Inhibitory effect of azelastine hydrochloride on synthesis and release of platelet activating factor from human alveolar macrophages

Prostaglandins, Leukotrienes and Essential FattyAcids (1997) 57(6), 561-566 © HarcourtBrace& Co Ltd 1997

Inhibitory effect of azelastine hydrochloride on synthesis and release of platelet activating factor from human alveolar macrophages K. Shindo, M. Machida, Y. Hirai, M. Fukumura The First Department of Internal Medicine, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan

The effect of azelastine hydrochloride (azelastine) on synthesis and release of platelet activating factor (PAF) in alveolar macrophages obtained from asthmatic and non-asthmatic subjects was examined. Alveolar macrophages (AMs) were preincubated with or without azelastine and stimulated with f-Met-Leu-Phe (fMLP,10 ~M) for 15 min. PAF activity was detected by aggregation of washed guinea pig platelets. PAF activity released from alveolar macrophages (AMs) from asthmatics without preincubation of azelastine was 15.97 [2.17] (mean [SD], ng/107 cells) in supernatants and 42.52 [10.16] in cell pellets. After preincubation with 10-8, 10-~, and 10-4 M of azelastine, PAF activity reduced to 10.71 [2.73] (mean [SD], ng/107 cells), 7.86 [0.94], and 3.52 [0.31] in the supernatants, and 35.58 [7.37], 21.57 [4.36], and 14.77 [0.99] (n = 15) in the cell pellets, respectively. PAF activity in non-asthmatic subjects without preincubation of azelastine was 8.55 [1.16] (mean [SD], ng/107 cells) in supernatants and 32.64 [3.37] in cell pellets. After preincubation with 10-8, 10-6, and 10--4 M of azelastine, PAF activity reduced to 6.68 [0.78] (mean [SD], ng/107 cells), 4.47 [0.51], and 2.97 [0.36] in the supernatants, and 29.53 [3.75], 14.78 [1.95], and 6.16 [0.55] (n = 20) in the cell pellets, respectively. Our results showed that preincubation with azelastine caused a dose-dependent inhibition of intra- and extracellular PAF activity from asthmatic and non-asthmatic macrophages in the same manner.

Summary

INTRODUCTION

The alveolar macrophage (AM) is the predominant cell type within the alveolus, and undoubtedly serves as the first line of host defence against inhaled organisms and soluble and particulate molecules? ,2 AMs possess low affinity IgE receptors 3 and can be activated and release mediators following IgE-dependent challenge. 4.s As noted above, there have been reasons for suggesting an AM as a candidate for a primary cell in bronchial asthma. Thus, it is necessary and useful to investigate the sensitivity of AMs to a therapeutic agent to evaluate the effectiveness of the agent on an asthmatic attack. Azelastine hydrochrolide (azelastine) is an orally effective and long-acting antiallergic agent. 6-s This drug not only strongly inhibits the release of leukotrienes (LTs) Received 2 December 1996 Accepted 30 January 1997 Correspondence to: Kunihiko Shindo, Tel. 00 45 787 2511; Fax. 00 45 787 2509

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 rabbits5 *° We have reported azelastine to have the inhibitory effect on the synthesis and release of PAF-like activity from human eosinophils n and neutrophils. ~2 However, there have been no data about the effect of azelastine on synthesis and release of PAF-like activity from human AMs, especially AMs obtained from patients with bronchial asthma. The objective of this study, therefore, was to investigate the effect of azelastine on synthesis and release of PAF activity from AMs obtained from asthmatic patients to know the effectiveness of the agent against an asthmatic attack. MATERIALS AND METHODS Subjects

