Role of lipoxins A4 and B4 in the generation of arachidonic acid metabolites by rat mast cells and their effect on [3H]serotonin release

Immunology Letters, 32 (1992) 117- 124

Elsevier IMLET 01769

Role of lipoxins A4 and B4 in the generation of arachidonic acid metabolites by rat mast cells and their effect on [3H]serotonin release P i o C o n t i 1, M a r c e l l a R e a l e l, R e n a t o C. B a r b a c a n e l, M a r i a R. P a n a r a l, M a u r o B o n g r a z i o 1 a n d T h e o h a r i s C. T h e o h a r i d e s 2 IImmunology Division, Institute of ExperimentalMedicine, University ofChieti, Chieti, Italy and2Department of Pharmacology, Tufts University Medical School, Boston, MA, USA

(Received 5 September 1991; revision received 15 November 1991; accepted 20 January 1992)

1. Summary Basophils located in tissues are called mast cells and are found in connective tissue. Many different compounds are secreted from basophil granules upon appropriate stimulation. Products such as heparin, histamine, serotonin (5-hydroxytryptamine, 5-HT), and membrane-derived materials which give rise to arachidonic acid metabolites, such as prostaglandins and leukotrienes, are some of the more important compounds released by mast cells. These compounds, when released after stimulation with a variety of molecules, such as IgE, specific antigen anaphylotoxin, as well as the compound 48/80 (C48/80) or calcium ionophore A23187, cause contraction of endothelial cells and mediate

Key words: Mast cell; Serotonin; Arachidonic acid; Leukotri-

ene; Prostaglandin; Lipoxin Correspondence to: Pio Conti, Immunology Division, Inst. of Experimental Medicine, Universityof Chieti, 66100 Chieti, Italy. Abbreviations: LT, leukotriene; SRS-A, slow reacting sub-

stance of anaphylaxis; LXA4 and B4, lipoxin A4 and B4; RPMC, rat peritoneal mast ceil; [3H]5HT, [3H]serotonin; A23187, calcium ionophore; RBL, rat basophilicleukemiacells; 48/80, compound 48/80; AA, arachidonic acid; PG, prostaglandin; 5 or 15-HETE, 5 or 15S-hydroxy-5,8,11-cis-13-transeicosatetraenoic acid; DMSO, dimethylsulfoxide; RIA, radioimmunoassay; NDGA, nordihydroguaiaretic acid.

atopic or anaphylactic hypersensitivity. In this report, we study the generation of some arachidonic acid products, namely leukotrienes C4, D4, E4, and B4 and the prostaglandins D2 and E2 by rat peritoneal mast cells (RPMC), using calcium ionophore A23187 as a degranulating agonist. We have also studied the new lipoxygenase products, lipoxins A4 and B4, on R P M C secretion using C48/80 as a secretagogue. A rat basophilic leukemia cell line (RBL) was also used to compare results with RPMC. In this paper we have demonstrated that R P M C stimulated with A23187 release LTC4, LTD4, LTE4 and LTB4 and also P G D 2 but not PGE2. These results were also confirmed when RBLs were used. In addition, we have shown that mast cells pretreated with LTC4, LTD4, LTE4 or 15-HETE do not modify the release of [3H]5HT exerted by C48/80 (0.5/~g/ml) or A23187 (5/~g/ml). When LXA4 or B4 was used, mast cells were inhibited slightly (not statistically significant) from degranulating after the secretagogue treatment. However, when LXA4 or B4 was used at the highest concentration (1 /~M) for 15 min, followed by the addition of the secretagogue for I0 min, a little but significant inhibition was found. In this report, we show that the generation of LTC4, D4 and E4 by mast cells certainly has no autocrine influence, since we found a non-significant effect of these lipoxygenase products on mast cell secretion. However, since LTB4 is generated by R P M C and other cells [l 1], it is possible that mast cells together with neu-

