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Do nasal mast cells release histamine on stimulation with substance P in allergic rhinitis?
Clinical and Experbnental Allergy, 1994, Volume 24, pages 922-929
D o nasal mast cells release histamine on stimulation with substance P in allergic rhinitis? G. B R A U N S T E I N , A. B U V R Y , J. L A C R O N I Q U E , N. D E S J A R D I N S and N. FROSSARD Laboratoh-e de physiologic respiratoire et service de pneunlologie, UFR Cochin-Port-Royal, Paris. France Summary The effects of nasal administration of increasing doses of exogenous substance P have been studied in patients with allergic rhinitis treated with placebo or with the H~ antagonist cetirizine (10 mg twice daily for 3 days). Responses to substance P were assessed by posterior rhlnomanometry (measuring nasal airway resistance) and by measure of histamine, protein and albumin production and cell recovery in nasal lavage fluids before and after challenge. Substance P induced a dose-dependent increase in nasal airway resistance which was similar after treatment with either cetwizine or placebo (maximal increase in nasal airway resistance was 4.2-fold greater than the baseline with the placebo and 4-7-fold greater than the baseline with cetinzine). No histamine release was observed. Similar increases in protein and albumin production were observed after stimulation with substance P along with the placebo (protein: from 0"35 + 0- l 1 to 3-31 + 0.62 mg and albumin: from 0"09 ___0.04 to 2"0g + 0-39 rag) and when combined with cetirizine treatment (proteins: from 0-42_+009 to 3-62_+0.77 and albumin: from 0.17+0-04 to 2.19+0.51 rag). After stimulation with substance P, percentages of neutrophils recovered in nasal flmds increased from 26.2+_ 11.5 to 54.5 + 9"5 with the placebo and from 35"5 -+ 11 "0 to 53.6-+ 9 5 with cetmzme. Eosmophils were inconsistently found after substance P stimulation during both treatments. In conclusion, nasal response to substance P is not modified by cetinzme which suggests that the effect of substance P is not secondary to histamine release m the nose in man.
Clinical and Experimental Allergy, Vol. 24, pp. 922-929. Submitted 20 July 1993; revised 28 September 1993; accepted 4 February 1994. Introduction Substance P, a mediator of sensory C fibres, has been localized in the nose o f m a n [I,2]. A role for substance P in allergic rhinitis has been suggested since (i) substance P is released during antigen challenge and recovered in nasal lavage fluids [3,4], (it) specific substance P receptors are present in the nasal mucosa [2] whose activation is linked to measurable nasal events such as a decrease in nasal patency [5], exudation of proteins and albumin and influx of inflammatory cells in nasal lavage fluids [6] and (rio response to substance P is greater in patients suffering from allergic rhinitis than in normal subjects [5]. Substance P activates rat peritoneal mast cells as shown by histamine release in vitro [7,8]. In man, intradermic injection of substance P induces a weal-and-flare reaction Correspondence: N. Frossard, Neurolmmunopharmacologle INSERM CJF 91-05, BP24, F-67401 lllklrch Cedex, France. By courtesy of the authors and pubhshers.
[9-11] partially inhibited by a pre-treatment with the antihistamine agent, chlorpheniramine [12]. Isolated cutaneous mast cells also degranulate on stimulation with substance P [ 13,14]. These interactions between mast cells and substance P, together with the fact that mast cells are found closely associated with sensory nerve endings in various tissues including the airways [I 5,16], suggest that part of the effect of substance P may be due to mast cell activation. Whether this occurs in the nose in man can be studied either by the biochemical measurement o f histamine release after stimulation with substance P, as has been described previously [6] or by pharmacological modulation of the effect o f substance P by antihistamine, whmh is the approach we have chosen in this study. Ceurizine is a highly selective and potent H r-antihistamine agent [17]. Oral administration of cetirizine for 3 days leads to concentration of the drug in the nasal mucosa leading to an inhibition of the local effect of
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exogenous histamine [18]. We reasoned that if substance P induced histamine release in the nose then the response to the peptide would be altered by cetirizine pre-treatment. Therefore, the aim of our study was to compare the nasal response to substance P in allergic rhinitic patients in the presence of cetirizine or a placebo.
Subjects and methods Subjects Twelve patients suffering from allergic rhinitis (mean age+sD: 28-0_+4.2; seven males and five females) were included. They all had (i) at least a 4-year past history of sneezing, secretion and nasal obstruction during natural exposure to allergen, (11) a positive skin-prick test to mixed grass pollen (nine patients) or Dermatophagoides pteronyssinus (Stallerg6nes, Paris, France) (three patients) and (iii) a positive nasal challenge to one of these allergens as assessed by occurrence of nasal symptoms and at least a 100% increase in nasal airway resistance (NAR) within 15 min after intranasal administration of the antigen [6]. Patients were free of nasal symptoms and of treatment for at least 1 month before inclusion in the study. They were informed of the risks and purpose of the study and gave their informed written consent to participate. The study was approved by the Ethics Committee of our hospital.
Study design The study was double-blind, randomized, cross-over and placebo-controlled with two treatment periods of 3 days separated by a wash-out period of 11 days. During each treatment period, subjects took cetirizine (10 mg tablets) or matched placebo b.i.d, at 21:00 h and 9:00 h. Nasal challenge to substance P was performed during the afternoon of the third day, between 13:00 h and 18:00 h, 4 h ( + 0.5 h) after the last administration of the treatment which was delayed to ensure compliance with the 4 h interval. To verify treatment compliance at the end of each treatment period, subjects returned the pill-bottles and histamine skin-prick tests (Phazet ®, Pharmacia, Uppsala, Sweden) were performed after each challenge with substance P by an independent person not involved in any part of the study. Diameters of weal-and-flare reactions were measured 10 rain after the prick test.
Nasal challenge to substance P Substance P from a stock solution (400 nmol/100 #1; stored at -80JC) was diluted just before use in saline to obtain three different isotonic solutions of substance P, i.e. 2-5 nmol, 10 nmol and 40 nmol of substance P per 100/~1.
