Effects of inhibitors of arachidonic acid metabolism on serotonin release from rat basophilic leukemia cells

Immunopharmacology, 25 (1993) 131-144 Elsevier Science Publishers B.V.

131

I M P H A R 00630

Effects of inhibitors of arachidonic acid metabolism on serotonin release from rat basophilic leukemia cells Yasushi Igarashi a, Jens D. Lundgren b, James H. Shelhamer b, Michael A. Kaliner a and M a r t h a V. White a,1 aAllergic Diseases Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, and b Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA (Received 17 January 1992; accepted 26 August 1992)

Abstract: Mast cells can release arachidonic acid (AA) metabolites as well as preformed mediators with IgE mediated stimulation, and these mediators are considered to play an important role in allergic reactions. The coincident release of preformed mediators and AA metabolites suggests that AAmetabolism is related to mast cell degranulation. To clarify the relationship between mast cell degranulation and AA metabolism, the effects of various AA cascade inhibitors on rat basophilic leukemia cell (RBL) mediator release induced by either anti-IgE or A23187 were examined. 5,8,11,14-eicosatetraynoic acid (ETYA) inhibited both PGD 2 and LTCa/D 4 generation, and partially inhibited serotonin release. Nordihydroguaiaretic acid (NDGA) caused complete inhibition of LTCa/D 4 generation, and partial inhibition of PGD 2 generation and serotonin release. The cyclooxygenase inhibitor, indomethacin, and the specific 5-1ipoxygenase inhibitor, L-651,392 completely inhibited PGD 2 and LTC4/D4 generation, respectively, without affecting release of other mediators. Both PGD 2 and LTCa/D 4 generation were abolished by the combination of indomethacin and L-651,392, however, serotonin release remained intact. HPLC analysis showed that no shift to other AA metabolites occurred after the treatment with these inhibitors. Mepacrine, a phospholipase A 2 inhibitor, completely inhibited PGD 2 and LTCa/D 4 generation, as well as AArelease itself, without affecting serotonin release. Therefore, neither AA metabolism nor AA release is necessary for RBL degranulation. Key words:

Mast cell; Mediator release; Cyclooxygenase; Lipoxygenase

Introduction Correspondence to: M. White, Allergic Diseases Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bldg. 10, Room 11C207, 9000 Rockville Pike, Bethesda, MD 20892, USA. Abbreviations: RBL, rat basophilic leukemia cells; AA, arachidonic acid; PG, prostaglandin; LT, leukotriene; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; RP-HPLC, reverse-phase high performance liquid chromatography; RIA, radioimmunoassay; ODS, octadecylsilane-silica; UV, ultraviolet; OD, optical density; MEM, minimal essential media; HBSS, Hanks' balanced

Mast cells/basophils play an important role in inflammation (Marone et al., 1989). In immediate

salt solution; HEPES, N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid; LDH, lactate dehydrogenase; DMSO, dimethyl sulfoxide; TFA, trifluoroacetic acid; ETYA, 5,8,11,14-eicosatetraynoic acid; NDGA, nordihydroguaiaretic acid; IC50, 50% inhibitory concentration. 1 Recipient of the Merrell Dow Scholar in Allergy Award.

132 allergic reactions, the interaction between antigen and IgE-antibody attached on the surface of these cells results in the release and generation of various bioactive mediators, which evoke allergic symptoms (Ishizaka, 1981, rat serosal mast cell). The mechanisms involved in mast cell mediator release are being elucidated, and involve Ca 2+ influx (Ishizaka et al., 1980, rat serosal mast cell), and the activation of phospholipase A2 (Kennerly et al., 1979, rat serosal mast cell), phospholipase C (Cunha-Melo et al., 1989, rat basophilic leukemia cell (RBL-2H3)), phospholipase D (Gruchalla et al., 1990, rat serosal mast cell) and protein kinase C (Warner et al., 1989a, human basophil). Further elucidation of the relationships among these biochemical phenomena may help clarify the complicated mechanism of mast cell mediator release. Stimulated mast cells release both preformed mediators and newly generated mediators such as arachidonic acid (AA) metabolites (eicosanoids). Although some mast cell secretagogues induce only preformed mediator release (Levi-Schaffer et al., 1989, rat serosal mast cell; Warner et al., 1989b, human basophil; Eggleston et al., 1990, human basophil and lung mast cell; Igarashi et al., 1992, RBL-2H3), both degranulation and eicosanoids generation occur simultaneously following antigen challenge in vivo (Murray et al., 1986; Creticos et al., 1984; Naclerio et al., 1985, human airways) and in vitro (Lewis et al., 1982, rat serosal and human lung mast cell; Peters et al., 1984, human lung mast cell; Heavey et al., 1988, rat intestinal mast cell; Razin et al., 1982, mouse bone marrow-derived mast cell). It is not clear whether eicosanoid generation is necessary for immunologically mediated degranulation. McGivney et al. (1981)reported that inhibitors of phospholipase A2 inhibited histamine release as well as AA liberation from RBL-2H3. The same authors observed that indomethacin blocks P G D 2 generation without affecting histamine release, but that both PGD2 generation and histamine release were inhibited by ETYA, which also block the 5-1ipoxygenase pathway. Others observed that the irreversible 5-1ipoxygenase inhibitor 5,6-dehydroarachidonic

