Specific leukotriene D4 receptors on guinea-pig alveolar macrophages

PROSTAGLANDINS

SPECIFIC LEUKCTRIENE D RECEPTORS ON GUINEA-PIG ALVEOLAR fsIAcROPHAGES *

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J.P. Cristol, B. ProvenGal, P. Borgeat and P. SiroisDept. Pharmacology,Faculty of Medicine, University of Sherbrooke, Sherbrooke,P.Q. Canada JlH 5N4 and *CHUL, Quebec, P.Q., Canada GlV 4G2 ABSTRACT: 3 ( H)-leukotriene D4 (LTD4) were Specific binding sites for identified 3on guinea-pig alveolar macrophages (GPAMs) using high specific activity ( H)-LTD4, in the3 presence or absence of unlabelled LTD4. The time required for ( H)-LTD4 binding to reach equilibrium was approximately 15 min at OOC. The binding was saturable, reversible and specific. The dissociation constant (Kd) and 6site density (Bmsx) mre found to be 2.3e.38 nM and 560-$3 fmol/lO cells, respectively,as determined from Ssatchard analysis. In competition studies for the displacement of ( H)-LTD4 from binding sites, leukotrienes B4, C4, and E and the peptidoleukotrieneantagonist FPL-55712 revealed an D4 order of 4htency of LTD (Ki 3.9 nM) > LTE4 (Ki 243.9 nM)> LTC4 (Ki 796.9 nM) > FPL-55712 &i 17.6 uM). Concentrationsof LTB4 up to 10 UM by did not displace the ( H)-LTD4 binding. Bioconversion of LTD determined by Reverse-Phase High-Performance L 4quid GPAMS, as Chromatography (RP-HPLC), was less than 3% in 30 min incubationperiods. It is concluded that these binding sites may be receptors for LTD on 4 GPAMS. Since LTD4 is produced by GPAMs, it is postulated that endogenous LTD4 may modulate thromboxanesynthesis and lung constriction. INTRODUCTION. Peptidoleukotrienes C4 (LTC~), ~~ (LTD~) and ~~ (LTE~) are the bioactive components of slow-reactingsubstance of anaphylaxis (SRS-A) (l-3). They are formed from arachidonic acid following exposure to several types of immunologicaland non-immunologicalstimuli by a variety of cells including bronchoalveolar macrophages (4,5). They all contract the guinea-pig lung strip, trachea (6) and bronchus (7). Injections of LTD4 in the circulation of guinea-pig perfused lung produce a large release of prostagpdins (PGs) and thromboxanes (TXs) (8,9). More recentLy, using ( H)-LTD4, a specific LTD receptor has been identified in crude membrane preparationsfrom gu4nea-pig lung homogenate (10-14) and on human alveolar macrophages obtained by bronchoalveolar Lavage (15). Since treatment of macrophageswith LTC and LTD induces PGs and TXs release (16), the aim of the present study bs to in$estigate whether specific LTD4 receptors are present on the guinea-pig alveolar macrophages (GPAMs). Footnote:

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Author for correspondence.

