Eosinophil Supernatant Causes Hyperresponsiveness of Airway Smooth Muscle in Guinea Pig Trachea 1 , 2

T. AIZAWA, K. SEKIZAWA, T. AIKAWA, N. MARUYAMA, S. ITABASHI, G. TAMURA, H. SASAKI, and T. TAKISHIMA

Introduction

In vitro studies have demonstrated that eosinophils contain various biologic substances such as histaminase (1), eosinophil peroxidase (2), eosinophil cationic protein (3), and major basic protein (4). Eosinophils produce sulfidopeptide leukotrienes (LTs) (5, 6) and platelet-activating factor (7). Of these, LTs cause bronchoconstriction, and platelet-activating factor (PAF) is able to induce bronchial hyperresponsiveness (8). Major basic and eosinophil cationic proteins are reported to cause epithelial damage to the airways (3, 9). However, the role of eosinophils in asthma remains unclear, although their association with hypersensitivity reactions is wellestablished. Some investigators have emphasized that eosinophil infiltration is associated with suppression of allergic response (10), whereas others have suggested that the eosinophil's presence is associated with proinflammatory processes (11) and tissue damage (12, 13). In the present study, we examined the interaction between the supernatant from eosinophils activated with calcium ionophore A23187 and airway smooth muscle and the mechanisms responsible for supernatant-induced effects on airway smooth muscle contraction. Methods Isolation of Eosinophils To obtain eosinophil-rich peritoneal fluid, male Hartley strain guinea pigs weighing 300 g were immunized by injecting the pigs with 2 ml of horse serum intraperitoneally once a week for 2 months. The percentage of eosinophils to total cell number in peritoneal lavage fluid increased gradually (9.9 ± 2.60/0 in 4 wk, 32.9 ± 7.9% of 6 wk, and 53.0 ± 6.7% in 8 wk). We performed peritoneal lavage 48 h after the last injection of horse serum (14, 15). To purify eosinophils, Percoll discontinuous gradients were prepared by modifying Gartner's method (16). The stocked Percoll solution (1.112 g/ml of density) was serially diluted with 24 mM PIPES physiologic solu-

SUMMARY To study the role of eoslnophils In airway hyperresponslveness, we studied the effect of supernatant obtained from activated eoslnophlls on the responses of isolated guinea pig tracheal smooth muscle segments to histamine. Eoslnophils obtained from guinea pig peritoneal fluid were purified and activated with Ca2 + lonophore A23187using a two-stage reaction. Supernatant obtained from different eosinophil cell numbers (3 x 105 to 107 cells) did not alter resting tension but potentiated the contractile response to histamine In a cell number-dependent fashion. Thus, at a cell number of 107 , the supernatant decreased the mean (± SE) log histamine concentration producing 50% of maximum contraction significantly from a control value of -5.62 ± 0.10 to -5.99 ± 0.07 M (p < 0.05). The potentiating effect of the supernatant (107 cells) was not altered by either removal of tracheal epithelium or by pretreatment with Indomethacin when cells were activated. However, pretreatment with AA 861 completely inhibited the supernatant (107 cells)-induced potentiating effect associated with inhibition of leukotriene C., 0., and E. release in the supernatant. The concentrations of exogenous authentic leukotrienes chosen to match concentrations in the supernatant mimicked the supernatant-Induced potentiating response to histamine. These results suggest that leukotrienes released from eoslnophlls cause hyperresponsiveness of airway smooth muscle in vitro. AM REV RESPIR DIS 1990; 142:133-137