acid, inhibits the formation of 5-HETE without appreciably affecting mast cell degranulation (Razin et al. 1984, mouse bone marrow-derived mast cell). The latter study suffered from the use of a 5-1ipoxygenase inhibitor that also had nonspecific effects on cell function. Further, both of those studies examined the effect on degranulation of blocking only one pathway of AA metabolism. Recently, newer more specific inhibitors of the 5-1ipoxygenase pathway have become available. Therefore, to clarify the relationship between mast cell degranulation and the generation and subsequent metabolism of AA, the effect of various inhibitors of AA metabolism on mediator release from RBL-2H3, a cultured cell line felt to most closely resemble mucosal mast cells (Seldin et al., 1985), was examined.

Materials and Methods Materials

Hanks' balanced salt solution (HB S S) (GIBCO, Grand Island, NY); Eagle's minimum essential medium with Earle's balanced salt (EMEM) without L-glutamine, heat-inactivated fetal calf serum, L-glutamine, Trypsin/VerseneT M solution (in HBSS) (Biofluids Inc., Rockville, MD]; calcium ionophore A23187 (A23187), dimethyl sulfoxide (DMSO), indomethacin, nordihydroguaiaretic acid (NDGA), mepacrine, fatty acid free bovine serum albumin (BSA) (Sigma Chemical Co., St. Louis, MO); 5,8,11,14-eicosatetraynoic acid (ETYA) (Calbiochem Co., San Diego, CA); 5-(1,2-3H(N))-hydroxytryptamine binoxalate ([3H]serotonin), [14,15-3H(N)]-LTB4, [14,153H(N)]-LTC4 (New England Nuclear, Boston, MA); [ 1-14C]arachidonic acid, prostaglandin D2 [3H] assay system, (Amersham Co., Arlington Heights, IL); LTC 4 standard, L T C 4 / D 4 antibody (Advanced Magnetics Inc., Cambridge, MA) were purchased from the manufacturers. RBL (Dr. Reuben Siraganian, NIDR) and 4-bromo-2,7-dimethoxy-3H-phenothiazine-3-one (L-651,392) [Dr. Joshua Rokach, Merck Frosst Canada Inc.] were each generous gifts.

133 R B L cell culture The 2H3 subline of RBL was maintained in 175-cm a tissue culture flasks in E M E M supplemented with 4 mM glutamine and 15~o heatinactivated fetal calf serum (Barsumian et al., 1981). The cells were treated with Trypsin/Versene T M solution, resuspended in medium, passaged every 3-7 days, and maintained in a 5 ~o CO2 humidified atmosphere at 37 ° C. Radiolabeling of R B L Degranulation of RBL was measured by~o release of radiolabeled serotonin. For serotonin release experiments, 2 x 105 RBL/ml were radiolabeled overnight with culture medium containing [3H]serotonin (1 #Ci/ml). For AA release experiments or AA metabolites determination by highperformance liquid-chromatography (HPLC), RBL were radiolabeled with [1-14C]arachidonic acid (0.05 /zCi/ml) overnight. Unlabeled cells were used for radioimmunoassay (RIA) of PGD2 and L T C 4 / D 4. Mediator release assay 5 × 104 RBL/well were placed in a 48-well culture plate and allowed to adhere overnight. The wells were washed three times with 4 °C Assay Buffer which consists of HBSS with 3 mM calcium (1 m M free calcium as determined with a calcium electrode), 2 m M magnesium, 20 mM HEPES, and 0.1 ~o gelatin pH 7.4. Cells were triggered with 90 /~1 anti-IgE (raised in rabbit; 1:1000 dilution in Assay Buffer) or A23187 (0.3/~g/ml). In experiments employing anti-IgE, RBL were sensitized with IgE IR-162 (raised in rat; 1 /~g/104 cells) in culture medium for 1 h before starting the release assay. A23187 (1 mg/ml stock solution in D M S O ) was diluted in Assay Buffer prior to use. In AA release assays, gelatin in the Assay Buffer was replaced with fatty acid free BSA. After a 30 min incubation with secretagogues, reactions were stopped by the addition of 200 #1 of 4 °C Assay Buffer to each well. The supernatants were immediately centrifuged at 9000 x g for 2 min to remove unattached/detached cells, and the supernatants assayed for