0165- 2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved

1 17

trophils participate in the increasing leukocyte adhesion to endothelium in small venules. We report for the first time the influence of LXA4 and B4 on mast cell secretion. The results are very inconsistent, however, evidencing a slight inhibitory potential of these two 15-HETE derivatives on mast cell secretion.

cells using as an agonist the classical secretagogue, the polycationic compound 48/80 (C 48/80) and calcium ionophore A23187 as a secretagogue for rat basophilic leukemia (RBL) cells. In addition, we have determined the generation of some arachidonic acid products by rat mast cells such as LTC4, LTD4, LTE4, LTB4, and PGD2 and PGE2.

2. Introduction

3. Materials and Methods

Mast cells are cells which are found in a variety of tissues and organs and store many mediators which are secreted upon appropriate stimulation, such as cross-linkage of the Fcz receptor [1]. In fact, after cross-linkage, mast cells secrete biologically active substances, such as histamine and arachidonic acid metabolites [2, 18]. Recent studies confirm that rat mast cells synthesize, as a dominant product, prostaglandin D2, which in some pathophysiological phenomena may be as much as or more than histamines or serotonin [2, 16]. Moreover, mast cells generate slow-reacting substance of anaphylaxis (SRSoA), i.e., leukotrienes. It is well known that SRS-A is composed of three distinct sulfidopeptide leukotrienes, a major constituent being LTC4, from which the other two molecules of the series, LTD4 and LTE4 are produced by enzymatic depredation. These leukotrienes form one major class of products of the 5-1ipoxygenase pathway of arachidonic acid metabolism which exhibit reactive and spasmogenic activities. Recently, other new compounds were additionally generated from arachidonic acid, lipoxins A4 (5,6,15-trihydroxy-7,9,11,13-eicosatetraenoic acid) and LXB4 (5D, 14,15-trihydroxy-6,8,10,12-eicosatetraenoic acid) (LXA4, LXB4). These compounds have proven to have different activities from those of other eicosanoids, such as smooth muscle contraction, thromboxane stimulation, LTB4 inhibition and down-regulation of natural killer cell activity [ 3 - 5]. With these observations in mind and considering the fact that certain arachidonic acid metabolites are involved in the delicate hypersensitivity reaction and histamine release, it is possible that arachidonic acid metabolites can affect rat peritoneal mast cell (RPMC) degranulation in vitro. In this study we have endeavoured to determine the influence of arachidonic acid products in releasing [3H]serotonin ([3H]5HT) by rat peritoneal mast

Rat peritoneal mast cells were obtained from peritoneal cavity lavage of male Sprague-Dawley rats, approximately 350 g in weight, in Hepes-buffered Locke's solution, pH 7.2 (150 mM NaC1/5 mM KCI/5 mM Hepes/2 mM CaC12/1 g dextrose 1-1/ 1 g bovine serum albumin l - 1, pH 7.2). Cells were purified ( > 9 0 % p u r i t y ) o v e r 22.5% metrizamide [15] and resuspended in the same buffer. The cells were loaded with [3H]serotonin ( 1 5 - 30 Ci/mmol, New England Nuclear, Boston, MA) for 1 h at 37°C, Were washed twice and were then resuspended in the indicated buffer (4 × 105/ml) in tubes (Falcon 2063) with a total sample volume of 0.5 ml/ tube and preincubated for 15 min with the compounds to be tested at 37 °C and then adding the classic secretagogue 48/80 or calcium ionophore A23187 for 10 min. At the end of the incubation, the cells were pelleted by centrifugation at 100 × g. The supernatant was removed, 2% Triton X-100 was added to the pellet to lyse the cells, and both supernatant and pellet radioactivity were quantified by a beta-counter. The release was expressed as percent of total [3H]serotonin released calculated as that present in the supernatant over that in the pellet and the supernatant combined. The percentage release was calculated using the formula:

