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. 2001 Dec;126(3):384–389. doi: 10.1046/j.1365-2249.2001.01646.x

Differential effect of phosphodiesterase inhibitors on IL-13 release from peripheral blood mononuclear cells

N Yoshida *, Y Shimizu *, K Kitaichi †, K Hiramatsu *, M Takeuchi *, Y Ito *, H Kume *, K Yamaki *, R Suzuki ‡, E Shibata †, T Hasegawa †, K Takagi †
PMCID: PMC1906235  PMID: 11737051

Abstract

Increased cyclic AMP (cAMP)-phosphodiesterase (PDE) activity in peripheral blood leucocytes is associated with the immunological inflammation that characterizes allergic diseases, such as atopic dermatitis and allergic rhinitis. Recently, it has been found that IL-13 has similar biological functions to IL-4. The aim of this study was to investigate the possible involvement of cAMP-PDE activity on IL-13 release from peripheral blood mononuclears cells (PBMC) from atopic asthma patients. Phytohaemagglutinin (PHA)-induced IL-13 release from PBMC was concentration-dependently inhibited by rolipram, a type 4 PDE inhibitor, as well as by dibutyryl cAMP, a membrane-permeant cAMP analogue. However, theophylline, a non-specific PDE inhibitor, and cilostazol, a type 3 PDE inhibitor, failed to inhibit IL-13 release. The inhibitory effect of rolipram was enhanced by the addition of forskolin (10−4 m), an adenylyl cyclase stimulator. PHA itself did not alter the intracellular cAMP level. Rolipram concentration-dependently increased cAMP level in PHA-stimulated PBMC, and this increase was synergistically facilitated by the addition of forskolin (10−4 m). These results suggest that type 4 PDE inhibitors, alone or synergistically in combination with forskolin, inhibit PHA-induced IL-13 release from PBMC of atopic asthma patients by elevating intracellular cAMP concentrations. These inhibitors have the potential to exert an anti-inflammatory effect by inhibiting IL-13 production in allergic diseases such as atopic asthma.

Keywords: IL-13, allergy, asthma, phosphodiesterase inhibitor, cAMP

INTRODUCTION

Recent evidence convincingly suggests that activated T-lymphocytes, particularly the CD4+ population, are involved in allergic asthma [1]. It is known that airway eosinophilia is a major characteristic of allergic asthma and the close association between CD4+ cells and eosinophilia indicates that the airway eosinophilia seen in asthma is orchestrated, in part, by lymphocytes [1]. A functional subset of CD4+ cells, namely Th2 cells, has recently been identified and it is hypothesized that several cytokines, including IL-4 and IL-5, are released from these cells and play important roles in the eosinophil-rich bronchial mucosal inflammation that characterizes clinical asthma [1,2]. IL-5 is a prominent Th2 cytokine that has important roles in immune modulation of inflammatory disease [2]. IL-5 is closely associated with eosinophilia because it regulates eosinophil differentiation, maturation, activation, degranulation and endothelial adherence [2]. Another important cytokine produced by Th2 cells is IL-4. IL-4 induces isotype switching of B cells to IgE synthesis, as well as modulating the expression of vascular cell adhesion molecule-1 (VCAM-1), an adhesion molecule for eosinophils, on endothelial cells [3–5].

Similar to IL-4, IL-13 induces Ig class switching and IgE secretion, up-regulates the expression of MHC class II molecules and the low affinity receptor for IgE (CD23) on B cells and induces the expression of VCAM-1 on endothelium [4–8]. IL-13 action is mediated by the IL-13 receptor, a heterodimer consisting of an IL-13-receptor α subunit and an IL-4-receptor α subunit which shares signalling pathways with IL-4 [4–6]. Recent evidence suggests that IL-13 is a crucial mediator of immune diseases, especially allergic asthma [4]. For example, a genetic variant of IL-13 was found in high amounts in patients with asthma [9] and the IL-13 receptor was prominently expressed in bronchi from asthmatic patients [9]. The elevated expression of IL-13 mRNA has also been observed in the bronchial mucosa of both atopic and non-atopic asthma patients [10–12]. Furthermore, BAL levels of IL-13 are elevated in atopic asthmatics [10,13], while IL-13 secretion is increased in basophils from atopic asthmatic subjects [10,14] and in peripheral blood mononuclear cells (PBMC) from atopic rhinitis patients following allergen challenge [15]. Thus, the pharmacological control of IL-13 signalling might be a novel therapeutic and preventive strategy for controlling asthma and atopy.

