Original Paper Int Arch Allergy Immunol 1992;98:361-369
Research Institute for Diseases of the Chest, Faculty o f Medicine, Kyushu University, Fukuoka, Japan
Key Words Human PMN Lipoxygenase metabolites Superoxide anion 5-Lo inhibitor (AA861) 15-HETE Arachidonic acid C5a Negative feed-back
Effects of a 5-Lipoxygenase Inhibitor, AA861, on Lipoxygenase Metabolism and Superoxide Anion Generation by Human Polymorphonuclear Leukocytes Potentiation of Superoxide Anion Generation by LTB4
Abstract We studied the influence of a selective 5-lipoxygenase inhibitor, AA861, on the generation of the superoxide anion (O5) and the lipoxygenase metabolites by human polymorphonuclear leukocytes (PMN). PMN produce O3 in a dosedependent manner following stimulation with arachidonic acid (AA), leukotriene B4 (LTB4), or C5a. When PMN were stimulated with one of those three agents in the presence of high doses of AA861 (1-10 pg/ml), a significant reduc tion of O3 release was observed. In contrast, the generation of O5by PMN stim ulated by LTB4 was potentiated at lower concentrations of AA861 (0.025-0.25 pg/ml). However, stimulation with AA or C5a did not influence O2 generation in the presence of AA861 at the same concentration range. Furthermore, treat ing the PMN with the cyclooxygenase inhibitor, acetylsalicylic acid, did not po tentiate the generation of Oj by stimulation with LTB4 over a wide range of concentrations. Quantification of lipoxygenase metabolites by reverse-phase high-performance liquid chromatography revealed that a high concentration of AA861 (0.5-5 pg/ml) completely inhibited the production of LTB4 and its omega-oxidative metabolites by PMN following stimulation with 100 pM AA, but only partially inhibited that of 5-hydroxyeicosatetraenoic acid (5-HETE). AA861 at a concentration of 5 pg/ml significantly increased the production of 15-HETE by PMN following the same stimulation. AA861 did not influence ca tabolism of LTB4 added to the reaction mixture to its omega-oxidative prod ucts by PMN over a wide range of concentrations. These findings suggest that the inhibition of 5-lipoxygenase metabolism may stimulate 15-lipoxygenase in human PMN. The potentiation of Oi production by stimulation with LTB4, but not by AA or C5a, may indicate an interaction between arachidonate lipoxyge nase metabolism and O2 release at the LTB4 receptor level.
Correspondence lo: Dr. Masayoshi Abe Research Institute for Diseases of the Chest Faculty of Medicine, Kyushu University 3-1*1 Maidashi, Higashi-ku Fukuoka 812 (Japan)
© 1992S. Karger AG, Basel 1018— 2438/92/0984—0361 $ 2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/3/2018 7:16:42 AM
Kiyoko Tanaka Masayoshi Abe Nobuaki Shigematsu
Introduction Human polymorphonuclear leukocytes (PMN) contain 5-lipoxygcnase and 15-lipoxygcnasc activity [1, 2]. When the arachidonic acid is the substrate of 5-lipoxygcnasc re action in PMN, various metabolites can be synthesized in cluding sulfidopeptide leukotrienes, leukotriene B4 and 5-hydroxyeicosatetraenoic acid (5-HETE) [3], Eosinophil leukocytes generate mainly sulfidopeptide leukotrienes (LTs), while neutrophil leukocytes produce mainly LTB4, via the unstable intermediate LTA4, through the terminal enzyme activities, LTC4 synthase and LTA4 hydrolase, re spectively [4, 5], The generated sulfidopeptide LTs pro duce smooth muscle contraction and plasma leakage [6]. LTB4 shows a potent chemotactic activity for PMN and monocytes, and for the release of lysosomal enzymes and Superoxide anion [7, 8]. A variety of agents that act on arachidonate metabo lism have been synthesized, and their pharmacological role and possible adverse reactions have been investigated [9-11], A potent, selective 5-lipoxygcnase inhibitor, 2-(12hydroxydodeca-5,10-diynyl)-3,5,6-trimcthyl-l,4-benzoquinone (AA861), has been evaluated extensively in various experimental models [12-14], Eicosanoids and oxygen radicals, which are important mediators released by PMN, may play a role in defending against infection and may also contribute to the acute in flammatory reaction [15], To see whether there is any in teraction between arachidonate metabolism and superox ide anion release by PMN, in the present study we exam ined the effects of AA861 on arachidonate lipoxygenase metabolism and the release of superoxide anion by poly morphonuclear leukocytes from humans.
