ELSEVIER Immunopharmacology 30 (1995) 191-198

Immunopharmacology

Effect of phosphodiesterase inhibition on IL-4 and IL-5 production of the murine Tn2-type T cell clone D10.G4.1

Jiirgen Schmidt a,*, Armin Hatzelmann b, Sandra Fleil3ner a, Irene Heimann-Weitschat a, Roland Lindstaedt a, Istvan Szelenyi a

a ASTA Medica AG, Department of Pharmacology, Frankfurt/Main, Germany b Byk Gulden, Department of Biochemistry, Konstanz, Germany

Received 20 February 1995; revised 22 May 1995; accepted 1 June 1995

Abstract

The effect of various phosphodiesterase (PDE) inhibitiors on anti-CD3 induced interleukin-(IL) -4 and IL-5 production of the murine T helper cell clone of type 2 phenotype D10.G4.1 (D10) has been investigated in vitro. D10 cells were incubated in the presence of drugs and anti-CD3 mAb for 16 h before measurement of cytokines in the cell supernatants by ELISA. Whereas all PDE inhibitors tested exerted minimal effects on anti-CD3 induced IL-4 production, a marked increase in IL-5 production by the non-selective PDE inhibitors IBMX, theophylline and enprofylline was observed. The action of these non-selective PDE inhibitors was mimicked by the PDE IV-selective inhibitor rolipram and in part by the PDE Ill-selective inhibitors motapizone and milrinone, whereas the PDE V-selective inhibitor zaprinast was inactive. Rolipram and motapizone enhanced IL-5 production in a synergistic fashion. In support of the functional importance of PDE III and IV for IL-5 synthesis in intact murine D10 cells, we have found PDE III and IV to be the predominant isoenzyme activities in corresponding cell lysates. The stimulatory effect of rolipram on IL-5 production was almost totally reversed by the protein kinase A inhibitor KT-5720. In addition, the membrane-permeable cAMP analogue 8-bromo-cAMP mimicked the stimula- tory effect of PDE inhibitors on IL-5 production while leaving IL-4 levels unaffected. Both results support the view that the action of the PDE inhibitors on murine D10 cells is mediated via an elevation of intracellular cAMP.

Keywords: Phosphodiesterase inhibitor; Murine T helper lymphocyte; Interleukin-4; Interleukin-5; Mice

Abbreviations: APC, antigen presenting cells; cAMP, cyclic 3',5'-adenosine monophosphate; cGMP, cyclic 3',5'-guanosine monophos- phate; D10, cells from the TH2-type cell clone D10.G4.1; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme linked immunosorbent assay; IBMX, 3-isobutyl-l-methyl-xanthine; IL, interleukin; MHC, major histocompatibility complex; PBS, phosphate buffered saline; PDE, phosphodiesterase; PKA, protein kinase A; S.D., standard deviation; T H 2, T helper cells of the type 2 phenotype

* Corresponding author. Present address: Department of Experimental Cancer Research, ASTA Medica AG, P.O. Box 100105, D-60001 Frankfurt/Main, Germany. Tel: +49 (069) 4001-1060; fax: +49 (069) 4001-2745.

0162-3109/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0162 -3109( 95 )00022-4

192 J. Schmidt et al. / Immunopharmacology 30 (1995) 191-198

1. Introduction

Airway eosinophilia is a major characteristic of allergic bronchopulmonary diseases like asthma (Holgate et al., 1991). Recent evidence suggests that T helper cells of the type 2 phenotype (T n 2) play an important role in allergic inflammation, such as the recruitment of eosinophils in lung tissue (Corrigan and Kay, 1992). One feature of TH2 cells is the production of IL-4 and IL-5 following T cell recep- tor activation (Mosmann and Coffman, 1989). By secretion of these cytokines T n 2 cells are thought to orchestrate the allergic inflammatory late-phase reac- tion. IL-4 is a multifunctional cytokine which is of particular importance for isotype switching of B-cells to IgE synthesis (Coffman et al., 1986) and for the expression of VCAM-1 (Schleimer et al., 1992), an adhesion molecule for eosinophils, on endothelial cells. IL-5 is closely related to eosinophilia because it is associated with eosinophilopoiesis (Sanderson et al., 1985), eosinophil chemoattraction (Walsh et al., 1990), prolongation of eosinophil survival (Rothen- berg et al., 1989), and eosinophil activation (Lopez et al., 1988).