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Initial NAR was measured. Four pre-challenge nasal lavages were then performed for 10 min. Vehicle (isotonic saline; 100/~1 per nostril) and successive increasing doses of substance P (2.5, 5, I0, 20, 40 and 80 nmol) were nebulized every 8 min into each nostril during which the subject held his/her breath for 30 s to avoid inhalation of substance P into the subglottic airways. For each administration, one or two puffs of solutions were given per nostril using a plastic hand-held nebulizer delivering 100 #1 (95-110/A) at each actuation (Stallerg6nes). Ninety seconds, 3 min, 5 rain and 7 5 min after each nebulization of isotonic saline or substance P, NAR, heart rate, systolic and diastolic blood pressure were measured. A nasal lavage was performed 10 min after saline and again 10 min after the last dose of substance P.
Assessment o f the responses NAR was measured by posterior rhinomanometry [6,19]. Briefly, the subjects breathed through the nose into a mask covering the nose. Nasal airflow was measured by a pneumotachograph (Fleisch No 1; Fleisch, Lausanne, Switzerland) connected to a differential pressure transducer (Validyne Model MP 45-1-1871; Validyne Corp., Northridge, CA, USA) placed on the mask. Transnasal pressure is the difference between pressures in the mask and at the tip of a plastic catheter placed over the tongue into the oropharynx, measured using an identical differential pressure transducer. Airflow and transnasal pressure were measured at 10 ms intervals between the onset of inspiration and maximal inspiratory airflow and were monitored using an X-Y recorder and results were fed in parallel into a microcomputer. NAR was calculated at each airflow and the linear regression of NAR as a function of airflow was determined (Mediprom rhinomanometer, Paris, France). NAR was calculated by interpolation at an airflow of 150 ml/s. Blood pressure was measured using a sphygmomanometer cuff placed around the arm. Pre- and post-challenge nasal lavages were performed with saline (0.9% NaC1; 37°C). Five 1 ml aliquots were injected using a soft-tipped catheter into each nostril, past the vestibule. After each injection, the subject expelled the fluid by gently blowing his/her nose into a plastic tube. The volume of fluid recovered was measured. Biochemical analyses were performed on the last pre-challenge nasal lavage, on the post-sahne nasal lavage and on the post-challenge lavage. The vortexed fluid (2 ml) was centrifuged at 1300×g for 10 mln (Beckman TJ-6 centrifuge; Beckman Instruments, Fullerton, CA, USA) The supernatant (1 ml) was sonicated and used for protein analysis [20] and measurement of albumin by the nephelemetric technique (Behring, RueiI-Malmaison,
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All subjects completed the study. One subject had a large increase in initial N A R on one study day compared with the other and was therefore excluded from the efficacy analysis, thus based on 11 subjects. Analysis of safety of cetirizine (and placebo) treatment was established with the 12 subjects. Pre- and post-saline values did not differ between the two study days for all parameters. Saline had no effect on any parameter. Post-saline values were therefore presented as baseline in the results below. Fluid recovery was similar within and between periods (range 9.0-9.5).
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Fig. 1. Dose-response curves to substance P on nasal airway resistance during treatment with cetinzine (O) or placebo (o). Values are means_+sEM (bars) of 11 experiments
France). Perchloric acid (1 ml: 0"8 N) was added to the remaining supernatant. The tubes were stored at - 2 0 ° C until histamine was measured using an automated fluorometric technique sensitive to 0.5 ng/ml [21]. The lavage fluids (2 ml) were aliquoted for cytological analyses. Total cell count was performed using a Malassez cell. After centnfugation at 850 x g for 5 min, cells were resuspended in Hank's solution and cytocentrifuged for 5 min at 550 rpm (Shandon cytocentrifuge), processing 30 000 cells per shde. Cells'were fixed by air before Diff-Quick ® staining (Dade, Diidingen, Switzerland). Cells were identified according to usual cytological criteria.
Expression of the results and statistical analyses The highest N A R value (cm H20.s. 1 - 1) measured on each dose of substance P was used to construct the doseresponse curve. Total amount of protein (mg), albumin (rag) and histamine (ng) recovered m the nasal lavage fluid were calculated before and after saline and after stimulation with substance P. Total cell count per ml of lavage fluid was calculated for each sample. Differential cell counts were based upon examination of 400 cells. Absolute values for all parameters are presented in the figures. Diameters of skin responses are expressed in mm. All results are presented as group average +_SEM. Comparison between cetirizine and placebo was made using Friedman's non-parametric analysis of variance. Wilcoxon rank test was used for paired comparisons. A P value less than 0'05 was considered significant.
Rev fi Allergol, 1995.35.4
With the placebo, nasal stimulation with substance P induced a dose-dependent increase in N A R (Fig. 1). The mean maximal increase was 4.2-fold basal N A R (range 1'8-8'3). Individual dose-response curves scattered on a large scale. With cetirizine, a similar effect of substance P was observed (Fig. 1). The mean maximal increase in N A R was 4-7-fold basal N A R (range 1-6 10.4; P > 0 . 2 between treatments). Scattering of individual doseresponse curves was also observed. Compartson of the dose-response curves produced during treatment with cetirizine.or placebo did not show statistical difference.
Clinical effects Substance P did not reduce sneezing either during treatment with placebo or cetirizine. A dose-dependent flush induced by substance P was observed with both treatments occurring within 30-45 s and disappearing 3 min after dosing. Mean starting dose (placebo. 21.4 + 3-9 and cetirizine: 17.2+_6.4 nmol) did not differ between treatments ( P > 0.2). A slight and sigmficant fall in systolic blood pressure induced by substance P was observed with both treatments (P<0-05). Maximal changes in blood pressure after stimulation with the highest dose of substance P were not significantly different with both treatments (placebo: - 9 + 3 mm Hg and cetirlzine: - 6 +_ 2 mm Hg). Diastohc blood pressure fell significantly after substance P stimulation on placebo ( - 6 +_2 mm Hg; P < 0.05) but the change did not achieve significance after substance P with cetirizine treatment ( - 1 + 2 mm Hg). A shght increase in heart rate was similar with placebo. + 8"6 +_4.0 and cetirizine: + 8.0 +_ 3.0 beats per min.
