Immunology Letters, 24 (1990)237-242
Elsevier IMLET 01391
Lipoxins A4 and B4 inhibit leukotriene B4 generation from human neutrophil leukocyte suspensions P i o C o n t i , M a r c e l l a Reale, R e n a t o C. B a r b a c a n e , M a u r o B o n g r a z i o a n d M a r i a R. P a n a r a Immunology Division, Institute of Experimental Medicine, University of Chieti Medical School, Chieti, Italy
(Received 5 March 1990; accepted 19 March 1990)
1. Summary
2. Introduction
Lipoxins A4 and B4 (5,6,15L-trihydroxy-7,9,11,13eicosatetraenoic and 5D,14,15-trihydroxy-6,8,10,12eicosatetraenoic acids, respectively) were examined in several biological systems and have proven to have many different activities from those o f other eicosanoids. Cultured human polymorphonuclear leukocytes were preincubated with LXA and B and their ability to inhibit leukotriene B4 generation was assessed after incubation with the calcium ionophore A23187. The preincubation time of neutrophils with lipoxin A4 and B4 was 15 min. After that time the cells were incubated for 6 min with A23187 (5/~M) for the release of LTB4. We found that the pretreatment of neutrophils with lipoxins inhibited the release of LTB4 by A23187-stimulated PMNs. Nordihydroguaiaretic acid (NDGA) (10/~M), used as a control, strongly inhibited the generation of LTB4. Since LTB4 has been shown to be a modulator of cellular immunity, our data suggest that lipoxin A4 and B4 can contribute to the immunosuppression via inhibition of LTB4 generation. Moreover, the inhibition of LTB4 by lipoxins in neutrophils could have an important regulatory role in inflammation.
Many different studies have suggested that initial lipoxygenation at C-15 can lead to the formation of substances of biological interest called lipoxins [1]. Lipoxins are trihydroxytetraenes, a novel series of oxygenated derivatives formed from arachidonic acid in human leukocytes [2]. The structures of the major compounds were established and proved to be 5S,6R,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid and 5D,14,15-trihydroxy-6,8,12-eicosatetraenoic acid. Recently, many isomers of LXA4 and B4 have been isolated, for example, 7-cis-ll-trans LXA4, l l - t r a n s LXA4, 6 - c i s - L X B 4 and 6-cis-8t r a n s - L X B 4 , and have been found to have opposite biological effects. 7-cis forms of LXA4 evoked a dose-dependent contraction o f guinea pig lung strip, while the 6-cis forms of LXB4 relaxed this preparation [3-5]. The addition of these compounds to human polymorphonuclear leukocytes or human natural killer ceils evokes selective responses different from those observed with leukotrienes [6-8]. In previous studies we have indicated that eicosanoids and their metabolites are involved in the function of leukocytes [9-13]. Recently we have found that lipoxin A and B play a pro-inflammatory role by increasing the granuloma formation in experimentally induced chronic inflammation [14]. In addition, we have also found that human PMN leukocytes exposed to LXA stimulate cyclooxygenase products, and that the addition of indomethacin to the cell suspensions blocks this formation. Based on these observations, and since certain isomers of LXA4 cause contraction of guinea pig lung strip, which seem to involve
Key words: Lipoxin; Leukotriene; Polymorphonuclear leuko-
cyte; Calcium ionophore Correspondence to: Dr. Pio Conti, Cattedra di Immunologia,
Istituto di Medicina Sperimentale, Universita' di Chieti, Via dei Vestini, 66100 Chieti, Italy. Abbreviations: LX, lipoxin; LT, leukotriene; PMN, polymor-
phonuclear; A23187, calcium ionophore; NDGA, nordihydroguaiaretic acid.
