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Azelastine hydrochloride inhibits platelet activating factor-like activity in human eosinophils
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 217-221 © PearsonProfessionalLtd 1996
Azelastine hydrochloride inhibits platelet activating factor-like activity in human eosinophils Kunihiko Shindo, Motonori Fukumura First Department of Internal Medicine, Yokohama City University School of Medicine, 3-9, Fukuura, Kanazawa-ku, Yokohama 236, Japan.
Summary We investigated the inhibitory effect of azelastine hydrochloride (azelastine), an anti-asthmatic drug, on platelet-activating factor (PAF)-Iike activity in eosinophils obtained from asthmatic and non-asthmatic patients. Eosinophils were preincubated with or without azelastine and stimulated with f-Met-Leu-Phe (fMLP, 10 lamol) for 15 min. PAr-like activity was detected by aggregation of washed guinea-pig platelets. PAr-like activity released from asthmatic eosinophils without preincubation of azelastine was 2.36 [1.02] (mean [SD], rig/107 cells) in supernatants and 13.87 [4.77] in cell pellets. After preincubation with 10-8, 10~, and 10-4 M azelastine, PAF-like activity reduced to 1.85 [0.46] (mean [SD], ng/107 cells), 1.11 [0.14], and 0.88 [0.09] (n = 15) in the supernatants, and 11.83 [2.93], 8.32 [1.41], and 6.27 [1.25] (n = 15) in the cell pellets, respectively. PAr-like activity in non-asthmatic eosinophils without preincubation of azelastine was 2.01 [0.86] (mean [SD], ng/107 cells) in supernatants and 7.44 [0.99] in cell pellets. After preincubation with 10-8, 10-e, and 10-4 M azelastine, PAr-like activity reduced to 1.73 [0.64] (mean [SD], rig/107 cells), 1.12 [0.23], and 0.84 [0.17] (n = 20) in the supernatants, and 6.26 [2.08], 4.65 [0.88], and 3.02 [0.43] (n = 20) in the cell pellets, respectively. Our results showed that preincubation with azelastine caused a dosedependent inhibition of intra and extracellular PAr-like activity from asthmatic and non-asthmatic eosinophils in the same manner.
INTRODUCTION Azelastine hydrochrolide (azelastine), (4-[p-chlorobenzyl]2- [ h e x a h y d r o - l - m e t h y l - 1 H-az epine-4-yl]- 1 -[2/4]phthalazinone hydrochloride), a H1 histamine receptor antagonist, is an orally effective and long-acting antiallergic agent. 1-3 This drug not only strongly inhibits the release of leukotrienes (LTs) from neutrophils and eosinophils, but also antagonizes these substances. Azelastine also inhibits and antagonizes chemical transmitters, such as histamine, released during allergic reactions in experiments using rats and rabbits. 4,5 An in vitro study has shown that azelastine inhibits PAF-induced paw edema in rats, PAF-induced platelet aggregation in platelet-rich human plasma, and PAFinduced bronchoconstriction in anesthetized guinea-
Received 1 December Accepted 21 December Correspondence to: Kunihiko Shindo, Tel. 045-787-2511; Fax. 045-787-2509.
pigs. 6 However, the effects of azelastine on the release of platelet-activating factor (PAF) from eosinophils obtained from patients with bronchial asthma are not known. We attempted to elucidate the regulatory effect of azelastine on human eosinophil function by investigating the effect of azelastine on PAF-like activity in eosinophils obtained from patients with bronchial asthma. MATERIALS AND METHODS Eosinophil separation Eosinophils were separated from a mixture of eosinophils and granulocytes by two subsequent centrifugations of a granulocyte preparation in isotonic Percoll solutions with densities of 1.082 and 1.085 g/ml at 1000 x gfor 10 min at room temperature. Over 98% pure neutrophfls appeared at the top of the 1.082 g/ml Percoll solution, while the eosinophils and some neutrophils were present as a small pellet on the bottom. After resuspension of the eosinophfl pellet in a 1.085 g/ml Percoll solution and centrifugation 217
218
Shindo & Fukumura
at 1000 x g for 10 min at room temperature, over 90% pure eosinophfls appeared on the bottom of the tube. Recovery rate for eosinophils was 67.41 [10.31% (mean [SD], n = 12). More than 95% cell viability was confirmed in all experiments by the trypan blue dye exclusion method.7,8 Human eosinophils and neutrophfls were isolated from the peripheral blood of 35 different donors, none of whom were ingesting aspirin, other non-steroidal antiinflammatory drugs, or corticosteroids. 15 donors were diagnosed as having bronchial asthma (atopic type); their peripheral eosinophfl levels ranged from 7.7 to 25% (mean [SD] of 14 [7]0/0). These eosinophils were used for asthmatic eosinophils in this study. 20 donors without diagnosed underlying disorders had 4.3 [1.21 (mean [SD])% eosinophilia. These eosinphils were used for non-asthmatic eosinophils.