118

CPM supernatant × 100 = % Release CPM supernatant + C P M pellet (precipitate)

In other experiments, we used rat basophilic leukemia (RBL) cells which have been shown to be homologous to the mast cell [14]. The cell line was washed twice and then resuspended in Locke's solution ready for culture. Arachidonic acid metabolites were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Lipoxins A4 and B4 were generous gifts from Charles N. Serhan, Hematolo-

TABLE 1 LTC4, LTD4, LTE4, and LTB4 detected ( + SD) in RPMC suspensions (106/ml) following the addition of indomethacin or NDGA for 15 min and after, A23187 (5 #M) for an additional 15 min. RPMC treatment

LTB4 (pg/ml)

LTC4 (pg/ml)

LTD4 (pg/ml)

LTE4 (pg/ml)

Vehicle(control) A23187 (5 #M) Indo. (10 -6 M)+A23187 NDGA (10 #M)+A23187

150+_ 62 6300_+ 1060 8400+- 1180 2080± 953

120_+ 52 3100_+610 6500+-720 1350_+ 410

110+ 48 2800+-450 5900_+580 1100_+ 390

N.D. 2200_+ 152 3900± 319 809_+ 111

gy Di~:~ion, Harvard Medical School, Boston, MA). Nordihydroguaiaretic acid (NDGA; Sigma) was prepared in DMSO to a final concentration of 10 #M. Standard leukotrienes were kind gifts from J. Rokach (Merck Frosst, Canada). Radioimmunoassay (RIA) was performed [13] and the kits for PGE2 and PGD2 were purchased from NEN (New England Nuclear, Boston, MA). Indomethacin was a kind gift from Merck, Sharp and Dohme, Rome, Italy). Calcium ionophore A23187 (Sigma, St. Louis, MO, USA) was dissolved in dimethylsulfoxide (DMSO) (Sigma) at 50 mg/ml. Dilutions of the ionophore were made directly in Locke's medium with various final concentrations. In separate tubes, in each experiment, cells were exposed to the vehicle alone (i.e., DMSO or ethanol) at identical concentrations, to determine non-specific formation of arachidonic acid metabolites on mast cell release. The generation of LTC4, LTD4, LTE4, LTB4, PGD2, and PGE2 was detected in RPMC or RBL suspensions (10 -6 cells/ml) and assayed by radioimmunoassay as described [6]. Results are expressed as pg/ml. Each test and all controls were done in triplicates with each series of rats. In some experiments, RBLs were loaded with [14C]arachidonic acid ([14C]AA) (New England Nuclear) for 1 h at 37 °C, washed and resuspended at

106/ml and then treated with calcium ionophore A23187 for 1 h at 37 °C. The °70 release of [14C]AA was calculated after counting in a gamma-counter. Each table and figure is a representative experiment of at least four experiments in triplicate. 4. Results

Since mast cells are the primary effectors of immediate hypersensitivity reactions, initiated by the surface binding of IgE, in this report we have studied RPMC and RBL degranulation under the influence of arachidonic acid metabolites, with and without a classic secretagogue. Mast cell function is not only limited to degranulation, but also to the oxidative metabolism of arachidonic acid following perturbation of the cell membrane. We have studied the generation of some arachidonic acid metabolites using as an agonist calcium ionophore A23187 on rat peritoneal mast cells and on a cell line, rat basophilic leukemia cells. Table 1 shows the generation of LTC4, LTD4, LTE4, and LTB4 (pg/ml) from RPMC before and after 15 min treatment with A23187 (5 #M). The production of eicosanoids by the cells was higher when RPMC were pretreated for 15 min with indomethacin 10 -7 M and then treated with A23187

TABLE 2 LTC4, LTD4, and LTE4 production ( + SD) and % [14C]AA release in RBL suspensions (106 cells/ml) were pretreated with dexamethasone for 15 min and after, A23187 for an additional 15 min for LTC4, LTD4, and LTE4 and [14C]AA release was calculated after A23187 treatment for 1 h. RBL treatment