Biochemical effects Histamine There was no significant change m the amount of histamine recovered in the nasal lavage fluid after 371
Nasal mast cells and substance P
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Fig. 2. Effect of substance P on production of protein (a, b) and albumin (e, d) in nasal lavage fluid during treatment with placebo (a, c) or cetirizine (b, d). Individual values before (pre) and after (post) stimulation with substance P are indicated (o). Means are represented by the box.
substance P with placebo (before: 13.2-+1.3, after: 17.3 -+ 1-6 ng) or with cetirizine (before: 11.5 _+ 1-0, after: 12.4-+ 1.1 ng). Pre- and post-challenge values did not differ between periods (P > 0.3 for both).
mg (median 3.1 ; range 0.7-7.8) with cetinzine (P < 0-05 for both). Substance P-induced increase in protein output did not differ between treatments. Albumhl Similar changes were observed for albumin (Fig.
Protebls Protein output significantly increased in nasal
lavage fluid of all subjects with both placebo and cetirizine (Fig.2). Total amount of protein increased from 0"35_+0'11 mg (median 0.3; range 0.0-1.3) to 3-31 +0.62 mg (median 3.1; range 0.8-7.0) with placebo and from 0.42_+ 0-09 mg (median 0.4; range 0.0-0-9) to 3-62___0.77
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2). Albumin output increased in the lavage fluid of all subjects on both treatments. During placebo treatment, total albumin output increased from 0.09+0.04 mg (median 0.11; range 0-0-0.47) to 2.08_+0.39 mg (median 1-64; range 0.55-4.23) and with cetirizine from 0.17-t-0"04 mg (median 0-14; range 0.0-0.41) to
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Fig. 3. Effect of substance P on recovery of polymorphonuclear cells (a, b) and of eosinophds (c, d) m nasal lavage fluid during treatment with placebo (a, c) or cetirlzme (b, d). Individual values before (pre) and after (post) stimulation with substance P are indicated (e). Means are represented by the box.
2.19±0.51 mg (median 1.27; range 0-66-5-25) ( P < 0 . 0 5 for both)i Increases did not differ between treatments. Expressed~ as percentage of total protein, albumin increased from 17_+ 5 to 64_+ 2'g, with placebo and from 41_+ 12 to 62__+5% with cetirizine ( P < 0 - 0 5 for both). Increases were similar for both treatments.
Cellular responses Total cells Substance P induced a significant increase in the total number of cells recovered in the nasal lavage fluid: with placebo, from 37.3+ 17.0 to 82.6+37.8 × 103 cells ( P < 0 . 0 5 ) and-with cetirizine from 33-6+11.0 to 7 1 . 8 ± 2 2 . 8 × 103 cells ( P < 0 ' 0 5 ) . These increases were similar for both treatments.
Rev.fr. Allergol.. 1995.35, 4.
Epithelial cells Substance P did not alter the number of epithelial cells either with placebo (before: 26.6 +- 12-2 and after: 14.6_+2.8x 103 cells) or with cetirizine (before: 12.9+_2.7 and after: 15.6+ 3.6 x 103 cells). Pre- and postchallenge values did not differ between treatments. Epithelial cells expressed as percentage of total ceils s~gnificantly decreased on stimulation with substance P during placebo treatment (from 72.6+ 11.4 to 4 4 . 0 + 9 . 3 % ) and during cetirizine treatment (from 63.2+11"0 to 44.3+_9.5%). Potymorphomtclear cells The percentage of P M N recovered in the lavage fluid increased from 26.2___ 11.5 before to 54.5 + 9 ' 5 % (P < 0"05) after stimulation with substance
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Nasal mast cells and substance P
P with placebo and from 35-5+11-0 to 53.6-1-9.9°/,, ( P < 0 . 0 5 ) with cetirizine. These increases did not differ between treatments. Considering the absolute number of cells, the same pattern of results was observed (Fig. 3). Eosinophils Changes in the number o f eosinophils in the nasal lavage fluid were not consistent in all subjects. With the placebo, an increase in the eosinophil count was observed in only four subjects and in only six with cetirizine. Therefore, the group average eosinophil counts expressed either as percentage or total cell number did not vary significantly when substance P was administered during either treatment periods (Fig. 3). Histamine skin-prick tests With the placebo, a weal-(9.0+0.3 mm) and flare(44.2-I- 3"3 mm) reaction was observed, which was almost totally abolished by cetirizine treatment (weal 0-8_+ 0.3 and flare 9' 1 _+ 1.1 mm). Adverse events No serious adverse events occurred during the study. One subject experienced drowsiness rated as slight during cetirizine treatment. Discussion
Our results show that the nasal response to exogenous substance P is not influenced by a pre-treatment with the specific and potent HL-receptor antagonist cetirizine in allergic rhinitis-suffering patients. The results obtained with the placebo confirm and extend to a larger group the nasal effects of substance P described previously. These effects include nasal obstruction, albumin extravasation, influx of polymorphonuclear cells and, in some cases, influx of eosinophils [6], a reproducible effect [22] which was not observed in response to repeated administration of saline [6]. Cetirizine did not modify any of these responses. The dose of cetirizine used in these experiments was identical to the dose required to reduce nasal responses to exogenous histamine. Nasal obstruction to low doses of histamine was totally blocked by cetirizine but still occurred at high doses [18]. Hence, in the present study, since nasal obstruction due to substance P was not reduced with cetirizine treatment, only large amounts of histamine could explain such a lack of effect by cetirizine. However, histamine was not recovered in the lavage fluid after substance P stimulation ([6] and this report). Altogether, these results demonstrate that substance P does not release histamine m the nose. This is streng-
