Elevated intracellular cyclic AMP inhibits chemotaxis in human eosinophils

Cellular Signalling Vol. 7, No. 5, pp. 527-534, 1995.

Elsevier ScienceLtd Printed in Great Britain

rergamon

0898-6568(95)00023-2

ELEVATED INTRACELLULAR

CYCLIC AMP INHIBITS CHEMOTAXIS

IN HUMAN

EOSINOPHILS T A K E S H I KANEKO,* R O B E R T A L V A R E Z , t IRIS F. UEKI* and JAY A. NADEL*$ *Cardiovascular Research Institute and Department of Medicine, University of California San Francisco, San Francisco, CA 94143-0130, U.S.A. and tSyntex Discovery Research, Palo Alto, CA 94304, U.S.A. (Received 27 August 1994; sent for revision 21 October 1994; and accepted 14 January 1995) Abstract--Elevated intracellular cyclic AMP is associated with the inhibition of many inflammatory cellular responses. In this study, we examined the effect of cyclic AMP on eosinophil chemotaxis. Eosinophils were isolated from healthy human volunteers using an immunomagnetic method. Eosinophils were treated with agents that elevate intracellular cyclic AMP and evaluated for chemotactic responses to platelet-activating factor (PAF; 10-6 M) and to complement factor 5a (C5a; 10-8 M) in microchemotaxis chambers. Forskolin, prostaglandin E~ (PGE0, and a phosphodiesterase (PDE) IV-selective inhibitor inhibited eosinophil chemotactic responses. The mean per cent inhibition of eosinophil chemotaxis in response to PAF by forskolin, PGE~, and the PDE IV-selective inhibitor (10-5 M) was 16.8 _+5.3, 26.6 _+9.5, and 35.1 _+ 6.1%, respectively (n = 5). The corresponding values for C5a were 17.5 _+7.9, 20.8 _+ 10.7, and 39.5 + 5.0%. An exogenous cyclic AMP analogue (dibutyryl cyclic AMP, 10-3 M) also inhibited eosinophil chemotaxis by 69.4 _+ 12.8 and 66.9 _+ 11.6% in response to PAF and C5a, respectively (n = 5). We conclude that elevated intracellular cyclic AMP inhibits eosinophil chemotaxis. Key words: Cyclic AMP, eosinophils, chemotaxis, platelet-activating factor, complement factor 5a, phosphodiesterase inhibitor.

INTRODUCTION

agents that elevate intracellular cyclic A M P on eosinophil chemotaxis in response to platelet-activating factor (PAF) and to complement 5a (C5a). T h e a g e n t s s t u d i e d i n c l u d e d : f o r s k o l i n and p r o s t a g l a n d i n El (PGE1), w h i c h are a d e n y l y l cyclase agonists and thus elevate intracellular cyclic AMP, and a phosphodiesterase (PDE) IVselective inhibitor (RS-25344), which prevents the enzymatic hydrolysis of cyclic AMP. At least seven different gene families of cyclic nucleotide PDE have been identified [5, 6]. The isoenzymes that preferentially hydrolyse cyclic AMP are PDE III and PDE IV. PDE III has a high affinity for cyclic A M P and is inhibited by cyclic GMP [6]. In contrast, PDE IV is specific for the hydrolysis of cyclic AMP and is insensitive to inhibition by cyclic GMP. We used a PDE IV-selective inhibitor because this isoenzyme appears to be predominant in eosinophils [7, 8]. We also examined the effect of an exogenous cyclic A M P ana-