°/0[14C]AA. release

LTC4 (pg/ml)

LTD4 (pg/ml)

LTE4 (pg/ml)

Vehicle (control) A23187 (5 #M) Dexamethasone (10 -6 M)+A23187 (5/tM)

10_+ 5 56 _+13 30+ l0

N.D. 2800 + 450 650+210

N.D. 2620 + 860 1110+280

N.D. 2580 + 600 1080+205

119

TABLE 3

70

PGE2 and PGD2 detected (_+SD) in RPMC or RBL (106 cells/ml) suspensions following the addition of A23187 (5 #M) for 15 min.

50

Treatment

PGE2 (pg/ml)

PGD2 (pg/ml)

40

RPMC RBL + RPMC RBL +

N.D. N.D. 152+62 115_+50

208_+ 92 133_+ 56 9560_+2900 1980_+ 301

30

60

x

~3

T

°o

T

T

E

F

G

8

+ vehicle (control) vehicle (control) + A23187 (5 #M) A23187 (5 #M)

o

v

v T

Z

z

z

T

T

20 10

or- -n (15 min) compared to A23187 treatment alone. The pretreatment of cells with NDGA (10 ~M) for 15 min, followed by treatment with A23187 for 15 min, strongly inhibited the production of the leukotrienes under observation. The results in Table 2 show the amount of LTC4, LTD4, LTE4 detected in RBL suspensions following the addition of calcium ionophore A23187 (5/~M). In addition, here the RBL were assayed for [14C]AA release in the presence or absence of A23187 (5 tzM). Calcium ionophore significantly increases leukotriene and [14C]AA release. These effects were inhibited when the cells were pretreated with dexamethasone for 15 min and then treated with A23187 (5/zM) for 1 h. Table 3 reports the amount of PGE 2 and PGD 2 detected in RBL and RPMC after treatment with A23187 (5/xM). The effect of A23187 was great for

8O

RPMC +_3.6 -+2.5

z3a W

T

T

-F-

6O _+2.0

<

"-1'-W

40 I-:2: kq I I

_+3.5

2O -'1.5

o

c

0.125

0.25

0.5

1

2

C481a0 pg/ml Fig. 1. Effect of C48/80 on [3H]5HT °70 release by RPMC in culture for 10 min. A dose-dependent release is observed. The mean and standard deviation of pH]5HT release are displayed for each dose of C48/80.

120

A

B

C

D

H

Treatment

Fig. 2. [3H]5HT percent release from RPMC (4×105/ml) ( _+SD) following the addition of PGD2 at different concentrations of LTC4, LTD4, or LTE4 for 15 min and after, C48/80 (0.5/zg/ml) for I0 min. P values (Student's t-test) are calculated by comparing PGD2 + C48/80; LTs + C48/80-treated cell with C48/80 (*) alone. A, control (vehicle); B, C48/80 (0.5 /xg/ml); C, PGD2 (10-6M) + C48/80 (0.5 #g/ml); D, PGD2 (10-7M) + C48/80(0.5 #g/ml); E, PGD2 (10 -8 M) + C48/80(0.5 #g/ml); F, LTC4 (1000 nM) + C48/80 (0.5 #g/ml); G, LTD4 (1000 nM) + C48/80 (0.5 ~g/ml); H, LTE4 (1000 nM) + C48/80 (0.5 /~g/ ml).