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Cells obtained from asthmatic patients and nonasthmatic patients were divided into four groups. Three groups of cells were preincubated with 10-8, 10-6, or 10-4 tool of azelastine for 15 rain, washed three times with Hanks' balanced salt solution without calcium or magnesium (HBSS), and suspended at a density 10 z cells in 500 gl of modified Tyrode's buffer, pH 7.8, containing O.1% gelatin, 1 mM calcium, 5 mM magnesium, 0.3 mM potassium, and 20ram L-serine, and then stimulated with N-formyl-L-methionyl-L-leucyl-~-phenylalanine (f-MLP) (10 gM) for 15 rain at 37°C. The remaining groups of cells obtained from asthmatic patients and non-asthmatic patients served as controls.
it was serially diluted until no activity was detected. Aggregation curves obtained with the synthetic AGEPC were determined by plotting the concentration (ng/ml) of synthetic AGEPC against the percent platelet aggregation (Fig. 1)?°
Measurement of PAF-like activity
Extraction
PAF-like activity was measured by platelet aggregation using washed guinea-pig platelets. Guinea-pig platelets were isolated by a modification of the Ficoll-Paque separation technique described by Pinckard et alY Synthetic compounds containing known amounts of authentic 1O-hexadecyl-2-acetyt-sn-glycero-3-phosphocholine (AGEPC; hexadecyl PAF) were dispersed in a mixture containing 0.9% sodium chloride and 0.25% bovine serum albumin to yield a phospholipid concentration of 10-2 to 10-3M. To initiate the aggregation process, a 10 gl sample was added to 100 gl of washed guinea-pig platelets (1.25 x 109 cells/ml) suspended in Tyrode's buffer solution, pH 6.5, and then mixed with 400 gl of Tyrode's gelatin mixture, pH 7.2, containing 1.33 mM calcium chloride. The reaction was started by stirring at 9000 rpm at 37°C. The change in light transmission through the suspension was monitored for 5 to 10 min with an aggregometer. If the sample showed aggregating activity,
After stopping the cell incubation, the samples were extracted according to the method of Nigh and Dyer. I1 1 ml each of chloroform and water were added to the supernatant or the cell pellet fraction, and vortexed. The chloroform phase was removed after centrifuging the mixture at 2000 x gfor 4 min, and 2 ml of chloroform was added to the water methanol phase. After vortexing, the mixture was centrifuged at 2000 x g for 2 min. The chloroform phase was removed and combined with the previous chloroform phase. The extract was applied to SEP-PAK silica column once and eluted with chloroform and methanol. The elute was applied to silica 60 TLC plate and AGEPCs were separated with CHC13:CH3OH:H20. The AGEPCs fraction was reextracted according to Bligh and Dyer as described above. The chloroform phase was evaporated under a nitrogen stream and the residue was resuspended in 0.2 ml Tris buffer solution containing (0.25% w/v) bovine serum albumin (Tris-BSA). 12
%
aggregation
of
washed guinea pig platelets
Fig. 1 Graphic representation of aggregation assay for hexadecyl PAF; Plot of molarity of hexadecyl PAF versus percent aggregation (change in transmittance). A reasonable evaluation of the concentration of an unknown PAF-like activity can be measured by the curve.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 217-221
© Pearson Professional Ltd 1996
Inhibitory effect of azelastine on PAF activity
219
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Fig. 2 Changes of aggregation curves induced by intracellular PAF-like activity in asthmatic eosinophils stimulated with fMLP (10 gM) are demonstrated: (1) control (without preincubation of azelastine); (2) intracellular PAF-like activity in stimulated asthmatic eosinophils after preincubation with 104 M of azelastine; (3) intracetlular PAF-like activity after preincubation with 10-e M of azelastine; (4) intracellular PAF-like activity after preincubation with 10-~ M of azelastine. The heights in vertical lines, which mean % aggregation of washed guinea pig platelets (transmittance of a piatelet suspension), were reduced with increasing concentrations of azelastine used in preincubations.