We evaluated 13 patients (male) with bronchial asthma, 561

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mean age 35.2 years (range 29-45 years), and 11 control subjects (male) without bronchial asthma, mean age 30.7 years (range 31-47 years). None of these subjects had ever smoked, and none had taken medication for 2 months prior to the study. Patients with bronchial asthma met the diagnostic criteria proposed by the American Thoracic Society;13 i.e., a history of episodic wheezing and a greater than 20% reversibility of the resting FEV1 after inhaling 400 mg of salbutamol. All patients had atopic asthma. Atopy was defined by the presence of a greater than 3 mm weal in response to skin-prick testing with at least two common airbome allergens vs that caused by the diluent control. Allergens were cat fur, mixed grass pollens, dog hair, feathers, a mould mixture, Dermatophagoides pteronyssinus, and Dermatophagoidesfarinae (Bencard, Brentford, UK). The group with bronchial asthma was clinically stable at the time of the study. Patients were excluded if: (a) their forced expiratory volume in 1 second (FEV1) was < 1.5 litres; 09) they showed evidence of active pulmonary infection. This study was approved by Yokohama City University's human subjects committee, and informed consent was obtained from all subjects before their participation. Topical anaesthesia to undergo bronchoscopy was achieved by administering lidocaine by nebulizer and by direct topical application. Meperidine and/or midazolam was administered to induce sedation. The bronchoscope (Olympus BF PIO) (Olympus Co. Ltd, Tokyo, Japan) was introduced through the nares. The anterior portion of the right middle lobe of the lung was lavaged with four aliquots of normal saline of 25 ml each. We collected the bronchoalveolar lavage fluid (BALF) and immediately placed it on ice. Cell counts were performed on unprocessed BALF using a haemocytometer and viability was determined by exclusion of a 0.04% solution of Trypan blue. Cells were prepared by cytocentrifugation and stained with May-Grunwald Giemsa. Differential cell counts were performed by counting 500 cells. The ceils in the fluid aspirated were separated from the lavage fluid by centrifugation (250 x ~; 10 min, 4°C) and resuspended in tissue culture medium RPMI 1640 (GIBCO, Grand Island, New York) (pH 7.4) supplemented with 10% FCS. Cells were plated at a density of 1 x 106 cells/35mm diameter well of tissue culture dishes. After incubation for 2 h at 37°C, the non-adherent cells were removed and adherent cells were washed once with serum-free RPMI. The cells (> 98% alveolar macrophages, as determined by non-specific esterase staining) were incubated at 37°C for 24 h in RPMI-1640 containing 1% FCS before use. Experimental procedure

Cells obtained from asthmatic patients and non-

asthmatic subjects were divided into each four groups, respectively. Each three groups of cells were preincubated with 10-8, 10-6, or 10-4 M of azelastine for 15 min, washed three times with Hanks' balanced salt solution without calcium or magnesium (HBSS), and suspended at a density 106 cells in 500 ~I of modified Tyrode's buffer, pH 7.8, containing 0.1% gelatin, 1 mM calcium, 5 mM magnesium, 0.3 mM potassium, and 20 mM L-serine, and then stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (10 ktM) for 15 min at 37°C. The remaining groups of cells obtained from asthmatic patients and non-asthmatic subjects served as controls. Measurement of PAF activity

PAF 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 al.14 Synthetic compounds containing known amounts of authentic 1-0-hexadecyl-2-acetyl-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 10-3 M. To initiate the aggregation process, a 10-~tl sample was added to 100~tl of washed guinea pig platelets (1.25x 109 cells/ml) suspended in Tyrode's buffer solution, pH 6.5, and then mixed with 400 ~tl 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, it was serially diluted until no activity was detected. Aggregation curves obtained with the synthetic AGEPC were determined by plotting the concentration (ng/1Td) of synthetic AGEPC against the percentage of platelet aggregation.1 ~,15 Extraction

After stopping the cell incubation, the samples were extracted according to the method of Bligh and Dyer. 16 One ml each of chloroform and water were added to the supernatant and vortexed. The chloroform phase was removed after centrifuging the mixture at 2000 x g for 4 min, and 2 ml of chloroform was added to the watermethanol 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 CHCL3:CH3OH:H20.The AGEPCs fraction

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pellets. After preincubation with 10-8, 10-6, and 10-4 M of azelastine, PAF activity reduced to 10.71 [2.73] (mean [SD], ng/107 cells), 7.86 [0.94], and 3.52 [0.31] in the supernatants, and 35.58 [7.37], 21.57 [4.36], and 14.77 [0.99] (n = 15) in the cell pellets, respectively. PAF activity in non-asthmatic subjects without preincubation of azelastine was 8.55 [1.16] (mean [SD], ng/lO 7, cells) in supernatants and 32.64 [3.37] in cell pellets. After preincubation with 10-s, 10-6, and lO-4M of azelastine, PAF activity reduced to 6.68 [0.78] (mean [SD], ng/10 z cells), 4.47 [0.51], and 2.97 [0.36] in the supernatants, and 29.53 [3.75], 14.78 [1.95], and 6.16 [0.55] (n = 20) in the cell pellets, respectively. Azelastine at concentrations above 10-SM caused dose-dependent inhibitions in platelet aggregation in the supematants and the cell pellets obtained from asthmatics (Fig. 2A) and non-asthmatic subjects (Fig. 2B) in the same manner. There was no significant difference in the inhibitory effect between the two groups. The PAF activity in stimulated AMs obtained from asthmatics not preincubated with azelastine was significantly higher than that in stimulated non-asthmatic subjects not preincubated with azelastine (P
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).17'18 Materials

