Development and application of a membrane receptor ... - Springer Link

Agentsand Actions,vol. 27, 3/4 (1989)

0065-4299/89/040469-04 $1.50+ 0.20/0 9 1989Birkh~iuserVerlag, Basel

Development and application of a membrane receptor assay for leukotriene B 4 D.M. Patelunas 1, A.P. Agins z, R.E. Zipkin 3 and J. B. Smith 1 1 TempleUniversity,Philadelphia, PA 19140,USA;z BrownUniversityProvidence,RI 02912,USA; 3 BiomolResearchLaboratories, Plymouth Meeting, PA 19462,USA

Abstract

5(S),12(R)-dihydroxy-6-cis-8,10 trans-14-cis-eicosatetraenoic acid (LTB4) is a potent inflammatory mediator generated by human cells. A receptor assay using membranes from cultured HL-60 cells has been developed to quantitate LTB 4 with a range of sensitivity from 10 pg to 5 ng. The initial metabolite of LTB4, 20-OH LTB4, has a cross reactivity of 28 % while other lipoxygenase products do not significantly compete. This assay has been used to study ionomyocin-induced formation of LTB 4 by human neutrophils. The use of membranes from HL-60 cells for the measurement of LTB 4 provides a sensitive and highly selective alternative to radioimmunoassay for the determination of the levels of this important eicosanoid in biological fluids and should be useful in the development of antagonists of the LTB 4 receptor.

Introduction

Materials and methods

5(S), 12(R)-dihydroxy-6-cis-8,10 trans-14-cis-eicosatetraenoic acid (LTB4) is a potent inflammatory mediator generated by different cells including neutrophils, monocytes and alveolar macrophages. The formation of LTB 4 involves the release of arachidonic acid (AA) from phospholipids in the cell membrane and its conversion to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE) via the lipoxygenase pathway. The 5-HPETE is then converted to an unstable intermediate, LTA4, by LTA 4 synthetase and then undergoes subsequent hydrolysis to form LTB 4. LTB 4 is known to be an important mediator of polymorphonuclear leukocyte (PMN) aggregation, chemotaxis and lysosomal enzyme release [1 4]. We have developed a simple method for quantifying LTB 4 using membranes from HL-60 cells. This assay provides a novel alternative to radioimmunoassay and possibly the study of LTB 4 receptor antagonism.

Reagents - Dimethylformamide (DMF), bovine 7-globulin (Cohn fraction II), Histopaque 1077 and polyethylene glycol 8000 (PEG) were purchased from Sigma (St. Louis, MO). Dexamethasone was obtained from Upjohn (Kalamazoo, Mich.). LTB 4 and analogs were synthesized as reported in [8]. [3H]-LTB4 (133 Ci/mmol) was purchased from NEN Research Products (Boston, Mass.). All reagents were dissolved in Hanks' balanced salt solution without calcium and magnesium (Hanks' buffer). HAWP filters (0.45 ~m) were obtained from Millipore (Bedford, Mass.). Ionomycin (Calbiochem, La Jolla, CA) was dissolved in dimethylsulfoxide (DMSO). Dextran was obtained ;rom ICN Biomedicals (Lisle, IL). Scintillation fluid, Liquiscint, was purchased from National Diagnostics (Manville, N J). LTB 4 assays were performed in polypropylene tubes.