Materials
The synthetic AGEPC was obtained from Sigma Chemical Japan (Tokyo, Japan). Azelastine hydrochloride was obtained from Eisai Pharmaceutical Co. (Tokyo, Japan). Statistical analysis Values are expressed as m e a n + / - SD or m e d i a n v a l u e
and range. PAT-like activity induced by preincubation with azelastine was analyzed by one-way ANOVA with repeated measures and Scheffe's F-test Differences between means were analyzed by a two-tailed paired t-test confirming to normal distribution. A P value of < 0.05 was considered significant. RESULTS
Figure 2 demonstrates changes of aggregation curves induced by intracellular PAT-like activity in asthmatic eosinophils preincubated with 10-8, 10-6, and 10-4M azelastine. The light transmissions decreased with the dose of azelastine. PAF-like activity released from asthmatic eosinophfls without preincubation of azelastine was 2.36 [1.02] (mean [SD], ng/107 cells) in supematants and 13.87 [4.77] in cell pellets. After preincubation with 10-8, 10-*, and lO-4M azelastine, PAT-like activity reduced to 1.85 [0.46](mean [SD], ng/lO z cells), 1.11 [0.14], and 0.88 [0.09] in the supematants, and 11.83 [2.93], 8.32 [1.41], and 6.27 [1.25] (n = 15) in the cell pellets, respectively. PAT-like activity in non-asthmatic eosinophfls without preincubation of azelastine was 2.01 [0.86](mean [SD], ng/10 z cells) in supernatants and 7.44 [0.99] in cell pellets. After preincubation with 10-8, 10-6, and 10-4 M azelastine, PAT-like activity reduced to 1.73 [0.64](mean [SD], ng/10 z cells), 1.12 [0.23], and 0.84 [0.171 in the supernatants, and 6.26 [2.08], 4.65 [0.88], and 3.02 [0.43] (n = 20) in the cell pellets, respectively. © Pearson Professional Ltd 1996
Azelastine at concentrations above 10-8 M caused dosedependent inhibitions in platelet aggregation in the supernatants and the cell pellets obtained from asthmatic (Fig. 3a) and non-asthmatic eosinophfls (Fig. 3b) in the same manner. There was no significant difference in the inhibitory effect between the two groups. The PAT-like activity in stimulated asthmatic eosinophils not preincubated with azelastine was significantly higher than that in stimulated non-asthmatic eosinophils not preincubated with azelastine (P < 0.05). Azelastine achieved ICs0 at a concentration of 10-6 M for both intracellular and extracellular PAF-like activity in asthmatic and non-asthmatic eosinophils. The platelet aggregation by hexadecyl-PAF (10-z M) in the presence of azelastine (10 -4 M) (a) was reduced by 7.1 (1.5-8.2)% (median (range)) (n = 4) relative to the aggregation in the absence of azelastine (b), as shown in Figure 4. Thus, azelastine itself had only a slight inhibitory effect on platelet aggregation induced by authentic hexadecylPAF. However, the reduction induced by azelastine itself was significantly smaller than that produced by supernatant and cell-associated PAF derived from f-MLPstimulated cells after preincubation of azelastine (10 -4 M).
DISCUSSION
Azelastine inhibited f-MLP-induced PAT-like activity in human asthmatic and non-asthmatic eosinophils in a dose-dependent manner. There is no significant difference in the inhibitory effect between asthmatic and nonasthmatic eosinophfls. PAT-like activity was significantly higher in f-MLP-stimulated asthmatic eosinophils than in non-asthmatic eosinophils. Although mass spectrometric analysis 13-~5provides the most persuasive evidence of PAT presence, its routine application is not practicable due to the tedious procedure of analysis, the great skill demanded of the operator and the high cost of the equipment. A new method for
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 217-221
220
Shindo & Fukumura
(a) 80
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Fig. 4 The platelet aggregation by hexadecyI-PAF (10-7 M) in the presence of azelastine (10-~ M) (a) and in the absence of azelastine (b).
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Fig, 3 Effects of azelastJne on f-MLP-induced intracellular (cell pellet) and extracellular (supernatant) PAF-like activity in human asthmatic (a) and non-asthmatic (b) eosJnophils. Data represent the means+/-SD of 6 (a) and 10 (b) separate experiments and are displayed as percentage of inhibition.