Eosinophils are the predominant leukocytes present at inflammatory sites in allergic diseases such as asthma [1], and these cells are believed to play important pathogenic roles in these diseases [2, 3]. I n h i b i t i o n o f e o s i n o p h i l r e c r u i t m e n t in these inflammatory sites might provide a novel therapeutic strategy. Previous studies have shown that agents that elevate intracellular cyclic A M P suppress the release of mediators in eosinophils [4]. In neutrophils, elevated intracellular cyclic A M P inhibits not only mediator release but also chemotaxis. We r e a s o n e d that elevated intracellular c y c l i c A M P m i g h t i n h i b i t c h e m o t a x i s in eosinophils. Therefore, we examined the effect of S A u t h o r to w h o m c o r r e s p o n d e n c e should be a d d r e s s e d at the C a r d i o v a s c u l a r R e s e a r c h Institute, B o x 0130, University o f C a l i f o r n i a S a n Francisco, San Francisco, C A 9 4 1 4 3 - 0 1 3 0 , U.S.A.

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528 logue (dibutyryl chemotaxis.

cyclic AMP) on eosinophil

MATERIALS AND METHODS

Materials Dextran, bovine serum albumin (BSA), C5a, forskolin, calmodulin, calmidazolium, dibutyryl cyclic AMP (N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate), Histopaque-1077, trichloroacetic acid, dimethyl sulphoxide (DMSO), bovine heart PDE (activator-deficient, P-0520), and Tris-HC1 buffer were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). PAF (L-~-phosphatidylcholine-~-acetyl-~/-o alkyl) was purchased from Behring Diagnostics (La Jolla, CA, U.S.A.). PGE~ was purchased from Upjohn (Kalamazoo, MI, U.S.A.). Diff-Quik was purchased from Baxter Healthcare Corporation (McGraw Park, IL, U.S.A.). Lympholyte M was purchased from Cedar Lane Laboratories Ltd (Hornby, Ontario, Canada). [3H]cyclic AMP and [3H]-cyclic GMP were purchased from New England Nuclear Corp. (Boston, MA). MgSO4 was purchased from Mallinckrodt (Paris, NY, U.S.A.). CaCI 2 was purchased from J. T. Baker (Phillipsburg, NJ, U.S.A.). Hanks' balanced salt solution containing Ca 2÷ and Mg 2+ (HBSS ÷) or Ca 2+- and MgZ+-free HBSS (HBSS-) was purchased from the Tissue Culture Facility, University of California, San Francisco. RPMI 1640 was purchased from GIBCO (Grand Island, NY, U.S.A.). Nu-Serum was purchased from Collaborative Research Inc. (Bedford, MA, U.S.A.). The PDE IVselective inhibitor, RS-25344: 8-aza-l-(3-nitrophenyl)3-(4-pyridylmethyl)-2,4-[1H, 3H]-quinazoline dione and the PDE III inhibitors, RS-82856: N-cyclohexyl-Nm e t h y l - 4 - [( 1 , 2 , 3 , 5 - t e t r a h y d r o - 2 - o x o i m i d a z o [ 2 , 1 b]quinazolin-7-yl)-oxy] butyramide, rolipram and trequinsin were provided by Dr Robert Wilhelm (Syntex Discovery Research). The structure, pharmacological properties and chemistry of RS-82856 were published previously [9, 10]. The 48-well microchemotaxis chambers and cellulose nitrate filters (5 ~tm pore size) were purchased from Neuro Probe (Cabin John, MD, U.S.A.). A magnetic cell sorter (MACS), MACS separation columns, and magnetic microbeads coated with antibodies to CD16 were purchased from Miltenyi Biotec Inc. (Sunnyvale, CA, U.S.A.).

separated from mononuclear cells by density gradient centrifugation with Histopaque-1077 at 400 g for 20 min. Contaminating red blood cells were removed by hypotonic lysis and then remaining cells were washed twice with HBSS . This cell fraction was incubated at 4°C for 30 min with magnetic microbeads coated with antibodies to CD16, and then the eosinophils were purified on separation columns in the magnetic field of a magnetic cell sorter. This method negatively selects eosinophils by retaining magnetically labelled neutrophils in a column made of a ferromagnetic matrix. The isolated eosinophils were > 99% pure as determined by differential counts of cytospun cells stained with Diff-Quik, and were > 98% viable as determined by trypan blue dye exclusion. The isolated eosinophils were washed with HBSS and resuspended in HBSS ÷ containing 1% BSA. The eosinophil concentration for each chemotaxis assay was adjusted to 1.5 x l06 eosinophils/ml.

