Effects of cigarette smoke on Toll-like receptor (TLR) activation of chronic obstructive pulmonary disease (COPD) macrophages.

Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal innate immune response. We have investigated the changes in the innate immune response of COPD alveolar macrophages exposed to both cigarette smoke and Toll-like receptor (TLR) stimulation. COPD and control alveolar macrophages were exposed to cigarette smoke extract (CSE) followed by TLR-2, -4 and -5 ligands [Pam3CSK4, lipopolysaccharide (LPS) and phase I flagellin (FliC), respectively] or non-typeable Haemophilus influenzae (NTHi). CSE exposure suppressed TLR-induced tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and regulated on activation, normal T cell expressed and secreted (RANTES) production in both COPD and control alveolar macrophages, but had no effect on interleukin 8 (CXCL8) production. Similarly, CSE suppressed NTHi-induced TNF-α but not NTHi-induced CXCL8 production in COPD alveolar macrophages. Gene expression analysis showed that CSE suppressed LPS-induced TNF-α transcription but not CXCL8 transcription in COPD alveolar macrophages. The dampening effect of CSE on LPS-induced cytokine production was associated with a reduction in p38, extracellular signal regulated kinase (ERK) and p65 activation. In conclusion, CSE caused a reduced innate immune response in COPD alveolar macrophages, with the exception of persistent CXCL8 production. This could be a mechanism by which alveolar macrophages promote neutrophil chemotaxis under conditions of oxidative stress and bacterial exposure.

Characterisation of a refined rat model of respiratory infection with Pseudomonas aeruginosa and the effect of ciprofloxacin.

BACKGROUND: We sought to characterise a refined rat model of respiratory infection with P. aeruginosa over an acute time course and test the antibiotic ciprofloxacin. METHODS: Agar beads were prepared ± SPAN(®)80. Rats were inoculated with sterile agar beads or those containing 10(5) colony forming units (cfu) P. aeruginosa via intra-tracheal dosing. Bacterial load and inflammatory parameters were measured. RESULTS: Differing concentrations of SPAN(®) 80 modified median agar bead diameter and reduced particle size distribution. Beads prepared with 0.01% v/v SPAN(®)80 were evaluated in vivo. A stable lung infection up to 7 days post infection was achieved and induced BALF neutrophilia 2 and 5 days post infection. Ciprofloxacin (50mg/kg) significantly attenuated infection without affecting the inflammatory parameters measured. CONCLUSION: SPAN(®) 80 can control the particle size and lung distribution of agar beads and P. aeruginosa-embedded beads prepared with 0.01%v/v SPAN(®)80 can induce infection and inflammation over 7 days.

Exposing rodents to a combination of tobacco smoke and lipopolysaccharide results in an exaggerated inflammatory response in the lung.

BACKGROUND AND PURPOSE: Acute exacerbations of chronic obstructive pulmonary disease (COPD), which are often associated with respiratory infections, are defined as a worsening of symptoms that require a change in medication. Exacerbations are characterized by a reduction in lung function, quality of life and are associated with increased pro-inflammatory mediators in the lung. Our aim was to develop an animal model to mimic aspects of this exaggerated inflammatory response by combining key etiological factors, tobacco smoke (TS) and bacterial lipopolysaccharide (LPS). EXPERIMENTAL APPROACH: Rats were exposed to TS for 30 min twice a day for 2 days. On day 3 animals were exposed to LPS for 30 min followed by exposure to TS 5 h later. Inflammation, mucus and lung function were assessed 24 h after LPS. KEY RESULTS: Neutrophils, mucus, oedema and cytotoxicity in lung and/or bronchoalveolar lavage was increased in animals exposed to combined LPS and TS, compared with either stimulus alone. Lung function was impaired in animals exposed to combined LPS and TS. Inflammatory cells, oedema and mucus were unaffected by pretreatment with the corticosteroid, budesonide, but were reduced by the phosphodiesterase 4 selective inhibitor roflumilast. Additionally, lung function was improved by roflumilast. CONCLUSIONS AND IMPLICATIONS: We have established an in vivo model mimicking characteristic features of acute exacerbations of COPD including lung function decline and increased lung inflammation. This model may be useful to investigate molecular and cellular mechanisms underlying such exacerbations, to identify new targets and to discover novel therapeutic agents.