determination of PAF has been introduced, using the PAF-induced serotonin release assay, 16 but still it is not commercial. Thus, we used bioassay, which is widely used for the determination of PAF, 12'1z-~9to evaluate the efficacy of azelastine. An in vitro study showed that azelastine inhibits PAF-induced paw edema in rats, PAF-induced platelet aggregation in platelet-rich human plasma and PAFinduced bronchoconstriction in anesthetized guinea pigs. a Additionally, the study showed that 100 gmol/1 of azelastine not only inhibited aggregation, but also initiated the disaggregation. ~ These findings suggest that inhibition of aggregation may have been caused by a direct action of azelastine rather than by azelastineinduced inhibition of PAF-like activity. However, in the present study, we washed cells three times with HBSS to
eliminate any direct inhibitory effect of azelastine on PAFinduced platelet aggregation. In addition, the reduction induced by incubation with azelastine was significantly smaller than that in the f-MLP-stimulated cells with preincubation of azelastine. Thus, it seems unlikely that the inhibition of aggregation in the present study was due to a direct inhibitory effect of azelastine. Azelastine has been found to inhibit histamine release from rat peritoneal mast ceils, even when it is added immediately before or simultaneously with the secretory stimuli,4,2°,21 suggesting that azelastine acts rapidly on basophil and mast-cell membranes, interfering with the generation of the stimulus-secretion coupling process. It is possible that azelastine also acts on the eosinophil membrane, inhibiting production of PAF from arachidonic acids in the cell membrane. However, the precise mechanism of the inhibitory action of azelastine remains to be clarified. Possible mechanisms of the azelastine-induced reduction in PAF release may include interference with the influx of Ca 2+ into cells and membrane-stabilizing activity. Further study is needed to determine how azelastine inhibits f-MLP-induced release of PAt: from human eosinophils. In the present study, the ICs0 of azelastine was 10-aM when f-MLP-stimulated asthmatic eosinophfls were preincubated for 15 min. This concentration is approximately the same as the plasma concentration 2 h after administration of oral azelastineY Thus, results suggest that oral azelastine may inhibit PAF-like activity in vivo. We conclude that azelastine inhibits PAF-like activity release from human asthmatic and non-asthmatic eosinophils.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 217-221
© Pearson Professional Ltd 1996
Inhibitory effect of azelastine on PAF activity
REFERENCES 1. Zechel H.J., Brock N., Lenke D., Achterrath-Tukermann U. Pharmacological and toxicological properties of azelastine, a novel antiallergic agent. Arzneimi#elforschung 1981; 31: 1184-1193. 2. Chand N., Nolan K., Diamantis W., Perhach J. L., Sofia R. D. Inhibition of leukotriene(SRS-A)-mediated allergic bronchospasm by azelastine, a novel, orally effective antiasthmatic drug. JAllergy Clin Immuno11983; 71: 149-156. 3. Katayama S., Akimoto N., Shionoya H., Morimoto T., Katah Y. Antiallergic effect of azelastine hydrochloride on immediate type hypersensitivity reactions in vivo and in vitro. Arzneimittelforschung 1981; 31:1196-1203. 4. Fields D. A. S., Pillar J., Diaanmantis W., Perhach J. L., Sofa R. D., Chand N. Inhibition by azelastine of non-allergic histamine release from rat peritoneal mast cells. JAllergy Clin Immunol 1984; 73: 400-403. 5. Chand N., Pillar J., Diammantis W., Sofa R. D. Inhibition of allergic histamine release by azelastine and selected antiallergic drugs from rabbit leukocytes. Int Arch Allergy AppI Immunol 1985; 77: 451-455. 6. Achterrath-Tuckermann U., Weischer C. H., Szelenyi I. Azelastine, a new antiallergic/antiasthmatic agent, inhibits PAFacether-induced platelet aggregation, paw edema and bronchoconstriction. Pharmacology 1988; 36: 265-271. 7. Verhagen J., Bruynzeel P. L B., Koedam J. A. et al. Leukotriene formation by purified human eosinophils and neutrophils. FEBS Le# 1984; 168: 23-28. 8. Bruynzeel P. L B., Kok P. T. M., Vietor KJ., Verhagen J. On the optimal conditions of LTC4 formation by human eosinophils in vitro. Prostaglandins Leukot Med 1985; 20:11-22. 9. Pinckard R. N., Farr R. N., Hanahan D.J. Physicochemical and functional identity of rabbit platelet-activating factor (PAF) released in vivo during IgE anaphylaxis with PAl: released in vitro from IgE sensitized basophils. J Immuno11979; 123: 1847-1857. 10. Hanahan D.J., Weintraub S. T. Platelet-activating factor, isolation, identification, and assay. Methods Biochem Anal 1986; 31: 195-219. 11. Bligh E. G., Dyer W.J. A rapid method of total lipid extraction and purification. CanJBiochem Physio11959; 37:911-917.