tion, pH 7.3, containing 125mM NaCl, 5 mM KCI, and 0.03% human albumin (PA solution) providing a series of different densities of Percoll solutions (1.090, 1.085, 1.080, and 1.070 g/ml). Densities of each Percoll solution were measured by refractometer (Atago Co. Ltd., Tokyo, Japan) and the standard curve. The standard curve was obtained from different densities of Percoll solution determined by pyknometer (Tokyo Garasukikai Co. Ltd., Tokyo, Japan). Using a peristaltic pump (Micro Tube Pump MP-3; Tokyo Rikakikai, Tokyo, Japan), we layered four different densities of Percoll solution carefully and successively into a 25-ml conical polycarbonate tube (Beckman Instruments, Inc., Fullerton, CA) (from bottom to top: 3 ml of 1.112 g/ml, 10 ml of 1.090 g/ml, 5 ml of 1.085 g/ml, and 3 ml of 1.080 g/ml Percoll solution, respectively) (17).The cells obtained were resuspended in 2 ml of 1.070 g/ml Percoll solution and cells (from 1.5 x 108 to 4.0 X 108 cells) were layered on top of the Percoll discontinuous gradients. The tube was then centrifuged (900 x g for 15 min at 18° C). The cell suspension was separated into three distinct bands containing approximately 30%, 70%, and 95% eosinophils from top to bottom, respectively. Therefore, to obtain highly purified eosinophils, we used the eosinophils of over 1.090 g/ml density from the lowest band. In preliminary studies, we found that peritoneal eosinophils obtained from guinea pigs sensitized with horse serum showed two density peaks (1.078 ± 0.002 g/ml and 1.085 ± 0.002

g/ml) as did peripheral blood eosinophils (1.076 ± 0.002 g/ml and 1.085 ± 0.002 g/ml). Therefore, eosinophils used in the present study were compatible with normal density eosinophils as described for human eosinophils (18). Cells were washed twice with PA solution, and an eosinophil pellet was obtained. Cells were examined with a WrightGiemsaair-dried smear, and purity of the eosinophils was within the range of 90 to 98% (mean 95%). These Percoll-purified eosinophils were invariably contaminated with mononuclear cells.

Production of Eosinophil Supernatant Cells were activated with Ca 2 + ionophore A23187 using a two-stage reaction (19),so that A23187 would not be present in the supernatant added to the muscle bath. Replicate aliquots of cells (either 3 x lOS, 106 , 3 X 106 , or 107 cells) were suspended in a Ca2+-free and Mg-t-free PA solution (1 ml) at 4° C. The cells werethen incubated with Ca 2+ ionophore (1 ug/ ml; 20 min) at 4° C, washed three times with (Received in original form June 25, 1989 and in revised form December 12, 1989) 1 From the First Department of Internal Medicine, Tohoku University of Medicine, Sendai, Japan. 2 Correspondence and requests for reprints should be addressed to T. Takishima, M.D., Professor and Chairman, First Department of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo machi Sendai 980, Japan.

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Ca 2+- free and Mg-t-free PA solution at 4° C, and resuspended in 1 ml of complete PA solution at 37° C. In preliminary studies, we found that the amount of LTC4 release from eosinophils reached maximum between 20 and 30 min. Therefore, we incubated the cells for 30 min at 37° C. After incubation for 30 min, the reaction was stopped by reducing the temperature to 4° C, the tubes were centrifuged at 200 g at 4°C for 8 min, and the supernatants from different cell numbers were obtained. The supernatants were stored in the dark under Argone gas at - 20° C. To study the mechanisms responsible for eosinophil supernatant-induced effects on airway smooth muscle contraction, the cells were preincubated with indomethacin (10-5 M; 30 min) or AA 861 (a selective 5-lipoxygenase inhibitor; 10-5 M; 30 min) (20) inhibiting the release of cyclooxygenase or lipooxygenase products. The supernatants were used for measuring content of cyclooxygenase and lipooxygenase products and for studies in the muscle bath. Supernatants were prepared in batches, and each series of experiment supernatant from the same batch was used. Cell viability was evaluated by trypan blue exclusion and was greater than 950/0.