PMN Preparations Heparinized venous blood was drawn from at least 20 healthy sub jects and exposed to dextran sedimentation for 30 min. The leuko cyte-rich supernatant was then collected and layered on an LSM. Fol lowing centrifugation for 30 min at 4(H)g, the pellet was washed with Ilepes buffer. To lyse the erythrocytes, the cells were exposed to dis tilled water for 20 s, and then an equal volume of 1.8% saline was add ed. Following centrifugation, the polymorphonuclear leukocytes (PMN) recovered in the pellet were resuspended in Hepes buffer. The numcr of cells was counted and their viability was determined to be at least 90% in the presence o f 0.1% trypan blue. Superoxide Anion Production by PMN Superoxidc anion ( O j ) was measured by the superoxide dismutase inhibitable fcrricytochrome C reduction method using arachi donic acid (10-100 pM), human C5a (0.05-5 pg/ml) or leukotriene B4 (0.15-1.5 \\M) as the triggers [16). Briefly, the reaction mixtures (total volume = 1 ml), containing 4 x IIP PMN and 100 \xM ferricytochrome C (type VI, Sigma) with or without drugs in the Hepes buffer were preincubatcd at 3 7 °C for 7 min. The stimulant was then added to the reaction mixture. The change in absorbancy at 550 nm referenced to that o f 540 nm was followed on a recorder attached to a Hitachi 556 double-beam spectrophotometer. The release o f superoxidc was de termined using a molar absorption coefficient for reduced-minusoxidized ferricytochrome C o f 19.1 x 105 Af 'x c n r 1. The maximum rate o f O, production was expressed as nmol cytochrome C rcduccd/4 x 105 PMN/min by using the initial rate of the reaction. Generation o f Lipoxygenase Products from PMN Each tube containing 5.0 x 106 PMN was preincubated at 37 °C for 3 min in the absence or presence o f the test agent. After adding the calcium ionophore, A23187 (final concentration = 10 pM) or arachi donic acid (final concentration = 100 pAi, less than 0.5% ethanol), each tube (total volume = 650 pi) was further incubated at 37 °C for 15 min. The reaction was terminated by adding 520 pi o f cold isopro panol. Next, 100 pmol o f prostaglandin (PGB,) were added to each tube as the internal standard. Lipoxygenase products were extracted by diethyl ether following acidification with formic acid as reported previously [16]. The recovery rates o f LTs. FIETEs. and PGB, ob tained by ether extraction all exceeded 85%. The ether was evap orated and the residue was resuspended in 100 pi of solvent (A). An aliquot o f 50 pi was injected onto the Novapak C18 column for re verse-phase high-performance liquid chromatography (RP-HPLC).
Materials and Methods
362
HPLC fo r Lipoxygenase Products The HPLC system consisted o f a model 510 pump, a U-6K in jector, and models 441 and 490 absorbance detectors (Waters Associ ates). We used a Novapak C18, 5-pm column (Waters Associates, 0.39x15 cm). As solvent (A) we used acetonitrile/methanol/water/ acetic acid (29:6:65:1 v/v), solvent (B) was 33.6:5.4:61:) (v/v), and solvent (C) 50:5.4:44.6:0.73, (v/v) [17.18]. All solvents were adjusted to pH 5.6 with tricthylaminc (Sigma). The mobile phase started at solvent (A) and was changed to solvent (B) at 2 min. Solvent (B) was changed to solvent (C) at 27 min. The flow rate was I ml/min, and elu tion was monitored simultaneously at 280 and 235 nm. Retention times for LTC4. D4, PGB,, LTE4, LTB„ 15-, 12-, and 5-HETE were ap proximately 11.5, 13.5,15.5,16,23,41.5,44.5, and 45.5 min, respectively. The other LT metabolites, i.e., the w-COOH-, w-OH-LTB4, 6-transand l2-cpi-6-/ram-LTB4 diastereoisomers, were eluted at approxi mately 2.1, 3.5, 20, and 21 min, respectively. These metabolites were
Tanaka/Abe/Shigematsu
AA86I and Potentiated O; Generation by LTB4
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/3/2018 7:16:42 AM
Materials The following agents were used: LSM (lymphocyte separation medium; Organon Teknika, N.C.), calcium ionophore A23187 (Calbiochem, Calif.), L-glutamine (Wako Chem. Kyoto), cytochrome c (Sigma, SL), cytochalasine E (Sigma, SL), arachidonic acid (Nakarai Chem. Kyoto), and prostaglandin B, (Sigma, SL). Standard LTC4, D4, E4, B4, 5-, 12-, and 15-HETEs were gifts from the Ono Pharmaceutical Company (Osaka, Japan). AA861 [2- (12-hydroxydodeca - 5,10 - diynyl)-3,5.6-trimethyl -1.4-benzoquinone]waskindlysuppliedbyTakeda Chemical Industry (Osaka , Japan). Purified human C5a was a gift o f Dr. Tony E. Hugh (Research Institute of Scripps Clinic, La Jolla, Calif.). All the organic solvents used were of HPLC quality; the water was distilled and filtered using Puric-R (Organo, Japan).