470 C e l l s " - HL-60 cells were grown in suspension culture in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum (heat-inactivated), penicillin (100U/ml) and streptomycin (100 gg/ml). Cultures were maintained at 37 ~ in a humidified atmosphere of 5% CO 2 in air. Cells were generally seeded at 2 - 3 x 105 per milliliter and differentiation was induced by culturing the cells in the presence of 0.8% (v/v) DMF and 10 gM dexamethasone for 5 days. Cell viability was assessed by trypan blue exclusion and was always >95%. Cells were harvested by centrifugation, washed twice in Hanks' balanced salts and resuspended to a final concentration of 0.5 x l0 s per milliliter in 0.01 M potassium phosphate buffer (pH 7.0) containing 20% (v/v) glycerol and 1.0 m M EDTA. Cells were lysed by sonication and the tysate centrifuged at 400 x 9 to remove residual intact cells and nuclear debris. The supernatant from the low speed centrifugation was then centrifuged at 100000 x 9 for 1 hour. The high-speed supernatant was decanted and pellet (P2) was resuspended in the same buffer as above. Protein was determined by the BioRad dye binding method using bovine 7-globulin as standard. Typically, 2 4 mg protein were recovered in the P2 fraction from 10s cells. assay - The binding assay for LTB 4 was performed at 4 ~ and was modified from the procedure of Gorman and Lin [5]. To each vial, 0.03 ng of [3H]-LTB4 (approximately 6000 cpm) was added. In general 0.20 mg protein from P2 membranes was added to each vial except for the background samples. Next, different concentrations of unlabeled LTB, (10 p g - 5 ng) were added. The samples were diluted with Hanks' buffer to field volume of 325 gl and were incubated overnight at 4~ Samples were adjusted to 5% (w/v) PEG and 600 gg of 7-globulin and then after 20 minutes the entire incubation mixture was rapidly filtered through a Millipore HAWP filter. The filters were washed with 20 ml of ice-cold 5% PEG and then dissolved in scintillation fluid and counted. Binding

Neutrophilpreparation - Blood was drawn by venipuncture from human volunteers. Neutrophils were purified by standard techniques employing dextran sedimentation followed by Histopaque gradient and ammonium chloride lysis [6]. Neutrophils had a viability and purity of greater than 97%. Neutrophils were suspended at a concentra-

Agentsand Actions,vol.27, 3/4 (1989) tion of 30 x 1 0 6 cells/ml in Hanks' balanced salt solution without calcium and magnesium. Samples were preincubated at 37 ~ with 1 m M magnesium (final concentration) and 1.3 m M calcium for two minutes with continuous stirring and then either stimulated with 10 gM ionomycin for different time intervals or treated with equivalent volumes of DMSO (0.5%) for five minutes. The cells were lysed with an equal volume of distilled water, frozen and thawed twice, and centrifuged (12000 x 9) for 2 minutes. The supernatants were removed and stored at - 7 0 ~ until assayed for LTB 4. Results

Initially, the protein concentration of the P2 membrane fraction required to achieve maximal binding with 0.03 ng [3H]-LTB4 was determined (data not shown). This binding of [3H]-LTB4 to HL-60 membranes was linear between 0.02mg and 0.20 mg of protein per sample and then reached saturation. In the presence of 0.03 ng [3H]-LTB4 and 0.2 mg membrane protein, 30% binding was obtained. The data in Figure 1 shows the specific binding of [3H]-LTB4 (0.03 ng) to HL-60 membranes (0.20 nag/sample) in the presence of different concentrations of unlabeled LTB 4 at 4~ Nonspecific binding was determined in the presence of excess unlabeled LTB 4 (1 gg) and specific binding was calculated by subtracting nonspecific binding (12%) from the total binding. The amount of LTB 4 in unknown samples was then interpolated from the standard curve in Figure 1. Unknown samples were diluted with Hanks' buffer so that the values fell on the linear portion of the graph. The only compound which showed competitive binding activity for the LTB 4 receptor was 20-OH LTB4, but even this was considerably less active than LTB 4 itself. The cross reactivity of 20-hydroxy LTB 4 in the receptor binding assay was determined to be 28%. 12(R)-HETE, LTD4, 14,15-LTD4, and LTB 4aminopropylamide exhibited a cross reactivity of 0.5% or less. Each of the following had a cross reactivity of less than 0.2%: 20-carboxy LTB 4, arachidonic acid, 5(S)-HETE, 12(S)-HETE, 5(S), 12(S)-Di-HETE, 5(S),12(R)-Di-HETE, 8(S), 15(S)Di-HETE, 5(S),6(R)-Di-HETE, LTC4, 14,15LTC 4 and LTE4. The percentages of cross reactivity were calculated from the mass required to displace 50% bound [3H]-LTB4.