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12. Court E. N., Kingston W. P. A sensitive assay for plateletactivating factor using guinea-pig platelets. BrJ Pharmacol 1987; 91: 409. 13. Oda M., Satouchi K., Yasunaga K., Saito tC Molecular species of platelet-activating factor generated by human neutrophils challenged with ionophore A23187. J Immunol 1985; 134: 1090-1093. 14. Yamada K., Asano O., Yoshimura T., Katayama K. Highly sensitive gas chromatographic-mass spectrometric method for the determination of platelet-activating factor in human blood. J Chromatogr 1988; 433: 243-247. 15. Shindo IC, Hashimoto Y. Quantitative analysis of platelet activating factor treated with pentafluorobenzoyl chloride using gas chromatography/negative ion chemical ionization mass spectrometry. Drugs ExptI Clin Res 1991; 17: 343-349. 16. Muller S., Nigam S. Development of a sensitive and specific 14C-serotonin release assay for platelet-activating factor (PAF) in human neutrophils. J Lipid Mediat 1990; 2: 329-341. 17. McManus L M., Hanahan D.J., Pinckard R. N. Human platelet stimulation by acetyl glyceryl ether phosphorylcholine. J Clin Invest 1981; 67: 903-906. 18. Valone F. H., Coles E., Reinhold V. IL, Goetzl E.J. Specific binding of phospholipid platelet-activating factor by human platelets. Jlmmuno11982; 129: 1637-1641. 19. Camussi G., Pawlowski L, Bussolino F., Caldwell P. K B., Brentjens J., Andres G. Release of platelet activating factor in rabbits with antibody-mediated injury of the lung: the role of leukocytes and of pulmonary endothelial cells. J Immunol t983; 131: 1802-1807. 20. Diamantis W., Chand N., Harrison J. E., Pillar J., Perhach J. L. Jr, Sofia R. D. Inhibition of release of SRS-A and its antagonism by azelastine (A), an H1 antagonist-antiallergic agent. Pharmacologist 1982; 24: 200-203. 21. Chand N., Pillar J., Diamantis W., Perhach J. L. Jr, Sofia R. D. Inhibition of calcium ionophore (A23187)-stimulated histamine release from rat peritoneal mast cells by azelastine. Implications for its mode of action. EurJPharmaco11983; 96: 227-233. 22. Magnussen H., Reuss G., Jorres R., Aurich K The effect of azelastine on exercise-induced asthma. Chest 1988;
93: 937-940.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 217-221
Azelastine hydrochloride inhibits platelet activating factor-like activity in human eosinophils Kunihiko Shindo, Motonori Fukumura First Department of Internal Medicine, Yokohama City University School of Medicine, 3-9, Fukuura, Kanazawa-ku, Yokohama 236, Japan.
Summary We investigated the inhibitory effect of azelastine hydrochloride (azelastine), an anti-asthmatic drug, on platelet-activating factor (PAF)-Iike activity in eosinophils obtained from asthmatic and non-asthmatic patients. Eosinophils were preincubated with or without azelastine and stimulated with f-Met-Leu-Phe (fMLP, 10 lamol) for 15 min. PAr-like activity was detected by aggregation of washed guinea-pig platelets. PAr-like activity released from asthmatic eosinophils without preincubation of azelastine was 2.36 [1.02] (mean [SD], rig/107 cells) in supernatants and 13.87 [4.77] in cell pellets. After preincubation with 10-8, 10~, and 10-4 M azelastine, PAF-like activity reduced to 1.85 [0.46] (mean [SD], ng/107 cells), 1.11 [0.14], and 0.88 [0.09] (n = 15) in the supernatants, and 11.83 [2.93], 8.32 [1.41], and 6.27 [1.25] (n = 15) in the cell pellets, respectively. PAr-like activity in non-asthmatic eosinophils without preincubation of azelastine was 2.01 [0.86] (mean [SD], ng/107 cells) in supernatants and 7.44 [0.99] in cell pellets. After preincubation with 10-8, 10-e, and 10-4 M azelastine, PAr-like activity reduced to 1.73 [0.64] (mean [SD], rig/107 cells), 1.12 [0.23], and 0.84 [0.17] (n = 20) in the supernatants, and 6.26 [2.08], 4.65 [0.88], and 3.02 [0.43] (n = 20) in the cell pellets, respectively. Our results showed that preincubation with azelastine caused a dosedependent inhibition of intra and extracellular PAr-like activity from asthmatic and non-asthmatic eosinophils in the same manner.
INTRODUCTION Azelastine hydrochrolide (azelastine), (4-[p-chlorobenzyl]2- [ h e x a h y d r o - l - m e t h y l - 1 H-az epine-4-yl]- 1 -[2/4]phthalazinone hydrochloride), a H1 histamine receptor antagonist, is an orally effective and long-acting antiallergic agent. 1-3 This drug not only strongly inhibits the release of leukotrienes (LTs) from neutrophils and eosinophils, but also antagonizes these substances. Azelastine also inhibits and antagonizes chemical transmitters, such as histamine, released during allergic reactions in experiments using rats and rabbits. 4,5 An in vitro study has shown that azelastine inhibits PAF-induced paw edema in rats, PAF-induced platelet aggregation in platelet-rich human plasma, and PAFinduced bronchoconstriction in anesthetized guinea-
Received 1 December Accepted 21 December Correspondence to: Kunihiko Shindo, Tel. 045-787-2511; Fax. 045-787-2509.