Table 1. Production of lipoxygenase m e tabolites by human PMN stimulated with 10 \iM A 23187 or 100 \iM A A at 37 °C for 15 min
Agent and donor
A23187 Case 1 Case 2 Case 3
15-HETE
w-COOHltb4
w-OHltb4
ltb4
5-HETE
37 12 35
73 40 90
62 48 76
150 87 147
0 0 0
5 9 17
8 10 30
13 10 53
62 84 284
22 53 53
AA Case 1 Case 2 Case 3
PMN from the same 3 donors were stimulated by the calcium ionophore A23187 or AA. After extraction o f the incubation mixtures, lipoxygenase metabolites were separated on RP-HPLC and quantitated by peak area ratios between PGB, and each metabolite. Each number is denoted as p m o l/5 x l0 6 PMN/15 min. Same case number indicates the same do nor.
identified on the basis of (a) comigration with authentic standards and (b) specificity of UV absorbance. Quantification o f LTs and HETEs was based on the peak area ra tios between PGB, (internal standard) monitored at 280 nm and LTs at 280 nm, or HETEs at 235 nm. Statistical analysis was performed using the Student t test. The level of significance was considered to be p
Research Institute for Diseases of the Chest, Faculty o f Medicine, Kyushu University, Fukuoka, Japan
Key Words Human PMN Lipoxygenase metabolites Superoxide anion 5-Lo inhibitor (AA861) 15-HETE Arachidonic acid C5a Negative feed-back
Effects of a 5-Lipoxygenase Inhibitor, AA861, on Lipoxygenase Metabolism and Superoxide Anion Generation by Human Polymorphonuclear Leukocytes Potentiation of Superoxide Anion Generation by LTB4
Abstract We studied the influence of a selective 5-lipoxygenase inhibitor, AA861, on the generation of the superoxide anion (O5) and the lipoxygenase metabolites by human polymorphonuclear leukocytes (PMN). PMN produce O3 in a dosedependent manner following stimulation with arachidonic acid (AA), leukotriene B4 (LTB4), or C5a. When PMN were stimulated with one of those three agents in the presence of high doses of AA861 (1-10 pg/ml), a significant reduc tion of O3 release was observed. In contrast, the generation of O5by PMN stim ulated by LTB4 was potentiated at lower concentrations of AA861 (0.025-0.25 pg/ml). However, stimulation with AA or C5a did not influence O2 generation in the presence of AA861 at the same concentration range. Furthermore, treat ing the PMN with the cyclooxygenase inhibitor, acetylsalicylic acid, did not po tentiate the generation of Oj by stimulation with LTB4 over a wide range of concentrations. Quantification of lipoxygenase metabolites by reverse-phase high-performance liquid chromatography revealed that a high concentration of AA861 (0.5-5 pg/ml) completely inhibited the production of LTB4 and its omega-oxidative metabolites by PMN following stimulation with 100 pM AA, but only partially inhibited that of 5-hydroxyeicosatetraenoic acid (5-HETE). AA861 at a concentration of 5 pg/ml significantly increased the production of 15-HETE by PMN following the same stimulation. AA861 did not influence ca tabolism of LTB4 added to the reaction mixture to its omega-oxidative prod ucts by PMN over a wide range of concentrations. These findings suggest that the inhibition of 5-lipoxygenase metabolism may stimulate 15-lipoxygenase in human PMN. The potentiation of Oi production by stimulation with LTB4, but not by AA or C5a, may indicate an interaction between arachidonate lipoxyge nase metabolism and O2 release at the LTB4 receptor level.