The synthetic AGEPC was obtained from Sigma Chemical Japan (Tokyo, Japan). Azelastine hydrochloride was obtained from Eisai Pharmaceutical Co. (Tokyo, Japan). Statistical analysis

PAF 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. A P value of < 0.05 was considered significant. Values are expressed as mean + SD or median value and range. RESULTS

Figure 1 demonstrates changes of aggregation curves induced by intracellular PAF activity in AMs from asthmatics preincubated with 10-s, 10-6, and l O-4M of azelastine. The light transmissions decreased with the dose of azelastine. PAF activity released from AMs from asthmatics without preincubation of azelastine was 15.97 [2.17] (mean [SD], ng/10 z ceils) in supernatants and 42.52 [10.16] in cell

DISCUSSION

Azelastine inhibited fMLP-induced PAF-like activity in AMs obtained from asthmatic patients and non-asthmatic

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Fig. 1 Changes of aggregation curves induced by intracellular PAF activity in asthmatic AMs stimulated with fMLP(10 ~M) are demonstrated. (1): Control (without preincubation of azelastine). (2): Intracellular PAF-like activity in stimulated asthmatic AMs after preincubation with 10-~ M of azelastine. (3): Intracellular PAF-like activity after preincubation with 104 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 platelet suspension), were reduced with increasing concentrations of azelastine used in preincubations. © Harcourt Brace & Co Ltd 1997

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subjects in a dose-dependent manner. There is no significant difference in the inhibitory effect between the two groups. PAF-like activity was significantly higher in fMLP-stimulated AMs from asthmatic patients than in fMLP-stimulated AMs from non-asthmatic subjects. The question of what role macrophage-derived platelet activating factor might play in inflammatory diseases of the lung is an intriguing one. Pulmonary macrophages are present in the interstitium and in the alveoli. These cells therefore are ideally positioned to respond to systemic or inhaled antigenic challenges to the lung. The release of a potent chemotactic factor by pulmonary macrophages could be important not only in the initiation of acute inflammatory reactions but also in the pathogenesis of certain chronic lung diseases. On the other hand, it has been well known that PAF has a potent chemotactic activity for migration of eosinophilsJ9 As indicated in the present study, once alveolar macrophages are activated, they release more amount of platelet activating factor from macrophages in asthmatic patients. Thus, when alveolar macrophages in asthmatic airways are exposed to antigen, they could play an

important role in eosinophil transmigration of bronchial epithelium by releasing the greater amount of PAF and in initiation of bronchial responsiveness. Two enzymatic pathways have been documented for the biosynthesis of PAF. One catalyzes the conversion of the biologically inactive 1-alkyl-2-1yso-sn-glycero-3-phosphocholine (1-alkyl-2-1yso-GPC) to bioactive alkylacetylGPC by acetly-CoA:alkyllyso-GPC acetyhransferase,2° and the other transfers the phosphocholine moiety from CDPcholine to 1-alkyl-2-sn-glycerol by a specific cholinephosphotransferase.2l These data in the present study do not explain sufficiently the mechanism by which azelastine inhibits the synthesis and release of PAF from AMs. However, it is, at least, likely that azelastine acts on which process for production of PAF. An in vitro study showed that 100 pmol/L of azelastine not only inhibited aggregation of platelet, but also initiated the disaggregation,n The finding suggested that inhibition of aggregation may have been caused by a direct action of azelastine rather than by azelastine-induced inhibition of PAF-like activity. We also demonstrated azelastine to have a direct inhibitory effect on aggregation of platelets. 11,12The platelet aggregation by hexadecylPAF (10-7M) in the presence of azelastine (10-4M) was reduced by 7.1 (1.5-8.2)% (median(range)) relative to the aggregation in the absence of azelastine. 11 Thus, azelastine itself had only a slight inhibitory effect on platelet aggregation induced by authentic hexadecyl-PAF. In the present study, we washed ceils three times with HBSS to eliminate any direct inhibitory effect of azelastine on PAF-induced platelet aggregation. In addition, the reduction induced by incubation with azelastine was significantly smaller than that in the fMLP-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. We reported a linear-regression curve to quantitate PAF in the previous studies. 1],15 The curve was shown by plotting concentrations of synthetic hexadecyl-PAF against the percentage of platelet aggregations. Thus, the concentration of PAF in a sample including several kinds of PAF was expressed as a concentration of hexadecylPAF. Likewise, it was done in the present study. When eosinophil preparations were incubated with 10-7 M fMLP for 1 and 15rain in Hanks' balanced salt solution in the absence of Ca2÷and Mg2÷and presence of 5 mM EDTA, the activity of acetyltransferase is known to be inhibited.23 We used HBSS including Ca2+/Mg2÷ when the AMs are stimulated with fMLP. Thus, we think that the preparations used in this study do not affect the activity of acetyhransferase in the AMs. Previous reports 24,25 showed that patients with bronchial asthma, but none of those with emphysema or control subjects, exhibited PAF in the BALF.The presence