pigs. 6 However, the effects of azelastine on the release of platelet-activating factor (PAF) from eosinophils obtained from patients with bronchial asthma are not known. We attempted to elucidate the regulatory effect of azelastine on human eosinophil function by investigating the effect of azelastine on PAF-like activity in eosinophils obtained from patients with bronchial asthma. MATERIALS AND METHODS Eosinophil separation Eosinophils were separated from a mixture of eosinophils and granulocytes by two subsequent centrifugations of a granulocyte preparation in isotonic Percoll solutions with densities of 1.082 and 1.085 g/ml at 1000 x gfor 10 min at room temperature. Over 98% pure neutrophfls appeared at the top of the 1.082 g/ml Percoll solution, while the eosinophils and some neutrophils were present as a small pellet on the bottom. After resuspension of the eosinophfl pellet in a 1.085 g/ml Percoll solution and centrifugation 217
218
Shindo & Fukumura
at 1000 x g for 10 min at room temperature, over 90% pure eosinophfls appeared on the bottom of the tube. Recovery rate for eosinophils was 67.41 [10.31% (mean [SD], n = 12). More than 95% cell viability was confirmed in all experiments by the trypan blue dye exclusion method.7,8 Human eosinophils and neutrophfls were isolated from the peripheral blood of 35 different donors, none of whom were ingesting aspirin, other non-steroidal antiinflammatory drugs, or corticosteroids. 15 donors were diagnosed as having bronchial asthma (atopic type); their peripheral eosinophfl levels ranged from 7.7 to 25% (mean [SD] of 14 [7]0/0). These eosinophils were used for asthmatic eosinophils in this study. 20 donors without diagnosed underlying disorders had 4.3 [1.21 (mean [SD])% eosinophilia. These eosinphils were used for non-asthmatic eosinophils.
lO -7 M
o
•
10 -8 M' • •
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• •
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•
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Experimental procedure
io
fo
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Cells obtained from asthmatic patients and nonasthmatic patients were divided into four groups. Three groups of cells were preincubated with 10-8, 10-6, or 10-4 tool of azelastine for 15 rain, washed three times with Hanks' balanced salt solution without calcium or magnesium (HBSS), and suspended at a density 10 z cells in 500 gl of modified Tyrode's buffer, pH 7.8, containing O.1% gelatin, 1 mM calcium, 5 mM magnesium, 0.3 mM potassium, and 20ram L-serine, and then stimulated with N-formyl-L-methionyl-L-leucyl-~-phenylalanine (f-MLP) (10 gM) for 15 rain at 37°C. The remaining groups of cells obtained from asthmatic patients and non-asthmatic patients served as controls.
it was serially diluted until no activity was detected. Aggregation curves obtained with the synthetic AGEPC were determined by plotting the concentration (ng/ml) of synthetic AGEPC against the percent platelet aggregation (Fig. 1)?°
Measurement of PAF-like activity
Extraction
PAF-like activity was measured by platelet aggregation using washed guinea-pig platelets. Guinea-pig platelets were isolated by a modification of the Ficoll-Paque separation technique described by Pinckard et alY Synthetic compounds containing known amounts of authentic 1O-hexadecyl-2-acetyt-sn-glycero-3-phosphocholine (AGEPC; hexadecyl PAF) were dispersed in a mixture containing 0.9% sodium chloride and 0.25% bovine serum albumin to yield a phospholipid concentration of 10-2 to 10-3M. To initiate the aggregation process, a 10 gl sample was added to 100 gl of washed guinea-pig platelets (1.25 x 109 cells/ml) suspended in Tyrode's buffer solution, pH 6.5, and then mixed with 400 gl of Tyrode's gelatin mixture, pH 7.2, containing 1.33 mM calcium chloride. The reaction was started by stirring at 9000 rpm at 37°C. The change in light transmission through the suspension was monitored for 5 to 10 min with an aggregometer. If the sample showed aggregating activity,
After stopping the cell incubation, the samples were extracted according to the method of Nigh and Dyer. I1 1 ml each of chloroform and water were added to the supernatant or the cell pellet fraction, and vortexed. The chloroform phase was removed after centrifuging the mixture at 2000 x gfor 4 min, and 2 ml of chloroform was added to the water methanol phase. After vortexing, the mixture was centrifuged at 2000 x g for 2 min. The chloroform phase was removed and combined with the previous chloroform phase. The extract was applied to SEP-PAK silica column once and eluted with chloroform and methanol. The elute was applied to silica 60 TLC plate and AGEPCs were separated with CHC13:CH3OH:H20. The AGEPCs fraction was reextracted according to Bligh and Dyer as described above. The chloroform phase was evaporated under a nitrogen stream and the residue was resuspended in 0.2 ml Tris buffer solution containing (0.25% w/v) bovine serum albumin (Tris-BSA). 12
%
aggregation
of
washed guinea pig platelets
Fig. 1 Graphic representation of aggregation assay for hexadecyl PAF; Plot of molarity of hexadecyl PAF versus percent aggregation (change in transmittance). A reasonable evaluation of the concentration of an unknown PAF-like activity can be measured by the curve.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 217-221
© Pearson Professional Ltd 1996
Inhibitory effect of azelastine on PAF activity
219
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Fig. 2 Changes of aggregation curves induced by intracellular PAF-like activity in asthmatic eosinophils stimulated with fMLP (10 gM) are demonstrated: (1) control (without preincubation of azelastine); (2) intracellular PAF-like activity in stimulated asthmatic eosinophils after preincubation with 104 M of azelastine; (3) intracetlular PAF-like activity after preincubation with 10-e M of azelastine; (4) intracellular PAF-like activity after preincubation with 10-~ M of azelastine. The heights in vertical lines, which mean % aggregation of washed guinea pig platelets (transmittance of a piatelet suspension), were reduced with increasing concentrations of azelastine used in preincubations.