PGD 2 generation in RPMCs, while it had no effect on the generation of PGE 2. However, RBLs treated with A23187 released significantly less than the RPMCs. Fig. 1 shows a dose-response curve of the °7o amount of [3H]5HT released by RPMC after addition of compound 48/80 at varying concentrations (0.125, 0.25, 0.5, 1 and 2/~g/ml). The amounts of [3H]5HT released by the cells after 10 min incubation was dose-dependent, maximum at 0.5, 1, and 2/zg/ml. Since the [3H]5HT release was not significantly different among the concentrations of 0.5, 1 and 2/zg/ml of 48/80, for all subsequent experiments we used the more appropriate concentration of 0.5/~g/ml. Fig. 2 gives the % of [3H]5HT release from RPMC following different treatment regimens, such as PGD 2 at different concentrations, LTC4, LTD4, and LTE4 for 15 min, subsequently adding C48/80 alone or in combination with the arachidonic acid products. In the controls the cells were exposed to the vehicle alone (i.e., ethanol). PGD 2 significantly inhibited [3H]5HT release and the effect was dose-dependent. No effect was found

TABLE 4 [3H]5HT 070 release by RPMC (4 x 105 cells/ml) (_+ SD) following the addition of increasing concentrations of LXB4 or 15HETE or LXB4+PGD2, PGD2 or 15-HETE+PGD2 for 15 min and then C48/80 (0.5 ~tg/ml) for 10 min. Pvalues (Student's t-test) are calculated by comparing the arachidonic acid metabolites plus C48/80-treated cells with C48/80 (*) alone. RPMC treatment

070 release + SD

P value

Control (vehicle) LXB4 (10 nM) LXB4 (100 nM) LXB4 (1000 nM) 48/80 (0.5 ttg/ml) LXB4 (1000 nM) + 48/80 LXB4 (100 nM)+ 48/80 LXB4 (10 nM)+ 48/80 15-HETE (1000 nM)+48/80 15-HETE (100 nM)+48/80 15-HETE (10 nM) + 48/80 PGD2 (10 -6 M) + 48/80 LXB4 (1000 nM)+PGD2+48/80 LXB4 (100 nM)+ PGD2+48/80 LXB4 (10 nM)+PGD2+48/80 15-HETE (1000 nM)+ PGD2+ 48/80

3.88 _+1.9 5.42_+2.4 5.84+_2.9 3.47 _+2.5 56.86_+ 2.4 50.10 _+3.0 52.51 _+3.5 56.19 + 2.6 57.48_+ 3.4 58.20+_3.1 58.27 _+2.9 42.44 _+3.2 45.76_+3.3 45.64_+2.1 46.10_+2.6 42.16+3.0

(*) < 0.05 N.S. N.S. N.S. N.S. N.S. < 0.01 <0.01 <0.01 <0.01 <0.01

when RPMC were pretreated with leukotrienes C4, D4, and E4 followed by C48/80. Table 4 shows the percent release of [3H]5HT following different treatments. LXB4, alone, at 10 and 100 nM did not influence significantly the percentage [3H]5HT release, nor was it affected after

treatment with C48/80 (0.5/~g/ml). When LXB4 was used at high concentrations (1 /~M) and then adding 48/80, some inhibition of [3H]5HT was observed. These effects did not influence the inhibitory action of PGD 2 (10 -6 M). 15-HETE added in a dose-dependent manner did not influence either 48/ 80 alone or PGD 2 plus 48/80. Table 5 shows the degranulation of RBL using LXB4, which did not influence the percentage of release of [3H]5HT. When A23187 (5/~M) was added after RBL pretreatment with LXB4, at various concentrations, a little, if any, inhibition was found when LXB4 was used at 10 or 100 mM. When the RBLs were pretreated for 15 min with 1000 nM of LXB4 and then added A23187 (5/~g/ml) for 10 min, a little significant inhibition was found. Table 6 shows the percentage of [3H]5HT release of RPMC and RBL after treatment with LXA4 at the concentrations of 10, 100 and 1000 nM. We used C48/80 as a secretagogue for RPMC and C48/ 80 for RBL. The results indicate that LXA4 alone did not affect the release of [3H]5HT. However, when LXA4 (1000 nM) was added to the cells with the addition of C48/80 or A23187, a small but significant inhibition of [3H]5HT release was seen. The inhibition was not significant when LXA4 was used at 10 or 100 nM followed by the two agonists. Nevertheless, the two secretagogues, by themselves, strongly affected the [3H]5HT °-/orelease of both cell populations, RPMC and RBL.