0165-2478 / 90 / $ 3.50 © 1990 Elsevier Science Publishers B.V.(Biomedical Division)
237
sites similar to leukotriene receptors, it is possible that lipoxins can affect PMNs in their production of lipoxygenase metabolites. LTB4 affects smooth muscle, cells with contractile capacity, and specifically stimulates a number of leukocyte functions, including the migration of leukocytes [15-17]. In this study, we endeavoured to determine the influence of LXA4 and B4 on the release of LTB4 by human peripheral blood-derived neutrophils in vitro. 3. Materials and Methods
3.1. Preparation of human P M N leukocytes Highly purified suspensions of PMNs were prepared from blood obtained from healthy donors and collected in polypropylene tubes (Falcon) containng sufficient heparin (Biochrom, Berlin, ER.G.) to obtain a final concentration of 20 U/ml. Tests were performed immediately after collection. PMN leukocytes were isolated as in ref. [18]. Briefly, whole blood was centrifuged over Ficoll-Hypaque (Pharmacia) in 2% dextran for 30 min at room temperature [18]. After washing the cells twice with buffered saline, the red cells were lysed by distilled water, the remaining cells were washed and resuspended in Gey's buffer (Eurobio, Paris) and viability examined by trypan blue-exclusion method. The cells were incubated at 5 × 106/ml at 37 °C in triplicate. The cell suspensions consisted of > 9 7 % PMN leukocytes. Prior to culture the cells were counted in a Biirker counting chamber. Samples were treated with LXA4 and B4 at different concentrations (10 -6, 10-9 M), the controls received methanol at the same concentration of LXA and B (10-9) and LTB4 was determined utilizing the conditions as described in ref. [19]. The incubations were performed in triplicate, and LTB4 was determined by radioimmunoassay of the resulting supernatants [19]. 3.2. Calcium ionophore A23187 Ionophore (Sigma, St. Louis, MO, U.S.A.) was dissolved in dimethylsulfoxide (DMSO) (Sigma, St. Louis, MO, U.S.A.) at 50 mg/ml. Dilutions of the ionophore were made directly in 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, 238
to determine non-specific formation or inhibition of LTB4 generation by the vehicle. The generation of LTB4 was assessed by radioimmunoassay as described [19]. Results are expressed as ng/ml. Each test and all controls were done in triplicate with each donor. 3.3. Tested drugs Nordihydroguaiaretic acid (NDGA; Sigma), was prepared in DMSO to a final concentration of 10/zM. The small amount of DMSO employed as a vehicle did not affect either cell viability or arachidonic acid product (LTB4) release.
3.4. Preparation of lipoxins LXA was a generous gift from Charles N. Serhan, Hematology Division, Harvard Medical School, Boston. LXB was a generous gift from Dr. J. Rokach, Merck Frosst Canada Inc. LXA was isolated from suspensions of mixed human leukocytes (i.e., neutrophils, eosinophils, basophils, etc.) as described [23]. H u m a n leukocytes prepared from healthy donors were warmed to 37 °C (200 ml of 100x 106 cells/ml). Next, 15-HETE (80-100/~M) and A23187 (2.5 #M) were added simultaneously in ethanol (1% final, v/v), and incubations were continued for 20 min. Incubations were stopped by addition of 2 vols. methanol, extracted with ether, and subjected to silicic acid chromatography. Materials present in the ethyl acetate fraction were combined with ll,12,15-trihydroxy-5,8,13-1-[14C]eicosatrienoate and ll,14,15-trihydroxy-5,8,12-eicosatrienoate, treated with diazomethane, and then subjected to thin-layer chromatography. Thin-layer chromatography was carried out with plates coated with silica gel G and ethyl acetate-2,2,4-trimethylpentane (5:1, v/v) as solvent. The regions containing methyl esters exhibiting tetraene UV spectra but no radiolabel were collected, and the material was eluted from the gel with methanol. Samples were pooled and injected onto a reverse-phase HPLC column Altex ultrasphere-ODS 10 mm × 25 cm eluted with 65:35 (v/v) at 30 ml/min, with the UV detector set at 301 nm. After re-chromatography in the same HPLC system, an aliquot of material was subjected to straight-phase HPLC employing a Bakerbond chiral column (J. T. Baker) with hexane/isopropanol
as solvent (90:10, v/v). Free acids were prepared by LiOH saponification and ether extraction followed by reverse-phase HPLC using M e O H / H 2 0 / a c e t i c acid (70:30:0.01, v/v). The final purity of L X A was assessed by reverse-phase HPLC and proved to be 95°70 L X A with 5o70 of its ll-trans-isomer (ll-transLXA). UV spectra of separated components were recorded in methanol with the use of a HewlettPackard 8450A spectrophotometer and an absorption coefficient of 50000 to determine concentrations. The compound was subjected to analysis by gas chromatography/mass spectrophotometry.
