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Allergen inhalation challenge induces decrease of serum neutral endopeptidase (NEP) in asthmatics
Peptides 21 (2000) 359 –364
Allergen inhalation challenge induces decrease of serum neutral endopeptidase (NEP) in asthmatics N. Tudorica,*, M. Zhangb, M. Kljajic–Turkaljc, J. Niehusb, B. Cvorisceca, K. Jurgovskyb, G. Kunkelb a
Department of Internal Medicine, University Hospital Dubrava, Zagreb, Croatia Department of Clinical Immunology and Asthma-Clinic, Charite´, Virchow-Klinikum Humboldt University, Berlin, Germany c Children’s Hospital Zagreb, Zagreb, Croatia
b
Received 14 July 1999; accepted 6 December 1999
Abstract There is accumulating evidence that tachykinins are implicated in inflammation, including asthma. Therefore, we hypothesized that the neutral endopeptidase (NEP), under challenge conditions, could be affected. Serum from 21 asthmatics and six healthy volunteers was sampled before, 30, and 120 min after allergen challenge. NEP-IR was determined using an ELISA and was found in all subjects. Compared to prechallenge, no difference was seen between asthmatics and controls; however, under challenge conditions, NEP-IR in asthmatics was significantly lower (30 min, P ⫽ 0.058; 120 min, P ⫽ 0.0017, respectively). This finding supports indirectly the hypothesis that tachykinins are released during allergen exposure, and suggests a regulatory role of NEP. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Allergen; Bronchoprovocation; Neutral endopeptidase
1. Introduction Numerous studies, mainly in animals, have investigated the regulatory role of the nonadrenergic, noncholinergic (NANC) nervous system in the respiratory tract (for review see Refs. [2] and [3]). NANC fibers, often called capsaicinsensitive sensory nerves, were found to contain tachykinins as putative neurotransmitters, of which the best known are substance P and neurokinin A and B [2,3,24 –26]. In rodents, it has been shown that airway tachykinins mediate bronchospasm, mucus secretion, cough, and vascular permeability. Different mechanical and chemical stimuli, including an allergen challenge, can activate noncholinergic excitatory nerves to release tachykinins locally [2,3]. Once released, peptides encounter membrane-bound and soluble peptidases, where neutral endopeptidase (NEP, also known as neutral metalloendopeptidase, enkephalinase, or EC 3.4.24.11) has the major role in tachykinin degradation [7,9,29,38]. Being located in the airway epithelium, [5–7, 36] submucosa, and smooth muscle, NEP seems to modu* Corresponding author. Tel.: ⫹385-1-290-2488; fax: ⫹385-1-264249. E-mail address: [email protected] (N. Tudoric)
late peptide-mediated neural responses in a similar way in which acetylcholinesterase modulates acetylcholine-induced effects. However, NEP was also detected in neutrophils [33] and macrophages [19], and the stimuli that recruit these cells to the airways may increase the amount of NEP locally [7]. The widespread distribution and physiologic effects of neuropeptides, including tachykinins, suggested their important role in the pathophysiology of bronchial asthma [2,3]. Despite the clear findings that allergen challenge, nonimmunologic stimulation of mast cells with compound 48/80, or mediators of the immediate immune response, such as histamine and leukotrienes [4,15,20,28,34,40,41], do induce the release of tachykinins in rodents, their role in human asthma is still controversial. However, a rich supply of peptide-containing nerve fibers and the occurrence, distribution, and coexistence pattern of an array of neuropeptides have been reported in human airways [27]. Furthermore, it has been shown that inhibition of endogenous NEP increases respiratory response to exogenously administered tachykinins in humans, including asthmatics [11–13,27]. It has been hypothesized that decreased NEP activity has unmasked the effects of tachykinins, which then can potentially induce airway hyperresponsiveness [12,13].
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To obtain more information on the possible role of NEP in bronchial asthma, we decided to measure the serum level of NEP in patients with bronchial asthma in basal conditions and after specific allergen inhalation challenge with a newly developed ELISA [42]. We hypothesized that specific allergen challenge would, in relation to released tachykinins, induce changes in serum NEP. According to previous findings, tachykinins, released during the allergic reaction, would be degraded mainly by the airway epithelium membrane-bound NEP. However, NEP soluble in tissue fluids or associated with cellular membranes of neutrophils and macrophages may be an additional mechanism of significant importance.