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MATERIELS AND METHODS. MATERIELS. (14-15 3H)-LTD (1.54 TBq/maol) was obtained from Amersham International. Synthetic LTD$, LTE4, LTC4, met: esters were obtained from Biomega, Inc ( ontreal, Canada). LTB was suppli:d as a generous gift from Dr J. Rokach of Merck Frosst Ltd, an FPL-55712 from Dr P. Sheard of Fisons Ltd (Loughborough, U.K.). Irmnediatly before use methyl ester LTs were hydrolysed by treatmentwith Na2C03 5% for lh. BRONCHOALVEOLARLAVAGE. Guinea-pig alveolar macrophageswere obtained as described by Maxwell et al. (17). The animals were sacrified by cervical dislocation and -exsanguinated by sectioning the dorsal aorta. The trachea was cannulated and phosphate-buffered saline (PBS, 80 ml, 37OC) was infused into the lung in 10 ml aliquots, and reaspiratedafter gentle massage. After lysis of red blood cells by hypotonic shock, the remaining cells were resuspended in buffer A (Hepes 25 mM, NaCl 109 mM, KC1 4.7 mM, KHP04 1.1 mM, glucose 5 mM). The pH was adjusted to 7.4 with NaOH. The n~ber of cells in the suspension and the cell viability were determined by th% trypan blue exclusion method. The cell yield averaged 1625x10 cellsflavage and the viability was 93+5% (n=20). Mononuclear cells were purified from the recovered cells- by continuous Percoll density centrifugation and the harvested cells contained more than 95% GPAMs as assessed by cytocentrifugationand Wright and Giemsa stains. (3H)-LTD4 BINDING ASSAY. Ligand binding assays were performed at O'C, in 130 ul of buffer B (buffer A3 with CaC12 2 mM, MgS04 1.2 mM), containing 0.5x106 cells / ml and ( H)-LTD4 (2.5 KIM), in the presence or absence3 of LTD4 or other competing ligands. Following incubation, bound ( H)-LTD4 was separated from unbound ligand by vacuum filtration through glass fiber filters (MSI, Fisher Scientific,Montreal, Canada). The filters were then washed 5 times with cold buffer A in less than 10 sec. The radioactivity remaining on the filters was measured in 10 ml of scintillatignfluid (Ready-solv MP, Bechnan, Montreal, Canada). Non-specific ( H)-LTD4 binding was determined in the presence of an excess of unlabelled ligand. In order to determine the saturability,affinify and density of LT binding sites, GPAMs were incubated with 2.5 nM ( H)-LTD4 and in the presence of increasing concentrations of unlabelled ligand. Computer analysis method (EBDA Mac Pherson) (18) was used to analyse the results obtained from saturation and competitionexperiments. METABOLISM OF LTD BY GUINEA PIG ALVEOLAR MACROPHAGES. Leukotriene (3 uM) was incubated in 200 ul of uffer B, at O'C, g for 30 and 60 !?n, in the presence or absence of 0.5xl.O cells/ml. After incubation, the cells in the reaction mixture were sedimented by microcentrifugation (2 min, 1200xg). Half volume of methanol was added to the supernatantand the pellet was resuspendedin 100 ul of methanol. Both the analysed by reverse-phase supernatant and the pellet were high-performance liquid chromatography (RF'-HPLC) using PGB2 and

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19-OH-PGB2 as internal standards (25 ng each) as previously described (19, 20). Briefly, each sample was centrifugedat 3OOOxg for 30 min to remove particulate material. The supernatants were acidified to pH 3, diluted with 1.3 ml of water and 1.7 ml was injected onto a Radial Pack C-18 cartridge (Water Ass., 100 x 8 w, 10 um particules) without prior derivatization or fractionation. Separation of the various compounds was achieved using gradients of organic solvents (methanol and acetonitrile) and a pH change, and elution was monitored by ultraviolet photometry at 280 nm and 229 nm.

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48

24

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Fig.1. Time course of (3H)-LTD, specific binding to GPAMs. Association of (3H)-LTD4 (2.5 nM) was measured as a function of time and adding unlabelled 4TD4 (8 uN dissociation was induced by at equilibrium. Data are expressed as a percentage of ( H)-LTD4 specific binding at equilibrium. Each point represents the mean-MM of three separate experiments.

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RESULTS.

REVERSIBILITY OF (3H)-LTD4 BINDING TO GUINEA-PIG TIME COURSE AND ALVEOLAR MACROPHAGES. The first series of experimen s were undertaken to study the 5 time-course and reversibility of ( H)-LTD4 biqding. When incubation was performed at 0°C in the presence of ( H)-LTD4 (2.5 nM) the a time-dependent manner, reaching specific binding increased in equilibrium within 15 min and remaining stable for at least 90 min. When a

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Fig.2. Saturation and Scatchard aoalgses of LTD4 receptor under the fqllowing conditions. GPAMs (0.5ti.o cells/ml) were incubated with ( H)-LTD4 (2.5 nM) and increasing concentrations of LTD4 (up to 30 nM) at O°C for 30 min, in a volume of 130 ul of incubationbuffer. The non-specific binding was determined by including the unlabelled IX,TD& (at concentration 1000 fold higher than the radioligand under identical conditions). The specific binding was determined by substractingthe non-specific binding from the total binding after equilibriumwas reached. At 2.5 nM, the non-specificbinding constituted 34% of the total binding. Each point represents the mean+sEy of five separate experiments.Scatchard analysis (insert). The ratio of specifically bound ligand to the free ligand was plotted as a function of specificallybound ligand.

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large excess (8 uM) of unlabelled ligand was added at equilibrium (30 min), a rapid decrease in the radioactivecomplex was observed showing the reversibility of the binding (T l/2 dissociation: 6 min) (Fig. 1). Non-specific binding at equilibrium represented 34% of the total binding. EQUILIBRIUM BINUING PARAMETERS OF (3H)-LTD BINDING. 5i ding parameters of LTD4 to GP were determined by incubating g !+ 0.5xLo cells/ml at O"C, with ( H)-LTD4 (2.5 nM) in isotopic dilution up to 30 nM for 30 min. Non-specific binding was determined in the presence of 3 or 6 UM of unlabelled ligand. As shown in Fig. 2 the specific binding of LTD4 was saturable, reaching the maximal binding at 13 nM of free ligand. Scatchard representation resulted in a linear plot correspondingto a single population of hi&h affinity binding sites. The mean Kd and Bmax values, derivgd from five different experiments,were 2.335.38 nM and 56of48 fmol/lO cells respectively (Fig. 2 insert).