Recent evidence suggests that cyclic AMP (cAMP)-phosphodiesterase (PDE) activity is altered in some immune disease states and can have important effects on lymphocyte function [16,17]. For example, more rapid degradation of cAMP by PDE was observed in lymphocytes from patients with atopic diseases than control subjects [18–21], with this degradation being sensitive to PDE inhibitors [19–21]. Thus far, at least 10 subtypes of PDE have been cloned [22,23]. Among them, type 4 PDE appears to be critical for regulation of cAMP-PDE activity [19–21], as well as for cytokine production [18,19] in lymphocytes. Thus, it is expected that inhibition of PDE activity, with the associated increase in cAMP, may inhibit IL-13 production in lymphocytes. Indeed, an earlier study revealed that a type 4 PDE inhibitor preferentially decreased allergen-stimulated IL-13 production in human T lymphocytes clones [16], although the relationship to cAMP level was not investigated.

Accordingly, in the present study, we investigated the effect of isoenzyme-selective PDE inhibitors on IL-13 release and cAMP accumulation in phytohaemagglutinin (PHA)-stimulated PBMC from atopic asthma patients.

MATERIALS AND METHODS

Reagents and media

The following reagents were used: PIPES, RPMI 1640, theophylline, rolipram, cilostazol, dibutyryl cAMP, forskolin, dexamethasone (SIGMA Immunochemicals, St. Louis, MO, USA); fetal calf serum (FCS; Filtron Pty Ltd, Brooklyn, Australia); LSM®-lymphocyte separation medium (LSM; ICN Pharmaceuticals, Inc., Costa Mesa, CA, USA); and phytohaemagglutinin (PHA; WAKO Pure Chemical Industries Ltd, Osaka, Japan). The 10 × PIPES buffer contained 250 mm PIPES, 1·10 m NaCl, 50 mm KCl, 0·1% glucose and 0·003% HSA, pH 7·47. The PBMC culture medium consisted of RPMI 1640 supplemented with 10% FCS, 25 mm HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mm non-essential amino acids. Pharmaceutical compounds were dissolved in dimethyl sulphoxide (DMSO) as a 10−1 m solution and were diluted appropriately with culture medium. The final DMSO concentration was lower than 0·1%, which had no effect on PHA-stimulated cytokine production.

Patients

We obtained PBMC from 13 adult atopic asthma patients (13 men, 41·8 ± 2·3 years of age). All patients had mild to moderate bronchial asthma with elevated serum IgE levels and IgE specific for the house dust mite (Dermatophagoides pteronyssinus: Der p). The patients' total serum IgE and mite (Der p)-specific IgE were measured by outside commercial laboratories. Mite-specific IgE levels were expressed by RAST scores (score 0: < 0·35 U/ml, score 1: 0·35–0·7 U/ml, score 2: 0·7–3·5 U/ml, score 3: 3·5–17·5 U/ml, score 4: 17·5–50 U/ml, score 5: 50–100 U/ml, score 6: > 100 U/ml). The mean mite RAST score was 3·1 ± 0·3 and the total IgE in the serum derived from patients was 331·1 ± 125·2 U/ml. At the time of the study the patients' symptoms were controlled using appropriate therapy, including occasionally inhaled β-agonist, regularly inhaled corticosteroid (beclomethasone dipropionate) and oral theophylline. Patients who were taking systemic corticosteroids were excluded from this study. Informed consent was obtained from each patient for donation of blood.