Animal models of cough: literature review and presentation of a novel cigarette smoke-enhanced cough model in the guinea-pig.

A wealth of literature describes the approaches that investigators have used to develop animal models of cough. The relevance of the models to cough in man and disease is still unknown. Furthermore, the choice of animal model that is used will depend on the purpose of the investigation and what questions are being asked. Cigarette smoke is known to cause COPD and cough is a principle symptom where patients demonstrate an increased cough response to citric acid or capsaicin. This paper describes the development of exacerbated cough to these agents in the guinea-pig following cigarette smoke exposure and pharmacological profiling of these models. Male Dunkin-Hartley guinea-pigs were exposed to air or cigarette smoke (4 or 5 research cigarettes daily for the capsaicin and citric acid studies, respectively) for a 3 s puff every 30 s, for up to 10 days. At selected time points conscious, unrestrained animals were placed in a plethysmograph chamber and challenged with an aerosol of 0.3 M citric acid (10 min) or 10 microM capsaicin (7 min). Cough and Penh area under the curve (AUC) were recorded during the exposure and for a further 10 min (citric acid) or 8 min (capsaicin) after exposure. Compounds were administered on day 3 or 11 for citric acid or capsaicin, respectively. Significant enhancement of citric acid-induced cough was evident 24 h (12+/-2 to 24+/-4* coughs) after a single exposure and further enhanced after 2 days (13+/-3 to 36+/-4* coughs). Enhanced cough to capsaicin was not reliable until after 10 days of cigarette smoke exposure (2+/-1 to 14+/-3** coughs). Data are expressed as mean+/-s.e.mean (n=10), *p<0.05, **p<0.01 vs. air-exposed animals (Mann-Whitney rank-sum test). The minimum effective doses to inhibit citric acid-induced cough were 10, 10, 3 and 0.3 mg/kg for codeine (p.o. -30 min), a selective NK(1)/NK(2) antagonist, DNK333 (p.o. -2 h), terbutaline (s.c. -1 h) and atropine (s.c. -1 h), respectively. The minimum effective doses to inhibit capsaicin-induced cough were 3, 1, 0.3 and 0.3 mg/kg for codeine, DNK333, terbutaline (p.o. -2 h) and atropine, respectively. The VR1 antagonists capsazepine and iodo-resiniferatoxin (IRTX) did not inhibit cough in either model. Differences in sensitivity between citric acid and capsaicin to pharmacological agents may be partly explained by the difference in magnitude of response to these agents. Clinically used compounds such as codeine and terbutaline have shown activity in both models, however the relevance of the models to cough in man and disease for potential new therapies is unknown.

Identification and quantification of phosphodiesterase 4 subtypes in CD4 and CD8 lymphocytes from healthy and asthmatic subjects.

In the present study, for the first time, PDE4 subtypes were identified and semi-quantified in both CD4 and CD8 lymphocytes from healthy and asthmatic individuals. CD4 and CD8 lymphocytes from healthy and mild asymptomatic asthmatic subjects (receiving beta-agonist therapy only) were isolated from peripheral venous blood using appropriate antibody coated paramagnetic beads. PDE4 subtypes and beta-actin were identified by digoxigenin (DIG)-labelling reverse transcriptase-polymerase chain reaction and semi-quantified by DIG-detection enzyme-linked immunosorbance assay. In CD4 and CD8 lymphocytes PDE4A, PDE4B and PDE4D were detected, with no significant differences observed between healthy and asthmatic groups. In CD8 lymphocytes, enzyme subtype expression was lower and showed more intersubject variability. In functional studies investigating the effects of various PDE inhibitors on PHA-induced proliferation of mononuclear cells from healthy and asthmatic subjects, CDP840 (0.03 - 10 microM), rolipram (0.1 - 10 microM) and theophylline (10 microM - 1 mM) inhibited PHA-induced proliferation of mononuclear cells from healthy and asthmatic subjects in a concentration-dependent manner, although no significant difference was observed between the groups investigated. In additional studies, total monocyte cyclic AMP PDE activity was investigated in cells isolated from asthmatic subjects both prior to and 24 h after allergen challenge. Total monocyte cyclic AMP PDE activity remained unaffected following challenge of asthmatic subjects with either house dust mite or cat dander and was inhibited in a concentration-dependent manner by rolipram (0.01 - 100 microM) both before and after allergen challenge.

