Gen. Pharmac. Vol. 24, No. 5, pp. 1207-1211, 1993 0306-3623/93 $6.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1993 Pergamon Press Ltd

EFFECT OF TYB-2285 ON ANTIGEN-INDUCED HISTAMINE RELEASE FROM MOUSE BONE

MARROW-DERIVED PERSISTING CELLS (P-CELLS) PRIMED WITH INTERLEUKIN-3-CONTAINING

CONDITIONED MEDIUM (IL3-CM)

AKIHIKO WATANABE, l , TAKANARI TOMINAGA, I HARUKI HAYASHI, ~ YUKIYOSHI YANAGIHARA 2 and

AKIHIDE KODA 3

t Department of Pharmacology, Pharmaceuticals Research Center, Toyobo Co. Ltd, 2-chome Katata l-1 Ohtsu Shiga, 520-02 [Fax 775(21) 1493], 2Department of Immunopharmacology, Sagamihara National Hospital, Sakuradai Sagamihara Kanagawa 228 and 3Department of Pharmacology, Gifu Pharmaceutical

University, Mitahorahigashi Gifu 502, Japan

(Received 2 February 1993)

Abstract--1. The present study was carried out to investigate the effect of TYB-2285 [3,5-bis(acetoxy- acethylamino)-4-chlorobenzonitrile] on histamine release from persisting cells (P-cells) primed with interleukin-3-containing conditioned medium (IL3-CM).

2. IL3-CM enhanced antigen-induced histamine release from P-cells, although 1L3-CM did not affect spontaneous histamine release.

3. TYB-2285 inhibited antigen-induced histamine release in the presence of IL3-CM, although it did not inhibit histamine release in the absence of IL3-CM.

4. The enhancement of histamine release by IL3-CM was reproduced by recombinant IL-3 (1 100 U/ml).

5. These results demonstrate that TYB-2285 inhibits the histamine release primed with IL-3 and also suggests that TYB-2285 might regulate allergic inflammation in vivo by the suppression of mediator release primed with IL-3.

INTRODUCTION

TYB-2285 inhibits allergic reactions in vivo (Wa tanabe

et al., 1989) such as passive cu taneous anaphylaxis

(PCA), passive per i toneal anaphylaxis and lung ana-

phylaxis. However, this c o m p o u n d does not inhibi t

ant igen- induced his tamine or peptide-leukotr ienes (p-

LTs) release f rom peri toneal exudate cells and it does

not an tagonize chemical media tors in vitro.

Recently, it has been shown tha t r ecombinan t cy-

tokines such as interleukin-3 (IL-3) and granulocyte

monocyte colony s t imulat ing factor ( G M - C S F ) en-

hance his tamine release and p-LTs release (Hirai et al.,

1988; Kur imo to et al., 1989; Schleimer et al., 1989).

In the present study, the effect of TYB-2285 on

his tamine release f rom mouse bone marrow-der ived

persist ing cells (P-cells) in the presence/absence of

IL-3-condi t ioning medium ( IL3-CM) was investi-

gated.

Reagents

To culture P-cells and spleen cells, 2-mercaptoethanol (2-ME; Sigma), fetal bovine serum (FBS; Hyclone), Eagle's or-minimum essential medium (~t-MEM; Flow Labs), streptomycin sulfate (Flow Labs), and NaHCO 3 (Nakarai Tesque) were used. Ovalbumin (OA; Sigma), bovine serum albumin (BSA; Sigma), recombinant IL-3 (rlL-3; Genzyme) disodium cromoglycate (DSCG, Intal~; Fujisawa) and TYB-2285 were used for histamine release. TYB-2285 was synthesized in the Pharmaceuticals Re- search Center of Toyobo Co. Ltd. DSCG and TYB-2285 were dissolved in dimethylsulfoxide (final concentration of DMSO: 0.5%).

Preparation of IL3-CM

IL3-CM was prepared by a modification of the method described by Parker and Metcalf (1974). The concentration of mitogen used to stimulate spleen ceils was 1 : 300 dilution of poke weed mitogen (PWM). Spleen cells (2 × 106 nucle- ated cells/ml) were incubated in ct-MEM supplemented with 10% FBS, 100/~M 2-ME and PWM at 37°C in a humidified atmosphere flushed with 5% CO 2 in air for 5 days. Conditioned medium was centrifuged, filtered through a 0.22 gm filter, and stored at -80°C.

MATERIALS AND METHODS

Animals

WBB6F I + / + male mice (Japan SLC Inc, Shizuoka) were used for the preparation of bone marrow-derived P-cells and WBB6F~ + / + female mice (Japan SLC Inc) were used for the preparation of IL3-CM.