Correspondence lo: Dr. Masayoshi Abe Research Institute for Diseases of the Chest Faculty of Medicine, Kyushu University 3-1*1 Maidashi, Higashi-ku Fukuoka 812 (Japan)
© 1992S. Karger AG, Basel 1018— 2438/92/0984—0361 $ 2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/3/2018 7:16:42 AM
Kiyoko Tanaka Masayoshi Abe Nobuaki Shigematsu
Introduction Human polymorphonuclear leukocytes (PMN) contain 5-lipoxygcnase and 15-lipoxygcnasc activity [1, 2]. When the arachidonic acid is the substrate of 5-lipoxygcnasc re action in PMN, various metabolites can be synthesized in cluding sulfidopeptide leukotrienes, leukotriene B4 and 5-hydroxyeicosatetraenoic acid (5-HETE) [3], Eosinophil leukocytes generate mainly sulfidopeptide leukotrienes (LTs), while neutrophil leukocytes produce mainly LTB4, via the unstable intermediate LTA4, through the terminal enzyme activities, LTC4 synthase and LTA4 hydrolase, re spectively [4, 5], The generated sulfidopeptide LTs pro duce smooth muscle contraction and plasma leakage [6]. LTB4 shows a potent chemotactic activity for PMN and monocytes, and for the release of lysosomal enzymes and Superoxide anion [7, 8]. A variety of agents that act on arachidonate metabo lism have been synthesized, and their pharmacological role and possible adverse reactions have been investigated [9-11], A potent, selective 5-lipoxygcnase inhibitor, 2-(12hydroxydodeca-5,10-diynyl)-3,5,6-trimcthyl-l,4-benzoquinone (AA861), has been evaluated extensively in various experimental models [12-14], Eicosanoids and oxygen radicals, which are important mediators released by PMN, may play a role in defending against infection and may also contribute to the acute in flammatory reaction [15], To see whether there is any in teraction between arachidonate metabolism and superox ide anion release by PMN, in the present study we exam ined the effects of AA861 on arachidonate lipoxygenase metabolism and the release of superoxide anion by poly morphonuclear leukocytes from humans.
PMN Preparations Heparinized venous blood was drawn from at least 20 healthy sub jects and exposed to dextran sedimentation for 30 min. The leuko cyte-rich supernatant was then collected and layered on an LSM. Fol lowing centrifugation for 30 min at 4(H)g, the pellet was washed with Ilepes buffer. To lyse the erythrocytes, the cells were exposed to dis tilled water for 20 s, and then an equal volume of 1.8% saline was add ed. Following centrifugation, the polymorphonuclear leukocytes (PMN) recovered in the pellet were resuspended in Hepes buffer. The numcr of cells was counted and their viability was determined to be at least 90% in the presence o f 0.1% trypan blue. Superoxide Anion Production by PMN Superoxidc anion ( O j ) was measured by the superoxide dismutase inhibitable fcrricytochrome C reduction method using arachi donic acid (10-100 pM), human C5a (0.05-5 pg/ml) or leukotriene B4 (0.15-1.5 \\M) as the triggers [16). Briefly, the reaction mixtures (total volume = 1 ml), containing 4 x IIP PMN and 100 \xM ferricytochrome C (type VI, Sigma) with or without drugs in the Hepes buffer were preincubatcd at 3 7 °C for 7 min. The stimulant was then added to the reaction mixture. The change in absorbancy at 550 nm referenced to that o f 540 nm was followed on a recorder attached to a Hitachi 556 double-beam spectrophotometer. The release o f superoxidc was de termined using a molar absorption coefficient for reduced-minusoxidized ferricytochrome C o f 19.1 x 105 Af 'x c n r 1. The maximum rate o f O, production was expressed as nmol cytochrome C rcduccd/4 x 105 PMN/min by using the initial rate of the reaction. Generation o f Lipoxygenase Products from PMN Each tube containing 5.0 x 106 PMN was preincubated at 37 °C for 3 min in the absence or presence o f the test agent. After adding the calcium ionophore, A23187 (final concentration = 10 pM) or arachi donic acid (final concentration = 100 pAi, less than 0.5% ethanol), each tube (total volume = 650 pi) was further incubated at 37 °C for 15 min. The reaction was terminated by adding 520 pi o f cold isopro panol. Next, 100 pmol o f prostaglandin (PGB,) were added to each tube as the internal standard. Lipoxygenase products were extracted by diethyl ether following acidification with formic acid as reported previously [16]. The recovery rates o f LTs. FIETEs. and PGB, ob tained by ether extraction all exceeded 85%. The ether was evap orated and the residue was resuspended in 100 pi of solvent (A). An aliquot o f 50 pi was injected onto the Novapak C18 column for re verse-phase high-performance liquid chromatography (RP-HPLC).