100 90 ~;80
Z
~4o 30' 2(>
101.5
3.
6.
; ,,
4. Results Lipoxin A4 and B4 are tetraenes formed by the transformation of 5 and 15-HETE by activated leukocytes [1, 2, 5] (Fig. 1). Before lipoxins were used in our studies a UV spectrum analysis was made for each compound to test the efficiency of these two new lipoxygenase products (Fig. 2). Human P M N leukocytes were pretreated for 15 min with LXA4 and LXB4 and assayed for LTB4 generation after treatment with calcium ionophore A23187 for 6 min. Fig. 3 shows the amounts of LTB4 (ng/ml)
~
H
112 TIME
(M IN.)
Fig. 2. UV spectrum of the conjugated tetraenes, lipoxin A4 and lipoxin B4, obtained from a spectrophotometer (Ultrospec II, LKB). All UV spectra were recorded in methanol.
released by human PMN cells after addition of calcium ionophore A23187 (5/zM) for various lengths of time (1.5, 3, 6 and 12 min). The amounts of LTB4 released by the PMNs were maximal at 3 and 6 min (52.0_+10.9 and 59.0+11.1, respectively) and minimum at 1.5 and 12 min (32.2_+9.6 and 37.3+12.2, respectively). The release of LTB4 at 12 min incubation was less; the neosynthesised LTB4 may have been retained by the PMNs and then subsequently metabolised via ~-oxydation to 20-hydroxy-LTB4
%.
OH 15- HETE
~
A
.
~).8 ~H
U H
Z
Elsevier IMLET 01391
Lipoxins A4 and B4 inhibit leukotriene B4 generation from human neutrophil leukocyte suspensions P i o C o n t i , M a r c e l l a Reale, R e n a t o C. B a r b a c a n e , M a u r o B o n g r a z i o a n d M a r i a R. P a n a r a Immunology Division, Institute of Experimental Medicine, University of Chieti Medical School, Chieti, Italy
(Received 5 March 1990; accepted 19 March 1990)
1. Summary
2. Introduction
Lipoxins A4 and B4 (5,6,15L-trihydroxy-7,9,11,13eicosatetraenoic and 5D,14,15-trihydroxy-6,8,10,12eicosatetraenoic acids, respectively) were examined in several biological systems and have proven to have many different activities from those o f other eicosanoids. Cultured human polymorphonuclear leukocytes were preincubated with LXA and B and their ability to inhibit leukotriene B4 generation was assessed after incubation with the calcium ionophore A23187. The preincubation time of neutrophils with lipoxin A4 and B4 was 15 min. After that time the cells were incubated for 6 min with A23187 (5/~M) for the release of LTB4. We found that the pretreatment of neutrophils with lipoxins inhibited the release of LTB4 by A23187-stimulated PMNs. Nordihydroguaiaretic acid (NDGA) (10/~M), used as a control, strongly inhibited the generation of LTB4. Since LTB4 has been shown to be a modulator of cellular immunity, our data suggest that lipoxin A4 and B4 can contribute to the immunosuppression via inhibition of LTB4 generation. Moreover, the inhibition of LTB4 by lipoxins in neutrophils could have an important regulatory role in inflammation.