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Fig.3. Specificity ap order of potency of LTD receptor agonists and antagon sts. The ( H)-LTD (2.5 nM) was kcubated with GPAMs A (0.5xl.O cells/ml) in 140 ul of incubation buffer in increasing concentrations of LTBfs ( A 1, LX ( Cl 1, d&lled L7D4 ( 0 1, LTE4 ( l ) and FPL- 5712 ( n ). fiachpoint represents the mean&SEM of three separate experiments.

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SPECIFICITYOF (3H)-LTD4 BINDING. Competition experiments were performed in the presence of 2.5 nM of increasing concentrations of each competing ligand. (3H)-LTD4 with LTC4 was tested in the presence of serine borate complex (15 mM) to prevent its metabolism to LTD (21). As shown in Fig. 3, unlabelled LTD4 displaced the specific btnding 0f (3H)-~~~ with a Ki value of 3.9 nM, which is close to the Kd value determined ty Scatchard analysis. LTE4 (Ki 244 r&i), LTC (Ki 797 nM) and the SRS-A antagonistFPL-55712 (Ki 17.6 uM) also displaced the specific bpding. Concentrationsof LTB4 up to 10 uM did not displace the binding of ( H)-LTD4 from its sites. METABOLISM OF LTD4 BY ALVEOLAR MACROPHAGES. In this last set of experiments, the metabolism of LTD4 by GPAMs was analysed by RP-HPLC to evaluate its degradation in our experimental conditions. In the control samples (incubationwithout cells for 30 and 60 min) LTD4 remained stable and no LTE was detected. In the presence of cells, at O°C for 30 and 60 min, Ld remained the major component in the supernatant (more than 97%), and 4a small amount (3%) of LTE4 was recovered. In the pellet LTE4 was not detected. DISCUSSION. These results show that GPA% have high affinity (Kd 2.3e.38 nM), saturable (Bmax = 560+48 fmol/lO cells), reversible, and selective binding sites for LTD4. The binding of (3H)-LTD4 is rapid, reaching equilibrium in min at OOC. The Ki of other less than 15 peptidoleukotrienes, LTE4 and LTC4 are 63 and 200 times that of antagonist FPL-55712 displaced the respectively. The LTD LTD4, 17.6 uM, whereas LTB4 was inactive. The specific binding with a Ki 04 RP-HPLC profiles of the cell-bound LTs and free LTs, incubated with GPAMs, showed that LTD represented the main LT ( > 95%) found in the supernatant, and &at no LTE was detected in the cell fraction. This 4 binding site, therefore, appears to possess the properties of a specific receptor. Specific binding sites have also been reported in the membrane fractions isolated from guinea-pig lung homogenate (lO-14), and on human alveolar macrophages obtained by bronchoalveolarlavage (15). The range of Kd in the guinea-pig lung homogenate was between 0.25.03 nM (10) and 5.44+1.87 r&l (12). The kd value of the human alveolar macrophages was 3.8 nM. -In our experiments the Kd (2.3e.38 nM) value is close to that previously reported (lO-15). The number of sites is more important60nthe fmol/lO cells GPAMs than on thg human alveolar macrophages (56e8 versus 90 fmol/lO cells). Binding sites for LTs have also been found in homogenate of guinea-pig lung membranes using unphysiologicalbuffer to a oid monovalent cation inhibition of the specific binding of (yfl)-LTD (14). In our study, living cells and a physiologicalbuffer were usett. Our competition studies using LTC4 and FPL-55712 agree with the results of others (lO-13) and with the concept of two different receptors for LTC4 and LTD4 in the lung (13). On the GPAMs, LTE4 was found to

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Fig.4. Metabolism of LTD4 by GPAMs as analysed by RP-HPLC. Elution was monitored by ultraviolet photometry at 229 nm (0) (for HETEs) and 280 run and 19-OH and 19-OH PGB ). PGB ) (for LTD LTE PGB (A PGB2 were used as ?kerna14itanda?ds. Graph A shows2the elueion times of standards. Graph B was obtained with 3 uM LTD4 in LTD4 and LTE absence of cfiellsafter 30 min incubation at O'C. Graph C gnd D were obtained after incubation of 3uM LTD4 in presence of 0.5x10 cells/ml separated by were and supernatants cells 0°C. The at microcentrifugation. Graph C shows the BP-HPLC profile of the supernatants and graph D the profile of the cellular fractions.