2. Methods 2.1. Subjects Twenty-one nonsmoking, atopic asthmatic subjects (8 women and 13 men), with a mean age 30.3 years (range, 20 –58 years) who met the diagnostic criteria of the American Thoracic Society for bronchial asthma, and six healthy nonatopic volunteers (three women and three men; mean age, 24.5 years; range, 21–30 years) were recruited for the study. All asthmatic subjects had a history of episodic dyspnea, wheezing, or chest tightness, positive skin prick test to house dust mite allergen, and specific IgE antibodies against Dermatophagoides pter., classes 3 or 4 (CAP-Pharmacia, Sweden). According to the results of metacholine bronchoprovocation tests, the patients had moderate to severe airway hyperresponsiveness, reacting with a 20% fall in FEV1 at metacholine concentration lower than 2 mg/ml. During the 2 weeks before allergen bronchoprovocation, the patients were symptom-free and were not taking any therapy with the exception of -2 agonists, which were withheld for at least 2 days before the test. They were not studied within 1 month of upper-respiratory tract infections. All provocation tests were started at 8 a.m. Specific bronchial provocation in asthmatics was performed as part of the regular diagnostic procedure. The study was approved by the local Ethics Committee of the ‘Sveti Duh’ Hospital. All participants signed a written informed consent. 2.2. Design All asthmatic subjects underwent the standard diagnostic procedures that included skin tests, metacholine challenge, and determination of specific IgE. On the day of the allergen challenge, all participants were physically examined. Blood samples, taken before the challenge, 30, and 120 min after a 20% fall in FEV1, were obtained. Samples were centrifuged at 2000 g for 10 min, then serum was collected and stored at ⫺70°C. All samples were processed simultaneously.
2.3. Allergen inhalation challenge Solutions for allergen inhalation were prepared from the stock solution (obtained from the Institute of Immunology, Zagreb, Croatia) that has been characterized as the Croatian national standard Dermatophagoides pter. allergen extract [39]. Briefly, dried house-dust mite Dermatophagoides pter. was extracted in the ratio 1:100 w/v in PBS. The centrifugation supernatant was dialyzed and filtrated. One-milliliter portions of the filtrate were freeze-dried in ampoules. Each ampoule contained 2.7 mg of dry residue, 0.8% of moisture, and 0.32 mg of proteins, with a relative potency of 6.5 ⫻ 105 IU/ampoule or 2.16 ⫻ 106 IU/mg protein, determined versus WHO International Standard by RAST inhibition assay. A concentration of allergen extract provoking a wheal of the same size as that of histamine 10 mg/ml was estimated by regression line analysis using log/log model determining Ch1 of 0.049. This meant that an allergen extract with a concentration of 0.049 mg protein/ml contained 10 000 BU/ml, or that one ampoule of our allergen extract contained 65 300 BU/ampoule. Stock solution was diluted with sterile PBS containing 0.5% phenol to obtain provocation solutions. The weakest allergen solution contained 1.5 g of allergen extract per ml, which represents about 32.2 BU/ml, whereas the highest concentration contained 21 400 BU/ml. These concentrations meet (considering the number of breaths and total aerosol output) the Official Statement of the European Respiratory Society [37]. Allergen was administered as an aerosol generated using an Asthma Provocation System (APS dosimeter, Jaeger, Wu¨rzburg, Germany), which exposes the nebulizer to compressed air at 1.6 bar (22.8 psi.) and duration of 0.6 s from the start of each breath. Under these conditions the output for each breath was 5.02 l with the particle size mass diameter of 1.9 m. The FEVl measurements were performed in triplicate (Body Plethysmograph, Jeager) after inhalation of a diluent and each concentration of allergen. Subjects inhaled five breaths (total output 25.1 l) of diluent or an allergen solution, starting each inhalation at functional residual capacity and terminating it at approximately 70% baseline vital capacity. A 5-s breathhold was maintained at the end of each inhalation. FEV1 was measured at 3 min, and then at 15 min intervals for 45 min. If a 20% fall of FEV1 was not achieved the concentration of allergen in the nebulizer was increased and the protocol was repeated. Bronchoconstriction was reversed after the 20% fall of FEV1 by a -agonist after blood samples were drawn. During the next 24 h, the patients were observed in the hospital and PEFR was measured every 2 h. 2.4. NEP ELISA This method, fully described in Ref. [26], was used with some modifications. Briefly, polystyrene microtitre plates (A/S Nunc, Roskilde, Denmark) were coated with 100 g/
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361
Table 1 Subject characteristics Subject no.