Preparation of PBMC

Peripheral blood (20 ml) from each donor was anticoagulated with 10 mm EDTA and mixed with the same volume of physiological salt solution. It was then layered onto LSM and centrifuged at 400 g for 20 min. Mononuclear cells at the interface were harvested and washed twice with 1× PIPES and RPMI 1640. Cells were resuspended at a density of 5 × 105 cells/ml in RPMI 1640 supplemented with 10% FCS, 25 mm HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mm non-essential amino acid. After PHA stimulation (0·1–1000 µg/ml), cell cultures were carried out in sterile 96-well culture plates (200 µl/well) (Becton Dickinson Co., Lincoln Park, NJ, USA) at 37°C in a humidified atmosphere with 5% CO2 for 24 h. The doses of PHA used were selected as described elsewhere [24]. In the case of drug inhibition experiments, each drug was preincubated for 10 min before PHA stimulation. Cell-free supernatant was then harvested following high-speed centrifugation and stored at −30°C.

Measurement of IL-13

IL-13 was quantified by ELISA (CLB, Amsterdam, Netherlands) according to the manufacturer's instructions. The assay sensitivity for IL-13 was less than 0·5 pg/ml.

Preparation of samples for cAMP analysis

The intracellular cAMP level was measured according to the method of Verghese et al. [23]. Briefly, after a 30-minute incubation period, the reaction was terminated by snap-freezing in liquid nitrogen and the cells were stored at −30°C. At the time of measuring cAMP, the cells' membranes were destroyed by ultrasound vibration. Then, after acetylating samples, a cAMP EIA kit (Amersham Int. plc. Buckinghamshire, UK) was used to measure the intracellular cAMP level. Values were expressed as the percent elevation of cAMP level at each time point.

Statistical evaluation

The data are shown as mean and range or mean ±SEM, and the means of each group were compared using the paired Student's t-test. Differences associated with probability of P < 0·05 were considered significant.

RESULTS

Dose response of IL-13 production by PBMC

As shown in Fig. 1a, PHA concentration-dependently increased IL-13 release from PBMC and was maximally effective at 100 µg/ml. From these results, we decided that 10 µg/ml of PHA, which is a suboptimal-concentration for IL-13 release, would be an ideal PHA concentration for subsequent experiments.

Fig. 1.

Fig. 1

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(a) PHA-induced IL-13 release by PBMC. Cells were stimulated with various concentrations of PHA (0·1–1000 µg/ml) for 24 h. The values of spontaneous IL-13 release (S) were 5·13 ± 1·74 pg/106 PBMC. Each result is the mean ±SEM of eight experiments; (b) the time–course of the effect of PHA on IL-13 release from PBMC. PBMC were stimulated with PHA (10 µg/ml) and supernatants were collected at each time point indicated. Each result is the mean ±SEM of seven experiments. Spontaneous (○); PHA (•).

Kinetics of IL-13 production by PBMC

Figure 1b shows the time–course effect of PHA on IL-13 release from PBMC. IL-13 was detected 4 h after PHA stimulation (10 µg/ml) and secretion increased for 48 h after the stimulation. Since there was a large amount of IL-13 production at 24 h, we used this time point for subsequent experiments.

Inhibitory effect of various drugs on IL-13 production by PBMC

Rolipram, a type 4 PDE inhibitor, and dibutyryl cAMP, a membrane-permeant cAMP analogue, suppressed PHA-induced IL-13 production in a concentration-dependent fashion (Fig. 2a,c). In contrast, the nonselective PDE inhibitor, theophylline, and a type 3 PDE inhibitor, cilostazol, failed to suppress PHA-induced IL-13 production (Fig. 2b). Dexamethasone (10−6 m) also exerted almost complete inhibition of PHA-induced IL-13 production from PBMC (Fig. 2c).

Fig. 2.