Recently, the potential utility of cyclic nucleotide phosphodiesterase (PDE) inhibitors in the treatment of bronchial asthma has been reconsidered. This has arisen predominantly from the acknowledged thera- peutic effectiveness of theophylline, which has pro- vided the mainstay in the treatment of bronchial asthma for over 50 years. Its therapeutic value is derived from a combination of antiinflammatory and bronchodilatory activities. Recently, it was also shown that low-dose oral theophylline inhibited eosinophil infiltration into the lungs of asthmatics (Sullivan et al., 1994). Of theophylline's many phar- macological activites, its PDE inhibition (Beavo and Reifsnyder, 1990) is suggested to be most important. PDE's comprise a family of at least five isoenzyme subclasses, which are differentially expressed and regulated in different cell types (Beavo and Reifsny- der, 1990). Because the inhibition of PDE by theo- phylline is non-selective and also relatively weak, a promising approach for future asthma therapy seems to be the development of isoenzyme-selective PDE inhibitors with higher potency and fewer side effects than theophylline (reviewed in Giembycz and Dent, 1992; Torphy and Undem, 1992).

In the present study we examined the effect of different non-selective as well as isoenzyme-selec- tive PDE inhibitors on IL-4 and IL-5 production of murine T H 2 cells.

2. Materials and methods

2.1. Reagents and media

Complete medium was RPMI 1640 containing HEPES (Boehringer Mannheim, Germany) supple- mented with 2 mM glutamine (Boehringer Mannheim), 50/zM mercaptoethanol (Sigma Chemie GmbH, Deisenhofen, Germany), 100 IU/ml peni- cillin, 100 /xg/ml streptomycin (Biochrom, Berlin, Germany), and 10% fetal calf serum (Boehringer Mannheim). Enprofylline, Ro 20-1724 and 8- bromo-cAMP sodium salt (8-bromo-cAMP) were ob- tained from RBI (Natick, MA), KT-5720, and rolipram from Biomol (Hamburg, Germany), and motapizone from Nattermann (K/51n, Germany). Theophylline, 3-isobutyl-l-methyl-xanthine (IBMX), milrinone, and zaprinast were purchased from Sigma Chemie GmbH. The hamster anti-mouse-CD3 mAb (145-2Cll) was obtained from Cedar Lane Labora- tories (Hornby, Ontario, Canada). All other chemi- cals and supplies were from standard commercial sources.

2.2. Cell culture

The TH2 clone D10.G4.1 (D10) is a major histo- compatibility complex (MHC)-restricted, antigen- specific helper T cell line. The line is specific for the antigen egg white conalbumin in the context of H-2k and was originally derived from an AKR/J mouse. The line was obtained from American Type Culture Collection (ATCC), Rockville, MD. It was stimu- lated every 10 days with the antigen conalbumin (100 /xg/ml) and mitomycin C treated syngeneic splenocytes as antigen presenting cells (APC) as previously described (Schmidt et al., 1994a).

2.3. Exposition of cells and stimulation by anti-CD3 mAb

Cells were cultured in 96-well microtiter plates (4 X 10 4 cells/well) for 3.5 h in complete medium.

J. Schmidt et al. / Immunopharmacology 30 (1995) 191-198 193

Stock solutions of the various drugs were made in dimethyl sulfoxide (DMSO) and subsequently di- luted in complete medium. Drug solutions and sol- vent, respectively, were added to the cultures and incubated for 30 min. Cells were stimulated by an anti-CD3 mAb and further incubated for 16 h. Super- natants were centrifuged and stored at - 7 0 ° C until measurement of cytokines.

2.4. Determination of cytokines

The supernatants were assayed by using commer- cially available ELISA kits for murine IL-4 and IL-5 (Endogen Inc., Boston, MA).

2.5. Measurement of PDE activity

Cells (0.7-1 × 107/ml) were suspended in PBS- HEPES buffer consisting of PBS, 1 mM MgC12, 10 mM HEPES, 1 mM EDTA, and 1 mM 2- mercaptoethanol. The suspension was sonified for 3 min at 4°C and immediately centrifuged (g = 100,000 m / s 2 for 1 h at 4°C). The supernantant and pellet (membranes) were separated and stored at - 7 0 ° C until measurement of PDE activity.