Eosinophil chemotaxis assay Assays of eosinophil chemotaxis were performed using 48-well microchemotaxis chambers [12]. Eosinophils (7.5 x l04 cells in 50 ~tl of HBSS* containing 1% BSA) were placed in each well of the upper compartment and allowed to migrate through a nitrocellulose filter of 5 ~tm pore size towards PAF (104 M) or C5a (10 -s M) in the well of the lower compartment for 30 min at 37°C. PAF and C5a were dissolved in HBSS ÷ containing 1% BSA. All experiments were performed in duplicate or triplicate. The concentrations of PAF and C5a used were chosen because they caused maximal eosinophil chemotaxis in our preliminary dose-response studies and in previous studies [13, 14]. Eosinophil chemotaxis was determined by a leading front method [15]. The distance of eosinophil migration was measured using a fixed microscope by the difference of the vernier in five high-power fields of each well. Eosinophil chemotactic response is expressed as distance %tm) beyond random migration. Random migration is migration to HBSS* containing 1% BSA. Chemotaxis of eosinophils treated with cyclic AMPstimulatory agents or the exogenous cAMP analogue is expressed as per cent of control chemotaxis. Control chemotaxis is chemotaxis of vehicle-treated eosinophils in response to PAF or C5a, and is designated as 100%. Per cent inhibition of chemotaxis was calculated as the reciprocal of per cent chemotaxis.

Eosinophil isolation Eosinophils were isolated from whole blood of healthy human volunteers who had normal to slightly elevated eosinophil counts, using an immunomagnetic method [11]. After dextran sedimentation, the fraction of blood containing neutrophils and eosinophils was

Determination of intracellular cyclic AMP in eosinophils Eosinophils were isolated as described above and then suspended in HBSS + containing 1% BSA at a concentration of 1.5 x l06 eosinophils/ml. Aliquots of

Intracellular cAMP inhibits chemotaxis in human eosinophils eosinophils were then incubated for 15 rain at 37°C with cyclic AMP-stimulatory agents or vehicle (control). The incubation was terminated by adding cold trichloroacetic acid (4°C, final concentration 6%) and eosinophils were sonicated and stored at -20°C. On the day of the assay, all samples, were defrosted and centrifuged at 8000 g for 15 min at 4°C, and the supernatants recovered. The supernatants were washed four times with water-saturated diethyl ether. The aqueous extracts were dried under a stream of nitrogen at 60°C and dissolved in a suitable volume of assay buffer provided in the assay system. All samples and standards were then acetylated. Concentrations of cyclic AMP were determined in duplicate using an enzyme immunoassay kit (Amersham Life Science, Arlington Heights, IL, U.S.A.).

Study design To determine whether elevated cyclic AMP inhibits eosinophil chemotaxis, purified human eosinophils were incubated with different concentrations of each of three cyclic AMP-stimulatory agents, an exogenous cyclic AMP analogue or vehicle. All agents were dissolved in DMSO; the final concentration of DMSO was 1% in HBSS + containing 1% BSA. Incubations were performed for 15 min at 37°C. Eosinophils were then assayed for chemotaxis in response to PAF and to C5a for 30 min at 370C. The cyclic AMP-stimulatory agents used were forskolin, PGE~ and a PDE IV-selective inhibitor (RS-25344). In preliminary studies, the effect of two PDE III inhibitors (anagrelide and RS-82856) on PAF-induced human eosinophil chemotaxis was examined and it was found that they do not inhibit eosinophil chemotaxis (data not shown). Studies were focused on PDE IV-selective inhibitor because this isoenzyme is the predominant PDE in guinea pig eosinophils [7, 8]. As an exogenous cyclic AMP analogue, l f f 3 M dibutyryl cyclic AMP was chosen, which has been used commonly in other studies [4, 16]. PAF and C5a were chosen to stimulate eosinophil chemotaxis because they are potent eosinophil chemotactic factors [13, 14]. To confirm that the effect of each cyclic AMP-stimulatory agent on eosinophil chemotaxis was due to the elevation of intracellular cyclic AMP in eosinophils, intracellular cyclic AMP in eosinophils after incubation of eosinophils with each cyclic AMP-stimulatory agent was measured. Data are expressed as the means _+ S.E.M. for five subjects. All data are analysed by Student's paired t-test. P < 0.05 is considered significant.