Fig. 2

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The effects of various drugs on PHA-stimulated IL-13 release from PBMC. PBMC were incubated with various concentrations of drugs for 10 min and then stimulated with PHA (10 µg/ml) for 24 h. Values are expressed as the percent inhibition of PHA-stimulated IL-13 release. (a) Rolipram (•, n = 8), forskolin (□, n = 4–5) and rolipram with 10−4 m forskolin (▪, n = 5); (b) theophylline (▵, n = 5) and cilostazol (▴, n = 5); (c) dibutyryl cAMP (▿, n = 5–6) and dexamethasone (▾, n = 7). The mean net values of PHA-stimulated IL-13 for the effect of theophylline, rolipram, cilostazol, dibutyryl cAMP, forskolin and dexamethasone were 230·1 ± 59·5, 149·8 ± 44·4, 230·1 ± 59·5, 188·3 ± 67·4, 230·1 ± 59·5 and 159·3 ± 50·1 pg/106 PBMC, respectively (spontaneous IL-13 were 0·0 ± 0·0, 0·64 ± 0·37, 0·0 ± 0·0, 0·44 ± 0·44, 0·0 ± 0·0, and 0·38 ± 0·31 pg/106 PBMC, respectively).

By itself, forskolin failed to significantly inhibit PHA-induced IL-13 (Fig. 2a). However, forskolin (10−4 m) treatment augmented the inhibitory effect of rolipram (Fig. 2a).

Kinetics of intracellular cAMP levels in PBMC

Figure 3 shows the kinetics of PBMC intracellular cAMP levels. In all treatment regimens, the intracellular cAMP levels were increased immediately reagent incubation, stabilizing 3 h later. PHA stimulation did not alter the cAMP level, compared with spontaneous levels. In contrast, PHA plus rolipram (10−5 m) resulted in significantly higher levels of intracellular cAMP compared with spontaneous or PHA-only stimulated PBMC, over the whole time course (Fig. 3). From these results, we selected a 30-minute incubation time for the detection of intracellular cAMP levels.

Fig. 3.

Fig. 3

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Time–course of the effect PHA and/or rolipram on cAMP levels in PBMC. PBMC were incubated with rolipram (10−5 m) for 10 min and stimulated with PHA (10 µg/ml). Supernatants were collected at each time point indicated. Data are expressed as net values: (PHA-induced value) – (spontaneous value). Each result is the mean ±SEM of four experiments. Spontaneous (○); PHA alone (▴); PHA with rolipram (▪).

Effects of rolipram on intracellular cAMP levels

Rolipram (10−8 −10−5 m) concentration-dependently increased the cAMP level in PHA-stimulated PBMC (Table 1). Rolipram (at 10−8 m, a concentration which does not significantly increase cAMP levels) in combination with forskolin (10−4 m) did not significantly alter the cAMP level, compared with PHA-stimulated vehicle (P > 0·05, Table 1). This is consistent with our preliminary results that forskolin (10−4 m) by itself does not alter the cAMP level (data not shown); however, addition of forskolin (10−4 m) at higher doses of rolipram synergistically elevated the rolipram-induced augmentation of cAMP levels (Table 1). Forskolin (10−4 m) showed statistically significantly increases in cAMP levels at rolipram concentrations 10−6−10−5 m (Table 1).

Table 1.

The effects of rolipram (10−8–10−5 m) with or without forskolin (10−4 m), and dexamethasone (10−6 m) on PHA-induced cAMP levels of PBMC

Treatments cAMP (fmol/106 cells)
Spontaneous
 Vehicle 10·6 ± 3·4
PHA stimulation
 Vehicle 11·1 ± 3·3
 Rolipram 10−8m 12·8 ± 3·4e
10−7m 13·8 ± 4·0e
10−6m 19·3 ± 4·6e
10−5m 28·0 ± 6·0b
Dexamethazone 10−6m 6·8 ± 2·4e
PHA stimulation with forskolin (10−4m)
 Rolipram 10−8m 18·4 ± 10·0e
10−7m 25·6 ± 9·8a
10−6m 36·8 ± 10·4b,c
10−5m 56·0 ± 12·1b,d
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 PBMC were incubated with the various drugs for 10 min and then stimulated with PHA (10 µg/ml) for 0·5 h. Each result is the mean ±SEM of 5 experiments.

a

P < 0·05,

b

P < 0·01 vs. PHA-stimulated vehicle;

c

P < 0·05,

d

P < 0·01 vs. corresponding rolipram with PHA;

e

no significant difference from PHA-stimulated vehicle (Student's t-test).