Evidence to suggest that the phosphodiesterase 4 isoenzyme is present and involved in the proliferation of umbilical cord blood mononuclear cells.

BACKGROUND: The type 4 phosphodiesterase (PDE) isoenzyme is the main isoenzyme of PDE involved in the control of adult mononuclear cell proliferation. OBJECTIVE: To establish whether PDE isoenzymes are present in umbilical cord blood mononuclear cells by the use of selective PDE inhibitors, and to identify which PDE isoenzymes are involved in controlling the proliferation of cord blood mononuclear cells. METHODS: Cord blood was obtained from normal deliveries and mononuclear cells isolated as described previously [1] with some modifications. Mononuclear cells were then stimulated to proliferate with phytohaemagglutinin (PHA) (2 microg/mL) in the presence of selective PDE inhibitors. Proliferation was measured by [3H]-thymidine incorporation. RESULTS: The type 4 PDE inhibitors (CDP840, rolipram and RO 20-1724), and the mixed PDE3/4 inhibitor, zardaverine, produced a concentration-related inhibition of PHA-stimulated cord blood mononuclear cell proliferation (P < 0.05, ANOVA). The non-selective PDE inhibitor, theophylline, also produced a concentration-related inhibition of proliferation (P < 0.05, ANOVA). In contrast, the PDE1 inhibitor, vinpocetine, the PDE3 inhibitor, siguazodan, and the PDE5 inhibitor, zaprinast, were unable to inhibit cord blood mononuclear cell proliferation. CONCLUSION: PDE4 is present in umbilical cord mononuclear cells and is involved in the control of cord blood mononuclear cell proliferation.

Possible Contribution of Prostaglandin E2 to the antiproliferative effect of phosphodiesterase 4 inhibitors in human mononuclear cells.

Phosphodiesterase (PDE) 4, mixed PDE3/4, and non-selective PDE inhibitors have been shown to inhibit the proliferation of human peripheral blood mononuclear cells (HPBM). The aim of the present study was to examine whether endogenous prostaglandins, in particular prostaglandin E2 (PGE2), are involved in mediating the antiproliferative actions of PDE inhibitors, by comparing their effects with drugs which elevate or mimic adenosine 3',5'-cyclic monophosphate (cAMP) through mechanisms other than PDE inhibition. Indomethacin significantly reduced the antiproliferative effects of the PDE4 inhibitors rolipram and CDP840 and the mixed PDE3/4 inhibitor zardaverine, increasing the IC50 values from 2.51 microM to >10 microM, 0.81 microM to 2.82 microM, and 1.58 microM to 4.82 microM, respectively (P < 0.05), but did not alter the effects of theophylline. Forskolin, PGE2, and dibutyryl cAMP also inhibited HPBM proliferation, and in the presence of indomethacin the effects of forskolin and dibutyryl cAMP were reduced (although this was not significant), whereas PGE2 was not affected. Rolipram, CDP840, zardaverine, and dibutyryl cAMP all produced a concentration-related increase in PGE2 production (P < 0.05, ANOVA), but theophylline significantly increased PGE2 production only at the highest concentration examined, 1000 microM. The ability of indomethacin to reduce the antiproliferative effects of rolipram, CDP840, and zardaverine, together with the fact that these drugs can stimulate PGE2 production, suggests that their antiproliferative actions may be mediated in part by stimulation of endogenous PGE2 production. In contrast, it appears that endogenous PGE2 is not critical for the antiproliferative actions of theophylline, forskolin, and dibutyryl cAMP in HPBM. These results establish the importance of co-ordinated regulation of the cAMP phosphodiesterase and cyclooxygenase-PGE2 systems for the regulation of lymphocyte function in man, and have clinical implications for therapeutic approaches to diseases associated with lymphocyte dysregulation.