mediator release. The adherent cells were lysed by incubation at 37 °C with HBSS containing 1 ~o Triton X-100, scraped out from the experimental wells, and used for total cell radioactivity counts. All release assays were performed in duplicate, and the results were averaged. For statistical analyses, the averaged values were treated as single data. Serotonin and AA were assayed by radioactive counting of 3H and ~4C, respectively, and~o release (ratio of counts in supernatant and counts in cells plus supernatant corrected for background counts) calculated. In experiments for HPLC, 1 x 107 RBL were incubated at 37 °C overnight in a 175 cm 2 tissue culture flask, and the release assay was performed as described above except that RBL were triggered with 10 ml of 0.3/~g/ml A23187. If not used immediately, samples were stored at -70 ° C until assayed. All results are expressed as net release (~o release from experimental cells minus ~o release from cells incubated with Assay Buffer) unless otherwise stated. Control serotonin and AA release was about 5~o and 1~o, respectively, regardless of the presence of inhibitors. RIA measurement for AA metabolites PGD2 and LTCn/D4 released into the supernatants were measured by a competitive RIA using commercially available reagents (Granstr~3m et al., 1978). Cross-reactivity of the P G D 2 antibody used in this study was 7 ~o with PGJ2 and less than 1 To with other major PG's and thromboxane. Cross-reactivity of the LTC4/D 4 antibody for L T C 4 and L T D 4 w a s 64~o and 100~o, respectively. Thus the amount of LTC4/D4 was expressed as LTC4 equivalents. Anti-IgE, A23187, and D M S O (<0.1~o) did not affect these RIA's. Lactate dehydrogenase (LDH) assay LDH in the supernatants and cells was assayed by measuring the consumption of N A D H during the reduction of pyruvate to lactate (Bergmeyer et al., 1965). The results, measured as the change in O D 3 4 o per min, were expressed as the~o of total cell LDH released.

134

Mediator release assay with inhibitors In some experiments, RBL were preincubated in Assay Buffer with or without various inhibitors of AA metabolism for 5 min at 3 7 ° C prior to exposure to anti-IgE or A23187 premixed with the same inhibitors, and mediator release was measured as described above. In these experiments, net release was corrected for that caused by inhibitors alone. In the concentrations used, none of the inhibitors significantly affected the spontaneous RBL mediator releases, except indomethacin, which reduced spontaneous PGD2 generations. ETYA was solubilized in ethanol. Because the concentration of ethanol present in the highest concentration of ETYA affected mediator release from stimulated RBL, results obtained with that dose of ETYA were corrected for the effects of ethanol alone. Other inhibitors were solubilized in D M S O , and used at 1000-fold or more dilutions, without any effect on mediator release. None of the inhibitors themselves nor their solvents affected the RIA. Sample preparation for HPLC Before assay by HPLC, samples were applied to Sep-Pak-C ~8 cartridges employing a modification of the method described by Verhagen (1986). Cartridges were prewashed with 15 ml of methanol, 5 ml of 5 mM ethylenediaminetetraacetic acid dipotassium (EDTA K2), and 10 ml of distilled water. Following sample application, the cartridges were washed with 10 ml of distilled water, and samples were extracted with 3 ml methanol, dried under nitrogen, and resuspended in 120 #1 of Solvent A [75 ~o(V/V) distilled water, 25% (V/V) acetonitrile, and 2.2 mM phosphoric acid]. HPLC separation of AA metabolites Reverse-phase-HPLC ( R P - H P L C ) analysis was performed using a method modified from Powell (1985). Briefly, after samples were applied to an octadecylsilane-silica (ODS) column (Ultrasphere 5 gm particle diameter, 0.46 x 25 cm), the column was washed with a mixture of Solvent A and Solvent B [60~o (v/v) methanol, 40~o(v/v)

acetonitrile, and 0.21 mM TFA] at a flow rate of 1.5 ml/min, using a combination of linear gradients shown in Figure 6A. Fractions were collected every 30 s, and the radioactivity in each fraction was counted. H P L C grade reagents were employed for all experiments.

Statistics The dose of inhibitor causing a 50~o reduction in mediator release (ICso) was estimated by linear interpolation between the two responses bracketing the 50~o point. Doses of the inhibitors, N D G A , indomethacin, and L-651,392 were transformed to logarithms since the data interpolation was linear in the logarithmic scale, whereas ETYA levels were expressed in the original scale. Percents were not transformed because it was found that, with one exception (anti-IgE stimulat i o n / N D G A treatment), sets of percentages were normally distributed. Normality was checked using the Shapiro-Wilk test included in the P R O C UNIVAR1ATE procedure in the Statistical Analysis System (SAS) library of programs. Mean ICs0's were based on ICs0's estimated from individual experiments so that the corresponding standard errors reflect experiment variability. Unbiased estimates of the (population) standard errors, including the correction for small numbers (N-I), were used. For the inhibitors, ETYA and N D G A , the ICso's associated with the three mediators (serotonin, PGD2, LTC4/D4) were compared in a one way analysis of variance using Tukey's multiple comparison test. Since data for the remaining inhibitors, indomethacin and L-651,392, included lower bounds on ICs0's (rather than actual values), a less formal method of comparison (confidence interval overlap) was used.