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Three experiments with at least five concentrations of RS-25344 in log order intervals were performed in triplicate. Maximum per cent hydrolysis was less than 15%. RS-25344 was dissolved in DMSO; the final concentration of DMSO in the enzyme assay was 1%. The PDE assay, including the secondary 5'-nucleotidase reaction and isolation of labelled adenosine, were performed in accordance with a previously described procedure [ 17].

Bovine heart PDE (PDE 1) Calmodulin (10 U) was added to the bovine heart stock solution containing 0.05 U/mg in 2 ml of 10 mM Tris-HC1, pH 7.7. The reaction medium contained 10 mM Tris-HC1 buffer, pH 7.7, 0.1 mM MgSO 4, 10 I.tM CaC12, 1 ~tM [3HI-cyclic GMP (0.2 ~tCi). Following addition of the enzyme, the contents were mixed and incubated for 15 min at 30°C. The reaction was terminated by immersing the tubes in a boiling water bath for 60 s.

Mouse splenocyte PDE (PDE 11) Spleens were dissected from 5-6 CD-(ICR) strain mice (euthanized by carbon dioxide asphyxiation) and homogenized with 2-3 ml RPMI 1640 using a glass homogenizer. A splenocyte pellet was obtained by centrifugation of the homogenate at 200 g for 10 min at 22°C. The cells in the pellet were washed, and then resuspended in 7 ml of RPMI 1640. The cell suspension was carefully layered over 3 ml of lympholyte M which was placed in the bottom of a 15 ml conical centrifuge tube. The lymphocyte layer which was on top of the lympholyte M was extracted after the sample tube was centrifuged at 1200 g for 20 min at 22°C. Splenocytes were washed twice with 10 ml of RPMI 1640 by centrifugation at 200 g for 10 min. The final pellet was resuspended in 10 ml ice cold 45 mM Tris-HC1, pH 7.7. The hypotonically lysed cell suspension was centrifuged at 12,000 g for 10 min at 4°C. The supematant fraction was used as the soluble PDE. When assayed with 1 ~tM cyclic AMP, this crude enzyme preparation contains PDE II, III and IV activity. When PDE III and IV activities are selectively blocked by 1 I.tM RS-82856 and RS-25344, the remaining activity was stimulated 2.1-fold by 10 ~M cyclic GMP (ECs0 = 0.2 ~tM). For inhibition studies, cyclic GMP (10 I.tM) was added to stimulate PDE II activity. RS-82856 (1 /,tM) and rolipram (10 ~tM) were added to inhibit PDE III and PDE IV activities, respectively. The PDE incubation medium contained 45 mM Tris-HC1 buffer, pH 7.7, 0.1 mM MgSO4 and 1 ~tM [3H]-cyclic AMP (0.2 ~tCi).

Cyclic nucleotide phosphodiesterase assays To examine the potency, isozyme and isoform selectivity for RS-25344, experiments were performed with PDE isoenzymes from different sources (see below).