Effects of dexamethasone on airway hyper-responsiveness to the adenosine A1 receptor agonist cyclo-pentyl adenosine in an allergic rabbit model.

1. New Zealand White (NZW) rabbits were immunized within 24 h of birth with Alternaria tenuis in aluminium hydroxide (Al (OH)3) (i.p.) or sham immunized (saline plus Al (OH)3 i.p.) and subsequently injected with the allergen (i.p.) or sham-immunized for the next 3 months. At 3 months of age, baseline airway responsiveness was assessed using cyclo-pentyl adenosine (CPA). Bronchoalveolar lavage (BAL) was performed in all animals and samples of peripheral blood were collected from some animals for estimation of dexamethasone levels. In some animals, blood was collected at the end of the experiment and cellular function was assessed by measurement of ex vivo proliferation of mononuclear cells in response to phytohaemagglutinin (PHA). 2. Allergen immunization significantly increased baseline airway responsiveness to inhaled CPA (P<0.05) in comparison with sham-immunized animals, at 3 months after immunization. Dexamethasone (0.5 mg kg(-1) day(-1)) treatment for 1 month did not modify this established airway hyper-responsiveness to CPA. Dexamethasone treatment did not affect either total or differential cell numbers in BAL fluid during the 4 week period, although significant plasma levels of dexamethasone were achieved in dexamethasone treated animals. 3. Treatment of rabbits with dexamethasone (0.1 mg kg(-1) i.p.), 6 h prior to each allergen injection from the neonatal stage, significantly reduced baseline airway hyper-responsiveness to CPA measured at 3 months (P<0.05). There was no significant difference in either total or differential cell numbers in BAL fluid, or any difference in mitogen-induced proliferation of mononuclear cells between dexamethasone and vehicle treated rabbits. 4. These results suggest that introduction of glucocorticosteroids in early life can prevent baseline airway hyper-responsiveness to inhaled CPA in allergic rabbits. However, once established, such underlying airway hyper-responsiveness is difficult to resolve, even with prolonged treatment with glucocorticosteroids.

The effect of selective phosphodiesterase 3 and 4 isoenzyme inhibitors and established anti-asthma drugs on inflammatory cell activation.

1. This study aimed to evaluate the effects of phosphodiesterase (PDE) inhibitors and currently prescribed anti-asthma drugs for their ability to inhibit inflammatory cell activation in vitro. 2. Alveolar macrophages and eosinophils were isolated from the bronchoalveolar lavage (BAL) fluid of ovalbumin (Ovalb)-sensitized guinea-pigs. Opsonized zymosan (OZ) and PAF stimulated leukotriene B4 (LTB4) release from eosinophils was measured by radioimmunoassay. Ovalb-induced superoxide generation was measured by reduction of cytochrome C. 3. Monocytes were separated from human peripheral venous blood and mast cells were dispersed from human lung fragments. Lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) release from monocytes was measured by ELISA and anti-IgE stimulated histamine release from mast cells was measured by a radioenzymatic method. 4. The beta 2 agonist, salbutamol inhibited TNF-alpha release from monocytes and histamine release from mast cells whilst having no effect on eosinophil-derived LTB4 release or macrophage superoxide generation. 5. The PDE 3 inhibitor, milrinone produced a concentration-related inhibition of TNF-alpha release from monocytes which achieved statistical significance at 10(-5) M but inhibited LTB4 release from eosinophils and superoxide generation from macrophages only at the highest concentration (10(-3) M) examined. Milrinone had no effect on histamine release from mast cells. 6. The selective PDE 4 inhibitors, denbufylline and rolipram and the corticosteroid, beclomethasone produced a concentration-related inhibition of LTB4 release from eosinophils, TNF-alpha release from monocytes and superoxide generation from alveolar macrophages whilst having no effect on histamine release from mast cells. 7. The mixed PDE 3/4 inhibitor, benzafentrine produced a concentration-related inhibition of LTB4 release from eosinophils, TNF-alpha release from monocytes, superoxide generation from alveolar macrophages and histamine release from mast cells. 8. In conclusion these data clearly show that both established anti-asthma medication as well as PDE inhibitors have the potential to inhibit inflammatory cell activation in vitro but that the anti-secretory actions of beta 2 agonists, corticosteroids and PDE inhibitors are distinct.