TABLE 5 [3H]5HT °70release ( _-!-SD) by RBL (4 × 105 cells/ml) following the addition of increasing concentrations of LXB4 for 15 min and after, A23187 (5 #g/ml) for 10 min. P values (Student's t-test) are calculated by comparing LXB4 plus A23187-treated cells with A23187 (5/~g/ml) (*) alone. RBL treatment

[3H]5HT 07o release_+ SD

P value

Control (vehicle) LXB4 (1000 nM) LXB4 (100 nM) LXB4 (10 nM) A23187 (5/~g/ml) LXB4 (1000 nM)+ A23187 (5 ttg/ml) LXB4 (100 nM)+A23187 (5 #g/ml) LXB4 (10 nM) + A23187 (5/~g/ml)

4.5_+2.2 5.2_+2.5 6.1 _+2.0 5.9+2.7 38.5_+4.5 30.9_+3.3 34.8_+3.5 37.7_+2.9

(*) < 0.05 N.S. N.S.

121

TABLE 6 [3H]5HT percent release by RPMC and RBL (4 × 105 cells/ml) following the addition of increasing concentrations of LXA4 for 15 min and after, C48/80 (0.5 #g/ml) for 10 min for RPMC or A23187 (5 #g/ml) for 10 min for RBLs. P values (Student's t-test) are calculated by comparing LXA4_+C48/80 or A23187-treated cells with C48/80 (*) or A23187 (*) alone. Treatment

RPMC

Control (vehicle) LXA4 (1000 nM) LXA4 (100 nM) LXA4 (10 nM) A23187 (5 #g/ml) 48/80 (0.5/~g/ml) LXA4 (1000 nM)+ A23187 (5 #g/ml) LXA4 (100 nM)+ A23187 (5 #g/ml) LXA4 (10 nM)+A23187 (5 #g/ml) LXA4 (1000 nM) + 48/80 (0.5 /zg/ml) LXA4 (100 nM) + 48/80 (0.5 #g/ml) LXA4 (10 riM) + 48/80 (0.5 #g/ml)

3.9 _+2.1 5.1 +3.1 5.8 _+3.4 6.1 _+2.4 60.8 _+4.5

(*)

52.4 _+3.4 56.9 + 2.4 60.4 _+3.6

< 0.05 (N.S.) (N.S.)

5. Discussion We have studied the interaction o f calcium ionophore A23187 in activating arachidonic acid metabolites in R P M C and R B L secretion. First, we confirm that mast cells produce cyclooxygenase and lipoxygenase products and degranulate after 4 8 / 8 0 challenge. The evidence provided by radiolabeled [14C]AA in RBLs suggests that stimulation with A23187 evokes a strong release o f [t4C]AA after 1 h incubation, this effect being significantly inhibited by dexamethasone (10-6 M). Interestingly the m a j o r cyclooxygenase and lipoxygenase product identified in the supernatants by r a d i o i m m u n o a s say was P G D 2 and LTC4 [7, 8, 12], thus providing further evidence that mast cells are the m a j o r source o f these eicosanoids. In addition, we used another model to study the activation-degranulation o f R P M C and R B L by following radiolabeled [3H]5HT and employing the two secretagogues C 4 8 / 8 0 and A23187. In these studies we f o u n d that the addition o f P G D 2 before the classic agonist strongly inhibits mast cell degranulation. The three leukotrienes LTC4, LTD4, and LTE4, which are c o m p o n e n t s o f SRS-A, or the leukotriene precursor 15-HETE, had no effect on mast cell degranulation. The process whereby mast cells release their activators and mediators has been extensively studied in vivo and in vitro [ 1 9 - 2 2 ] . The aim o f this 122