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thened by the absence of the classical effect of histamine, i.e. sneezing, after substance P administration in man. Albumin recovery increased on administration of exogenous substance P to our rhinitic patients. This is in contrast with results obtained during nasal challenge with capsaicin, a substance releasing endogenous substance P, which does not induce albumin extrusion [23,24]. These latter results were obtained in non-rhinitic patients, probably accounting for the difference in recovery of albumin. However, we cannot exclude that the difference may be due to the endogeneous vs exogeneous origin of substance P. Cetirizine did not affect albumin extrusion, indicating that microvascular leakage due to substance P, like vasodilatlon inducing nasal obstruction, is not mediated by histamine. Substance P induces histamine release from isolated human skin mast cells [13,14]. h7 vivo, in man, the cutaneous effect of substance P is mediated partially by mast cell degranulation since it is reduced by pretreatment with an antihistamine [12,25]. Therefore, mast cells from different tissues in the same species, i.e. the skin and the nose in man, have different sensitivities to substance P. This indicates the presence of different mast cell phenotypes in these tissues. Similar differential effects are observed with compound 48/80 which acts via the same mechanism of action as substance P [26]. Compound 48/80 induces degranulation of skin mast cells [14] whereas mast cells from biopsies of the nasal mucosa are insensitive to this product [27]. Also, in the lung, mast cells do not release histamine after treatment with substance P [28], suggesting a similar reactivity of the mast cell phenotypes along the airways. In mice, intradermic administration o f substance P leads to local infiltration of leucocytes, an effect diminished in animals which are genetically mast cell-deficient and which can be restored by mast cell repletion [29,30]. This links leucocyte recruitment on substance P stimulation to mast cell activation in the skin of mice. In the nose of man, we demonstrated a large influx of P M N on stimulation with substance P which was not inhibited by cetirizine and not associated with histamine release thus not secondary to mast cell activation. This suggests a direct effect of substance P on P M N and/or endothelial cells. Experiments in the rat have already shown the rapid adherence of neutrophils to the endothelium on stimulation with substance P [30] which might suggest an increased expression of adhesion molecules. An influx of eosinophils after substance P administration was not found consistently and when present was of low magnitude compared with P M N recruitment. Nevertheless, substance P may enhance the chemotactic effect of platelet-activatmg factor (PAF) and LTB4 on eosinophils [32] and has been shown to have a greater effect oil
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eosinophil recruitment in the presence o f chronic inflamm a t i o n o f the nasal mucosa [33]. C o o p e r a t i o n between m e d i a t o r s may explain the e n h a n c e d effect o f substance P d u r i n g chronic inflammation. Cetirizine, in addition to its antihistamine effect, can reduce the recruitment o f e o s i n o phils b o t h in vitro [34] a n d in vivo [35], In o u r experiments, eosinophil recruitment seldomly occurred with the placebo. Th~s condition was not optimal for the study-of a n eventual inhibitory effect of cetirizine. A role for substance P in the p a t h o p h y s i o l o g y of allergic rhinitis may be suggested by (i) a release o f substance P during antigen challenge in nasal lavage fluid [3], (ii) the localization o f specific substance P receptors on the nasal mucosa [2], (iii) a nasal physiological effect o f exogenous substance P activating these receptors ([6] a n d this report). In particular, a p r o - i n f l a m m a t o r y effect of substance P has been shown, which m a y underline s y m p t o m s of allergic rhinitis. Additionally, some effect o f substance P may be due to its active N-terminal m e t a b o lites as has been previously described [6]. Here we d e m o n s t r a t e that the nasal effect o f s u b s t a n c e P is not secondary to histamine release. In conclusion, the highly selective H~-antihistamine agent, cetirizine, does not modify the nasal responses to substance P m patients with allergic rhinitis. This, together w~th the absence of histamine release, d e m o n strates that nasal mast cells do not d e g r a n u l a t e after stimulation w~th substance P, at least for histaminec o n t a i n i n g granules, without precluding release of o t h e r mediators. This is in contrast with skin m a s t cells a n d illustrates mast cell heterogeneity in various tissues within species.
Acknowledgements W e express our gratitude to A. Weyer for histamine measurements, G. LeGall for technical assistance, G. Strauch (IRT), M. Melac a n d D. Milinkevitch for c o n s t a n t encouragement. This work was s u p p o r t e d by U C B P h a r m a , France.
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on mast cells and basophils from different species and tissues, lnt Arch Allergy Appl l m m u n o l 1986: 79:I21-4. Y a n o H, Wershil BK, Arizono N, Galli SJ. Substance Pinduced augmentation o f cutaneous vascular permeability and granulocyte infiltration in mice is mast celt dependent. J Clin Invest 1989; 84:1276-86. M a t s u d a H, Kawakita K, Kiso Y, N a k a n o T, Kitamura Y. Substance P induces granulocyte infiltration through degranulation of mast cells. J I m m u n o l 1989; 142:927-31. M c D o n a l d D. Neurogenic inflammation m the rat trachea. I. C h a n g e s in venules, leucocytes and epithelial cells. J Neurocytol 1988; 17:583-603. N u m a o T, Agrawal DK. Neuropeptides modulate h u m a n eosinophll chemotaxis. J l m m u n o l 1992" 149:3309-15. Fajac 1. Braunstein G, Buvry A e t al. Potentiation of nasal responses to substance P during pollen season in patients with allergic rhmltis. Eur Respir J 199t; 4:476S. Capron M, Leprevost C, De Vos C, T o m a s i m M, Capron A. In vitro acuvlty o f cetlrizine on eosinophils. Health Science Res 1989:3:5-7 Fadel R, Herpm-Richard N, Rihoux JP, Henocq E. Inhibitory effect ofceurizine 2HCI on eosinophd migration in vivo. Clin Allergy 1987; 17:373-9
Clinical and Experimental Allergy is published monthly (t volume per annum) and the subscription prices for 1995 are £297.00 (Europe), £326.00 (Rest of World), $506.00 (USA and Canada), in all cases post free. Subscribers in Canada must add 7% to allow for Canadian GST. Subscribers in Europe must quote thezr VAT registration number or state that they are not registered. Subscribers in The Netherlands who are not VAT registered must add 6% to the subscription price, to allow for VAT. Orders for current subscriptions and back issues should be sent to Blackwell Science Ltd, PO Box 87, Oxford OX2 ONE, all other business correspondence, including orders for offprints and advertising space, should be addressed to Blackwell Science Ltd, Osney Mead, Oxford OX2 0EL (telephone no. 01865 240201, telex 83355 Medbok G, fax 01865 721205) Members of the British Society for Allergy and Chnical Immunology receive the journal automancally This journal is covered by Current Contents, Biological Abstracts, ASCA, IS1/ BIOMED and CABS (Curre~zt Awareness ~n Btologzcal Sctenees).
Rev. fr. Allergol., 1995, 35, 4.