NEP-IR (pg/ml) 0 min
NEP-IR (pg/ml) 30 min
NEP-IR (pg/ml) 120 min
PC20 FEV1 metacholine*
PC20 FEV1 allergen**
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
935.0 277.1 516.4 279.3 484.2 205.8 516.1 1025.1 322.4 115.4 2100.2 1940.8 782.1 727.9 571.4 330.7 1135.6 412.8 399.5 298.2 142.3
1076.5 243.0 417.5 263.5 428.3 137.6 522.6 987.4 205.1 10.2 1502.8 1364.1 753.5 702.2 631.9 365.7 1302.8 292.7 307.1 310.7 136.2
1022.5 212.7 559.3 135.1 212.9 5.3 555.6 769.1 219.2 5.3 1686.5 1126.5 7171.6 612.8 661.8 292.7 987.6 296.3 253.8 323.4 85.6
0.102 0.521 0.977 0.446 0.206 1.372 0.161 0.054 3.359 0.297 0.457 0.466 0.333 1.189 0.214 0.456 0.731 0.109 0.035 0.059 7.511
127 94 111 100 110 233 127 300 380 25 49 49 339 110 102 79 95 55 82 148 76
* PC20FEV1 metacholine in mg/ml; ** PC20FEV1 allergen in BU/ml.
well of rat-anti-NEP mAb AL 2 (Dunn Labortechnik, Asbach, Germany) at a concentration of 2 g/ml in 0.05 M carbonate-buffered saline (pH 9.6) at 4°C overnight. Nonspecific binding was blocked with 1% BSA (300 m/well) in Tris-buffered saline [TBS, 0.1 M tris-(hydroxymethyl)aminomethane, 0.15 M NaCl, 0.02% NaN3, 0.05% Tween20, pH 7.6]. Either the sample (300 l/well) or recombinant NEP (15– 4000 pg/ml) as reference (kindly provided by KHEPRI-Pharmaceutical, San Fransicso, CA, USA), was added to the plates and incubated for 1 h. Mouse mAb against human NEP, ALB-1 (100 g/well) (Dianova, Hamburg, Germany) was added to the plates at a concentration of 1 g/ml; then the plates were incubated for 60 min and washed. Alkaline phosphatase-conjugated goat-anti-mouse IgG (100 l/well) (Dianova) was added (diluted 1:5000 in TBS with 0.2% BSA) and incubated for 1 h; then 100 l/well of substrate (AMPAK, DAKO Diagnostics, Ely, Cambs., UK) was added in a timed sequence and incubated for 20 min, followed by 100 l/well of reconstituted amplifier in the same sequence. The optical density was determined at 492 nm on an automated microtitre plate reader (Multiskan MCC/340 MK II, Flow Laboratories International SA, Lugano, Switzerland). The NEP values in samples were calculated by interpolation from the reference dilution curve using the P.Fit calculator program (Biosoft, Cambridge, UK). All incubations were performed at room temperature. Because it was only possible to detect recombinant NEP in Western Blot, NEP-values are expressed as NEP-like immunoreactivity (NEP-IR). Measurements of samples were performed in a blinded fashion.
2.5.Statistical analysis Significance of differences between NEP values at 0, 30, and 120 min after the challenge was determined using the Wilcoxon signed rank test. Correlation coefficients were calculated by use of the Spearman rank test. Regression analysis was performed with the software package of SPSS. The difference between groups was considered significant at P ⬍ 0.05. 3. Results NEP-like immunoreactivity (NEP-IR) was found in the serum of all studied persons (Table 1). Marked interindividual serum NEP-IR (pg/ml) differences were found. In the group of asthmatics, the serum NEP levels measured 30 and 120 min after allergen inhalation challenge were significantly lower (P ⫽ 0.058 and P ⫽ 0.0017, respectively), whereas in healthy volunteers, no significant change was seen. Comparing the serum NEP-IR levels of asthmatics with healthy volunteers at basal conditions, no significant difference was observed (Table 2). Furthermore, no correlation nor regression was found between the decrease of the serum NEP-IR level and PC20 for metacholine and/or allergen. 4. Discussion The results of the present study indicate that allergen inhalation challenge induces, in asthmatic patients, a signif-
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Table 2 The serum level of NEP-IR* in asthmatics and controls
Asthmatics Controls
NEP-IR (0 min) MED (25%–75%)
NEP-IR (30 min) MED (25%–75%)
NEP-IR (120 min) MED (25%–75%)
484.2 (298.2–782.5) 496.2 (321.5–758.8) NS
412.3 (263.8–753.4) 530.5 (339.2–796.3) P ⬍ 0.05
323.4 (212.9–717.7) 508.4 (349.2–769.7) P ⬍ 0.05
P ⬍ 0.0017 NS
* NEP-IR (pg/ml) is expressed as median value (MED) and 25th and 75th percentile (values in parentheses).