Human platelet PDE (PDE Ili) Platelet high-affinity cyclic AMP PDE was obtained from human blood in accordance with previously

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described procedures [17]. The PDE incubation medium contained 45 mM Tris-HC1 buffer, pH 7.7, 10 MgSO4, 1 ~tM [3H]-cyclic AMP (0.2 ~tCi) in a total volume of 0.2 ml. RS-82856 was used as a reference standard to estimate the percentage of the total activity that was due to PDE III. When assayed with 1 ~tM cyclic AMP, the majority (> 90%) of the PDE activity detected in human platelets is PDE III.

Human lymphocyte PDE (PDE IV) A human B cell line (43D), isolated by Dr Mary Mulkins (Syntex Discovery Research), was used as a source of PDE IV. This cell line appears to be normal except for probable infection with Epstein-Barr virus. The cells were cultured at 37°C in 7% CO2 in RPMI 1640 with L-glutamine and 10% Nu-Serum. Prior to the assay -1.5 × 108 cells were centrifuged at 250 g for 10 min in a table-top clinical centrifuge. The pellet was resuspended in 2-3 ml of 45 mM Tris-HC1 buffer, pH 7.4. The suspension was centrifuged at 48,000 g at 4°C for 10 min. The supernatant was diluted to 28 ml with Tris-HC1 buffer and stored at -20°C. Rolipram was used as a reference standard to estimate the percentage of the total PDE activity due to PDE IV. When assayed at 1 p.M cyclic AMP, the majority (> 80%) of the PDE activity detected in 43D cells was PDE IV. RS-82856 (1 ~tM) did not inhibit PDE activity. These results indicate the absence of PDE III activity in this enzyme preparation. The PDE incubation medium contained 45 mM Tris-HC1 buffer, pH 7.4, 0.1 mM MgSO 4, and 1.0 ~M [3H] cyclic AMP (0.2 I.tCi) in a total volume of 0.2 ml. After addition of the enzyme, the contents were mixed and incubated for 10 min at 30°C. The reaction was terminated by immersing the tubes in a boiling water bath for 60 s.

RESULTS

Eosinophi! chemotaxis T h e m i g r a t i o n o f vehicle-treated eosinophils to H B S S + containing 1% B S A ( r a n d o m migration) was variable a m o n g donors. T h e m e a n eosinophil net migration in response to P A F (67.2 _+ 3.6 I.tm) and in response to C5a (59.5 _+ 5.0 ~tm) w e r e similar. T r e a t m e n t o f eosinophils with cyclic A M P stimulatory agents did not affect r a n d o m migration (data not shown). Forskolin, P G E t, and the P D E I V - s e l e c t i v e inhibitor all inhibited eosinophil c h e m o t a x i s in response to both P A F and C 5 a in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r (Figs 1-3). C o m p a r i n g the inhibition caused by a IO 5 M concentration of each cyclic A M P - s t i m u l a t o r y agent s h o w e d that the P D E I V - s e l e c t i v e inhibitor was m o s t efficacious and forskolin was least efficacious. T h e m e a n per cent inhibition o f e o s i n o p h i l c h e m o t a x i s in response to P A F by 10 -5 M forskolin, PGE1 and the P D E I V - s e l e c t i v e inhibitor was 16.8 _+ 5.3, 26.6 _+ 9.5 and 35.1 -+ 6 . l % , respectively (n = 5). F o r C5a, the corresponding values were 17.5 _+ 7.9, 20.8 _+ 10.7, and

Eosinophil chemotaxis (% of Control) 100

91)

80 60

Human eosinophil PDE 1V Human eosinophils were isolated as described above and sonicated (Sonifier Cell Disruptor 200, Branson Sonic Power Co., Danbury, CT, U.S.A.) at the lowest setting for 5 s x 6 times on ice and then centrifuged at 8000 g for 15 min at 4°C. Under these conditions we confirmed that the majority of the PDE activity remains in the supernatant fraction. This is consistent with the recent findings of other researchers [18]. The supernatant (0.5 ml) from - 5 x 106 cells was diluted into 4.5 ml Tris-HC1 buffer, pH 7.7 containing 0.1 mg/ml BSA and used directly in the PDE assay. The PDE incubation medium contained 10 mM Tris-HC1 buffer, pH 7.4, 10 mM MgSO4, and 1.0 ~tM [3H] cyclic AMP (0.2 gCi) in a total volume of 0.2 ml. After addition of the enzyme, the contents were mixed and incubated for 30 min at 30°C. The reaction was terminated by immersing the tubes in a boiling water bath for 60 s. J