DISCUSSION

In the present study, we have shown that PHA stimulation caused the production of IL-13 from PBMC in a PDE-inhibitor-sensitive manner. Among the PDE inhibitors evaluated [cilostazol (a type 3 inhibitor), rolipram (a selective type 4 PDE inhibitor) and theophylline (a non-selective PDE inhibitor)], only rolipram elicited a concentration-dependent inhibition of the PHA-induced IL-13 release from PBMC. The addition of 10−4 m forskolin, which directly stimulates adenylyl cyclase activity and increases intracellular cAMP levels, synergistically enhanced the inhibitory effect of rolipram on PHA-induced IL-13 release from PBMC. Moreover, dibutyryl cAMP, a cAMP analogue, inhibited PHA-induced IL-13 release. In PHA-stimulated PBMC, rolipram significantly increased the cAMP level, and this was synergistically facilitated by forskolin. Taken together, these results suggest that rolipram inhibited PHA-induced IL-13 production in PBMC by increasing intracellular cAMP levels. This is the first report to show a possible relationship between cAMP accumulation and IL-13 production in PBMC.

Rolipram alone and rolipram in combination with forskolin are likely to have different modes of action toward cAMP accumulation and IL-13 release. For example, rolipram (10−6 m) alone and rolipram (10−8 m) plus forskolin showed almost similar effects on cAMP accumulation [rolipram (10−6 m) alone: 19·3 ± 4·6, rolipram (10 −8 m) with forskolin: 18·4 ± 10·0 fmol/10 6 cells, P > 0·05 (statistical difference), Table 1] although they revealed differential inhibitory effects on IL-13 release [rolipram (10−6 m) alone: 26·9 ± 9·5%, rolipram (10−8 m) plus forskolin: 0·86 ± 0·65%, P < 0·01 (statistical difference), Fig. 2]; whereas rolipram (10−6−10−5 m) in combination with forskolin significantly stimulated cAMP accumulation and synergistically inhibited IL-13 release compared with rolipram alone (Table 1 and Fig. 2). Thus, the selective inhibition of the PDE subtype in combination with or without the nonspecific stimulation of adenylyl cyclase might display unique/differential effects on cAMP levels and IL-13 release.

T lymphocytes [7] and basophils [25] are known to produce IL-13, whereas monocytes and platelets do not respond to PHA activation [26]. Mononuclear cells, separated from peripheral blood by LSM, contain mainly lymphocytes and a relatively small number of monocytes and platelets. Granulocytes, including basophils, should separate out during LSM and not be collected. Although it is impossible to avoid the possibility that the PBMC preparation contains a very small number of basophils as contaminants, it is likely that the basophils are not a major source of IL-13 in the preparation. Taken together, it suggests that the effect of rolipram most likely occurs in the T lymphocyte population in this preparation.

As for the inhibitory effect of dexamethasone, we did not observe an increase in cAMP levels in dexamethasone-treated PBMC, despite near complete inhibition of IL-13 production. Byron et al. have reported that hydrocortisone inhibited PHA-induced IL-4 production from PBMC and that hydrocortisone also decreased the transcription of IL-4 mRNA [27]. Moreover, Okayama et al. have reported that glucocorticoids suppressed production and gene expression of IL-5 by PBMC in atopic patients [28]. These observations indicate that the inhibitory effect of IL-13 production by dexamethasone might occur at the level of transcription.

Some earlier studies demonstrated that cAMP-PDE activity is elevated in PBMC from patients with atopic disease, such as atopic dermatitis [18–21] and allergic rhinitis [20]. The cAMP-PDE activity is likely to be important in regulating the function of T lymphocytes and PBMC. For example, dibutyryl cAMP, a cAMP analogue, activates the cAMP-dependent signalling pathway, and subsequently down-regulates IL-4 mRNA expression and decreases IL-4 protein levels in activated human T cells [29]. The inhibition of PDE activity by PDE inhibitors causes the accumulation of cAMP, subsequently (1) attenuating blastgenesis in T lymphocytes [30]; (2) suppressing IL-5 and interferon gamma (IFN-γ) gene expression [31], as well as cell-proliferation [19]; (3) inhibiting the release of IL-4 from PBMC [18,21]; and (4) inhibiting the release of arachidonate from mononuclear cells [32]. In the present study, rolipram, a type 4 PDE inhibitor, inhibited the release of IL-13 from PBMC and increased cAMP levels in PBMC. Considering these observations and our results, it suggests that the increase in cAMP levels is associated with the inhibitory effect of rolipram on PHA-induced IL-13 production from PBMC, and is likely to regulate other functions. This observation is, in part, consistent with the earlier study of Essayan et al. [16] that showed rolipram inhibits allergen (Amb a 1)-stimulated IL-13 release from human T lymphocytes clones, although these authors did not correlate their findings with cAMP levels.