icant decrease of the serum NEP-IR level. Based on evidence that NEP is the major enzyme responsible for tachykinin degradation [9,38,29,7], this finding favors the hypothesis that tachykinins are involved in the pathophysiological mechanisms of bronchial asthma in humans. NEP, also known as CALLA (common acute lymphocytic leukemia antigen), has been shown to be a membranebound molecule on cells of common acute lymphoblastic leukemia [22], neutrophil granulocytes [33], and many other organs including the kidney and brain [16,21,35]. More recently, studies described the role of peptidases in modulating peptide-induced responses in airways, and enkephalinase activity has been found in crude lung homogenates [23], causing more interest in the location and distribution of NEP in airways. The enzyme has been shown by enzymological methods to be present in the airway epithelium of the rat and ferret [5,6] and in the submucosa and airway smooth muscle of the ferret [6,36]. Human studies showed a significantly different distribution of NEP in the human respiratory tract. Using immunohistochemical staining of lung biopsy specimens, Johnson et al. [19] determined NEP predominantly associated with alveolar surface cells, possibly type I or type II epithelial cells, whereas Haver et al. found NEP associated with alveolar walls and the subepithelial cell layer [18]. In cultured lung cells, the same authors determined NEP in lung fibroblasts and in filamentous material that might be beneath the cells [19]. Our unpublished data corroborate these findings, as specific NEP immunoreactivity was found in lung biopsy specimens predominantly localized in subepithelial, filamentous connective tissue and elastic elements, alveolar epithelium cells, and intercellular connective tissue. These findings also agree with a report from Niehus et al. [31], who found specific NEP positive immunostaining in the cell layer under the basement membrane and in the connective tissue surrounding the submucosal glands of the human nasal mucosa. The authors believe that these cells may correspond to subepithelial myofibroblasts described in the lower airways by Brewster et al. [8]. In addition, the airway and lung distribution of NEP described in the cited studies [18,19], which appears to be similar to that in the upper airways [31], corresponds adequately to the distribution of sensory neuropeptides (substance P and calcitonin generelated peptide) in human airways [27].
To the cited differences in NEP localization, a simple extrapolation of the hypothesis regarding the role of tachykinins and NEP in neurogenic inflammation [2,3,24 –26] from animal studies to the pathophysiology of human bronchial asthma, is not allowed; however there are several intriguing reports. Thus, both the number and length of substance P-containing nerves were reported as being increased in asthmatic airways [32]. Furthermore, a specific NEP inhibitor was shown as having the ability to increase the bronchoconstrictive activity of LTD4, which suggests that this potent inflammatory mediator of asthma exerts its effect partly by secondary release of endogenous tachykinins [14]. A significantly higher level of substance P (SP) was found in BAL fluid obtained from atopics compared to normal controls [30], with increase of SP after allergen provocation only in asthmatics. More recently, the low plasma concentration of VIP and elevated levels of other neuropeptides during exacerbation of asthma were documented [10]. The findings, which clearly documented the uniform distribution of NEP in the human lungs and airways [18,19,31], together with the cited reports, make the hypothesis about the regulatory role of NEP in human asthma more likely. This hypothesis is supported by the findings that endogenous NEP is capable of degrading exogenously administered tachykinins [12,13,42]. We believe that the present finding corroborates this hypothesis. The decreased serum level of NEP may indicate its mobilization to the lungs as the site of increased neuropeptide activity. The important data regarding the functional