PDE activity was determined as described by Thompson and Appleman (1979) with some modifi- cations (Bauer and Schwabe, 1980). The assay mix- ture contained 40 mM Tris-HCl (pH 7.4), 5 mM MgC12, 0.5 /xM cAMP or cGMP, [3H]cAMP or [3H]cGMP (about 50.000 cpm/assay), appropiate concentrations of inhibitors and activators and aliquots (about 50 /xl) of the supernatant and pellet fraction, respectively, at a final assay volume of 200 /xl. The reaction was started by the addition of substrate and the assays were incubated for 30 min at 37°C. 50 /zl of 0.2 N HCI was added to stop the reaction and the assays were left on ice for about 10 min. Following incubation with 25 /xg 5'-nucleoti- dase (Crotalus atrox snake venom) for 20 min at 37°C, the assays were loaded on QAE Sephadex A-25 columns (Econo colums, Bio-Rad, 1 ml bed volume). The columns were eluted with 2 ml of 30 mM ammonium formiate (pH 6.0) and the eluate was counted for radioactivity. Results were corrected for blank values (measured either in the presence of denatured protein or buffer) which were below 2% of total radioactivity. The amount of cyclic nu- cleotide hydrolyzed did not exceed 20% of the origi-

nal substrate concentration. PDE activity was calcu- lated as p M / m i n / c e l l equivalents.

The isoenzyme activity pattern was determined using isoenzyme specific inhibitors and/or activa- tors. The inhibitors used were applied at concentra- tions which suppress > 95% of the PDE activity to be influenced but inhibit < 5% of the activity of the other PDE isoenzymes. Briefly, PDE III and IV activity was calculated as the difference of basal cAMP hydrolysis versus cAMP hydrolysis measured in the presence of the PDE Ill-selective inhibitor motapizone (1 /xM) and the PDE IV-selective in- hibitor rolipram (10 /xM), respectively. PDE V ac- tivity was determined as the difference of cGMP hydrolysis in the presence of motapizone versus cGMP hydrolysis measured in the presence of mo- tapizone and the PDE V-selective inhibitor zaprinast (10/xM). PDE I activity was calculated based on the increase of cGMP hydrolysis entailed by the addition of Ca 2÷ (1 mM) and calmodulin (150 nM). PDE II activity was defined as the elevation of cAMP hydro- lysis by cGMP (5 /xM) in the presence of rolipram and motapizone.

3. Results

3.1. Anti-CD3 induced cytokine production

Following 16 h incubation with various dilutions of anti-CD3 mAb IL-4 as well as IL-5 could be detected in the supernatants of D10 cells. The amounts of both of the cytokines were strictly depen- dent on the concentration (dilution) of anti-CD3 mAb (data not shown), and at the final dilution of 1/100 2647 _ 460 pg IL-4 /ml (mean _ S.D. from 16 independent experiments) and 324 _+ 66 pg IL- 5 / m l (mean _+ S.D. from 17 independent experi- ments) was measured. In the supernatants of unstim- ulated cells cytokine levels were below the detection limit of the ELISA. Anti-CD3 mAb at the final dilution of 1/100 was used in all further experi- ments.

Because stock solutions (1000 × concentrated) of all tested drugs were made in DMSO, the effect of DMSO on anti-CD3 induced cytokine production was also investigated. DMSO in concentrations up to 0.1% did neither affect IL-4 nor IL-5 production (data not shown).

194 J. Schmidt et al. / lmmunopharmacology 30 (1995) 191-198

3.2. Effect of non-selective PDE inhibitors

The anti-CD3 induced production of IL-4 and IL-5 was differentially affected by the non-selective PDE inhibitors tested (Fig. 1). IL-4 production was only marginally influenced, whereas IL-5 production was increased by all of the three PDE inhibitors. IBMX (Fig. 1A), and enprofylline (Fig. 1C) concen- tration-dependently increased (up to 250%) anti-CD3 induced IL-5 production. For comparison, the in- crease in IL-5 production by theophylline (Fig. 1B) was relatively weak (maximally 65% at 300 /xM).