Results

RBL, stimulated with anti-IgE or A23187, release the AA metabolites, P G D 2 and LTC4, as well as serotonin (Igarashi et al., 1992). In order to determine whether AA metabolism could be

135 inhibited independently from serotonin release, we examined the effect on RBL mediator release of various agents known to inhibit AA metabolism. RBL were preincubated for 5 min with buffer or inhibitors of AA metabolism, then exposed to either anti-IgE or A23187, and the release of serotonin, PGD2, and L T C 4 / D 4 was measured.

droguaiaretic acid (NDGA), a known lipoxygenase inhibitor (Levine, 1983), was next examined (Fig. 2). NDGA (0.1-10 #g/ml) induced dose

(A) 100 t

'~ W

Effects of non-selective A A inhibitors on R B L mediator release

The effect on RBL mediator release of 5,8,11,14eicosatetraynoic acid (ETYA), an acetylenic fatty acid structurally similar to arachidonic acid, which inhibits both cyclooxygenase and lipoxygenase activity (Ktihn et al., 1984; Ahern et al., 1970) was first examined (Fig. 1). In 3 experiments, anti-IgE induced 33.5~o serotonin release and the generation of 35.5 pmol P G D 2 / 1 0 6 RBL and 70 pmol LTC4/D4/10 6 RBL, while A23187 caused 32.4~o serotonin release and the generation of 61.5 pmol PGD2/106 RBL and 123.4 pmol LTCa/D4/10 6 RBL. Preincubation of RBL with 1.35-13.5 #M ETYA for 5 min caused dose-dependent inhibition of both PGD2 and L T C 4 / D 4 generation as well as inhibition of serotonin release from RBL stimulated with either anti-IgE or A23187. 13.5 #M ETYA caused 35~o, 98~o, and 65~o inhibition of anti-IgE induced release of serotonin, PGD2, and LTCg/D4, respectively. In contrast, ETYA was a more potent inhibitor of A23187 induced L T C 4 / D 4 generation and serotonin release, since 13.5 ~M ETYA caused 75 ~o, 99 ~o, and 95 ~/o inhibition of A23187 induced serotonin release, and PGD2 and L T C 4 / D 4 generation, respectively. Thus, ETYA effectively inhibits both AA metabolism and serotonin release. In these experiments, complete inhibition of PGD2 generation was achieved along with partial inhibition of L T C 4 / D 4 generation and minimal inhibition of serotonin release. To determine whether it was possible to selectively inhibit the lipoxygenase pathway without affecting either the cyclooxygenase pathway or serotonin release, the effect on RBL mediator release of nordihy-

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[ETYA(pM) Fig. 1. Inhibition of RBL serotonin release (circles) P G D 2 generation (triangles), and LTC4/D 4 generation (squares) by ETYA. 5 x 104 RBL were preincubated with various concentrations of ETYA for 5 min at 37 °C and stimulated with (A) anti-IgE (1:1000 dilution) or (B) A23187 (0.3 /~g/ml) for 30 min at 37 °C in the continued presence of ETYA. Net release was calculated by correcting for spontaneous release. In the absence of ETYA (control release), antiIgE induced 33.5_+5.2~ serotonin release, and the generation of 35.5 _+4.1 pmol/106 cell P G D 2 and 70.2 + 12.1 pmol/106 cell LTC4/D 4. A23187 induced 32.4__ 7.2% serotonin release, and the generation of 61.5 + 0.8 pmol/106 cell P G D 2 and 123.44_+ 17.85 pmol/106 cell LTC4/D 4. Mediator release obtained in the presence of ETYA is presented as net% control release• Data are expressed as mean + SEM of three different experiments.

136

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Fig. 2. Inhibition of RBL serotonin release (circles), PGD 2 generation (triangles), and L T C 4 / D 4 generation (squares) by NDGA. 5 x 10 4 RBL were preincubated with various concentrations of NDGA for 5 min at 37 °C, and stimulatedwith (A) anti-IgE (1:1000 dilution) or (B) A23187 (0.3 #g/ml) for 30 min at 37 °C in the continued presence of NDGA. In the absence of NDGA, anti-IgE induced 33.5 + 5.27o serotonin release, and the generation of 35.5 _+4.1 pmol/106 cell PGD 2 and 70.2+ 12.1 pmol/106 cell LTC4/D4. A23187 induced 34.3_+5.5% serotonin release, and the generation of 65.0 _+3.6 pmol/106 cell PGD 2 and 100.9 _+25.8 pmol/106 cell LTC4/D4. Mediator release obtained in the presence of NDGA is presented as net% control release. Data are expressed as mean+ SEM of three (anti-IgE) and four (A23187) different experiments.

related inhibition of anti-IgE and A23187 induced release of serotonin, PGD2, and LTC4/D 4. At 1/~g/ml, however, N D G A caused 97 and 67~o inhibition of anti-IgE and A23187 induced

LTC4/D~ generation, respectively, but only 37 and 41~/o inhibition of anti-IgE and A23187 induced serotonin release, respectively, and 39 and 17Yo inhibition of anti-IgE and A23187 induced generation of PGD2, respectively. The ICso for ETYA inhibition of IgE induced generation of either P G D 2 or LTC4/D 4 is significantly lower than that for serotonin release (Tukey's multiple comparison test, p < 0.05 and p < 0 . 0 1 , respectively; Table I). Likewise, the ICs0 for N D G A inhibition of LTC4/D 4 generation is significantly lower than that of PGD2 or serotonin release (Tukey's multiple comparison test, p < 0 . 0 1 ; Table I). However, at no dose of ETYA or N D G A could complete inhibition of PGD2 or LTC4/D 4 generation be achieved without concomitant inhibition of serotonin release, and both ETYA and N D G A when used in high doses are inhibitors of both the cyclooxygenase and lipoxygenase pathways, since complete inhibition of one pathway cannot be accomplished without partial inhibition of the other pathway.