state of the NEP determined are still not available. However, in cultured human endothelial cells, NEP-IR as well as its functional activity, measured as a specific inhibition by phosphoramidon, has been determined [17]. The present finding offers new details for the hypothesis of the regulatory role of tachykinins in allergic reactions. Based on early animal studies [15,27,28,40] and reports that inhibition of endogenous NEP increases respiratory responses to exogenously administered tachykinins [11–13], the existence of NEP in the human airway epithelium, smooth muscle, and blood vessel endothelium was presumed. Thus, released tachykinins might be degraded instantly by the NEP located in their close proximity. This hypothesis has been supported by immunohistological findings of NEP and NEP mRNA in human bronchi [1]. On the
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contrary, immunohistological studies of human lung periphery have shown no NEP at the mentioned locations [18,19]. If this is so, NEP soluble in tissue fluids or associated with cellular membranes of neutrophils [13] and macrophages [19] may be the mechanism of a greater importance via leakage and/or cell migration. Acknowledgment This work was supported by a research grant from the Deutscher Akademischer Austauschdienst. References [1] Baraniuk JN, Ohkubo K, Kwon OJ, Max J, Ali M, Davies R, Twort C, Kaliner M, Letarte M, Barnes PJ. Localization of neutral endopeptidase (NEP) mRNA in human bronchi. Eur Respir J 1995;9: 1458 – 64. [2] Barnes PJ, Baraniuk JN, Belvisi MG. Neuropeptides in the respiratory tract. Part I. Am Rev Respir Dis 1991;144:1187–98. [3] Barnes PJ, Baraniuk JN, Belvisi MG. Neuropeptides in the respiratory tract. Part II. Am Rev Respir Dis 1991;144:1391–9. [4] Bertrand C, Geppetti P, Graf PD, Foresi A, Nadel JA. Involvement of neurogenic inflammation in antigen-induced bronchoconstriction in guinea pigs. Am J Physiol 1993;265:L507–11. [5] Borson DB, Brokaw JJ, Sekizawa K, McDonald D, Nadel JA. Neutral endopeptidase and neurogenic inflammation in rats with respiratory infections. J Appl Physiol 1989;66:2653– 8. [6] Borson DB, Malfroy B, Gold M, Ramachandran J, Nadel JA. Tachykinins inhibit enkephalinase activity from tracheas and lungs of ferrets. Physiologist 1986;29:174. [7] Borson DB. Roles of neutral endopeptidase in airways. Am J Physiol 1991;260:L212–25. [8] Brewster CEP, Howarth PH, Djukanovic R, Wilson J, Holgate ST, Oche WR. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am Resp Cell Mol Biol 1990;3:507–11. [9] Bunnett NW. Postsecretory metabolism of peptides. Am Rev Respir Dis 1987;136:S27–34. [10] Cardel LO, Uddman R, Edvinsson L. Low plasma concentration of VIP and elevated levels of other neuropeptides during exacerbations of asthma. Eur Respir J 1994;7:2169 –73. [11] Cheung D, Bel EH, Den Hartigh J, Dijkman JH, Sterk PJ. The effects of an inhaled neutral endopeptidase inhibitor, thiorphan, on airway response to neurokinin A in normal humans in vivo. Am Rev Respir Dis 1992;145:1275– 80. [12] Cheung D, Timmers MC, Zwinderman AH, Den Hartigh J, Dijkman JH, Sterk PJ. Neutral endopeptidase activity and airway hyperresponsiveness to neurokinin A in asthmatic subjects in vivo. Am Rev Respir Dis 1993;148:1467–73. [13] Crimi N, Palermo F, Oliveri R, Polosa R, Magri S, Mistretta A. Inhibition of neutral endopeptidase potentiates bronchoconstriction induced by neurokinin A in asthmatic patients. Clin Exp Allergy 1994;24:115–20. [14] Diamant Z, Timmers MC, Van der Veen H, Booms P, Sont JK, Sterk PJ. Effect of an inhaled neutral endopeptidase inhibitor, thiorphan, on airway responsiveness to leukotriene D4, in normal and asthmatic subjects. Eur Respir J 1994;7:459 – 66. [15] Ellis JL, Undem BJ. Antigen-induced enhancement of noncholinergic contractile response to vagus nerve and electrical field stimulation in guinea pig isolated trachea. J Pharmacol Exp Ther 1992;262:646 –53. [16] Gafford J, Skidgel RA, Erdo¨s EG, Hersh LB. Human kidney “enkephalinase,” a neutral metalloendopeptidase that cleaves active peptides. Biochemistry 1983;22:3265–71.