Ovalbumin challenge following immunization elicits recruitment of eosinophils but not bronchial hyperresponsiveness in guinea-pigs: time course and relationship to eosinophil activation status.

Eosinophils are known to be present in the airways of allergic asthmatics, and have been suggested to contribute to the pathophysiological changes accompanying this condition, particularly hyperresponsiveness to airway spasmogens. However, a causal relationship between pulmonary eosinophil accumulation and bronchial hyperresponsiveness in asthma is not proven. In the present study, the time course of pulmonary cell influx was investigated in an immunized guinea-pig model. Eosinophil activation status was also determined together with the bronchial responsiveness to histamine. Guinea-pigs were sensitized [20 micrograms ovalbumin (OVA) per animal in A1(OH)3 (0.5 ml) i.p.] and subsequently challenged with aerosolized OVA (100 micrograms/ml) for 1 h 18-21 days later. At different time points (1 h to 72 h) after OVA challenge, bronchial responses to i.v. histamine were measured and bronchoalveolar lavage (BAL) was performed to assess pulmonary cell influx. Eosinophil peroxidase (EPO) and total protein levels were measured in BAL fluid supernatants. Exposure of sensitized animals to aerosolized OVA produced a significant increase (P < 0.05 vs. sham immunized) in eosinophil infiltration 24 h later which was sustained up to 72 h. Despite this, OVA challenge did not cause either eosinophil activation, as measured by EPO release, or bronchial hyperresponsiveness to histamine at any of the time points examined. These data show that allergen challenge of sensitized guinea-pigs can elicit airway eosinophil accumulation without accompanying airways hyperresponsiveness or eosinophil activation.

Effect of isoenzyme selective phosphodiesterase inhibitors on the proliferation of murine thymus and spleen cells.

The effects of isoenzyme selective phosphodiesterase inhibitors on mitogen-stimulated proliferation of murine thymus and spleen cells was determined. The type 4 phosphodiesterase inhibitors, (rolipram, RO 20-1724 and denbufylline) and the mixed type 3/4 inhibitors, (zardaverine and benzafentrine) produced a concentration-related inhibition of mitogen stimulated thymus and spleen cell proliferation. Combined addition of the type 4 inhibitor, rolipram and the type 3 inhibitor, SK&F 94836 had no antiproliferative effect additional to that of rolipram alone. The thymus cells were more sensitive to the type 4 inhibitors than the spleen cells. The type 3 phosphodiesterase inhibitor, SK&F 94836 significantly inhibited cell proliferation, but only at high concentrations. The type 1 (vinpocetine) and the type 5 (zaprinast) phosphodiesterase inhibitor had no significant effect on proliferation. These results suggest that thymus and to a lesser extent spleen cell proliferation is dependent on the activity of the type 4 phosphodiesterase isoenzyme.

Differential effect of phosphodiesterase 4 inhibitors on the proliferation of human peripheral blood mononuclear cells from normals and subjects with atopic dermatitis.