Assay of Leukotrienes To remove protein, ethanol (8 ml) was added to 2 ml of sample, mixed gently and centrifuged (2,000 x g for 15 min at 4° C). To partially extract hydrophobic substances, dichrolomethane (30 ml) and phosphate buffer (10 ml) were added to these supernatants, mixed and centrifuged (2,000 x g for 15 min at 4° C). The upper layer was then partially purified on an octadecylsilyl silica column (Sep-pak" C18 cartridge; Waters Chromatography Division, Millipore Corp., Milford, MA). These extracts were analyzed by means of reverse-phase, high-performance liquid chromatography (Waters Model 510) by monitoring absorbance at 235 nm and 280 nm (Waters 490 Wavelength Detector) (21, 22). Fractions eluting with the same retention time as LTC4 , D 4 , and E 4 standards and bracketing fractions were then collected, dried, and resuspended in phosphate-buffered saline. Concentrations of LTC4 , D 4 , and E 4 were assayed in duplicate by a specific sulfidopeptide leukotriene radioimmunoassay with antisera (Amersham Corp., Arlington Heights, IL) (23). This antisera showed cross-reactivity between LTC4 , D 4 , and E 4 in ratios of 100: 64:64. Based on the cross-reactivity ratio, the measured values of LTC4 , D 4 , and E 4 were corrected. Log transformations of the calibration curves for LTC4 were linear, between 10 and 200 pg. In this assay, LTC 4 , D 4 , and E 4 could be measured with high reproducibility in quantities ranging from 10 to 200 pg. When the measured values were over 200 pg, the samples were diluted. The mean recovery rates of LTC4 , D 4 , and E 4 were 60, 50, and 50%, respectively.

Assay of Cyc/ooxygenase Products Two milliliters of sample were diluted with

acidic water (pH 3). The samples were then passed through the reverse-phase cartridge column (Bond Elute C18 cartridge; Waters). The eluents were further eluted with ethyl acetate and directly loaded onto an ionexchanging column (Bond Elute DEA cartridge column; Analytichem International, USA). Cyclooxygenase products were recovered by elution with 0.1 M acetic acid in 60% methanol. Cyclooxygenase products were then separated by normal-phase preparative thin layer chromatogram (Merck Art 5721; Merck and Co., Inc., Rahway, NJ) and extracted with 0.5 % acetic acid in methanol. The developing solvents were chloroform/ethyl acetate/ acetic acid (20/20/4/1, v/v) for prostaglandin E 2 (PGE 2 ) and ethyl acetate/acetic acid (99/1, v/v) for PGF2a, thromboxane B2 (TXB 2) and 6-keto-PGFl a. The cyclooxygenase products were purified again with the reverse-phase cartridge column (Bond Elute C18 cartridge; Waters). The concentration of individual cyclooxygenase products was assayed in duplicate by radioimmunoassay with antiserum for each cyclooxygenase product (Ono Pharmaceutical Co. Ltd., Osaka, Japan).

Preparation of the Trachea Male Hartley strain guinea pigs (300 to 450 g) were anesthetized intraperitoneally with pentobarbital sodium (50 mg), and the trachea was removed. Transverse rings (5 mm long) with intact epithelium were cut from the trachea and mounted in chambers filled with 5 ml of Krebs-Henseleit solution of the following composition (in mM/L): NaCl, 118;KCL, 5.9; CaCI 2 , 2.5; MgS0 4 , 1.2; NaH 2P04 , 1.2; NaHC0 3 , 25.5 and glucose, 5.6. The solution was maintained at 37° C and aerated continuously by bubbling with a mixture of 95% O 2 to 5% CO 2 , The tracheal rings were connected to a strain gauge (TB-612 T; Nippon Koden, Tokyo, Japan) for continuous recording of isometric tension. The rings were initially stretched to a tension of 1 g for 30 s and were then allowed to equilibrate for 1 h with resting tension maintained at 0.5 g. Preliminary experiments showed that maximum responses to histamine (3 x 10-6 M) were obtained with 0.5 g to 0.8 g of resting tension.