Materials
The synthetic AGEPC was obtained from Sigma Chemical Japan (Tokyo, Japan). Azelastine hydrochloride was obtained from Eisai Pharmaceutical Co. (Tokyo, Japan). Statistical analysis Values are expressed as m e a n + / - SD or m e d i a n v a l u e
and range. PAT-like activity induced by preincubation with azelastine was analyzed by one-way ANOVA with repeated measures and Scheffe's F-test Differences between means were analyzed by a two-tailed paired t-test confirming to normal distribution. A P value of < 0.05 was considered significant. RESULTS
Figure 2 demonstrates changes of aggregation curves induced by intracellular PAT-like activity in asthmatic eosinophils preincubated with 10-8, 10-6, and 10-4M azelastine. The light transmissions decreased with the dose of azelastine. PAF-like activity released from asthmatic eosinophfls without preincubation of azelastine was 2.36 [1.02] (mean [SD], ng/107 cells) in supematants and 13.87 [4.77] in cell pellets. After preincubation with 10-8, 10-*, and lO-4M azelastine, PAT-like activity reduced to 1.85 [0.46](mean [SD], ng/lO z cells), 1.11 [0.14], and 0.88 [0.09] in the supematants, and 11.83 [2.93], 8.32 [1.41], and 6.27 [1.25] (n = 15) in the cell pellets, respectively. PAT-like activity in non-asthmatic eosinophfls without preincubation of azelastine was 2.01 [0.86](mean [SD], ng/10 z cells) in supernatants and 7.44 [0.99] in cell pellets. After preincubation with 10-8, 10-6, and 10-4 M azelastine, PAT-like activity reduced to 1.73 [0.64](mean [SD], ng/10 z cells), 1.12 [0.23], and 0.84 [0.171 in the supernatants, and 6.26 [2.08], 4.65 [0.88], and 3.02 [0.43] (n = 20) in the cell pellets, respectively. © Pearson Professional Ltd 1996
Azelastine at concentrations above 10-8 M caused dosedependent inhibitions in platelet aggregation in the supernatants and the cell pellets obtained from asthmatic (Fig. 3a) and non-asthmatic eosinophfls (Fig. 3b) in the same manner. There was no significant difference in the inhibitory effect between the two groups. The PAT-like activity in stimulated asthmatic eosinophils not preincubated with azelastine was significantly higher than that in stimulated non-asthmatic eosinophils not preincubated with azelastine (P < 0.05). Azelastine achieved ICs0 at a concentration of 10-6 M for both intracellular and extracellular PAF-like activity in asthmatic and non-asthmatic eosinophils. The platelet aggregation by hexadecyl-PAF (10-z M) in the presence of azelastine (10 -4 M) (a) was reduced by 7.1 (1.5-8.2)% (median (range)) (n = 4) relative to the aggregation in the absence of azelastine (b), as shown in Figure 4. Thus, azelastine itself had only a slight inhibitory effect on platelet aggregation induced by authentic hexadecylPAF. However, the reduction induced by azelastine itself was significantly smaller than that produced by supernatant and cell-associated PAF derived from f-MLPstimulated cells after preincubation of azelastine (10 -4 M).
DISCUSSION
Azelastine inhibited f-MLP-induced PAT-like activity in human asthmatic and non-asthmatic eosinophils in a dose-dependent manner. There is no significant difference in the inhibitory effect between asthmatic and nonasthmatic eosinophfls. PAT-like activity was significantly higher in f-MLP-stimulated asthmatic eosinophils than in non-asthmatic eosinophils. Although mass spectrometric analysis 13-~5provides the most persuasive evidence of PAT presence, its routine application is not practicable due to the tedious procedure of analysis, the great skill demanded of the operator and the high cost of the equipment. A new method for
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 217-221
220
Shindo & Fukumura
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Fig, 3 Effects of azelastJne on f-MLP-induced intracellular (cell pellet) and extracellular (supernatant) PAF-like activity in human asthmatic (a) and non-asthmatic (b) eosJnophils. Data represent the means+/-SD of 6 (a) and 10 (b) separate experiments and are displayed as percentage of inhibition.