Effects of selective AA inhibitors on R B L mediator release Although ETYA and N D G A partially inhibited serotonin release, the inhibitory effects of these agents on serotonin release could be unrelated to their effects on AA metabolism. Therefore the effects on R B L mediator release of the specific cyclooxygenase inhibitor, indomethacin (Levine, 1983), and the specific 5-1ipoxygenase inhibitor, L-651,392 (Ouindon etal., 1987), were examined. Indomethacin 0.001-0.1 #g/ml caused dose-related inhibition of both anti-IgE and A23187 induced P G D 2 generation with only minimal effects on serotonin release or LTC4/D 4 generation (Fig. 3). Indomethacin 0.1 #g/ml caused 100 and 90~o inhibition of anti-IgE and A23187 induced R B L P G D 2 generation, and less than 15~o inhibition of serotonin release and LTC4/D 4 generation. Moreover, indomethacin selectively inhibited the cyclooxygenase pathway, since it was over 100-fold more potent in inhibiting P G D 2 generation as compared with either serotonin release or LTC4/D 4 generation (99°~,

137 TABLE I ICso of inhibitors on RBL mediator release Stimulant

anti-lgE (1:1000)

Mediator

Serotonin PGD 2

LTC4/D 4

A23187 (0.3 #g/ml)

Serotonin PGD 2 LTC4/D4

ICso of inhibitor ETYA (/zM)

NDGA (/~g/ml)

Indomethacin (#g/ml)

L-651,392 ~M)

> 13.5 2.15"* (_+1.16)

10.0 (×/+ 1.79 (x/+

> 0.1

>1

0.001* ( x / + 1.55)

0.44 ( x / + 1.26)

6.06* (_+1.86)

0.08** ( x / - 1.53)

> 0.1

0.01 t (x/+

6.09 ( + 2.37) 2.34 (+0.63) 3.63 (_+0.66)

> 10

> 0.1

> 1

0.004 t ( x / + 1.12) > 0.1

> 1

4.14 (x/+ 0.60 (x/+

1.77) 1.27)

1.35) 1.79)

0.53 (x/-

1.75)

1.98)

ICsowas determined from the individual experiment. Shown are the mean (ETYA) or the geometric mean (NDGA, indomethacin, or L-651,392) of 3-5 separate experiments. In the case in which 50~o inhibition could not be achieved with the concentrations tested, values were expressed as greater than ( > ) the highest concentration. Values in parentheses represent the standard error of the mean (ETYA) or the relative standard error of the geometric mean (NDGA, indomethacin, or L-651,392). Symbols indicate significant differences as compared with the inhibitory effect on serotonin release (Tukey's multiple comparison test, *p < 0.05, **p < 0.01; Confidence interval overlap, t = 99~o confidence level).

confidence interval overlap; Table I). Thus, RBL serotonin release can proceed virtually unaffected by the complete inhibition of PGD2 generation. To determine whether serotonin release could also be dissociated from the 5-1ipoxygenase pathway, a specific 5-1ipoxygenase inhibitor, L-651, 392, was employed. L-651,392 was a more potent inhibitor of anti-IgE induced L T C 4 / D 4 generation as compared with A23187 induced generation (Fig. 4). At 1 0 - 7 M , L-651,392 induced almost complete inhibition of LTC4/D 4 generation with minimal effect on PGD2 generation and no significant effect on serotonin release. Thus, L-651,392, 1 0 - 7 M , caused 92 and 19~o inhibition of anti-IgE induced L T C 4 / D 4 and PGD2 generation, respectively, while serotonin release remained intact. The ICso for L T C 4 / D 4 generation was significantly lower than that for either P G D 2 o r serotonin release (99~o confidence interval overlap; Table I). Likewise, L-651,392,

1 0 - 6 M , caused 71~o inhibition of A23187 induced L T C 4 / D 4 generation without significantly inhibiting PGD2 generation or serotonin release. Thus, RBL serotonin release can proceed despite almost complete inhibition of L T C 4 / D 4 generation. In the experiments employing indomethacin and L-651,392, PGD2 o r L T C 4 / D 4 generation could be specifically prevented without affecting serotonin release. To determine whether it is possible to simultaneously inhibit both the cyclooxygenase pathway and the 5-1ipoxygenase pathway without affecting serotonin release, the effect of preincubation with both indomethacin and L-651,392 on stimulated RBL mediator release was examined (Fig. 5). The combination of either 0.01 /~g/ml indomethacin plus 10-TM L-651,392 or 0.1 /~g/ml indomethacin plus 1 0 - 6 M L-651,392 caused inhibition of both antiIgE induced P G D 2 and L T C 4 / D 4 generation