363
[17] Graf K, Kunkel G, Zhang M. Activation of adenylate cyclase and phosphodiesterase inhibition enhance NEP activity in human endothelial cells. Peptides 1995;16:1273– 8. [18] Haver K, Kobzik L, Shore SA. Neutral endopeptidase activity and localization in human lungs and airways. Am Rev Respir Dis 1993; 147: A15 (Abstr). [19] Johnson AR, Ashton J, Schulz WW, Erdo¨s EG. Neutral metalloendopeptidase in human lung tissue and culture cells. Am Rev Respir Dis 1985;132:564 – 8. [20] Kawano O, Kohrogi H, Yamaguchi T, Araki S, Ando M. Neutral endopeptidase inhibitor potentiates allergic bronchoconstriction in guinea pigs in vivo. J Appl Physiol 1993;75:185–90. [21] La Bien TW, McCormack RT. The common acute lymphoblastic leukemia antigen (CD10)— emancipation from a functional enigma. Blood 1989;73:625–35. [22] Letarte M, Vera S, Tan R, et al. Common acute lymphocytic leukemia antigen is identical to neutral endopeptidase. J Exp Med 1988;168: 1247–51. [23] Llorens C, Schwartz JC. Enkephalinase activity in rat peripheral organs. Eur J Pharmacol 1981;69:113– 6. [24] Lundberg JM, Brodin E, Saria A. Effects of distribution of vagal capsaicin-sensitive substance P neurons with special reference to the trachea and lungs. Acta Physiol Scand 1983;119:243–52. [25] Lundberg JM, Hoekfelt T, Martling C-R, Saria A, Cuello C. Sensory substance P-immunoreactive nerves in the lower respiratory tract of various mammals including man. Cell Tissue Res 1984;235:251– 61. [26] Lundberg JM, Saria A, Theodorsson–Norheim E, Brodin E, Hua X, Martling C-R, Gamse R, Hoekfelt T. Multiple tachykinins in capsaicin-sensitive afferents: occurrence, release and biological effects with special reference to irritation of the airways. In: Håkanson R, Sundler F, editors. Tachykinin antagonists. Amsterdam: Elsevier, 1985. pp. 159 – 69. [27] Luts A, Uddman R, Alm P, Basterra J, Sundler F. Peptide-containing nerve fibers in human airways: distribution and coexistence pattern. Int Arch Allergy Immunol 1993;101:52– 60. [28] Martins MA, Shore SA, Drazen JM. Release of tachykinins by histamine, metacholine, PAF, LTD4 and substance P from guinea pig lungs. Am J Physiol 1991;261:449 –55. [29] Nadel JA. Neutral endopeptidase modulates neurogenic inflammation. Eur Respir J 1991;4:745–54. [30] Nieber K, Baumgarten C, Witzel A, Rathsack R, Oehme P, Barunnee T, Kleine-Tebbe J, Kunkel G. The possible role of substance P in the allergic reaction, based on two different provocation models. Int Arch Allergy Immunol 1991;94:334 – 8. [31] Niehus J, Schnellbacher T, Mu¨ller S, De Baey C, Mir–Salim P, Kunkel G. Neutral endopeptidase (NEP) 3.4.24.11. in human nasal mucosa—an immunohistological study. Allergy Clin Immunol News 1994;6:85– 8. [32] Ollerenswah SL, Jarvis D, Sullivan CE, Woolcock AJ. Substance P immunoreactive nerves in airways from asthmatics and nonasthmatics. Eur Respir J 1991;4:673– 82. [33] Painter RG, Dukes R, Sullivan J, Carter R, Erdo¨s EG, Johnson AR. Function of neutral endopeptidase on the cell membrane of human neutrophils. J Biol Chem 1988;263:9456 – 61. [34] Saria A, Martling CR, Yan Z, Theodorsson–Norheim E, Gamse R, Lundberg JM. Release of multiple tachykinins from capsaicin-sensitive sensory nerves in the lung by bradykinin, histamine, dimethylphenyl piperazinium, and vagal nerve stimulation. Am Rev Respir Dis 1988;137:1330 –5. [35] Schwartz JC, Malfroy B, De La Baume S. Biological inactivation of enkephalins and the role of enkephalin-dipeptidyl-carboxypeptidase (“enkephalinase”) as neuropeptidase. Life Sci 1981;29:1715. [36] Sekizawa K, Tamaoki J, Graf PD, Basbaum CB, Borson DB, Nadel JA. Enkephalinase inhibitor potentiated mammalian tachykinin-induced contraction in ferret trachea. J Pharmacol Exp Ther 1987;243: 1211–7.
