw o-w ay analysis of variance . It w as thus possible to ex amine the w hole dose -response c urves obtaine d in these diffe rent groups of animals.

Results
Ex posure of anaesthe tize d guinea-pigs to the succe ssive ae rosols of ACh (50, 100, 200 and 500 m g/ml) induc ed a dose -re lated bronchopulmonary response. Whe n the animals w ere previously ex posed to OA, the dose -response curve to ACh w as significantly shifted to the left (P< 0.001) (Figs 1-4).
When the se nsitized guine a-pigs w ere pre treate d w ith Ro 20-1724 (30 mg/kg), and studied 48 h after OA, a signific ant reduction (P< 0.05) of the leftw ard shift of the dose -re sponse c urve to ACh w as note d (Fig. 1), w he re as no e ffec t w as observe d at the low er dose (10 mg/kg) (Fig. 2).

Discussion
The prese nt study de monstrated that the selec tive PDE4 inhibitor, Ro 20 -1724 and the se lec tive PDE3 inhibitor, milrinone , but not the se lective PDE5 inhibitor, zaprinast, are able to inhibit the deve lopme nt of airw ay hyperre sponsivene ss induced by antigen challenge in sensitized guinea-pigs.
Airw ay hyperre sponsivene ss induc ed by antigen challe nge is generally assoc iate d in humans and in ex pe rimental animals w ith an influx of inflammatory ce lls in lung tissue 1,16 and the ac tivation of re side nt pulmonary c ells such as alveolar mac rophages. 9 ,17 ,1 8 We have also reported that mac rophages recove red in the BAL fluid of e ithe r saline-ex posed or OA-challenge d se nsitized guinea-pigs ex hibit a PDE type 3 and a PDE type 4 is oe nzyme activity. 9 It c an there fore be suggeste d that the p rofile of PDE activities in mac rophage s may influe nce the ac tivity of PDE inhibitors. In this regard, w e observe d that the c ombination of milrinone and the sele ctive PDE4 inhibitors, rolipram or Ro 20 -1724 had a signific ant inhibitory effe ct on arachidonate re le ase. 9 We rec ently proposed that the reduced activity of alve olar macrophage s may be involved in the inhibition of the de ve lopme nt of airw ay hype rresponsiveness by both PDE3 and PDE4 inhibitors and suggest that activation of alveolar macrophage s is a ke y e vent in the bronchopulmonary alterations that follow antige n challenge.
Such a hypothe sis is not consis te nt w ith the fac t that the de ve lopme nt of airw ay hyperre sponsivene ss is closely associate d w ith eosinophil influx in airw ays. We and othe rs have previously demonstrated that PDE4 inhibitors, but not PDE3 inhibitors, are able to reduce the eosinophil recruitme nt induc ed either by antigen challenge in sensitize d guinea-pigs 5 ,19 or administration of che motac tic factor, such as PAF and IL-5 in naive guinea-pig s. 5,6 This w ould suggest a dissociation be tw e en eosinophil re cruitme nt and the development of airw ay hyp erresponsive ness. This dissociation take s into acc ount the fac t that eosinophils do not c ontain PDE3 isoenzyme , 4 and the n that PDE3 inhibitors are not able to block mediator release from these inflammatory ce lls. 1 9 This hypothe sis is also stre ngthene d by the re sults obtained w ith ae rosol ex posure of guine a-pigs to substance P. Inde ed, the development of airw ay hype rre sponsivene ss induce d by aerosol administration of substanc e P is assoc iate d w ith alveolar macrophage ac tivation rathe r than granulocyte re cruitment. 2 A propose d mechanism for the inhibition of antigen-induce d airw ay hyperresponsive ness by PDE inhibitors is the regulation of cyclic AMP by PDE3 and 4 c ontributing to the modulation of ne uronal sensitivity in the airw ays and of tachykinin release . 20 ,21 It is now generally ac cepted that se lective PDE4 inhibitors are able to reduc e airw ay hype rre sponsivene ss, and that this effec t is assoc iated w ith anti-Se le ctive ph o s ph o die s te ra s e in hibito rs a n d b ro n chia l hy pe rre s po n s ive n e s s  1 5 have rep orte d an inhibition of LPS-induc ed bronchial hyperre ac tivity in guinea-pigs, and Fujimura e t a l. 13 a reduced bronchial hyperresponsivene ss in asthmatic patients. Finally, Bardin e t a l. 14 have reported that the selec tive PDE3 inhibitor, MKS492, is able to pre ve nt early bronchoconstric tor resp onse in asthma and attenuates late response. Since PDE3 inhibitors have bronchodilator and bronchoprotective propertie s, 2 2 it can also be suggeste d that the bronchoprote c tive effe ct obse rved in these latter studies w as due to their re lax ant ac tivity on smooth muscles. 23 In the prese nt study, w e ex clude d such a possibility since the bronchopulmonary re sponse induce d by acetylcholine w as not modified by milrinone in unchallenge d animals.
In conclusion, the prese nt study show e d that PDE3 (milrinone ) and PDE4 inhibitors (Ro 20-1724), but not the PDE5 inhibitor (zap rinast), are able to reduc e the de velop ment of airw ay hype rre sponsivene ss in se nsitized guine a-p igs and suggests that this group of se le ctive compounds may have a role to play in the tre atment of asthma.