471

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Specificity of binding of LTB 4 to HL-60 membranes. H L - 6 0 m e m b r a n e s (0.2 m g protein/sample) were incubated with 0.03 ng [3H]-LTB4 in the presence of varying concentrations of unlabeled LTB 4 or 2 0 - O H LTB 4. All assays were c o n d u c t e d at 4 ~ D a t a are presented as mean o f four experiments in duplicate. .W

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Time course of LTB 4 p r o d u c t i o n in ionomycin-stimulatcd neutrophils (N). A total o f 30 • 106 N / m l were stimulated with 10 p M ionomycin at 37 ~ The control samples were treated with a n equivalent volume of D M S O for 5 minutes. The statistical significance of the d a t a were determined using a paired Student's t-test where *** p < 0.001 a n d ** p < 0.005 when c o m p a r e d against control values. The 1, 2, a n d 5 minute time points did not differ significantly (p >0.05) f r o m each other.

472

This assay was used to monitor the time-course of LTB 4 formation (Fig. 2) in ionomycin-stimulated neutrophils. The control samples contained 41+_4ng of LTB4/30x 10 6 cells (mean+SEM). The production of LTB 4 rapidly increased during the first minute following stimulation with ionomycin and then plateaued. The data in Figure 2 indicate that human neutrophils are capable of synthesizing 901+ 51 ng of L T B 4 / 3 0 • 10 6 cells within two minutes of ionomycin stimulation.

Discussion The use of membranes from HL-60 cells for the measurement of LTB 4 provides a highly selective alternative to radioimmunoassay for the determination of the levels of this important eicosanoid in biological fluids. This novel assay has been used to study the ionophore-induced formation of LTB 4 by human neutrophils. The value of 901 ng of LTB 4 release found here compares favorably with the results of Weller et al. [7] who found a release of 963 ng of LTB4/30• 10 6 cells with ionophore-stimulated human neutrophils. The receptor assay may also be useful for screening potential LTB 4 receptor antagonists.

Agents and Actions, vol. 27, 3/4 (1989)

References [1] A. W. Ford-Hutchinson, J. A. Bray, M. V. Doig, M. E. Shipley and M. J. H. Smith, Leukotriene B 4 is a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286, 264 265 (1980). [2] A. H. Lin, D. R. Morton and R. R. Gorman, Acetylglyceryl ether phosphorylcholine stimulates leukotriene B 4 synthesis in human polymorphonuclear leukocytes. J. Clin. Invest. 70, 1058-1065 (1982). [3] G. M. Bokoch and P. W. Reed, Evidence for inhibition of leukotriene A 4 synthesis by 5,8,11,14-eicosatetraynoic acid in guinea pig polymorphonuclear leukocytes. J. Biol. Chem 256, 5317 5320 (1981). [4] J. Palmblad, C. L. Malmsten, A. M. Uden, O. Radmark, L. Engstedt and B. Samuelsson, Leukotriene B 4 is a potent stereospec(fic stimulator of neutrophil chemotaxis and adherence. Blood 58, 658 661 (1981). [5] R. R. Gorman and A. H. Lin, Assay.for the leukotriene B 4. In Methods in enzymology, vol. 141. (Eds. S. P. Colowick and N. O. Kaplan) pp. 372-378, Academic Press 1983. [6] A. Boyum, Separation of leukocytes from blood and bone marrow: isolation of leukocytes from human blood further investigations: methylcellulose, dextran, and ficoll as erythrocyte aggregating agents. Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77 89 (1968). [7] P. F. Weller, C. W. Lee, D. W. Foster, E. J. Corey, K. F. Austen and R. A. Lewis, Generation and metabolism of 5-1ipoxygenase pathway leukotrienes by human eosinophils: Predominant production of leukotriene C4. Proc. Natl. Acad. Sci. U.S.A. 80, 7626 7630 (1983). [8] K. C. Nicolaou, R. E. Zipkin, R. E. Dolle and B. Harris, A general and stereocontrolled total synthesis of leukotriene B 4 and analogs. J. Am. Chem. Soc. 106, 3748-3751 (1984).