Materials and Methods
362
HPLC fo r Lipoxygenase Products The HPLC system consisted o f a model 510 pump, a U-6K in jector, and models 441 and 490 absorbance detectors (Waters Associ ates). We used a Novapak C18, 5-pm column (Waters Associates, 0.39x15 cm). As solvent (A) we used acetonitrile/methanol/water/ acetic acid (29:6:65:1 v/v), solvent (B) was 33.6:5.4:61:) (v/v), and solvent (C) 50:5.4:44.6:0.73, (v/v) [17.18]. All solvents were adjusted to pH 5.6 with tricthylaminc (Sigma). The mobile phase started at solvent (A) and was changed to solvent (B) at 2 min. Solvent (B) was changed to solvent (C) at 27 min. The flow rate was I ml/min, and elu tion was monitored simultaneously at 280 and 235 nm. Retention times for LTC4. D4, PGB,, LTE4, LTB„ 15-, 12-, and 5-HETE were ap proximately 11.5, 13.5,15.5,16,23,41.5,44.5, and 45.5 min, respectively. The other LT metabolites, i.e., the w-COOH-, w-OH-LTB4, 6-transand l2-cpi-6-/ram-LTB4 diastereoisomers, were eluted at approxi mately 2.1, 3.5, 20, and 21 min, respectively. These metabolites were
Tanaka/Abe/Shigematsu
AA86I and Potentiated O; Generation by LTB4
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/3/2018 7:16:42 AM
Materials The following agents were used: LSM (lymphocyte separation medium; Organon Teknika, N.C.), calcium ionophore A23187 (Calbiochem, Calif.), L-glutamine (Wako Chem. Kyoto), cytochrome c (Sigma, SL), cytochalasine E (Sigma, SL), arachidonic acid (Nakarai Chem. Kyoto), and prostaglandin B, (Sigma, SL). Standard LTC4, D4, E4, B4, 5-, 12-, and 15-HETEs were gifts from the Ono Pharmaceutical Company (Osaka, Japan). AA861 [2- (12-hydroxydodeca - 5,10 - diynyl)-3,5.6-trimethyl -1.4-benzoquinone]waskindlysuppliedbyTakeda Chemical Industry (Osaka , Japan). Purified human C5a was a gift o f Dr. Tony E. Hugh (Research Institute of Scripps Clinic, La Jolla, Calif.). All the organic solvents used were of HPLC quality; the water was distilled and filtered using Puric-R (Organo, Japan).
Table 1. Production of lipoxygenase m e tabolites by human PMN stimulated with 10 \iM A 23187 or 100 \iM A A at 37 °C for 15 min
Agent and donor
A23187 Case 1 Case 2 Case 3
15-HETE
w-COOHltb4
w-OHltb4
ltb4
5-HETE
37 12 35
73 40 90
62 48 76
150 87 147
0 0 0
5 9 17
8 10 30
13 10 53
62 84 284
22 53 53
AA Case 1 Case 2 Case 3
PMN from the same 3 donors were stimulated by the calcium ionophore A23187 or AA. After extraction o f the incubation mixtures, lipoxygenase metabolites were separated on RP-HPLC and quantitated by peak area ratios between PGB, and each metabolite. Each number is denoted as p m o l/5 x l0 6 PMN/15 min. Same case number indicates the same do nor.
identified on the basis of (a) comigration with authentic standards and (b) specificity of UV absorbance. Quantification o f LTs and HETEs was based on the peak area ra tios between PGB, (internal standard) monitored at 280 nm and LTs at 280 nm, or HETEs at 235 nm. Statistical analysis was performed using the Student t test. The level of significance was considered to be p