D o nasal mast cells release histamine on stimulation with substance P in allergic rhinitis? G. B R A U N S T E I N , A. B U V R Y , J. L A C R O N I Q U E , N. D E S J A R D I N S and N. FROSSARD Laboratoh-e de physiologic respiratoire et service de pneunlologie, UFR Cochin-Port-Royal, Paris. France Summary The effects of nasal administration of increasing doses of exogenous substance P have been studied in patients with allergic rhinitis treated with placebo or with the H~ antagonist cetirizine (10 mg twice daily for 3 days). Responses to substance P were assessed by posterior rhlnomanometry (measuring nasal airway resistance) and by measure of histamine, protein and albumin production and cell recovery in nasal lavage fluids before and after challenge. Substance P induced a dose-dependent increase in nasal airway resistance which was similar after treatment with either cetwizine or placebo (maximal increase in nasal airway resistance was 4.2-fold greater than the baseline with the placebo and 4-7-fold greater than the baseline with cetinzine). No histamine release was observed. Similar increases in protein and albumin production were observed after stimulation with substance P along with the placebo (protein: from 0"35 + 0- l 1 to 3-31 + 0.62 mg and albumin: from 0"09 ___0.04 to 2"0g + 0-39 rag) and when combined with cetirizine treatment (proteins: from 0-42_+009 to 3-62_+0.77 and albumin: from 0.17+0-04 to 2.19+0.51 rag). After stimulation with substance P, percentages of neutrophils recovered in nasal flmds increased from 26.2+_ 11.5 to 54.5 + 9"5 with the placebo and from 35"5 -+ 11 "0 to 53.6-+ 9 5 with cetmzme. Eosmophils were inconsistently found after substance P stimulation during both treatments. In conclusion, nasal response to substance P is not modified by cetinzme which suggests that the effect of substance P is not secondary to histamine release m the nose in man.
Clinical and Experimental Allergy, Vol. 24, pp. 922-929. Submitted 20 July 1993; revised 28 September 1993; accepted 4 February 1994. Introduction Substance P, a mediator of sensory C fibres, has been localized in the nose o f m a n [I,2]. A role for substance P in allergic rhinitis has been suggested since (i) substance P is released during antigen challenge and recovered in nasal lavage fluids [3,4], (it) specific substance P receptors are present in the nasal mucosa [2] whose activation is linked to measurable nasal events such as a decrease in nasal patency [5], exudation of proteins and albumin and influx of inflammatory cells in nasal lavage fluids [6] and (rio response to substance P is greater in patients suffering from allergic rhinitis than in normal subjects [5]. Substance P activates rat peritoneal mast cells as shown by histamine release in vitro [7,8]. In man, intradermic injection of substance P induces a weal-and-flare reaction Correspondence: N. Frossard, Neurolmmunopharmacologle INSERM CJF 91-05, BP24, F-67401 lllklrch Cedex, France. By courtesy of the authors and pubhshers.
[9-11] partially inhibited by a pre-treatment with the antihistamine agent, chlorpheniramine [12]. Isolated cutaneous mast cells also degranulate on stimulation with substance P [ 13,14]. These interactions between mast cells and substance P, together with the fact that mast cells are found closely associated with sensory nerve endings in various tissues including the airways [I 5,16], suggest that part of the effect of substance P may be due to mast cell activation. Whether this occurs in the nose in man can be studied either by the biochemical measurement o f histamine release after stimulation with substance P, as has been described previously [6] or by pharmacological modulation of the effect o f substance P by antihistamine, whmh is the approach we have chosen in this study. Ceurizine is a highly selective and potent H r-antihistamine agent [17]. Oral administration of cetirizine for 3 days leads to concentration of the drug in the nasal mucosa leading to an inhibition of the local effect of
Rev.fi" Allergol., 1995, 35, 4 : extraJts Presse internationale, 369-376
Nasal mast cells and substance P
exogenous histamine [18]. We reasoned that if substance P induced histamine release in the nose then the response to the peptide would be altered by cetirizine pre-treatment. Therefore, the aim of our study was to compare the nasal response to substance P in allergic rhinitic patients in the presence of cetirizine or a placebo.
Subjects and methods Subjects Twelve patients suffering from allergic rhinitis (mean age+sD: 28-0_+4.2; seven males and five females) were included. They all had (i) at least a 4-year past history of sneezing, secretion and nasal obstruction during natural exposure to allergen, (11) a positive skin-prick test to mixed grass pollen (nine patients) or Dermatophagoides pteronyssinus (Stallerg6nes, Paris, France) (three patients) and (iii) a positive nasal challenge to one of these allergens as assessed by occurrence of nasal symptoms and at least a 100% increase in nasal airway resistance (NAR) within 15 min after intranasal administration of the antigen [6]. Patients were free of nasal symptoms and of treatment for at least 1 month before inclusion in the study. They were informed of the risks and purpose of the study and gave their informed written consent to participate. The study was approved by the Ethics Committee of our hospital.
Study design The study was double-blind, randomized, cross-over and placebo-controlled with two treatment periods of 3 days separated by a wash-out period of 11 days. During each treatment period, subjects took cetirizine (10 mg tablets) or matched placebo b.i.d, at 21:00 h and 9:00 h. Nasal challenge to substance P was performed during the afternoon of the third day, between 13:00 h and 18:00 h, 4 h ( + 0.5 h) after the last administration of the treatment which was delayed to ensure compliance with the 4 h interval. To verify treatment compliance at the end of each treatment period, subjects returned the pill-bottles and histamine skin-prick tests (Phazet ®, Pharmacia, Uppsala, Sweden) were performed after each challenge with substance P by an independent person not involved in any part of the study. Diameters of weal-and-flare reactions were measured 10 rain after the prick test.
Nasal challenge to substance P Substance P from a stock solution (400 nmol/100 #1; stored at -80JC) was diluted just before use in saline to obtain three different isotonic solutions of substance P, i.e. 2-5 nmol, 10 nmol and 40 nmol of substance P per 100/~1.
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Initial NAR was measured. Four pre-challenge nasal lavages were then performed for 10 min. Vehicle (isotonic saline; 100/~1 per nostril) and successive increasing doses of substance P (2.5, 5, I0, 20, 40 and 80 nmol) were nebulized every 8 min into each nostril during which the subject held his/her breath for 30 s to avoid inhalation of substance P into the subglottic airways. For each administration, one or two puffs of solutions were given per nostril using a plastic hand-held nebulizer delivering 100 #1 (95-110/A) at each actuation (Stallerg6nes). Ninety seconds, 3 min, 5 rain and 7 5 min after each nebulization of isotonic saline or substance P, NAR, heart rate, systolic and diastolic blood pressure were measured. A nasal lavage was performed 10 min after saline and again 10 min after the last dose of substance P.