Preparation of P-cells

Mouse bone marrow cells (1 x 106 nucleated cells/ml) were cultured in ct-MEM supplemented with 10% IL3-CM, 10% FBS, 100/~M 2-ME at 37°C in a humidified atmos- phere flushed with 5% CO 2 in air. Half of the medium was replaced every 7 days. P-cells were used for experiments at 4-6 weeks after the beginning of the culture.

Ge Z4/.~--L 1207

1208 AKIHIKO WATANABE et al.

25

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O

O n "

O

-= 10 E

5

D i I I

$pon Spon Antigen +IL3-CM

. / / / /

/ / / / / . / / / # . / / / . / / / / ~ ¢ / / / /

/ / / / / r / / / ~

I / / / , , " / / / / f 1 1 1 ~ / / / / / ~ / / / I

/ / / I ~ f l / / ~

/ / / / ,

r i l l . ,

¢ / l i . , t 1 1 / . i / / / . , ~ / , / / / i ¢ 1 1 1 .

Antigen +IL3-CM

Fig. 1. Effect of IL3-CM on antigen-induced histamine release. P-cells were pretreated with IL3-CM for 5 min before antigen challenge. **P < 0.01; spon= spontaneous.

Histamine release from P-cells

P-cells sensitized with mouse anti-OA antibody (PCA titer >1000) were washed twice with BSA(+), Ca2+( - ) Tyrode's solution and were washed once with BSA( - ), Ca2+( + ) Tyrode's solution. P-cells were suspended in B S A ( - ), Ca2+( + ) Tyrode's solution at the concentration of 1 x 106 cells/ml. P-cells were incubated with OA for 15min following the incubation with IL3-CM or rIL-3. P-cells were preincubated with TYB-2285 or DSCG for 30sec before the addition of IL3-CM. TYB-2285 was tested at 100/aM unless other- wise specified. The reaction was stopped by the addition of iced saline. Supernatant was obtained by centrifugation (1500rpm, 4°C, 8min) and stored at -80°C. Residue was sonicated for 5min, boiled for 10min and stored at -80°C. All experiments were done in dupli- cate.

Statistical analysis

Data was analyzed with Student's unpaired t-test. Data in the figure represents the mean and SEM of each group.

RESULTS

The effect o f I L 3 - C M on antigen-induced histamine release

IL3-CM enhanced antigen-induced histamine

release, however it did not enhance spontaneous re-

lease, rather it decreased it (Fig. 1). The enhancement

was increased in a time-dependent manner (Fig. 2).

Effect o f TYB-2285 on histamine release in the pres- ence/absence o f l L 3 - C M

TYB-2285 inhibited histamine release in the

presence of IL3-CM at either incubation time (5

or 15 min), although it did not affect histamine release

in the absence of IL3-CM [Fig. 3(a and b)]. TYB-2285

inhibited histamine release primed with IL-3 irrespec-

tive of the concentration of antigen (Fig. 4).

TYB-2285 (0.01-1/IM) inhibited histamine

release in the presence of IL3-CM in a dose-dependent

manner to the level provoked by only antigen chal-

lenge. D S C G also inhibited histamine release in the

presence of IL3-CM in a dose-dependent manner,

although it did not inhibit significantly at a concen-

tration <0.1 # M (Fig.'5).

Effect o f r lL-3 on antigen-induced histamine release

Murine rlL-3 enhanced antigen-induced histamine

release at 0.1-100 U/ml in a dose-dependent manner (Fig. 6).

DISCUSSION

Histamine assay

Histamine was assayed according to the method of Shore et al. (1959).

TYB-2285 inhibits type I allergic reactions such as

PCA and lung anaphylaxis (Watanabe et al., 1989).

TYB-2285 is thought of as an inhibitor of mediator

250

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r r "

150

-1-

100

0 i i I

0 5 15 Treating time(rain) with IL3-CM

Fig. 2. Time-course of the enhancement of histamine release from P-cells by IL3-CM.

Histamine release from P-cells 1209

(a)

25

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• 15 Q

c lO E

tD

0 ,

Antigen + +

I L3 -CM -

T Y B - 2 2 8 5 - +

r

i

4- 4-

t ~ 35 1 , o | , s O • 2 0 D + E

" 5

, 0

+ Antigen

+ IL3 -CM + T Y B - 2 2 8 5

(b) [ - - - .IHF - -

I

+ + + +

-- - + 4-

- 4- - 4-

Fig. 3. Effect of TYB-2285 on antigen-induced histamine release from P-cells in the presence/absence of IL3-CM. IL3-CM was pretreated for 5(a) or 15min (b). **P <0.01.

release from previous studies (Watanabe et al., 1989, 1990) that TYB-2285 did not inhibit histamine- or serotonin-induced edema at 30 mg/kg, while it inhib- ited histamine release in PCA, histamine release by passive peritoneal anaphylaxis or the production of TxB2 in lung anaphylaxis at 3-30 mg/kg. However, the compound or its metabolites did not inhibit antigen-induced histamine release from rat peritoneal exudate cells even at 100#M in vitro (data not shown).