determination of PAF has been introduced, using the PAF-induced serotonin release assay, 16 but still it is not commercial. Thus, we used bioassay, which is widely used for the determination of PAF, 12'1z-~9to evaluate the efficacy of azelastine. An in vitro study showed that azelastine inhibits PAF-induced paw edema in rats, PAF-induced platelet aggregation in platelet-rich human plasma and PAFinduced bronchoconstriction in anesthetized guinea pigs. a Additionally, the study showed that 100 gmol/1 of azelastine not only inhibited aggregation, but also initiated the disaggregation. ~ These findings suggest that inhibition of aggregation may have been caused by a direct action of azelastine rather than by azelastineinduced inhibition of PAF-like activity. However, in the present study, we washed cells three times with HBSS to
eliminate any direct inhibitory effect of azelastine on PAFinduced platelet aggregation. In addition, the reduction induced by incubation with azelastine was significantly smaller than that in the f-MLP-stimulated cells with preincubation of azelastine. Thus, it seems unlikely that the inhibition of aggregation in the present study was due to a direct inhibitory effect of azelastine. Azelastine has been found to inhibit histamine release from rat peritoneal mast ceils, even when it is added immediately before or simultaneously with the secretory stimuli,4,2°,21 suggesting that azelastine acts rapidly on basophil and mast-cell membranes, interfering with the generation of the stimulus-secretion coupling process. It is possible that azelastine also acts on the eosinophil membrane, inhibiting production of PAF from arachidonic acids in the cell membrane. However, the precise mechanism of the inhibitory action of azelastine remains to be clarified. Possible mechanisms of the azelastine-induced reduction in PAF release may include interference with the influx of Ca 2+ into cells and membrane-stabilizing activity. Further study is needed to determine how azelastine inhibits f-MLP-induced release of PAt: from human eosinophils. In the present study, the ICs0 of azelastine was 10-aM when f-MLP-stimulated asthmatic eosinophfls were preincubated for 15 min. This concentration is approximately the same as the plasma concentration 2 h after administration of oral azelastineY Thus, results suggest that oral azelastine may inhibit PAF-like activity in vivo. We conclude that azelastine inhibits PAF-like activity release from human asthmatic and non-asthmatic eosinophils.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 217-221
© Pearson Professional Ltd 1996
Inhibitory effect of azelastine on PAF activity
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12. Court E. N., Kingston W. P. A sensitive assay for plateletactivating factor using guinea-pig platelets. BrJ Pharmacol 1987; 91: 409. 13. Oda M., Satouchi K., Yasunaga K., Saito tC Molecular species of platelet-activating factor generated by human neutrophils challenged with ionophore A23187. J Immunol 1985; 134: 1090-1093. 14. Yamada K., Asano O., Yoshimura T., Katayama K. Highly sensitive gas chromatographic-mass spectrometric method for the determination of platelet-activating factor in human blood. J Chromatogr 1988; 433: 243-247. 15. Shindo IC, Hashimoto Y. Quantitative analysis of platelet activating factor treated with pentafluorobenzoyl chloride using gas chromatography/negative ion chemical ionization mass spectrometry. Drugs ExptI Clin Res 1991; 17: 343-349. 16. Muller S., Nigam S. Development of a sensitive and specific 14C-serotonin release assay for platelet-activating factor (PAF) in human neutrophils. J Lipid Mediat 1990; 2: 329-341. 17. McManus L M., Hanahan D.J., Pinckard R. N. Human platelet stimulation by acetyl glyceryl ether phosphorylcholine. J Clin Invest 1981; 67: 903-906. 18. Valone F. H., Coles E., Reinhold V. IL, Goetzl E.J. Specific binding of phospholipid platelet-activating factor by human platelets. Jlmmuno11982; 129: 1637-1641. 19. Camussi G., Pawlowski L, Bussolino F., Caldwell P. K B., Brentjens J., Andres G. Release of platelet activating factor in rabbits with antibody-mediated injury of the lung: the role of leukocytes and of pulmonary endothelial cells. J Immunol t983; 131: 1802-1807. 20. Diamantis W., Chand N., Harrison J. E., Pillar J., Perhach J. L. Jr, Sofia R. D. Inhibition of release of SRS-A and its antagonism by azelastine (A), an H1 antagonist-antiallergic agent. Pharmacologist 1982; 24: 200-203. 21. Chand N., Pillar J., Diamantis W., Perhach J. L. Jr, Sofia R. D. Inhibition of calcium ionophore (A23187)-stimulated histamine release from rat peritoneal mast cells by azelastine. Implications for its mode of action. EurJPharmaco11983; 96: 227-233. 22. Magnussen H., Reuss G., Jorres R., Aurich K The effect of azelastine on exercise-induced asthma. Chest 1988;
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Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 217-221