Assessment o f the responses NAR was measured by posterior rhinomanometry [6,19]. Briefly, the subjects breathed through the nose into a mask covering the nose. Nasal airflow was measured by a pneumotachograph (Fleisch No 1; Fleisch, Lausanne, Switzerland) connected to a differential pressure transducer (Validyne Model MP 45-1-1871; Validyne Corp., Northridge, CA, USA) placed on the mask. Transnasal pressure is the difference between pressures in the mask and at the tip of a plastic catheter placed over the tongue into the oropharynx, measured using an identical differential pressure transducer. Airflow and transnasal pressure were measured at 10 ms intervals between the onset of inspiration and maximal inspiratory airflow and were monitored using an X-Y recorder and results were fed in parallel into a microcomputer. NAR was calculated at each airflow and the linear regression of NAR as a function of airflow was determined (Mediprom rhinomanometer, Paris, France). NAR was calculated by interpolation at an airflow of 150 ml/s. Blood pressure was measured using a sphygmomanometer cuff placed around the arm. Pre- and post-challenge nasal lavages were performed with saline (0.9% NaC1; 37°C). Five 1 ml aliquots were injected using a soft-tipped catheter into each nostril, past the vestibule. After each injection, the subject expelled the fluid by gently blowing his/her nose into a plastic tube. The volume of fluid recovered was measured. Biochemical analyses were performed on the last pre-challenge nasal lavage, on the post-sahne nasal lavage and on the post-challenge lavage. The vortexed fluid (2 ml) was centrifuged at 1300×g for 10 mln (Beckman TJ-6 centrifuge; Beckman Instruments, Fullerton, CA, USA) The supernatant (1 ml) was sonicated and used for protein analysis [20] and measurement of albumin by the nephelemetric technique (Behring, RueiI-Malmaison,
Rev.fr Allergol, 1995,35, 4.
G. Braunstein et al.
924
15
Results
~4 0 z" E
All subjects completed the study. One subject had a large increase in initial N A R on one study day compared with the other and was therefore excluded from the efficacy analysis, thus based on 11 subjects. Analysis of safety of cetirizine (and placebo) treatment was established with the 12 subjects. Pre- and post-saline values did not differ between the two study days for all parameters. Saline had no effect on any parameter. Post-saline values were therefore presented as baseline in the results below. Fluid recovery was similar within and between periods (range 9.0-9.5).
10 o
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France). Perchloric acid (1 ml: 0"8 N) was added to the remaining supernatant. The tubes were stored at - 2 0 ° C until histamine was measured using an automated fluorometric technique sensitive to 0.5 ng/ml [21]. The lavage fluids (2 ml) were aliquoted for cytological analyses. Total cell count was performed using a Malassez cell. After centnfugation at 850 x g for 5 min, cells were resuspended in Hank's solution and cytocentrifuged for 5 min at 550 rpm (Shandon cytocentrifuge), processing 30 000 cells per shde. Cells'were fixed by air before Diff-Quick ® staining (Dade, Diidingen, Switzerland). Cells were identified according to usual cytological criteria.
Expression of the results and statistical analyses The highest N A R value (cm H20.s. 1 - 1) measured on each dose of substance P was used to construct the doseresponse curve. Total amount of protein (mg), albumin (rag) and histamine (ng) recovered m the nasal lavage fluid were calculated before and after saline and after stimulation with substance P. Total cell count per ml of lavage fluid was calculated for each sample. Differential cell counts were based upon examination of 400 cells. Absolute values for all parameters are presented in the figures. Diameters of skin responses are expressed in mm. All results are presented as group average +_SEM. Comparison between cetirizine and placebo was made using Friedman's non-parametric analysis of variance. Wilcoxon rank test was used for paired comparisons. A P value less than 0'05 was considered significant.
Rev fi Allergol, 1995.35.4
With the placebo, nasal stimulation with substance P induced a dose-dependent increase in N A R (Fig. 1). The mean maximal increase was 4.2-fold basal N A R (range 1'8-8'3). Individual dose-response curves scattered on a large scale. With cetirizine, a similar effect of substance P was observed (Fig. 1). The mean maximal increase in N A R was 4-7-fold basal N A R (range 1-6 10.4; P > 0 . 2 between treatments). Scattering of individual doseresponse curves was also observed. Compartson of the dose-response curves produced during treatment with cetirizine.or placebo did not show statistical difference.
Clinical effects Substance P did not reduce sneezing either during treatment with placebo or cetirizine. A dose-dependent flush induced by substance P was observed with both treatments occurring within 30-45 s and disappearing 3 min after dosing. Mean starting dose (placebo. 21.4 + 3-9 and cetirizine: 17.2+_6.4 nmol) did not differ between treatments ( P > 0.2). A slight and sigmficant fall in systolic blood pressure induced by substance P was observed with both treatments (P<0-05). Maximal changes in blood pressure after stimulation with the highest dose of substance P were not significantly different with both treatments (placebo: - 9 + 3 mm Hg and cetirlzine: - 6 +_ 2 mm Hg). Diastohc blood pressure fell significantly after substance P stimulation on placebo ( - 6 +_2 mm Hg; P < 0.05) but the change did not achieve significance after substance P with cetirizine treatment ( - 1 + 2 mm Hg). A shght increase in heart rate was similar with placebo. + 8"6 +_4.0 and cetirizine: + 8.0 +_ 3.0 beats per min.
Biochemical effects Histamine There was no significant change m the amount of histamine recovered in the nasal lavage fluid after 371
Nasal mast cells and substance P
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substance P with placebo (before: 13.2-+1.3, after: 17.3 -+ 1-6 ng) or with cetirizine (before: 11.5 _+ 1-0, after: 12.4-+ 1.1 ng). Pre- and post-challenge values did not differ between periods (P > 0.3 for both).
mg (median 3.1 ; range 0.7-7.8) with cetinzine (P < 0-05 for both). Substance P-induced increase in protein output did not differ between treatments. Albumhl Similar changes were observed for albumin (Fig.