4O 20 0

-'6 Control

-~

-'4

Forskolin (log M)

Fig. 1. Inhibitory effect of forskolin on eosinophil chemotaxis in response to platelet-activating factor (open circles) and to complement factor 5a (closed circles) in five subjects. Data are presented as the means _+ S.E.M. The 100% (control) value represents chemotaxis in response to platelet-activating factor or complement factor 5a in eosinophils without forskolin. *P < 0.05 vs control.

Intracellular c A M P inhibits chemotaxis in human eosinophils

Eosinophil chemotaxis (% of Control) 100-

531

Eosinophil chemotaxis (% of Control) 100Q)

lid

80-

80.

60-

60.

40-

40-

20-

20-

0-

0.

¢,

-tt Control

Prostaglandin E1 (log M)

Fig. 2. Inhibitory effect of prostaglandin E l on eosinophil chemotaxis in response to platelet-activating factor (open circles) and complement factor 5a (closed circles) in five subjects. Data are presented as the means - S.E.M. The 100% (control) value represents chemotaxis in response to platelet-activating factor or complement factor 5a in eosinophils without prostaglandin El. *P < 0.05 vs control. 39.5 _+ 5.0%. The exogenous cyclic A M P analogue, dibutyryl cyclic A M P (10 -3 M), also inhibited eosinophil chemotaxis in response to PAF and C5a by 69.4 _+ 12.8, and 66.9 -+ 11.6%, respectively (Fig. 4).

Intracellular cyclic AMP concentrations in eosinophils Cyclic AMP-stimulatory agents (10 -s M) elevated intracellular cyclic A M P in eosinophils, although the value for forskolin did not reach statistical significance (Table 1). The order of efficacy was PDE IV inhibitor > PGE~ > forskolin. This order was consistent with the order of efficacy in inhibiting eosinophil chemotaxis at the same concentration (10 -5 M).

Control

The large elevation (6-fold) in intracellular cyclic A M P obtained in the presence of RS-25344 was unexpected. Thus, it was of interest to examine the effect of RS-25344 on PDE activity in human eosinophils. RS-25344 is a potent and selective inhibitor of PDE IV in human lympho-

-7

:5

Fig. 3. Inhibitory effect of a phosphodiesterase IVselective inhibitor, RS-25344 on eosinophil chemotaxis in response to platelet-activating factor (open circles) and complement factor 5a (closed circles) in five subjects. Data are presented as the means _+ S.E.M. The 100% (control) value represents chemotaxis in response to platelet-activating factor or complement factor 5a in eosinophils without RS-25344. *P < 0.05 vs control.

Eosinophil chemotaxis (% of Control) I

PAF

C5a I

I

I

100. 80 60 40 20

Control

Inhibition of cyclic nucleotide phosphodiesterases by RS-25344

.'9

Phosphodiesterase IV inhibitor RS-25344 (log M)

Dibutyryl cyclic AMP

Control

Dibutyryl cyclic AMP

Fig. 4. Inhibitory effect of an exogenous cyclic AMP analogue, dibutyryl cyclic AMP (10-3 M) on eosinophil chemotaxis in response to platelet-activating factor (PAF, open column) and complement factor 5a (C5a, closed column) in five subjects. Data are presented as the means _+ S.E.M. The 100% (control) value represents chemotaxis in response to PAF or C5a in eosinophils without dibutyryl cyclic AMP. *P < 0.05 vs control.