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less potent than LTD4 in displacing the (3H)-LTD specific binding. This result is in favour of distinct receptors f& LTE and LTD4 on the GPAMs instead of a unique receptor as hypothetized frok the results with guinea-pig lung homogenate membranes (22). Recently, it was suggested that some peptidoleukotrienehigh affinity binding sites were not receptors but were binding units of enzymatic proteins such as glutathione transferase or dipeptidase.In rat liver homogenate, Austen -et al. (23) have identifieda high affinity binding site for LTC as a subunit of glutathione transferase.Since competitive studies revea1ed different affi ity for each peptidoleukotrieneand since 9the FPL-55712 displaced the ( H)-LTD4 binding, it appears that this binding site is a putative receptor. Peptidoleukotrienes were shown to be potent myotropic substances on the guinea-pig lung parenchyma strips. The ED 50 of these products were around 0.5 and 1 TIM.The compound FPL-55712 produces dose-dependent(20 nM - 20 uM) antagonism of responses due to LTC4, LTD4 and LTE4 without affecting the contraction elicited by LTB (6,24). Our results with macrophages are in agreement with the resufts obtained on the myotropic on the lung parenchyma strips and with the potency of activity of LTD 4 FPL-55712. In this species, it has been demonstrated (9,25) that the contractile effect of LTD is partly due to the formation of TXs and PGs by the lung cells. In minsed guinea-pig lung, it was shown that LTD induced the 4 synthesis and release of prostanoids including TXs and prostacyclin.This secretion appeared dose-dependent, with an ED of l.w_2nM (26). On the other hand, macrophages which are rich in es 5?erified arachidonicacid (27) were shown to release PGs, TXs and LTs following stimulationwith A-23187 and zymosan as well as during phagocytosis(28). Among these eicosanoids, LTC4 and LTD could enhance the release of PGs and TXs by peritoneal macrophages ($6). Another study from our laboratory (unpublished data) showed that stimulation of adherent GPAMs with nanomolar concentration of LTD4 increases significantlythe secretion of TXS. The specific binding sites for LTD4 on macrophages described in this study could possibly be involved in the activation of macrophages reported previously. be

In conclusion, we have shown that the GPAMs bear specific receptors for LTD4. Since alveolar macrophages are an important source of LTD4, we suggest that endogenous LTD4 may modulate TXs synthesis and lung constriction. ACXNOWLEDGMENTS. The authors wish to thank Mr M. Lauziere and J.P. Pel6 for technical assistance and Ms L. Dion for typing the manuscript. REFERENCES. 1. Brocklekurst, W.E. The release of histamine and formation of a slowreacting substance (SRS-A) during anaphylactic shock. J. Physiol. -151:416. 1960.

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2. 3.