Protebls Protein output significantly increased in nasal
lavage fluid of all subjects with both placebo and cetirizine (Fig.2). Total amount of protein increased from 0"35_+0'11 mg (median 0.3; range 0.0-1.3) to 3-31 +0.62 mg (median 3.1; range 0.8-7.0) with placebo and from 0.42_+ 0-09 mg (median 0.4; range 0.0-0-9) to 3-62___0.77
372
2). Albumin output increased in the lavage fluid of all subjects on both treatments. During placebo treatment, total albumin output increased from 0.09+0.04 mg (median 0.11; range 0-0-0.47) to 2.08_+0.39 mg (median 1-64; range 0.55-4.23) and with cetirizine from 0.17-t-0"04 mg (median 0-14; range 0.0-0.41) to
Rev. fr. Allergol., 1995, 35, 4.
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G. Braunstein et al.
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Fig. 3. Effect of substance P on recovery of polymorphonuclear cells (a, b) and of eosinophds (c, d) m nasal lavage fluid during treatment with placebo (a, c) or cetirlzme (b, d). Individual values before (pre) and after (post) stimulation with substance P are indicated (e). Means are represented by the box.
2.19±0.51 mg (median 1.27; range 0-66-5-25) ( P < 0 . 0 5 for both)i Increases did not differ between treatments. Expressed~ as percentage of total protein, albumin increased from 17_+ 5 to 64_+ 2'g, with placebo and from 41_+ 12 to 62__+5% with cetirizine ( P < 0 - 0 5 for both). Increases were similar for both treatments.
Cellular responses Total cells Substance P induced a significant increase in the total number of cells recovered in the nasal lavage fluid: with placebo, from 37.3+ 17.0 to 82.6+37.8 × 103 cells ( P < 0 . 0 5 ) and-with cetirizine from 33-6+11.0 to 7 1 . 8 ± 2 2 . 8 × 103 cells ( P < 0 ' 0 5 ) . These increases were similar for both treatments.
Rev.fr. Allergol.. 1995.35, 4.
Epithelial cells Substance P did not alter the number of epithelial cells either with placebo (before: 26.6 +- 12-2 and after: 14.6_+2.8x 103 cells) or with cetirizine (before: 12.9+_2.7 and after: 15.6+ 3.6 x 103 cells). Pre- and postchallenge values did not differ between treatments. Epithelial cells expressed as percentage of total ceils s~gnificantly decreased on stimulation with substance P during placebo treatment (from 72.6+ 11.4 to 4 4 . 0 + 9 . 3 % ) and during cetirizine treatment (from 63.2+11"0 to 44.3+_9.5%). Potymorphomtclear cells The percentage of P M N recovered in the lavage fluid increased from 26.2___ 11.5 before to 54.5 + 9 ' 5 % (P < 0"05) after stimulation with substance
373
Nasal mast cells and substance P
P with placebo and from 35-5+11-0 to 53.6-1-9.9°/,, ( P < 0 . 0 5 ) with cetirizine. These increases did not differ between treatments. Considering the absolute number of cells, the same pattern of results was observed (Fig. 3). Eosinophils Changes in the number o f eosinophils in the nasal lavage fluid were not consistent in all subjects. With the placebo, an increase in the eosinophil count was observed in only four subjects and in only six with cetirizine. Therefore, the group average eosinophil counts expressed either as percentage or total cell number did not vary significantly when substance P was administered during either treatment periods (Fig. 3). Histamine skin-prick tests With the placebo, a weal-(9.0+0.3 mm) and flare(44.2-I- 3"3 mm) reaction was observed, which was almost totally abolished by cetirizine treatment (weal 0-8_+ 0.3 and flare 9' 1 _+ 1.1 mm). Adverse events No serious adverse events occurred during the study. One subject experienced drowsiness rated as slight during cetirizine treatment. Discussion
Our results show that the nasal response to exogenous substance P is not influenced by a pre-treatment with the specific and potent HL-receptor antagonist cetirizine in allergic rhinitis-suffering patients. The results obtained with the placebo confirm and extend to a larger group the nasal effects of substance P described previously. These effects include nasal obstruction, albumin extravasation, influx of polymorphonuclear cells and, in some cases, influx of eosinophils [6], a reproducible effect [22] which was not observed in response to repeated administration of saline [6]. Cetirizine did not modify any of these responses. The dose of cetirizine used in these experiments was identical to the dose required to reduce nasal responses to exogenous histamine. Nasal obstruction to low doses of histamine was totally blocked by cetirizine but still occurred at high doses [18]. Hence, in the present study, since nasal obstruction due to substance P was not reduced with cetirizine treatment, only large amounts of histamine could explain such a lack of effect by cetirizine. However, histamine was not recovered in the lavage fluid after substance P stimulation ([6] and this report). Altogether, these results demonstrate that substance P does not release histamine m the nose. This is streng-
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927
thened by the absence of the classical effect of histamine, i.e. sneezing, after substance P administration in man. Albumin recovery increased on administration of exogenous substance P to our rhinitic patients. This is in contrast with results obtained during nasal challenge with capsaicin, a substance releasing endogenous substance P, which does not induce albumin extrusion [23,24]. These latter results were obtained in non-rhinitic patients, probably accounting for the difference in recovery of albumin. However, we cannot exclude that the difference may be due to the endogeneous vs exogeneous origin of substance P. Cetirizine did not affect albumin extrusion, indicating that microvascular leakage due to substance P, like vasodilatlon inducing nasal obstruction, is not mediated by histamine. Substance P induces histamine release from isolated human skin mast cells [13,14]. h7 vivo, in man, the cutaneous effect of substance P is mediated partially by mast cell degranulation since it is reduced by pretreatment with an antihistamine [12,25]. Therefore, mast cells from different tissues in the same species, i.e. the skin and the nose in man, have different sensitivities to substance P. This indicates the presence of different mast cell phenotypes in these tissues. Similar differential effects are observed with compound 48/80 which acts via the same mechanism of action as substance P [26]. Compound 48/80 induces degranulation of skin mast cells [14] whereas mast cells from biopsies of the nasal mucosa are insensitive to this product [27]. Also, in the lung, mast cells do not release histamine after treatment with substance P [28], suggesting a similar reactivity of the mast cell phenotypes along the airways. In mice, intradermic administration o f substance P leads to local infiltration of leucocytes, an effect diminished in animals which are genetically mast cell-deficient and which can be restored by mast cell repletion [29,30]. This links leucocyte recruitment on substance P stimulation to mast cell activation in the skin of mice. In the nose of man, we demonstrated a large influx of P M N on stimulation with substance P which was not inhibited by cetirizine and not associated with histamine release thus not secondary to mast cell activation. This suggests a direct effect of substance P on P M N and/or endothelial cells. Experiments in the rat have already shown the rapid adherence of neutrophils to the endothelium on stimulation with substance P [30] which might suggest an increased expression of adhesion molecules. An influx of eosinophils after substance P administration was not found consistently and when present was of low magnitude compared with P M N recruitment. Nevertheless, substance P may enhance the chemotactic effect of platelet-activatmg factor (PAF) and LTB4 on eosinophils [32] and has been shown to have a greater effect oil
Rev.fr. Allergol., 1995, 35, 4.