Dose-response Effects of Eosinophil Supernatant on Contractile Response to Histamine To study the effect of eosinophil-derived mediators on the contractile response of tracheal smooth muscle, we added supernatants to the muscle bath by removing 0.5 ml of Krebs-Henseleit solution from a 5-ml reservoir and replacing it with 0.5 ml of eosinophil supernatant (or dilution thereof). In preliminary studies we found that a supernatant by itself did not alter the resting tension. Therefore, we studied whether eosinophil supernatant potentiates the response to histamine. We preincubated tissues with propranolol (10-6 M; 15 min), phentolamine mesylate (10-5 M; 15 min), and indomethacin (10-6 M; 30 min) to eliminate the possible effects of

alpha- and beta-adrenergic innervation and release of cyclooxygenase products from tissues, which might make it difficult to obtain reproducible responses to histamine. To determine the baseline response, we added 3 x 10-6 M histamine (the concentration that produced approximately 50% of the maximum contraction) to the organ bath and compared the baseline response with the response obtained after incubating tissues with eosinophil supernatants from different cell numbers (3 x 105 to 107 cells). The effects of eosinophil supernatant on the response to histamine reached maximum at 5 min and lasted about 20 min with few changes. The bath was rinsed before administration of the supernatant and each tissue was exposed to the supernatant obtained from each of the eosinophil numbers.

Effects of Eosinophil Supernatant on Dose-response Curve to Histamine Because we found that the potentiating effect of eosinophil supernatant on the contractile response to histamine was dependent on cell number, we studied supernatant obtained from 107 cells, the maximum number tested in the present study. To determine responsiveness to histamine, cumulative dose-response curves to histamine were obtained using concentrations ranging from 10-8 to 10-4 M. Each succeeding concentration of histamine was added after the previous contraction had reached a plateau. After completion of the first dose-response curve to histamine, eosinophil supernatant (107 cells) was added, and 5 min later a second dose-response curve to histamine was obtained.

Effects of Contaminant Cells on Contractile Response to Histamine To study whether contaminant mononuclear cells in purified eosinophils altered contractile response to histamine, we purified mononuclear cells in peritoneal lavage fluid by the Percoll gradient method. A sample of 3 x 106 cells containing 97% mononuclear cells and 3% eosinophils was activated in a manner similar to that described above and supernatants were obtained. Responses to histamine (3 x 10-6 M) were measured in the absence and then in the presence of mononuclear cell supernatant (3 x 106 cells).

Effects of Removal of Epithelium on Eosinophil Supernatant To determine whether removal of the epithelium altered the effects of eosinophil supernatant on the response to histamine, we performed parallel studies on paired tracheal rings from the same animal after removing the epithelium from one randomly chosen tracheal ring by gently rubbing the luminal surface with gauze held by a pair of fine forceps. Removal of the epithelium was confirmed histologically in all tracheal rings. Responses to histamine (3 x 10-6 M) were measured in the absence and then in the presence of eosinophil supernatant (107 cells).

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tiple range test, significance was accepted at p < 0.05.

Active tension (% control)

200 Results

** 150

100 j

3 X 105

I

3 X 106

Effects of Supernatants Obtained from Different Cell Numbers on Response to Histamine Supernatant used in this study did not alter resting muscle tension. However, supernatant potentiated the contractile response to histamine (3 X 10-6 M) in a cell number-dependent fashion (figure 1). In contrast to eosinophil supernatant, mononuclear cell supernatant did not alter contractile response to histamine (3 x 10-6 M) (103.7 ± 3.7frJo of control, p > 0.20, n = 3).

Eosinophil number Fig. 1. Effects of supernatant obtained from different eosinophil numbers on the contractile response to histamine (3 x 10 -6 M). Results are reported as mean ± SE of five guinea pigs. Significant differences from control values are indicated by **p < 0.01.