532

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Table 1. Effect of cyclic AMP elevating agents (10 5 M) on intracellular cyclic AMP concentration in eosinophils

al.

Percent inhibition 100.

Treatment

Cyclic AMP (pg/106 eosinophils)

Control Forskolin Prostaglandin E~ RS-25344

29.6 _+6.8 62.6 -+21.6 85.3 -+5.8* 178.9 _+29.1"

Values represent the means _+ S.E.M. for five different subjects in each group. *P < 0.05 compared with control.

80. 60. 40. 20.

0.

cytes (IC50 = 0.2 nM), with only w e a k inhibitory effects on P D E I, II and III (IC50 values > 100 ~ M ; T a b l e 2). R S - 2 5 3 4 4 was a m o r e potent inhibitor of the soluble P D E IV isolated f r o m h u m a n l y m p h o cytes than f r o m particulate or soluble P D E in eosinophils. The difference in the c o n c e n t r a t i o n response c u r v e for R S - 2 5 3 4 4 m a y represent inhibition o f two or m o r e f o r m s of P D E IV in eosinophils (Fig. 5). It is possible that there are different i s o f o r m s or splice variants o f P D E IV in the two cell types.

DISCUSSION In this study, we s h o w that e l e v a t e d intracellular cyclic A M P inhibits eosinophil chemotaxis. PGE~, forskolin and RS-25344 inhibited eosinophil c h e m o t a x i s in a c o n c e n t r a t i o n - d e p e n dent manner. A t e q u i m o l a r concentrations (10 -5 M), the rank order o f e f f i c a c y for these agents in inhibiting e o s i n o p h i l c h e m o t a x i s c o r r e s p o n d e d to their ability to increase intracellular cyclic A M P .

-11

-10

-9

-8

-7

-6

-5

-4

RS-25344 (log M)

Fig. 5. Inhibition of cyclic AMP phosphodiesterase activity in human lymphocytes (closed circles) and human eosinophils (closed squares) by RS-25344. Data are presented as the means _ S.E.M., n = 3. Most of the symbols of each value encompass the standard error bars because standard errors are small.

A n e x o g e n o u s cyclic A M P analogue also inhibited eosinophil chemotaxis. P h a r m a c o l o g i c a l agents with different sites of action are capable o f increasing cyclic A M P in intact cells. A d e n y l y l cyclase agonists elevate intracellular cyclic A M P by increasing its synthesis while P D E inhibitors p r e v e n t the e n z y m a t i c hydrolysis o f the nucleotide. P r e v i o u s studies h a v e demonstrated that PGE1 produces a receptorm e d i a t e d stimulation of adenylyl cyclase. In contrast, forskolin directly increases catalytic activity. P D E IV appears to be p r e d o m i n a n t in eosinophils [7, 8]. In our pilot study, P D E III inhibitors, both

Table 2. Inhibition of soluble cyclic nucleotide phosphodiesterases by calmidazolium, trequinsin, rolipram and RS-25344 0%0, btM) Cyclic nucleotide phosphodiesterase PDE I PDE II PDE III PDE IV

Calmidazolium 0.1 -6.2 22

Trequinsin 4.3 -0.00041 0.4

Rolipram

RS-25344

> 100

> 100 160 330 0.00028

> 100 0.15

The enzyme preparations were assayed in the presence of selective inhibitors or activators as described in the Methods section. Labelled cyclic GMP (1 p-M) was used as the substrate for PDE I. Labelled cyclic AMP (1 p.M) was used for PDE II, III, and IV. PDE I activity was stimulated with calmodulin. PDE II activity was stimulated with 1 p-M cyclic GMP. The concentration range studied was from 10 pM to 10 p-M in log intervals for the compounds exhibiting potent activity. Weak compounds were tested up to a maximum concentration of 10 mM. The data are from representative experiments with each concentration tested in triplicate. Standard deviations from the mean values were less than 5%. The experiments were repeated three times with similar results.