In contrast, cilostazol, a type 3 PDE inhibitor, and theophylline, a non-selective PDE inhibitor, did not inhibit the release of IL-13 from PBMC. The effect of cilostazol (up to 100 µm) and theophylline (up to 10 µm) on cAMP accumulation in PBMC will be elucidated in the near future but was not addressed in the present study because of their lack of effects on PHA-stimulated IL-13 release; however, high doses of cilostazol and theophylline are enough to increase cAMP level in other preparations [25,33,34]. For example, Yoshimura et al. [34] have demonstrated that theophylline (100 µm) and another type 3 PDE inhibitor milrinone (10 µm) increased cAMP levels in PBMC; however, theophylline (100 µm) and milrinone (10 µm) only inhibited PHA-stimulated Th1-related cytokine release (IFN-γ, IL-2) and not Th2-related cytokines (IL-4, IL-5). Th2 cells are a good source of IL-13 release [35]. Thus, it is likely that the inhibitory effect of rolipram against type 4 PDE activity mainly affects the function of Th2, not Th1, cells.

Several investigators have revealed that IL-13 release from PBMC or a T cell population was higher in atopic subjects than in normal subjects [15,16,36,37]. In order to investigate the difference in IL-13 release between normal and atopic subjects, most studies used specific but different allergens to stimulate the release of IL-13 [15,16,36,37], except for one study that used PHA [15]. The differences in stimulants and the selection of the doses and exposure time are likely to be important. For example, Li et al. [15] demonstrated that allergen-stimulated IL-13 production from PBMC in atopic dermatitis was higher than that in normal subjects, although PHA-stimulated cytokine release was not significantly different between the two groups. In their study, they applied 10 mg/ml of PHA for 3 days whereas we applied lower doses of PHA (0·1 µg/ml to 1 mg/ml; 10 µg/ml used in most studies) for 24 h in order to investigate PHA-induced IL-13 release. Similar to our present studies, previous studies [19,38,39] have also applied low doses of PHA to establish the effect of PDE inhibitors on PHA-induced cell-proliferation. Thus, to evaluate fully the underling mechanisms of IL-13 release, further studies including measuring the differences in cAMP-PDE activity between normal and atopic asthma subjects and the net involvement of cAMP-PDE activity by using an inhibitor of cAMP-dependent protein kinase should be carried out.

In summary, the regulation of cAMP level by inhibiting PDE activity, especially type 4 PDE activity, appears to be involved in the regulation of IL-13 release from atopic PBMC. This could be of significance for the treatment of diseases in which significant increases in IL-13 release have been reported, including atopic asthma [40,41]. Indeed, very recently, higher expression of the bronchial IL-13 receptor and genetic variation in IL-13 and its signalling have been reported in atopic asthma patients [4,9]. Although it has not yet been fully validated whether PDE inhibitors show differential effects on IL-13 release in normal and atopic subjects, highly selective type 4 PDE inhibitors could increase cAMP levels and decrease IL-13 release, thereby potentially improving atopic diseases.

Acknowledgments

The authors would like to thank Professors Tetsuo Hayakawa (Second Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan) and Hiroichi Nagai (Department of Pharmacology, Gifu Pharmaceutical University, Gifu, Japan) for their encouragement throughout the study and Dr Michitaka Shichijo (Department of Pharmacology, Gifu Pharmaceutical University, Gifu, Japan) for his cooperation and advice.

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