3.3. Effect of isoenzyme-selective PDE inhibitors

Motapizone has been described as a selective PDE III inhibitor with an IC50 of about 10 -7 M (Schudt et al., 1991). Motapizone slightly but con- centration-dependently increased IL-5 production at concentrations of 0.03-1.0 /~M (Fig. 2A). However, at higher concentrations of motapizone (3.0-30.0 /~M) no further increase could be detected (data not shown). Qualitatively similar to motapizone, the PDE III inhibitor milrinone concentration-dependently in- creased IL-5 production (Fig. 2B). The PDE IV inhibitor rolipram showed a strong increase in IL-5 production at concentrations >~ 10 nM (Fig. 2C). Comparable results were obtained with another PDE IV inhibitor Ro 20-1724 (data not shown). The PDE V inhibitor zaprinast (Fig. 2D) increased IL-5 pro- duction only at high concentrations, which are well above the concentrations required to inhibit PDE V activity but are known to exert PDE IV inhibitory activity (Schudt et al., 1991). Anti-CD3 induced IL-4 production was only marginally affected by all the isoenzyme-selective PDE inhibitors tested (Fig. 2).

As shown in Fig. 3, the combination of the PDE III inhibitor motapizone with the PDE IV inhibitor rolipram exerted a synergistic effect on IL-5 produc- tion.

3.4. PDE-isoenzyme pattern of DlO cells

The functional experiments described so far pointed to an important role of PDE isoenzymes III and IV in the action of PDE inhibitors on IL-5 production. In order to evaluate which PDE isoen-

zymes are expressed in D10 cells, we investigated the PDE isoenzyme activity pattern under cell-free conditions. For this purpose, we prepared both cy- tosolic and particulate (pellet) fractions of cell ho-

i

? 8

u

, o >

300 : ~A

2001 I

lOO I I

t

0

-100 ~

300

200

tO0 -

-100 !

0.1 0.3 1.0 3.0

IBMX t~M]

B

10.0 30.0 100.0

1.0 3.0 10,0 30.0 100.0 300.0 1000.0

Theophyline [/aM]

300 : i C

200]

100 ~ ;

_10oi 1.0 3.0

,H 10.0 30.0 100.0 300.0 1000.0

Enprofylllne [FcM]

Fig. 1. Histograms showing the effects on anti-CD3 induced IL-4 (open bars) and IL-5 (filled bars) production by IBMX (A), theophylline (B), and enprofylline (C). Values given represent the deviation in per cent+S.D. (n = 4) from anti-CD3 induced cy- tokine production of non-treated cells. In these four independent experiments non-treated cells produced 2959+429 pg IL-4/ml and 370 + 70 pg IL-5/ml (mean + S.D.).

J. Schmidt et al. / Immunopharmacology 30 (1995) 191-198 195

mogenates obtained by sonication. Fig. 4 demon- strates that PDE III and IV were the predominant PDE isoenzyme activities detected under the experi- mental conditions employed. No PDE II activity, and only trace amounts of PDE I and V activities were found. Whereas PDE IV was mainly localized in the cytosol, most of the PDE III activity was found membrane-associated.

3.5. Effect of 8-bromo-cAMP

To confirm that the increasing effect of PDE inhibition on IL-5 production was mediated by an increase of intracellular cAMP levels we loaded the cells by the membrane-permeable cAMP analogue 8-bromo-cAMP. At concentrations 100 /xM a con- centration-dependent increase in IL-5 production up to 500% was observed whereas IL-4 production was not affected (Fig. 5).

o l +

"~ i 400 ~

,~ ~ 3oo _

0 - 30 100 300

concentration [nM]

Fig. 3. Histogram showing the synergistic effect (hatched bars) of the PDE III inhibitor motapizone (open bars) and the PDE IV inhibitor rolipram (filled bars) on anti-CD3 induced IL-5 produc- tion. Values given represent the deviation in per cent_+S.D. (n = 3) from anti-CD3 induced IL-5 production of non-treated cells. In these three independent experiments non-treated cells produced 329 _+ 67 pg IL-5/ml (mean _+ S.D.).

-- 300 l .~, A

2oo

§~ o

I

-loo j 0.01 0.03 0.1 0.3 1.0

Motaplzone [/,dvfl

0.01 0.03 0.1 0.3 1.0 3.0 10.0 Rolipram [/,d~l]

.~ B

I~ '

o_

I

- 1 1 ~ 1 0.1 0.3 1.0 3 .0 10.0 30 .0 100.0

Milrinone [/zM]

D 300~

200-

100-

O-

0.3 1.0 3.0 10.0 30 .0 100.0 Zaprinast [/,+,M]

Fig. 2. Histograms showing the effects on anti-CD3 induced IL-4 (open bars) and IL-5 (filled bars) production by the PDE I|I inhibitors motapizone (A) and milrinone (B), the PDE IV inhibitor rolipram (C), and the PDE V inhibitor zaprinast (D). Values given represent the deviation in per cent _+ S.D. (n = 4) from anti-CD3 induced cytokine production of non-treated cells. In these four independent experiments non-treated cells produced 2590 _+ 549 pg IL-4/ml and 320 + 71 pg IL-5/ml (mean _+ S.D.).