Effects of Exogenous Administration of Authentic Leukotrienes Because we found that AA 861 inhibited eosinophil supernatant-induced potentiating response to histamine and that eosinophil supernatant contained significant amounts of LTC4 , D4 , and E4 , westudied exogenously administered LTC4 , D4 , and E 4 • To facilitate comparison of the effects of exogenous LTs with those produced by supernatant, the concentration of LTs was chosen to match those in the 5-ml bath containing 0.5 ml of supernatant (107 cells). Responses to histamine (3 x 10-6 M) were measured in the absence and then in the presence of LTs. Effects of PAF Antagonist on Eosinophil Supernatant To study whether eosinophil supernatantinduced potentiating response to histamine was caused by PAF, tissues were incubated with CV-3988, a specific PAF receptor antagonist (24) (10-5 M; 15 min) before the administration of eosinophil supernatant (107 cells). Drugs. The following drugs were used: propranolol, histamine, indomethacin, PAF, calcium ionophore A23187, EDTA, human albumin, PIPES, LTD4 , LTE4 (Sigma Chemical Co., St. Louis, MO), phentolamine mesylate (Ciba-Geigy, Basel, Switzerland), AA 861 (Takeda Pharmaceutical Co., Japan), horse serum (GIBCO Laboratories, Grand Island, NY), dimethyl sulfoxide (Wako Chemical Co., Ltd., Osaka, Japan), Giemsa solution, Wright eosine methylene blue (Merck, Japan). LTC4 and CV-3988 (the gifts from Takeda Chemical Industries, Ltd., Osaka, Japan). Statistical Analysis Data are expressed as mean ± SE. For statistical analysis of variance and Duncan's mul-

Dose-response Curves to Histamine Supernatant (107 cells) shifted the mean dose-response curve to the left (figure 2A) and significantly decreased log ED so of histamine concentration from a control value of - 5.62 ± 0.10to - 5.99 ± 0.07 M (p < 0.05), although dose-response curves to histamine were reproducible and did not change significantly with time (figure 2B).

Effects of Synthetic Inhibitors Indomethacin and AA 861 on Contractile Response to Histamine Supernatant (1Wcells) without the drug

Active tension (% control)

A

increased the contractile response to histamine by 158.4 ± 8.7frJo of control. Likewise, the supernatant pretreated with indomethacin increased the contractile response to histamine by 144.6 ± 5.7frJo of control, and the response did not differ significantly between the supernatant with and without indomethacin (p >0.10) (figure 3). However, cells (1W cells) activated in the presence of AA 861 failed to increase the contractile response to histamine (100.2 ± 6.2frJo of control, p > 0.50). Therefore, AA 861 completely inhibited the supernatant-induced effect.

Release of Leukotrienes in Supernatant in the Absence and Presence of AA 861 Cells activated in the absence of pretreatment with AA 861 contained significant amounts of LTC4 , D 4 , and E 4 (p < 0.01). Pretreatment with AA 861 significantly inhibited release of LTC4 , D 4 , and E 4 (figure 4). To establish the role of LTs suggested by the supernatant experiments more definitively, we studied the effects of authentic LTC4 , D 4 , and E 4 , in concentrations matching those in the supernatant bath (lW cells). Authentic LTsdid not alter resting tension, but they increased the contractile response to histamine (159.0 ± 1O.OfrJo of control, p < 0.01, n = 5). There was no significant differ-

Active tension (% control)

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o 8

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Fig. 2. (A) Effects of supernatant (107 cells) on dose-response curve to histamine. Results are reported as mean ± SE of seven guinea pigs. Maximum response in the control condition was 2.0 ± 0.4 g tension. Tension development was expressed as a percentage of the tension obtained at a concentration of 10-4 M histamine in each doseresponse curve and EDso was calculated from linear interpolation. Supernatant (closed circles) shifted the doseresponse curve from the control response (open circles) to the left and significantly decreased EDso values (p < 0.05). (8) Reproducibility of the histamine dose-response curve. Open circles show the first dose-response curve and closed circles the second one. Results are reported as mean ± SE of five guinea pigs. The second dosesresponse curve did not differ significantly from the first in terms of EDso and the maximum contraction (p > 0.50).