1. The aims of this study were to compare the effects of selective inhibitors of the type 3, type 4 and type 5 phosphodiesterase (PDE) isoenzymes on the phytohaemagglutinin (PHA)-stimulated proliferation of human peripheral blood mononuclear cells (HPBM) from normals and subjects with atopic dermatitis (AD). 2. Mononuclear cells were isolated from peripheral venous blood of normals and subjects with AD. A concentration-response curve was carried out with PHA (0.5-5 micrograms ml-1) and a concentration which produced a submaximal stimulation of proliferation (2 micrograms ml-1) was selected for further experiments. HPBM (10(5) cells per well) were stimulated with PHA (2 micrograms ml-1) in the absence or presence of PDE inhibitor (0.01 microM-10 microM) and 24 h later [3H]-thymidine (0.1 microCi per well) was added. Cells were incubated for an additional 24 h period and [3H]-thymidine incorporation measured. 3. The type 4 PDE inhibitors (rolipram, RO 20-1724 and denbufylline) produced a concentration-related inhibition of proliferation of HPBM from normal and AD subjects. The IC50 for rolipram was significantly (P < 0.05) lower in HPBM from AD patients 0.28 microM (95% confidence limits (CL): 0.158-0.499, n = 5) vs normal subjects 2.6 microM (95% CL: 0.867-7.05, n = 5, P < 0.05) as were the IC50 values for denbufylline: 0.26 microM (95% CL: 0.152-0.440, n = 5) vs 1.84 microM (95% CL: 0.467-7.23, n = 5, P < 0.05) respectively and RO 20-1724: 1.49 microM (95% CL: 0.61 microM-3.64 microM) vs 6.46 microM (95% CL: 2.03 microM-20.46 microM), respectively. 4. The mixed type 3/4 inhibitors (zardaverine and benzafentrine) produced a concentration-related inhibition of proliferation of HPBM from normal and AD subjects. The IC50 value for zardaverine in HPBM from normal subjects: 1.8 microM (95% CL: 0.43 microM-7.85 microM, n = 4) was similar to that in AD subjects: 1.03 microM (95% CL: 0.48 microM-2.28 microM) as was the IC50 value for benzafentrine in normal 3.8 microM (95% CL: 2.45 microM-5.9 microM) and atopic 5.5 microM (95% CL: 3.84 microM-7.78 microM) HPBM. The type 5 PDE inhibitor, zaprinast was ineffective at inhibiting the proliferation of normal HPBM. The type 3 PDE inhibitor, siguazodan only inhibited [3H]-thymidine incorporation at a concentration of 10 microM. 5. These results show that combined inhibition of the type 3 and 4 PDE isoenzymes in HPBM from normal subjects has a greater antiproliferative effect than inhibition of the type 4 isoenzyme alone. In addition these data indicate that the proliferative response of HPBM from AD subjects is more sensitive to PDE 4 inhibition than the proliferation of HPBM from normals.

Immunomodulatory actions of xanthines and isoenzyme selective phosphodiesterase inhibitors.

Theophylline and related xanthines have been used in the treatment of airways diseases such as bronchial asthma for over 50 years. The therapeutic effectiveness of this class of drugs was traditionally thought to be derived from their ability to elicit bronchodilation, however evidence is accumulating to suggest that theophylline in particular possesses immunomodulatory and anti-inflammatory activity. The molecular mechanisms of action of theophylline have not yet been clarified, although several putative mechanisms of action have been proposed. One of these suggests that theophylline via non-selective inhibition of the phosphodiesterase (cAMP) and cyclic guanosine monophosphate (cGMP) levels resulting in relaxation of airway smooth muscle and inhibition of inflammatory cell activation. To date seven different families of the PDE enzyme have been defined according to a variety of criteria including substrate specificity, sensitivity to selective inhibitors and the effect of allosteric modulators. The type IV isoenzyme is the predominant isoenzyme in most inflammatory cells. This article reviews some of the in vitro, in vivo and clinical studies which have demonstrated that theophylline and selective PDE inhibitors possess anti-inflammatory and immunomodulatory activity.

Theophylline and selective phosphodiesterase inhibitors as anti-inflammatory drugs in the treatment of bronchial asthma.

Theophylline has been in clinical use for the treatment of bronchial asthma and other respiratory diseases for well over 50 yrs. Over this time, a considerable body of evidence has accumulated to show that this drug has a wide range of pharmacological actions, in addition to the well-recognized action on airway smooth muscle function. Current evidence suggests that part of the therapeutic value of theophylline in the treatment of asthma is by virtue of an anti-inflammatory or immunomodulatory effect, although the actual mechanism of action remains unclear. It has been proposed that the observed anti-inflammatory effects of theophylline could be attributed to phosphodiesterase (PDE) inhibition, and recently the type III and IV isoenzymes have been characterized in a number of inflammatory cells. This article reviews the evidence that theophylline and the newer more selective type IV PDE isoenzyme inhibitors can inhibit the activation of inflammatory cell types, such as T-lymphocytes, eosinophils, mast cells and macrophages, in vitro. The evidence supporting the ability of theophylline and selective PDE IV isoenzyme inhibitors to modify allergic inflammation both in animal models and clinical asthma is also discussed. We conclude that theophylline possesses important anti-inflammatory and immunomodulatory activity and that, in light of this evidence, it is timely to reconsider the place of theophylline in the treatment of asthma.