364
N. Tudoric et al. / Peptides 21 (2000) 359 –364
[37] Sterk PJ, Fabbri LM, Quanjer PH, Cockcroft DW, O’Byrne PM, Anderson SD, Juniper EF, Malo J-L. Airway responsiveness. Standardized challenge testing with pharmacological, physical, and sensitizing stimuli in adults. Eur Respir J 1993;4(suppl. 16):53– 83. [38] Stimler–Gerard NP. Neutral endopeptidase-like enzyme controls the contractile activity of substance P in guinea pig lung. J Clin Invest 1987;79:1819 –25. [39] Trescec A, Kolevska T, Cvoriscec B, Krnic B, Stipic–Markovic A, Tudman Z, Dekaris D. Characterization and partial purification of the Croatian national standard Dermatophagoides pteronyssinus allergen extract. Allergy 1993;48:454 –9.
[40] Tudoric N, Coon RL, Bosnjak ZJ. Inhibition of neutral endopeptidase potentiates compound 48/80-induced constriction of guinea-pig tracheal smooth muscle. Pulm Pharmacol 1994;7:343–7. [41] Tudoric N, Coon RL, Bosnjak ZJ. Phosphoramidon augments contraction of guinea pig tracheal smooth muscle induced by histamine and leukotriene-D4. Int Arch Allergy Immunol 1994; 103:286 –92. [42] Zhang M, Niehus J, Schnellbacher T, Mu¨ller S, Graf K, Schultz KD, Baumgarten CR, Lucas C, Kunkel G. ELISA for the neuropeptide degrading endopeptidase 3.4.24.11. in human serum and leukocytes. Peptides 1994;15:843– 8.
Allergen inhalation challenge induces decrease of serum neutral endopeptidase (NEP) in asthmatics N. Tudorica,*, M. Zhangb, M. Kljajic–Turkaljc, J. Niehusb, B. Cvorisceca, K. Jurgovskyb, G. Kunkelb a
Department of Internal Medicine, University Hospital Dubrava, Zagreb, Croatia Department of Clinical Immunology and Asthma-Clinic, Charite´, Virchow-Klinikum Humboldt University, Berlin, Germany c Children’s Hospital Zagreb, Zagreb, Croatia
b
Received 14 July 1999; accepted 6 December 1999
Abstract There is accumulating evidence that tachykinins are implicated in inflammation, including asthma. Therefore, we hypothesized that the neutral endopeptidase (NEP), under challenge conditions, could be affected. Serum from 21 asthmatics and six healthy volunteers was sampled before, 30, and 120 min after allergen challenge. NEP-IR was determined using an ELISA and was found in all subjects. Compared to prechallenge, no difference was seen between asthmatics and controls; however, under challenge conditions, NEP-IR in asthmatics was significantly lower (30 min, P ⫽ 0.058; 120 min, P ⫽ 0.0017, respectively). This finding supports indirectly the hypothesis that tachykinins are released during allergen exposure, and suggests a regulatory role of NEP. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Allergen; Bronchoprovocation; Neutral endopeptidase
1. Introduction Numerous studies, mainly in animals, have investigated the regulatory role of the nonadrenergic, noncholinergic (NANC) nervous system in the respiratory tract (for review see Refs. [2] and [3]). NANC fibers, often called capsaicinsensitive sensory nerves, were found to contain tachykinins as putative neurotransmitters, of which the best known are substance P and neurokinin A and B [2,3,24 –26]. In rodents, it has been shown that airway tachykinins mediate bronchospasm, mucus secretion, cough, and vascular permeability. Different mechanical and chemical stimuli, including an allergen challenge, can activate noncholinergic excitatory nerves to release tachykinins locally [2,3]. Once released, peptides encounter membrane-bound and soluble peptidases, where neutral endopeptidase (NEP, also known as neutral metalloendopeptidase, enkephalinase, or EC 3.4.24.11) has the major role in tachykinin degradation [7,9,29,38]. Being located in the airway epithelium, [5–7, 36] submucosa, and smooth muscle, NEP seems to modu* Corresponding author. Tel.: ⫹385-1-290-2488; fax: ⫹385-1-264249. E-mail address: [email protected] (N. Tudoric)
late peptide-mediated neural responses in a similar way in which acetylcholinesterase modulates acetylcholine-induced effects. However, NEP was also detected in neutrophils [33] and macrophages [19], and the stimuli that recruit these cells to the airways may increase the amount of NEP locally [7]. The widespread distribution and physiologic effects of neuropeptides, including tachykinins, suggested their important role in the pathophysiology of bronchial asthma [2,3]. Despite the clear findings that allergen challenge, nonimmunologic stimulation of mast cells with compound 48/80, or mediators of the immediate immune response, such as histamine and leukotrienes [4,15,20,28,34,40,41], do induce the release of tachykinins in rodents, their role in human asthma is still controversial. However, a rich supply of peptide-containing nerve fibers and the occurrence, distribution, and coexistence pattern of an array of neuropeptides have been reported in human airways [27]. Furthermore, it has been shown that inhibition of endogenous NEP increases respiratory response to exogenously administered tachykinins in humans, including asthmatics [11–13,27]. It has been hypothesized that decreased NEP activity has unmasked the effects of tachykinins, which then can potentially induce airway hyperresponsiveness [12,13].