AIZAWA, SEKIZAWA, AIKAWA, MARUYAMA, ITABASHI, TAMURA, SASAKI, AND TAKISHIMA

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Discussion

(pg/m£)

Active tension (% controD

We conclude from these results that LTs released from eosinophils cause hyperresponsiveness of airway smooth muscle. Our conclusion is based on the observa500 ** tions that first, the supernatant obtained from activated eosinophils potentiated the contractile response to histamine in a cell number-dependent fashion al** though the supernatant obtained from Fig. 4. Release of leukotrienes in the supernatant in the absence (dotted columns) and presence of pretreatment contaminant mononuclear cells was 140 with AA 861, an inhibitor of 5-lipoxygenase (closed without effects; second, AA 861, an incolumns). Results are reported as mean ± SE of suhibitor of 5-lipoxygenase, inhibited the pernatants (107 cells) obtained from three guinea pigs. supernatant-induced potentiating reSignificant differences between results with and withsponse to histamine associated with inout AA 861 are indicated by * P < 0.05 and ** p < 0.01. hibition of LTs release in the supernatant; third, authentic LTs (concentrations matching those in the supernatant) mimEffects of PAF Antagonist on icked the supernatant-induced effects. Eosinophil Supernatant Cyclooxygenase products of arachi5 100 PAF receptor antagonist up to 10- M did No drug Indomethacin AA861 donate metabolism are known to modinot alter resting tension and it failed to + 7 fy airway smooth muscle contraction. Supernatant (l 0 cells) inhibit eosinophil supernatant-induced Athough eosinophils released cyclooxypotentiating response to histamine (160.4 Fig. 3. Effects of synthetic inhibitors of indomethacin genase products in the supernatant, cyand AA 861 on supernatant-induced increase in the con± 11.80/0 of control, p < 0.01, n = 5), clooxygenase products are not likely to tractile response to histamine (3 x 10- 6 M). Results and the percentage increase did not difbe responsible for the supernatant-inare reported as mean ± SE of five guinea pigs. Signififer significantly between that with and cant differences from control values are indicated by . duced potentiating response to histamine that without PAF antagonist (p > 0.50). ** P < om, and between conditions with and without because pretreatment of eosinophils with Furthermore, exogenously administered treatment with the drugs are indicated by ++ P < 0.01. indomethacin failed to inhibit superna5 8 PAF (10- to 10- M) did not significantly tant-induced effects. Eosinophils release alter resting tension, and PAF (10-5 M; 5 PAF (7) and PAF is known to cause bronmin) failed to potentiate the contractile chial hyperresponsiveness (8). However, ence between authentic LTs- and super- response to histamine (3 x 10-6 M) (101.5 PAF antagonist failed to inhibit eosinonatant-induced effects on the contractile ± 9.90/0 of control, p > 0.50, n = 3). phil supernatant-induced potentiating reresponse to histamine (p > 0.50). sponse to histamine. Furthermore, an exEpithelial Removal Effects of Indomethacin and AA 861 Removal of the epithelium slightly but ogenous PAF did not mimic supernatanton Release of Cyclooxygenase significantly enhanced the contractile re- induced effects. Thus, it is unlikely that Products in the Supernatant sponse to histamine (3 x 10-6 M) (0.79 PAF is responsible for the supernatantTable 1 shows that cells activated in the ± 0.10 g tension with epithelium versus induced potentiating response to histamine. Eosinophils are reported to release absence of pretreatment with either in- 1.20 ± 0.11 g tension without epithelium) domethacin or AA 861 contained signifi- (p < 0.05, n = 10). Supernatant signifi- LTC4 in response to various stimuli incant amounts of PGE 2 , PGF2a , TXB 2 , cantly increased the contractile respons- cluding A23187 (25, 26), platelet activatand 6-keto-PGF 1a (p < 0.01). Pretreat- es to histamine with (157.3 ± 9.3% of ing factor (27), and IgG-coated Sephment with indomethacin significantly control, p < 0.01, n = 5) and without arose beads (6). Of these, A23187 is the inhibited release of all cyclooxygenase epithelium (153.6 ± 10.2% of control, most potent stimulant producing LTC4 products. However, AA 861 did not sig- p < 0.01, n = 5) and the percentage in- (6,25,26). The concentration of A23187 nificantly inhibit release of any cyclo- crease did not differ significantly with used in the present study (1 ug/ml) was the maximum concentration producing and without epithelium (p > 0.50). oxygenase products (table 1). LTC4 without affecting cell viability (27, 28). The processing of LTC4 to LTD4 and LTE4 by peptide cleavage represents bioTABLE 1 conversion from one active mediator to EFFECTS OF INDOMETHACIN AND AA 861 ON RELEASE OF CYCLOOXYGENASE another and not a catabolic activation PRODUCTS IN THE SUPERNATANT process (29). These bioconversions are 6-keto-PGF 1u PGF2u TXB 2 PGE 2 mediated by y-glutamyl trans peptidase (pg/ml) (pg/ml) (pg/ml) (pg/ml) and dipeptidases in the cell membrane Without (30). Furthermore, eosinophil peroxidase 460 ± 46 1,177 ± 167 306 ± 87 inhibitors 1,806 ± 776 can degrade LTC4 and LTD4 (31). ThereIndomethacin fore, LTD4 and LTE4 are likely to be the 540 ± 130t (10- 5 M) 61 ± 17* 3 ± 1* 39 ± 15* metabolized products in the present AA 861 1,501 ± 55 266 + 100 (10- 5 M) 392 ± 21 882 ± 87 supernatant. LTC4 , LTD4 , and LTE4 are potent conData are reported as mean ± SE of supernatants (10 cells) obtained from three guinea pigs. Significant differences from results without inhibitors are indicated by * P < 0.01 and t p < 0.05. tractile agonists for guinea pig tracheal