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G. Braunstehr et al.
eosinophil recruitment in the presence o f chronic inflamm a t i o n o f the nasal mucosa [33]. C o o p e r a t i o n between m e d i a t o r s may explain the e n h a n c e d effect o f substance P d u r i n g chronic inflammation. Cetirizine, in addition to its antihistamine effect, can reduce the recruitment o f e o s i n o phils b o t h in vitro [34] a n d in vivo [35], In o u r experiments, eosinophil recruitment seldomly occurred with the placebo. Th~s condition was not optimal for the study-of a n eventual inhibitory effect of cetirizine. A role for substance P in the p a t h o p h y s i o l o g y of allergic rhinitis may be suggested by (i) a release o f substance P during antigen challenge in nasal lavage fluid [3], (ii) the localization o f specific substance P receptors on the nasal mucosa [2], (iii) a nasal physiological effect o f exogenous substance P activating these receptors ([6] a n d this report). In particular, a p r o - i n f l a m m a t o r y effect of substance P has been shown, which m a y underline s y m p t o m s of allergic rhinitis. Additionally, some effect o f substance P may be due to its active N-terminal m e t a b o lites as has been previously described [6]. Here we d e m o n s t r a t e that the nasal effect o f s u b s t a n c e P is not secondary to histamine release. In conclusion, the highly selective H~-antihistamine agent, cetirizine, does not modify the nasal responses to substance P m patients with allergic rhinitis. This, together w~th the absence of histamine release, d e m o n strates that nasal mast cells do not d e g r a n u l a t e after stimulation w~th substance P, at least for histaminec o n t a i n i n g granules, without precluding release of o t h e r mediators. This is in contrast with skin m a s t cells a n d illustrates mast cell heterogeneity in various tissues within species.
Acknowledgements W e express our gratitude to A. Weyer for histamine measurements, G. LeGall for technical assistance, G. Strauch (IRT), M. Melac a n d D. Milinkevitch for c o n s t a n t encouragement. This work was s u p p o r t e d by U C B P h a r m a , France.
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Rev #'. Allergol. 1995, 35, 4.
4 Mosimann BL, White MV, Hohman RJ et al. Substance P, calcltonin gene-related peptlde, and vasoactlve intestinal pept~de increase in nasal secretions after allergen challenge in atopic patients. J Allergy Clin Immuno11993, 92: 95-104. 5 Devilller P, Dessanges JF, Rakotosihanaka F et ah Nasal 'response to substannce P and methacholine in subjects with and without allergic rhimtls. Eur Resplr J 1988; 1:356--61. 6 Braunstein G,-Fajac I, Lacronique J, Frossard N. Chnical and inflammatory responses to exogenous tachykinins in allergic rhimtis. Am Rev Respir Dis. 1991; 144:630-5. 7 Mazurek N, Pecht I, Telchberg VI, Blumberg S. The role of the N-terminal tetrapeptide in the histamine releasing action of substance P. Neuropharmacol 1981: 20:1025-7. 8 Devilher P, Renoux M, Glroud JP, Regoh D. Peptides and histamine release from rat peritoneal mast cells. Eur J Pharmacol 1985:117.89-96. 9 H~igermark O, Hokfelt T, Pernow B Flare and itch induced by substance P m human skin. J Invest Derm 1978; 71:233-5. 10 Barnes P J, Brown M J, Doltery CT et ah Histamine is released from skin by substance P but does not act as the final vasoddator in the axon reflex. Br J Pharmacol 1986; 88:741-5. II Fuller RW, Conradson TB, D~xon CMS, Crossman DC, Barnes PJ Sensory neuropept~de effect on human skin Br J Pharmacol 1987; 92 781-8. 12 Foreman JC, Jordan CC, Oehme P, Renner H. Histamine release and vascular changes mduccd by neuropeptides Agents Action 1983, 13.105- 16 13 Lowman MA, Benyon RC, Church MK. Characterization of neuropeptlde-lnduced histamine release from human dispersed skin mast cells. Br J Pharmacol 1988:95'121 30. 14 Benyon RC, Robinson C, Church M. Differentml release of h~stamme and elcosanoids t¥om human skin mast cells actwited by IgE-dependent and non-immunologic stunuh. Br J Pharmacol 1989; 97:898-904 15 Bienenstock J, Perdue M, Blennerhassett M Inflammatory cells and the epithelium. Mast cell/nerve interactions m the lung in vttro and m vwo. Am Rev Resplr Dis 1988, 138 $31 $34. 16 Dometj S, Carls66 B, Dahlqvist A, Forsgren S Occurence of mast cells in relation to the d,stributlon of nerve fibers in the rat larynx. Acta Otolaryngol (Stockh) 1991; 111:981-9. 17 Snyder SH, Snowman AM. Receptor effects of cetirizine. Ann Allergy 1987, 59 4-8. 18 Braunstein G, Malaqum F, Fajac I, Melac M, Frossard N. Inhibition of hlstamme-mduced nasal obstruction by cetirizine in allergic rhimtls Br J Chn Pharmacol 1992, 33'445 8. 19 Ghaem A, Martineaud JP. Determination of nasal resistance by two rhinomanometry techniques m normal man Clin Respir Physlol 1985, 21:11-6. 20 Macart M, Gerbaut L An improvement of the Coomassle blue dye binding method allowing an equal sensttlvlty to various proteins: apphcation to cerebrospmal fluid Clin Chlm Acta 1982, 122.93-101 21 Slragaman RP. An automated continuous-flow system for the extraction and fluorometnc analysis of histamine. Anal Btochem 1974:57.383 94.
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Rev. fr. Allergol., 1995, 35, 4.