IntracellularcAMP inhibits chemotaxisin human eosinophils anagrelide and RS-82856, did not inhibit eosinophil chemotaxis. Thus, a selective inhibitor of PDE IV (RS-25344) was used to block enzyme activity. There have been few studies on the effects of cyclic AMP-stimulatory agents on eosinophil functions. Cyclic AMP-stimulatory agents including a non-selective PDE inhibitor reduce immunoglobulin-induced degranulation in human eosinophils, as assessed by the release of the eosinophil-derived neurotoxin [4]. A PDE Ill/IV mixed inhibitor has been shown to reduce the respiratory burst in human eosinophils [19] and PDE IV-selective inhibitors reduce respiratory burst in guinea pig eosinophils [7]. No literature is available on the effect of PDE IV-selective inhibitors on human eosinophil function. Recently, it has been shown that a PDE IVselective inhibitor reduces antigen-induced eosinophil recruitment as assessed by both bronchoalveolar lavage and by histological evaluation of the airways in guinea pigs [20, 21]. It is noteworthy that a PDE IV-selective inhibitor has been shown to reduce antigen-induced eosinophil recruitment even when administered as late as 12 h after antigen challenge [20]. These results suggest that the mechanism of the reduction of antigen-induced eosinophil recruitment into the airways by the PDE IV-selective inhibitor is not reduction of the release of chemotactic mediators from airways, which presumably had occurred before the administration of the drug. Possibly, the PDE IV-selective inhibitor reduced antigeninduced eosinophil recruitment by limiting eosinophil motility. In other inflammatory cells including neutrophils [22], lymphocytes [23], basophils and mast cells [24], elevated intracellular cyclic AMP has been shown to inhibit mediator release. Many of the mediators released from these inflammatory cells (e.g., histamine, PAF, eicosanoids) cause smooth muscle contraction and extravasation in airways, and some mediators cause further recruitment of inflammatory cells into the airways [25]. Thus, cyclic AMP-stimulatory agents might be effective in the treatment of bronchial asthma by inhibiting mediator release from airway inflam-

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matory cells, Chromatographic analysis combined with kinetic and pharmacological characterization shows that PDE IV is the major PDE isoenzyme responsible for catabolizing cyclic AMP in mast cells, basophils, and neutrophils, as well as eosinophils [26]. Therefore, PDE IV-selective inhibitors are potentially a promising new approach in the treatment of bronchial asthma [26, 27]. In support of this possibility, it has been shown that a PDE IV-selective inhibitor reduces antigen-induced bronchoconstriction and reduces airway hyperresponsiveness, probably as a result of reduction in the release of leukotrienes from the airways [28]. In the chemotaxis assay, we used PAF and C5a to induce eosinophil chemotaxis. PAF is not only chemotactic for eosinophils [13] but also induces eosinophil activation [29, 30]. Inhalation of PAF has been shown to induce bronchoconstriction and eosinophil recruitment into airways in baboons [31]. PAF is suggested to be an important mediator involved in allergic inflammatory reactions [32]. The complement system may also generate important mediators involved in allergic inflammatory reactions [33, 34]. In summary, we have shown that elevated intracellular cyclic AMP inhibits eosinophil chemotaxis. Because eosinophils are believed to play important roles in allergic diseases such as asthma, inhibition of eosinophil recruitment might provide an important therapeutic benefit in these diseases. In asthma, PDE IV-selective inhibitors might provide a novel and selective therapeutic approach. Acknowledgements--The authors thank Mimi Zeiger for editorial assistance, and Shauna McDonough and Natalie Holt for assistance in the preparation of the manuscript. This work was supported in part by NIH grant HL-24136. Dr Kaneko was supported by the Will Rogers Memorial Fund.

REFERENCES l. Weller P. F. (1984) J. Allergy Clin. Immun. 73, 1-10. 2. Frigas E. and Gleich G. J. (1986) J. Allergy Clin. lmmun. 77, 527-537.

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