138 with minimal effects on serotonin release. At the lower dose tested, PGD2, LTC4/D4, and serotonin release were inhibited 98, 95, and 8 ~o, respectively. Indomethacin 0.1 #g/ml + L-651,392

(A)

10-6M also inhibited A23187induced PGD2 (81 ~o) and LTC4/D 4 (70 ~o) without significant effect on serotonin release (8 ~o inhibition). Thus, the combination of indomethacin and L-651,392

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[INDOMETHAClN (pg/ml)] Fig. 3. Inhibition of RBL serotonin release (circles), P G D 2 generation (triangles), and LTC4/D 4 generation (squares) by indomethacin. 5 x 104 RBL were preincubated with various concentrations of indomethacin for 5 min at 37 °C, and stimulated with (A) anti-lgE ( h 1000 dilution) or (B) A23187 (0.3/~g/ml) for 30 min at 37 °C in the continued presence of indomethacin. In the absence of indomethacin, anti-lgE induced 31.1 ± 4.1% serotonin release, and the generation of 23.2 + 6.6 pmol/106 cell P G D 2 and 36.4 + 11.0 pmol/106 cell LTC4/D 4. A23187 induced 35.7+ 3.5% serotonin release, and the generation of 54.7+4.8 pmol/106 cell P G D 2 and 106.6 ± 22.4 pmol/106 cell LTC4/D 4. Mediator release obtained in the presence of indomethacin is presented as net% control release. Data are expressed as mean _+SEM of five (anti-IgE) or four (A23187) different experiments•

0

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[L-651,392 (M)] Fig. 4. Inhibition of RBL serotonin release (circles), P G D 2 generation (triangles), and LTC4/D 4 generation (squares) by L-651,392. 5 x 104 RBL were preincubated with various concentrations of L-651,392 for 5 rain at 37 °C, and stimulated with (A) anti-IgE (1:1000 dilution) or (B) A23187 (0.3 #g/ml) for 30 min at 37°C in the continued presence of L651,392. In the absence of L-651,392, anti-IgE induced 26.0+3.5% serotonin release, and the generation of 15.9+_4.8 pmol/106 cell P G D 2 and 18.2+- 9.1 pmol/106 cell LTC4/D 4. A23187 induced 45.3 +7.6% serotonin release, and the generation of 59.2 ± 11.6 pmol/106 cell P G D 2 and 74.7+- 11.4 pmol/106 cell LTC4/D 4. Mediator release obtained in the presence of L-651,392 is presented as net% control release• Data are expressed as mean +- SEM of four different experiments.

139

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Fig. 5. Inhibition of RBL serotonin release (circles), P G D 2 generation (triangles), and LTC4/D 4 generation (squares) by the combination of indomethacin and L-651,392. 5 x 104 RBL were preincubated with various concentrations of L-651,392 for 5 rain at 37°C, and stimulated with (A) anti-IgE (1:1000 dilution) or (B) A23187 (0.3 /~g/ml) for 30 rain at 37 °C in the continued presence of indomethacin and L-651,392. In the absence of indomethacin and L-651,392, anti-lgE induced 26.3 _+4.9~o serotonin release, and the generation of 15.1 + 6.7 pmol/106 cell P G D 2 and 21.8 + 11.7 pmol/106 cell LTC4/D 4. A23187 induced 36.0 + 3.1 ~o serotonin release, and the generation of 49.5 + 4.6 pmol/106 cell P G D 2 and 65.8 + 16.4 pmol/106 cell LTC4/D 4. Mediator release obtained in the presence of indomethacin and L-651,392 is presented as net~o control release. Data are expressed as mean _+SEM of three different experiments.

permits RBL serotonin release in the virtual absence of PGD z and LTC4/D 4 generation. In order to rule out the possibility that these inhibitors shifted AA metabolism to the production of metabolites other than PGD2 and

LTC4/D4, RBL were cultured overnight with [t4C]AA, and aliquots were preincubated with various inhibitors of AA metabolism prior to stimulation with A23187. The supernatants were then fractionated by RP-HPLC and free AA and its metabolites identified (Fig. 6). In the absence of inhibitors, A23187 stimulated RBL released PGD2, LTB4, LTC 4, and 5-HETE. Indomethacin caused selective inhibition of radioactivity co-eluting with the PGD2 standard, while N D G A and L-651,392 selectively inhibited the release of radioactive products co-eluting with the 5-1ipoxygenase products. The dose of N D G A used in these experiments was 1 #g, which is well below the ICs0 for inhibition of the cyclooxygenase pathway at this dose of NDGA. The inhibition of radioactive 5-1ipoxygenase products by N D G A was nearly complete. Both ETYA and the combination of indomethacin plus L-651,392 caused inhibition of both cyclooxygenase and 5-1ipoxygenase products. In no instance was enhancement of any AAmetabolite, with the possible exception of AA itself, noted. In these studies, however, complete inhibition of AA metabolism could not be demonstrated by any single inhibitor or combination of inhibitors.