Many different studies have suggested that initial lipoxygenation at C-15 can lead to the formation of substances of biological interest called lipoxins [1]. Lipoxins are trihydroxytetraenes, a novel series of oxygenated derivatives formed from arachidonic acid in human leukocytes [2]. The structures of the major compounds were established and proved to be 5S,6R,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid and 5D,14,15-trihydroxy-6,8,12-eicosatetraenoic acid. Recently, many isomers of LXA4 and B4 have been isolated, for example, 7-cis-ll-trans LXA4, l l - t r a n s LXA4, 6 - c i s - L X B 4 and 6-cis-8t r a n s - L X B 4 , and have been found to have opposite biological effects. 7-cis forms of LXA4 evoked a dose-dependent contraction o f guinea pig lung strip, while the 6-cis forms of LXB4 relaxed this preparation [3-5]. The addition of these compounds to human polymorphonuclear leukocytes or human natural killer ceils evokes selective responses different from those observed with leukotrienes [6-8]. In previous studies we have indicated that eicosanoids and their metabolites are involved in the function of leukocytes [9-13]. Recently we have found that lipoxin A and B play a pro-inflammatory role by increasing the granuloma formation in experimentally induced chronic inflammation [14]. In addition, we have also found that human PMN leukocytes exposed to LXA stimulate cyclooxygenase products, and that the addition of indomethacin to the cell suspensions blocks this formation. Based on these observations, and since certain isomers of LXA4 cause contraction of guinea pig lung strip, which seem to involve
Key words: Lipoxin; Leukotriene; Polymorphonuclear leuko-
cyte; Calcium ionophore Correspondence to: Dr. Pio Conti, Cattedra di Immunologia,
Istituto di Medicina Sperimentale, Universita' di Chieti, Via dei Vestini, 66100 Chieti, Italy. Abbreviations: LX, lipoxin; LT, leukotriene; PMN, polymor-
phonuclear; A23187, calcium ionophore; NDGA, nordihydroguaiaretic acid.
0165-2478 / 90 / $ 3.50 © 1990 Elsevier Science Publishers B.V.(Biomedical Division)
237
sites similar to leukotriene receptors, it is possible that lipoxins can affect PMNs in their production of lipoxygenase metabolites. LTB4 affects smooth muscle, cells with contractile capacity, and specifically stimulates a number of leukocyte functions, including the migration of leukocytes [15-17]. In this study, we endeavoured to determine the influence of LXA4 and B4 on the release of LTB4 by human peripheral blood-derived neutrophils in vitro. 3. Materials and Methods
3.1. Preparation of human P M N leukocytes Highly purified suspensions of PMNs were prepared from blood obtained from healthy donors and collected in polypropylene tubes (Falcon) containng sufficient heparin (Biochrom, Berlin, ER.G.) to obtain a final concentration of 20 U/ml. Tests were performed immediately after collection. PMN leukocytes were isolated as in ref. [18]. Briefly, whole blood was centrifuged over Ficoll-Hypaque (Pharmacia) in 2% dextran for 30 min at room temperature [18]. After washing the cells twice with buffered saline, the red cells were lysed by distilled water, the remaining cells were washed and resuspended in Gey's buffer (Eurobio, Paris) and viability examined by trypan blue-exclusion method. The cells were incubated at 5 × 106/ml at 37 °C in triplicate. The cell suspensions consisted of > 9 7 % PMN leukocytes. Prior to culture the cells were counted in a Biirker counting chamber. Samples were treated with LXA4 and B4 at different concentrations (10 -6, 10-9 M), the controls received methanol at the same concentration of LXA and B (10-9) and LTB4 was determined utilizing the conditions as described in ref. [19]. The incubations were performed in triplicate, and LTB4 was determined by radioimmunoassay of the resulting supernatants [19]. 3.2. Calcium ionophore A23187 Ionophore (Sigma, St. Louis, MO, U.S.A.) was dissolved in dimethylsulfoxide (DMSO) (Sigma, St. Louis, MO, U.S.A.) at 50 mg/ml. Dilutions of the ionophore were made directly in 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, 238
to determine non-specific formation or inhibition of LTB4 generation by the vehicle. The generation of LTB4 was assessed by radioimmunoassay as described [19]. Results are expressed as ng/ml. Each test and all controls were done in triplicate with each donor. 3.3. Tested drugs Nordihydroguaiaretic acid (NDGA; Sigma), was prepared in DMSO to a final concentration of 10/zM. The small amount of DMSO employed as a vehicle did not affect either cell viability or arachidonic acid product (LTB4) release.