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of PAF in t h e BALF of t h e a s t h m a t i c patients was signific a n t l y associated with low n e u t r o p h i l a n d h i g h l y m p h o cyte counts, a n d w i t h t h e m e t a b o l i c activity of t h e m a c r o p h a g e s , as assessed b y l u c i g e n i n c h e m i l u m i n e s cence. The p r e s e n t results suggest t h a t t h e p r e s e n c e of PAF in BALF of b r o n c h i a l a s t h m a t i c s may, at least in part, be d u e to t h e h i g h ability for PAF release b y AMs in a s t h m a t i c lung. Azelastine at c o n c e n t r a t i o n s a b o v e 10-SM i n h i b i t e d t h e platelet a g g r e g a t i o n in t h e s u p e r n a t a n t s a n d t h e cell pellets o b t a i n e d from a s t h m a t i c a n d n o n - a s t h m a t i c eosinophils, 11 neutrophils, 12 a n d AMs in t h e p r e s e n t s t u d y in a similar d o s e - d e p e n d e n t fashion. The ICs0s of azelasfine were 10-6M a n d 10-SM in eosinophils a n d n e u t r o phils. T h e ICs0 in t h e AMs also was 10 -6 M in t h e p r e s e n t study. The ICs0s of azelastine were lower in t h e eosinophils a n d t h e AMs t h a n in t h e neutrophils. W e c a n n o t explain t h e difference o n l y b y t h e cell sources; t h e AMs were o b t a i n e d from BALF a n d t h e eosinophils a n d n e u t r o p h i l s were o b t a i n e d from p e r i p h e r a l blood. W h a t differences in t h e cell f u n c t i o n m a y i n d u c e d t h e difference in r e s p o n s e to azelastine. At all events, we n e e d to f u r t h e r e x a m i n e to clear t h e differences. The overall i m p o r t a n c e of PAF in p u l m o n a r y disorders h a s b e e n e s t a b l i s h e d Y '2z The clinical efficacy of a n orally active PAF a n t a g o n i s t has r e c e n t l y b e e n e v a l u a t e d in a s t h m a t i c patients. 2s U n f o r t u n a t e l y s u c h t r e a t m e n t d i d n o t r e d u c e t h e r e q u i r e m e n t for i n h a l e d corticosteroid in atopic asthma. The precise role of PAF in t h e developm e n t of b r o n c h i a l a s t h m a r e m a i n s controversial. However, PAF m a y c o n t r i b u t e to t h e d e v e l o p m e n t of b r o n c h i a l asthma, b e c a u s e a g r e a t e r a m o u n t of PAF was released from t h e alveolar m a c r o p h a g e s o b t a i n e d from a s t h m a t i c patients vs n o n - a s t h m a t i c subjects. In addition, as n o t e d above, PAF d e m o n s t r a t e d in t h e BALF of b r o n c h i a l a s t h m a also m a y c o n t r i b u t e to t h e developm e n t of t h e disease, m a k i n g eosinophils m i g r a t e d from l u n g tissue to b r o n c h i a l l u m e n . It is t h e r e f o r e possible t h a t azelastine is effective to clinically p r e v e n t initiating b r o n c h i a l a s t h m a i n h i b i t i n g t h e s y n t h e s i s of PAF from A_Ms.

CONCLUSION W e c o n c l u d e t h a t azelastine inhibits s y n t h e s i s a n d release of PAF activity from AMs o b t a i n e d from a s t h m a t i c patients a n d n o n - a s t h m a t i c subjects.

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