Effect of

AA release inhibitor on R B L

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To confirm whether serotonin release could proceed without any AA generation and/or metabolism, the effect of mepacrine, an inhibitor of phospholipase A2 (Flower et al., 1975; Markus et al., 1969), was studied. Mepacrine, 10 5-10-4 M, dose-dependently inhibited anti-IgE-induced generation of PGD2 and LTC4/D4, with complete inhibition of PGD 2 and LTC4/D4 generation achieved by 10-aM mepacrine, without appreciable effects on serotonin release (Fig. 7A). To confirm that mepacrine was actually inhibiting the generation of AA, RBL were cultured overnight with [14C]AA. Aliquots of [14C]AAlabeled RBL were then preincubated with mepacrine or buffer prior to stimulation with anti-IgE and 14C release determined. Since the AA was labeled with 14C in the first carbon position,

140 (A) CONTROL

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Fig. 6. HPLC analysis of RBL eicosanoid release. RBL were allowed to incorporate []4C]AA (2.5 /~Ci/107 cells) overnight, preincubated for 5 rain with (A) Assay Buffer, (B) 13.5/~M ETYA, (C) 1.0/~g/ml NDGA, (D) 0.1 #g/ml indomethacin, (E) 10 6 M L-651,392, or (F) 0.1 #g/ml indomethacin + 10 -6 M L-651,392 at 37 °C, then stimulated with A23187 (0.3/~g/ml) for 30 rain. The supernatants were fractionated by RP-HPLC as described, the eluate collected every 30 s, and radioactivity counted. Shown are the results from a single HPLC run for each condition, representative of 3-4 other HPLC runs. All experiments shown were performed on the same day. Arrows show the retention times of standards. Line on graph (A) shows the gradients (Solvent A%) used in all elution.

released AA as well as its metabolites would be expected to contain 14C. Mepacrine, 10 -610-4M, caused dose-related inhibition of the release of [14C]AA/metabolites, and 10-4M mepacrine inhibited the release of [ 14C]AA/metabolites by 96 % without affecting serotonin release

(Fig. 7B). Mepacrine, 10 - 6 - 1 0 4 M, had no effect on mediator release caused by A23187 (data not shown). Thus, IgE mediated serotonin release from RBL is independent of both AA generation and its metabolism.

141 Discussion

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[MEPACRINE (M)I Fig. 7. (A) Inhibition of RBL serotonin release (circles),

PGD 2 generation (triangles), and LTC4/D4 generation (open squares) by mepacrine. 5 × 104 RBL were preincubated with various concentrations of mepacrine for 5 rain at 37 ° C, and stimulated with anti-IgE (1:1000 dilution) for 30 min at 37 ° C in the continued presence of mepacrine. In the absence of mepacrine, antilgE induced 26.4± 3.4% serotonin release, and the generation of 32.2_+ 8.0 pmol/106 cell PGD 2 and 17.4±9.0 pmol/106 cell LTC4/D ~. (B) Inhibition of RBL serotonin release (circles) and AA release (closed squares) by mepacrine. 5 x 104 RBL were preincubated with various concentrations of mepacrine for 5 min at 37 °C, and stimulated with anti-IgE ( 1:1000 dilution) for 30 min at 37 ° C in the continued presence of mepacrine. Percent serotonin release was measured by [3H]serotonin counting, and AA release was also measured by ~4C counting. In the absence of mepacrine, anti-IgE induced 28.5 ± 4.6 % net release of incorporated 13H]serotonin and 8.4 ± 0.2% net release of incorporated [ IaC]AA from RBL. Mediator release obtained in the presence of mepacrine is presented as net% control release. Data are expressed as mean _+SEM of three different experiments. At these concentrations, mepacrine did not inhibit A23187 induced mediator release, and higher concentrations (10 3M) of mepacrine were cytotoxic as assessed by LDH

Although McGivney et al. (1981) reported that mast cell histamine release required AAmetabolism, other reports show that in some conditions degranulation can occur in the absence of AA metabolism (Levi-Schaffer et al., 1989; Warner et al., 1989b; Eggleston et al., 1990; Churcher et al., 1990; Igarashi et al., 1992). To clarify the relationship between mast cell degranulation and AA metabolism, the effect of selective and nonselective inhibitors of AA metabolism on RBL mediator release was studied. In the experiments reported herein, degranulation of RBL was inferred from the non-cytotoxic release of serotonin from RBL. Evidence of degranulation was not sought by electron microscopy. However, others have shown that activation of RBL with either anti-IgE or calcium ionophore leads to degranulation (Bonifacino etal., 1986; Seldin etal., 1985). The authors feel that it is highly probable that serotonin release in these experiments was accompanied by degranulation, although it is conceivable that degranulation may not have occurred. Initially the effects of ETYA, the alkyne analog of AA, and N D G A were studied. ETYA inhibits the cyclooxygenase pathway as well as the lipoxygenase pathway (K~ihn et al., 1984), while N D G A is a lipoxygenase inhibitor with antioxidant properties (Levine, 1983). In agreement with previous reports (Shulman et al., 1989), both ETYA and N D G A caused inhibition of both PGD2 and LTC4/D4 generation, and to a lesser extent inhibited serotonin release as well. The ICs0 of ETYA (3.6 #M) and N D G A (0.6 /lg/ml) for L T C 4 / D 4 generation were comparable to those previously reported (Shuhnan et al., 1989). In these two sets of experiments, serotonin release remained mostly intact despite almost complete inhibition of the generation of either PGD2 or L T C 4 / D 4 alone, however, substantial inhibition of the generation of both PGD2 and L T C 4 / D 4 w a s accompanied by significant inhibition of serotonin release. These data suggested that AA metabolism could be required for IgE and A23187 mediated serotonin release and that