196 J. Schmidt et al. / Immunopharmacology 30 (1995) 191-198

7

o 300

I II Ill IV V

[ ~ soluble I [ ] part iculate

200

a. V= × _~ I00

Q.

t -r-- l

Fig. 4. Histogram showing the activity pattern of PDE isoenzymes of murine TH2 lymphocytes (D10.G4.1) in the cytosol (soluble) and I00,000 g pellet fraction (paniculate) of DI0 cell lysates. PDE isoenzyme activities were determined using isoenzyme- specific activators or inhibitors as described in Section 2. Results are given as mean_+ S.D. from four independent experiments.

3.6. Effect of protein kinase A inhibition

A previous report indicated that the enhancement of IL-5 production mediated by an increase in the level of intracellular cAMP activates the IL-5 promo- tor via the PKA pathway (Lee et al., 1993). In order to test the involvement of the PKA pathway in the action of PDE inhibitors we exposed the cells to rolipram in the presence of the PKA inhibitor KT- 5720 (Kase et al., 1987). As shown in Fig. 6 the stimulating effect of rolipram on IL-5 production

700 U

Z eoo ]

~ 500 -

.~ 8 4oo~

.~ 3°°i ~.~= ~ 200 .=-~I ~ lOO

o 4 ~ -100 3.0

. • _ J A 10.0 30.0 100.0 300.0

8 - B r o m o - c A M P tp.M] 1000.0

Fig. 5. Histogram showing the effects on anti-CD3 induced IL-4 (open bars) and IL-5 (filled bars) production by 8-bromo-cAMP. The values given represent the deviation in per cent + S.D. (n = 3) from anti-CD3 induced cytokine production of non-treated cells. In these three independent experiments non-treated cells produced 2448 + 414 pg IL-4 /ml and 320 -I- 55 pg IL-5 /ml (mean -I- S.D.).

300

_~ i~ "5

¢z~ 200 ~ ! =o ii

• - | 1 100 -

i

0 I _ _ c o n t r o l 0.03 0.1 0.3 1.0

R o l i p r a m [.,u./vl]

Fig. 6. Histogram showing the effect of on anti-CD3 induced IL-5 production by rolipram in the presence (filled bars) and absence (open bars) of the PKA inhibitor KT-5720 (10 /zM). Anti-CD3 induced IL-5 production of the group not treated with" rolipram and KT-5720 (control, open bar) was set at 100%. The values given represent the deviation in per cent+S.D, from anti-CD3 induced IL-5 production of non-treated cells. In these three inde- pendent experiments non-treated cells produced 286+38 pg IL- 5 / m l (mean + S.D.).

was almost totally blocked by KT-5720 (10 /zM). KT-5720 itself slightly decreased IL-5 production.

4. Discussion

In the present study we have shown that PDE inhibition in the murine TH2 cell clone D10.G4.1 resulted in an increase in anti-CD3 induced IL-5 production whereas the anti-CD3 induced IL-4 pro- duction was only marginally affected.

D10 cells were used in order to obtain repro- ducible experimental conditions with a pure cell type. This T cell clone represents a prototypic T a 2- type T cell which secrets IL-4, IL-5, but not IL-2 upon stimulation by anti-CD3 mAb. D10 cells re- quire antigen plus APC stimulation to survive in tissue culture and fulfill the criteria for a nontrans- formed, growth factor-dependent T cell clone. The stimulus used, anti-CD3 mAb, mimicks T cell recep- tor stimulation and may push the in vivo occuring stimulation pathway of T cells (Kaye et al., 1983).

The PDE isoenzymes functionally important in intact D10 cells appeared to be PDE III and IV which is in agreement with the predominance of these PDE isoenzymes in corresponding cell lysates.