The effect of selective phosphodiesterase inhibitors in comparison with other anti-asthma drugs on allergen-induced eosinophilia in guinea-pig airways.

The effects of isoenzyme selective phosphodiesterase (PDE) inhibitors and other anti-asthma drugs on antigen-induced eosinophil recruitment and activation in guinea-pig airways was studied. Guinea-pigs were sensitized and subsequently challenged with aerosolized ovalbumin (OVA). Bronchoalveolar lavage (BAL) was performed 24 h later. A significant increase in eosinophils and eosinophil peroxidase (EPO) was detected in BAL fluid and BAL fluid supernatant respectively from OVA immunized guinea-pigs compared with sham treated animals. Guinea-pigs were treated for 7 days prior to antigen challenge with either the following drugs or the appropriate vehicle (i.p.). The selective beta 2 agonist, salbutamol (0.3 mg/kg), the PDE III inhibitor, milrinone (15 mg/kg) and the non-selective PDE inhibitor, trequinsin (1 mg/kg) had no effect on eosinophil number or EPO levels. The PDE IV inhibitor, rolipram (15 mg/kg), the mixed type III/IV PDE inhibitor, benzafentrine (15 mg/kg) and the non-selective PDE inhibitor, aminophylline (31.5 mg/kg) had no effect on eosinophil number but reduced the amount of EPO detected. The anti-inflammatory glucocorticosteroids, beclomethasone (10 mg/kg) and betamethasone (4 mg/kg) and the type IV PDE inhibitor, RP 73401 (5 mg/kg) reduced both eosinophil numbers and EPO levels. These results suggest a role for the type IV PDE isoenzyme in the control of eosinophil recruitment and possibly activation in the airways.

Acute versus chronic administration of phosphodiesterase inhibitors on allergen-induced pulmonary cell influx in sensitized guinea-pigs.

1. The aims of this study were to determine which phosphodiesterase (PDE) isoenzymes are involved in the control of eosinophil accumulation in the airways of ovalbumin (OVA)-immunized guinea-pigs by the use of isoenzyme selective inhibitors and to compare the effects of acute versus chronic administration of PDE isozyme inhibitors on pulmonary cell influx in ovalbumin-immunized guinea-pigs. 2. Guinea-pigs were sensitized and subsequently challenged with aerosolized OVA. Twenty four hours later bronchoalveolar lavage (BAL) was performed to permit assessment of inflammatory cell accumulation. A significant increase in the number of eosinophils was observed in the lavage fluid from OVA-immunized (13.6 +/- 1.4 x 10(4) ml-1 in acute experiments and 10.1 +/- 1.4 x 10(4) ml-1 in chronic experiments) animals compared with sham-treated controls (5.6 +/- 0.6 x 10(4) ml-1 in acute experiments and 5.1 +/- 0.6 x 10(4) ml-1 in chronic experiments). There was no difference in neutrophil, mononuclear cell or total cell numbers between the two groups. 3. Acute administration of a high dose of selective and non-selective PDE inhibitors by the i.p. route had no significant effect on eosinophil accumulation in the airways. 4. Chronic administration of a low dose (3 mg kg-1, i.p., twice daily for 7 days) of the type IV PDE inhibitor, RO 20-1724, and the PDE III/IV inhibitor, zardaverine, produced a significant inhibition of eosinophil accumulation (46% and 59% respectively). 5. These results suggest that the type IV PDE isoenzyme plays a role in the control of allergen-induced eosinophil infiltration into the airways, but indicate that a period of low dose chronic treatment with a type IV or mixed type III/IV PDE inhibitor is necessary for eosinophil accumulation in the airways to be reduced.