142 metabolism through either the cyclooxygenase or the 5-1ipoxygenase pathway is sufficient to allow degranulation to proceed. To test this theory, the effects of the selective cyclooxygenase inhibitor, indomethacin, and the competitive 5-1ipoxygenase inhibitor, L-651,392, on RBL mediator release were studied. Indomethacin, 0.1 #g/ml, completely inhibited IgE mediated PGD2 generation and induced 90~,, inhibition of A23187 induced PGD2 generation while leaving both serotonin release and LTC4/D 4 generation 85-90~o intact. Likewise, L-651,392, 10 7M, caused 91~o inhibition of IgE mediated LTC4/D 4 generation and 19~o inhibition of PGD2 generation but had no inhibitory effect on serotonin release. Similarly, L-651,392, 10 6M, caused 71~o inhibition of A23187 stimulated LTC4/D 4 generation without inhibiting either P G D 2 generation or serotonin release. Thus, serotonin release stimulated by either antiIgE or A23187 is unaffected by near complete inhibition of either the cyclooxygenase or the 5-1ipoxygenase pathways. If degranulation were dependent upon AA metabolism, then complete inhibition of both the 5-1ipoxygenase and the cyclooxygenase pathways should inhibit serotonin release. Thus the effects of preincubation with the combination of indomethacin and L-651,392 were studied. Indomethacin plus L-651,392 caused over 95~o inhibition of PGD2 and LTCa/D 4 generation stimulated by antiIgE and 70-80~o inhibition of PGD 2 and LTC4/D4 generation induced by A23187. In these experiments, serotonin release remained at least 90% intact. Thus, RBL serotonin release can proceed in the virtual absence of PGD 2 and LTC4/D4 generation. These results were unexpected but agreed with similar experiments performed independently on human mast cells (Falkenhein et al., 1980). It was conceivable that the inhibition of P G D 2 and LTC4/D4 production by these inhibitors could have led to enhanced production of other AA metabolites which might have supported degranulation. To investigate this possibility, [14C]AA was incorporated into RBL, the cells

were preloaded with indomethacin plus L-651, 392, and then stimulated with A23187. The supernatants were then fractionated by RP-HPLC and AA and its metabolites identified as peaks of radioactivity co-eluting with known standards. The combination of indomethacin plus L-651, 392 lead to inhibition of all 5-1ipoxygenase and cyclooxygenase products. There were no increases in any AA metabolites resulting from preincubation with indomethacin plus L-651,392. Thus, the preservation of degranulation despite near complete inhibition of PGD2 and LTC 4 generation is not due to a shift in AA metabolism towards other products, suggesting that serotonin release is not dependent upon AA metabolism. The small increase in free AA liberation noted, however, suggested that perhaps the generation of AA itself may be necessary and sufficient to support degranulation. To test this hypothesis, the effect of mepacrine, an inhibitor of phospholipase A2, on stimulated RBL serotonin and AA release as well as PGD2 and LTC4/D 4 generation was examined. Mepacrine completely inhibited the generation of PGD2 and LTC4/D 4 and inhibited the release of [14C]AA by 95°o without affecting serotonin release. These results are in contrast to those reported by McGivney et al. (1981). However in the previous study, a longer preincubation time with mepacrine was employed, and the stimulant was antigen rather than anti-IgE. The present data suggest that the cleavage of AA by phospholipase A 2 is also not necessary for IgE mediated RBL degranulation. The data obtained using [14C]AA must be interpreted with caution, since much of the radiolabeled material might be incorporated into pools unrelated to IgE or A23187 mediated stimulation of AAmetabolism, or, alternatively, unmetabolized AA may remain trapped within the RBL. Indeed, when [14C]AA-prelabeled RBL were stimulated with anti-IgE, 9o/o of total cell [14C]AA was released in the presence of buffer containing fatty acid free BSA, while only 1.5}~, was released in the presence of regular buffer (data not shown). Taken as a whole,

143 however, the data suggest that IgE mediated serotonin release occurs independent of AA generation and metabolism. The data also suggest that A23187 induced serotonin release proceeds independently of AA metabolism, although, interestingly, mepacrine did not inhibit A23187 induced release of [ 1 4 C ] A A or the generation of PGD2 and L T C a / D 4 at the doses tested, and higher doses were cytotoxic. Since A23187 is a more potent inducer of AA metabolism than anti-IgE, the failure of mepacrine to inhibit A23187 induced AA liberation and metabolism may be dose related. However, an alternative explanation is that A23187 may induce the generation of AA through additional pathways, not involving phospholipase A2.

Acknowledgements The authors thank Dr. David Alling for excellent assistance in statistical analyses.

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