1000

180

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EOSINOPHIL AND AIRWAY SMOOTH MUSCLE INTERACTION

smooth muscle, with a molar ratio of the amount eliciting equivalent concentrations of 1:1 :0.1, respectively (32). In this study, we did not examine the relative contributions of LTC4 , LTD4 , and LTE4 to the contractile response to histamine. LTE4 is reported to potentiate the contractile response to histamine in guinea pig tracheal smooth muscle, whereas LTC4 and LTD4 do not (32). However, LTE4 induced potentiating responses to histamine were observed at a concentration of 10nM, which was 10times higher than that in the supernatant (107 cells). Therefore, LTs in the supernatant might potentiate the contractile response to histamine by synergistic action of the LTs on tracheal smooth muscle. A recent study showed that major basic protein (MBP) from human eosinophils potentiated the contractile response to acetylcholine and histamine without changing baseline tension in guinea pig trachea (33). The study suggested that MBP causes hyperresponsiveness of airway smooth muscle by inhibiting epithelial function (e.g., epithelial-derived relaxing factor). However, it is unlikely that MBP was responsible for the supernatant- induced potentiating response to histamine for the following reasons: the effects of MBP appeared 5 h after incubating tissues with MBP and were totally dependent on the presence of the epithelium. However, in the present study epithelial removal did not alter supernatant-induced potentiating response to histamine, and effects of the supernatants reached maximum 5 min after incubating tissues with supernatant. Furthermore, eosinophils stimulated with 1 ug/ ml of A23187 did not release eosinophilderived neurotoxin (34). Because MBP coexists with eosinophil-derived neurotoxin in granules (9, 34), it is unlikely that the supernatant used in the present study contained significant amounts of MBP. Thus, it seems that different substances produced by eosinophils may alter guinea pig airway smooth muscle responsiveness in at least two different ways: MBP may potentiate smooth muscle response by causing epithelial dysfunction (33), and LTs may cause changes in contractile responses of airway smooth muscle. Acknowledgment We thank Ms. R. Haryu for preparing the

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