J. Schmidt et al . / lmmunopharmacology 30 (1995) 191-198 197

Simultaneous inhibition of both isoenzymes by the two isoenzyme-selective inhibitors motapizone and rolipram exerted a synergistic stimulatory effect on anti-CD3 induced IL-5 production in comparison to the effect caused by inhibition of only PDE III or IV, respectively. Also compounds with non-selective PDE-inhibitory activity like IBMX and enprofylline exerted marked increases in IL-5 production. Surpris- ingly, the increase in IL-5 production by theo- phylline was relatively weak. Based on the known PDE inhibitory activity of theophylline (Beavo and Reifsnyder, 1990) a higher increase in IL-5 produc- tion would have been expected. Besides PDE inhibi- tion theophylline is also known as an adenosine receptor antagonist. That the adenosine receptor an- tagonism possibly counteracted the increase in IL-5 production mediated by theophyllines' inhibitory ac- tivity on PDE is suggested by the pronounced action of enprofylline, a xanthine-derivative lacking adeno- sine receptor antagonism (Persson, 1985). Adenosine exerts its effects via adenosine receptors which are closely linked to cAMP-generating adenylate cyclase in either an inhibitory (A1) or a stimulating (A2) manner. Experiments to clarify the role of adenosine and adenosine receptors, respectively, in the present cell system are in progress.

Inhibition of PDE activity results in an increase of intracellular cAMP levels which in turn modulate a variety of cell responses. Similar to PDE inhibition direct elevation of intracellular cAMP by exposure of the cells to membrane-permeable 8-bromo-cAMP led to an increase of anti-CD3 induced IL-5 produc- tion suggesting that the elevation of intracellular cAMP is also involved in the mechanism of PDE inhibitors.

These findings are in line with previously pub- lished data describing IL-5 increasing effects in murine as well as in human T cells by substances which elevate intracellular cAMP either by treatment with membrane-permeable cAMP analogues (Lee et al., 1993; Munoz et al., 1990; Snijdewint et al., 1993), by the adenylate cyclase activator forskolin (Betz and Fox, 1991; Schmidt et al., 1994b; Sni- jdewint et al., 1993) or by stimulation of adenylate cyclase-coupled receptors such as the prostanoid (PGE 2) receptor (Betz and Fox, 1991; Lee et al., 1993; Snijdewint et al., 1993), or the histamine He-receptor (Schmidt et al., 1994b). In addition, it

has been shown that IL-4 production was not in- creased by elevation of intracellular cAMP (Munoz et al., 1990; Betz and Fox, 1991; Lee et al., 1993; Schmidt et al., 1994b; Snijdewint et al., 1993), which is in agreement with the present results.

Recently, it has been demonstrated that an in- crease in intracellular cAMP augmented IL-5 pro- duction at the mRNA and the protein level in mouse thymoma line EL-4 cells. Using a transient transfec- tion assay it was demonstrated that cAMP probably exerts its action through the signaling pathway that involves PKA modulating the activity of the IL-5 promotor (Lee et al., 1993). In the present study, we could demonstrate that the increase in IL-5 produc- tion induced by PDE inhibition also involved the PKA pathway. Inhibition of PKA activity by KT- 5720 neutralized the IL-5 increasing effect of rolipram suggesting that the increase in IL-5 produc- tion by elevation of intracellular cAMP was tran- scriptionally mediated.

Although the observed in vitro increase in IL-5 production by elevation of intracellular cAMP in the mouse system is in line with other published results and comparable in vitro effects were published with freshly isolated human Tn2-type T cell clones (Snijdewint et al., 1993) open questions are remain- ing about the regulation of IL-5 synthesis by cAMP in vivo. In allergic challenged guinea-pigs, develop- ment of airway eosinophilia was effectively inhibited by treatment with both IL-5 neutralizing anti-IL-5 mAbs (Gulbenkian et al., 1992) as well as with PDE inhibitors (Underwood et al., 1993; Lagente et al., 1994) suggesting that in vivo IL-5 production in guinea-pigs is rather inhibited than enhanced by PDE inhibitors. In the in vivo system PDE inhibitors exert multiple effects not only on different lymphocyte subsets cells but also on many other immune cells, which may be dominating over the increasing effects on IL-5 production in T H 2 cells. For example, aller- gen induced expression of IL-5 mRNA was inhibited in peripheral blood mononuclear cells by the treat- ment with PDE inhibitors in vitro (Essayan et al., 1993) indicating that in an in vitro system, where antigen is presented by antigen presenting cells to the T cells in presence of other immune cells, an opposite effect to our observation was found. There- fore, care should be taken in jumping to conclusions for pharmacotherapy from our presented data, be-

198 J. Schmidt et al. / lmmunopharmacology 30 (1995) 191-198

cause these data are based on a single TH2 cell pulled out from the much more complex in vivo situation.

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