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Piper, P.J. and J.P. Seal@. Non-immunologicalrelease of Slow-reacting substance from guinea-pig lungs. Br. J. Pharmacol. 67:67. 1979. Borgeat, S. Hammarstrikaand R.C. Murphy. Samuelsson, B., P. Introduction of a nomenclature:Leukotrienes.Prostaglandins-17:785. 1979. Rankin, J.A., M. Hitchcock, W.W. Merril, M.K. Both, J.R. Brashler and P. w. Askenase. IgE dependent release of leukotriene C4 from alveolar macrophages.Nature -297:329. 1982. Michel, F.B., Ph. Godard, M. Damon, C. Chavis and Crastes de Paulet A. Chemical mediators of anaphylaxis released by alveolar macrophages in bronchial asthma. Eur. J. Respir. Dis. 69 supp1.146:189.1986. Sirois, P., S. Roy, J.P. TBtrault, P. Borgeat, S. Picard and E.J. Corey.. Pharmacological activity of- leukotrienes A4, B4, and c4 on selected guinea-pig, rat, rabbit and human smooth muscles. D4 ProstaglandinsMed. 7~327. 1981. Sirois, P., S. Pr%, P. ThBriault, G. Rouleau and M. LauziBre. The guinea pig bronchoalveolar model for icosanoid studies. Inflammation 11:447. 1987. Piper, P.J. and M.N. Samhoun. Stimulation of arachidonic acid metabolism and generation of thromboxane A.. by leukotrienes B,. C and D 4 in guinea-pig lung -in vitro. Bi. J: Pharmacol.-77:26?: 1582. Sirois, P., M. Chagnon, P. Borgeat and P. Vallerand. Role of cyclooxygenase products in the lung action of leukotrienesA4, B4, D4 and E . Pharmacology 31:225. 1985. c4' Mong, S., ft.-L. Wu, M.A.-lark, J.M. Stadel, .J.G.Gleason and S.T. Crooke. Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung. Prostaglandins 28:805. 1984. Gng, S.-S. and R.N. DeHaven. Characterizationof a leukotrieneD4 receptor in guinea pig lung. Proc. Natl. Acad. Sci. U.S.A. -80:74?5. 1983. Cheng, J.B. and R.G. Townley. Identification of leukotriene D4 receptor binding sites in guinea pig lung homogenatesusing (3H) leukotriene D4. Biochem. Biophys. Res. Commun. -118:20. 1984. Hogaboom, G.K., S. Mong, H.-L. Wu and S.T. Crooke. Peptidoleukotrienes: Distinct receptors for leukotriene C4 and D4 in the guinea-pig lung. Biochem. Biophys. Res. Commun. l-16:1136.1983. Mong, S., H.-L. Wu, G.K. Hogaboom, M.A. C1arm.M. Stadel and S.T. Crooke. Regulation of ligand binding to leukotriene D4 recptors: effects of cations and guanine nucleotides. Eur. J. Pharmacol. 106:241. 1985. Geer, F.A. and H.C. Hoogsteden. Characterization of specific receptors for leukotriene D4 on human alveolar macrophages. Prostaglandins28:183. 1984. Feuerstein, N., M. Foegh, P.W. Ramwell. Leukotrienes and D c4 induce prostaglandin and thromboxane release from rat peritonea4 macrophages. Br. J. Pharmacol. 72:389. 1981. Maxwell, K.W., T. Dietz and-S. Marcus. An in situ method for harvasting guinea pig alveolar macrophages.Am. Rev. Respir. Dis. -89: 579. 1964.

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18. MC Pherson, G.A. Analysis of radioligand binding experiments. A collection of computer programs for the IBM PC. J. Pharmacol. Methods, 14:213. 1985. 19. sirois, P., Y. Brousseau, M. Chagnon, J. Gentile, M. Gladu, H. Salari and P. Borgeat. Metabolism of leukotrienesby adult and fetal human lungs. Exp. Lung Res., 9:17. 1985. 20. Borgeat, P. Reversed-phase HPLC profiling and quantitation of lipoxygenaseproducts. Prostaglandins-27:356. 1984. 21. Cheng, J.B. and R.G. Townley. Effect of the serine borate complex on the relative abilit of leukotriene C 4'an3 ($ ?I ~,,,';ri;;~;t brain ( H) leukotriene D lung and binding: demonstration of the agonists' $otency order for leukotrieni D receptors. Biochem. Biophys. Res. commun. and leukotriene C 4 lff9:612.1984. 22. Mong, s., M.O. Scott, M.A. Lewis, H.-L. WU, G.K. Hogaboom, M.A. Clark and S.T. Crooke. Leukotriene binds specifically to LTD4 E4 receptors in guinea pig lung membranes. Eur. J. Pharmacol.-109:183. 1985. 23. Sun, F.F., L.Y. Chau and K.F. Austen. Binding of leukottieneC4 by glutathione transferase: a reassessmentof biochemical and functional criteria for leukotriene receptors.Fed. Proc. 46:204. 1987. 24. Sirois, P., S. Roy and P. Borgeat. Specificity of receptors for leukotrienes A4, B4, C#, D4, E4 and histamine on the guinea-pig lung parenchyma. ,ffect of FPL-55712 and desensitizationof the myotropic activity. Prostaglandins-26:91. 1983. 25. Piper, P.J. and M.N. Samhoun. The mechanism of action of leukotrienes in guinea-pig isolated perfused lung and parenchyma and D c4 strips of gtinea pig, rabbit and rat. Prostaglandins21: 793. 1981. 26. Mong, S., H.-L. Wu, M.A. Clark, J.G. Gleason zd S.T. Crooke. Leukotriene receptor-mediated synthesis and release of D4 arachidonic acid metabolites in guinea pig lung: Induction of thromboxane and prostacyclin biosynthesis by leukotriene D4. J. Pharmacol. Exp. Ther. 239:63. 1986. 27. Scott, W.A., J.M. Zrike, A.L. Hamill, J. Kempe and Z.A. Cohn. Regulation of arachidonic acid metabolites in macrophages.J. Exp. Med. -152:324. 1980. 28. Takemura, R. and Z. Werb. Secretory products of macrophages and their functions.Am. J. physiol.-246:Cl. 1984.

Editor:J.Roberts

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~eceived:ll-18-87

Accepted:3-15-88

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