3.4. Preparation of lipoxins LXA was a generous gift from Charles N. Serhan, Hematology Division, Harvard Medical School, Boston. LXB was a generous gift from Dr. J. Rokach, Merck Frosst Canada Inc. LXA was isolated from suspensions of mixed human leukocytes (i.e., neutrophils, eosinophils, basophils, etc.) as described [23]. H u m a n leukocytes prepared from healthy donors were warmed to 37 °C (200 ml of 100x 106 cells/ml). Next, 15-HETE (80-100/~M) and A23187 (2.5 #M) were added simultaneously in ethanol (1% final, v/v), and incubations were continued for 20 min. Incubations were stopped by addition of 2 vols. methanol, extracted with ether, and subjected to silicic acid chromatography. Materials present in the ethyl acetate fraction were combined with ll,12,15-trihydroxy-5,8,13-1-[14C]eicosatrienoate and ll,14,15-trihydroxy-5,8,12-eicosatrienoate, treated with diazomethane, and then subjected to thin-layer chromatography. Thin-layer chromatography was carried out with plates coated with silica gel G and ethyl acetate-2,2,4-trimethylpentane (5:1, v/v) as solvent. The regions containing methyl esters exhibiting tetraene UV spectra but no radiolabel were collected, and the material was eluted from the gel with methanol. Samples were pooled and injected onto a reverse-phase HPLC column Altex ultrasphere-ODS 10 mm × 25 cm eluted with 65:35 (v/v) at 30 ml/min, with the UV detector set at 301 nm. After re-chromatography in the same HPLC system, an aliquot of material was subjected to straight-phase HPLC employing a Bakerbond chiral column (J. T. Baker) with hexane/isopropanol
as solvent (90:10, v/v). Free acids were prepared by LiOH saponification and ether extraction followed by reverse-phase HPLC using M e O H / H 2 0 / a c e t i c acid (70:30:0.01, v/v). The final purity of L X A was assessed by reverse-phase HPLC and proved to be 95°70 L X A with 5o70 of its ll-trans-isomer (ll-transLXA). UV spectra of separated components were recorded in methanol with the use of a HewlettPackard 8450A spectrophotometer and an absorption coefficient of 50000 to determine concentrations. The compound was subjected to analysis by gas chromatography/mass spectrophotometry.
100 90 ~;80
Z
~4o 30' 2(>
101.5
3.
6.
; ,,
4. Results Lipoxin A4 and B4 are tetraenes formed by the transformation of 5 and 15-HETE by activated leukocytes [1, 2, 5] (Fig. 1). Before lipoxins were used in our studies a UV spectrum analysis was made for each compound to test the efficiency of these two new lipoxygenase products (Fig. 2). Human P M N leukocytes were pretreated for 15 min with LXA4 and LXB4 and assayed for LTB4 generation after treatment with calcium ionophore A23187 for 6 min. Fig. 3 shows the amounts of LTB4 (ng/ml)
~
H
112 TIME
(M IN.)
Fig. 2. UV spectrum of the conjugated tetraenes, lipoxin A4 and lipoxin B4, obtained from a spectrophotometer (Ultrospec II, LKB). All UV spectra were recorded in methanol.
released by human PMN cells after addition of calcium ionophore A23187 (5/zM) for various lengths of time (1.5, 3, 6 and 12 min). The amounts of LTB4 released by the PMNs were maximal at 3 and 6 min (52.0_+10.9 and 59.0+11.1, respectively) and minimum at 1.5 and 12 min (32.2_+9.6 and 37.3+12.2, respectively). The release of LTB4 at 12 min incubation was less; the neosynthesised LTB4 may have been retained by the PMNs and then subsequently metabolised via ~-oxydation to 20-hydroxy-LTB4
%.
OH 15- HETE
~
A
.
~).8 ~H
U H
Z