Controversial results between in vitro and in vitro experiments suggest that TYB-2285 might inhibit the histamine release enhanced by some factor in vivo, but not affect antigen-induced histamine release directly. It was reported that human rIL-3 enhanced the release of histamine

and p-LTs from human basophils (Hirai et ai.,

1988; Kurimoto et al., 1989; Schleimer et al., 1989).

In the present study, it was found that IL3oCM enhanced antigen-induced histamine release from P-cells, although it did not affect spontaneous his- tamine release. P-cells are rather like basophil cells in the point of their response to compound 48/80, but are like most cells morphologically (data not shown).

TYB-2285 inhibited histamine release from P-cells primed with IL3-CM, although it did not affect histamine release in the absence of IL3-CM. TYB- 2285 (0.01-1 #M) inhibited histamine release primed with IL3-CM in a dose-dependent manner. DSCG also inhibited histamine release in the presence of IL3-CM in a dose-dependent manner. Sakaguchi

120

_: 100

E

i eo

I E 20 o

m .~ o W

- 2 0

[ . - - I

3 10

D : Antloon+IL$-CM

~ ] : Antlgen+IL3-CM +TYB-2285

J SO 100

Coneontratlon(pg/ml) of Antigen

Fig. 4. Effect o f T Y B - 2 2 8 5 o n the e n h a n c e m e n t o f h i s t amine release b y I L 3 - C M at several an t i gen c o n c e n t r a t i o n s ( I L 3 - C M p r e t r e a t m e n t : 5 min) . * P < 0.05; **P < 0.01.

1210 AKIHIKO WATANABE et al.

30

2o

_=-

-1-

O E ~" - l O .=

UJ

- 2 0 , :l

0

• : T Y B - 2 2 8 5

_ • : DSCG

4H~

4HF

i i i

0.01 0.1 1

Concentrat ion of drugs(/JM)

Fig. 5. Dose-response of the inhibitory effect of TYB-2285 and DSCG on the enhancement of histamine release by IL3-CM (IL3-CM pretreatment: 5 min). **P < 0.01.

et al. (1992) demonstrated that histamine release from P-cells was enhanced by co-culture with fibroblast and also showed that DSCG inhibited histamine release from P-cells cultured in the presence of fibro- blast, although it did not inhibit histamine release from P-cells cultured in the absence of fibroblast. Our results are consistent with their results in the point that DSCG inhibited histamine release from P-cells enhanced by some factors, although it did not inhibit histamine release from P-cells directly.

We demonstrated that TYB-2285 inhibited his- tamine release from human basophils primed with rIL-3 (Tominaga et al., 1993) and also found that anti-CD1 lb inhibited histamine release from human basophils primed with rIL-3 (Watanabe et al., 1992). These results suggest that TYB-2285 may inhibit histamine release primed with IL-3 via suppression of the function of some adhesion molecules.

Lebel et aL (1990) found that IL-3-1ike activity was increased in blood and in hemopoietic organs by antigen challenge in actively sensitized mice. Burd et aL (1989), Plaut et al. (1989) and Filipowicz et ai.

(1989) reported that mast cells produced cytokines such as IL-3 by the stimulation of anti-IgE, antigen, or ionomycin. These findings suggest that IL-3 pro- duced by allergic reaction should augment allergic reaction and induce a bigger inflammation. Schleimer et al. (1989) also discussed the possibility that IL-3 might play an important role in the late phase allergic reaction.

In conclusion, the present study demonstrates that TYB-2285 inhibits histamine release from murine P-cells primed with IL3-CM and also suggests that this compound might regulate allergic inflammation by the suppression of mediator release primed with cytokines such as IL-3.

1R

a _e Q

n -

O c E

O E O O gg m

W

80

60

20

0

0 0.1 i r

10 100

Concentrat ion of rlL-3(U/ml)

Fig. 6. Dose-response of the enhancement of histamine release by rIL-3, rlL-3 was pretreated for 5 min.

Histamine release from P-cells 1211

Acknowledgements--We are grateful to Miss M. Hashimoto and Miss T. Nagao for their technical assistance and to Mr J. Tsuji for his helpful advice. We also appreciate Mr Y. Ebi and Dr Y. Kitamura very much for their kindness in teaching us the method of the culture of P-cells and the preparation of IL3-CM.

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