P value

RBL

P value

4.5 _+2.8 5.9_+3.5 6.2 _+3.0 6.4_+ 3.3 41.5 _+2.8 35.2 _+3.0 38.1 + 2.9 39.7_+2.6 -

(*) ( < 0.05) (N.S.) (N.S.)

work was to re-examine the role o f some arachidonic acid metabolites on mast cell [3H]serotonin release. In addition, in this report, for the first time we show the effect o f the novel trihydroxytetraenes lipoxin A 4 and B4 on mast cell degranulation. The effects o f lipoxins in this system produced very inconsistent results. Rat peritoneal mast cells were investigated for cell degranulation following treatment with the agonist C 4 8 / 8 0 after pretreatment with L X B 4 or L X A 4 for 15 min. Mast cells showed little inhibition (not statistically significant) o f degranulation after C 4 8 / 8 0 treatment. However, when L X B 4 or A 4 was used at the highest concentration (1 #M) for 15 min and the addition o f a secretagogue for 10 rain, a little but significant inhibition was found in all experiments. The low potency o f these two 5- and 15-HETE products has been confirmed in our previous study where L X A , only at high concentrations produced an increase in t h r o m b o x a n e release and inhibition o f LTB4 generation from h u m a n neutrophil suspensions [4, 3]. These results m a y be explained by the fact that hum a n leukocytes generate, physiologically, very small a m o u n t s o f L X A 4 and L X B 4 [9, 18]. The slight inhibition o f mast cell secretion by LXs was found either when it was used with A23187, which directly elevates intracellular calcium, or with C48/ 80, which does not require Ca 2 +. Degranulation by some chemical agents appears to mimic a part o f

IgE-induced processes. Calcium ionophore A23187 induces Ca 2÷ influx and c o m p o u n d 48/80 induces mobilization o f intracellular Ca 2÷ and Ca 2÷ influx [ 2 3 - 25]. It has recently been reported that calciu m / p h o s p h o l i p i d - d e p e n d e n t protein kinase C (PKC) and c A M P m a y be involved in mast cell degranulation and are necessary components for mast cell secretion [13]. Since P K C is an integral part o f the signal transduction p a t h w a y and is activated by calcium and phospholipids [17], it is possible that the eicosanoids lipoxins m a y be involved in this mechanism competing with phospholipids and interfering with P K C . This phenomenon, in vivo, m a y require different concentrations o f L X A 4 or B4 than in vitro. In this study, the enhancement o f leukotriene production by indomethacin (Fig. 1), a non-steroidal a n t i - i n f l a m m a t o r y c o m p o u n d which blocks the formation o f cyclooxygenase products (which are immunosuppressive) m a y explain the slight immunostimulatory effect o f this drug through the enhancement o f LTB4, which is reported to be an important mediator o f immunological functions [26, 27].

In conclusion, of the two secretagogues used in these experiments, we found that A23187 stimulates R P M C and R B L to release P G E D 2 and, to a lesser extent, P G E 2 [10, 28, 29]. The generation o f LTC4, D4, and E4 by R P M C and RBLs certainly has no autocrine influence, since we found a nonsignificant effect o f these lipoxygenase products on mast cell secretion. However, these c o m p o u n d s formed by the t r a n s f o r m a t i o n o f the instable epoxide intermediates LTA4 by the addition o f glutathione to generate LTC4 and then, the other two molecules LTD4 and E4, are potent b r o n c h o c o n strictors, increase vascular permeability in post capillary venules and stimulate mucus secretion [11]. These three leukotrienes, which are released f r o m lung tissue o f asthmatic subjects exposed to the specific allergens, certainly play a p a t h o p h y siological and p r o i n f l a m m a t o r y role in mediating hypersensitivity reactions, while the biological role o f the lipoxins A 4 and B4 in vitro and in vivo, remain to be determined.

References

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