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To obtain more information on the possible role of NEP in bronchial asthma, we decided to measure the serum level of NEP in patients with bronchial asthma in basal conditions and after specific allergen inhalation challenge with a newly developed ELISA [42]. We hypothesized that specific allergen challenge would, in relation to released tachykinins, induce changes in serum NEP. According to previous findings, tachykinins, released during the allergic reaction, would be degraded mainly by the airway epithelium membrane-bound NEP. However, NEP soluble in tissue fluids or associated with cellular membranes of neutrophils and macrophages may be an additional mechanism of significant importance.
2. Methods 2.1. Subjects Twenty-one nonsmoking, atopic asthmatic subjects (8 women and 13 men), with a mean age 30.3 years (range, 20 –58 years) who met the diagnostic criteria of the American Thoracic Society for bronchial asthma, and six healthy nonatopic volunteers (three women and three men; mean age, 24.5 years; range, 21–30 years) were recruited for the study. All asthmatic subjects had a history of episodic dyspnea, wheezing, or chest tightness, positive skin prick test to house dust mite allergen, and specific IgE antibodies against Dermatophagoides pter., classes 3 or 4 (CAP-Pharmacia, Sweden). According to the results of metacholine bronchoprovocation tests, the patients had moderate to severe airway hyperresponsiveness, reacting with a 20% fall in FEV1 at metacholine concentration lower than 2 mg/ml. During the 2 weeks before allergen bronchoprovocation, the patients were symptom-free and were not taking any therapy with the exception of -2 agonists, which were withheld for at least 2 days before the test. They were not studied within 1 month of upper-respiratory tract infections. All provocation tests were started at 8 a.m. Specific bronchial provocation in asthmatics was performed as part of the regular diagnostic procedure. The study was approved by the local Ethics Committee of the ‘Sveti Duh’ Hospital. All participants signed a written informed consent. 2.2. Design All asthmatic subjects underwent the standard diagnostic procedures that included skin tests, metacholine challenge, and determination of specific IgE. On the day of the allergen challenge, all participants were physically examined. Blood samples, taken before the challenge, 30, and 120 min after a 20% fall in FEV1, were obtained. Samples were centrifuged at 2000 g for 10 min, then serum was collected and stored at ⫺70°C. All samples were processed simultaneously.
2.3. Allergen inhalation challenge Solutions for allergen inhalation were prepared from the stock solution (obtained from the Institute of Immunology, Zagreb, Croatia) that has been characterized as the Croatian national standard Dermatophagoides pter. allergen extract [39]. Briefly, dried house-dust mite Dermatophagoides pter. was extracted in the ratio 1:100 w/v in PBS. The centrifugation supernatant was dialyzed and filtrated. One-milliliter portions of the filtrate were freeze-dried in ampoules. Each ampoule contained 2.7 mg of dry residue, 0.8% of moisture, and 0.32 mg of proteins, with a relative potency of 6.5 ⫻ 105 IU/ampoule or 2.16 ⫻ 106 IU/mg protein, determined versus WHO International Standard by RAST inhibition assay. A concentration of allergen extract provoking a wheal of the same size as that of histamine 10 mg/ml was estimated by regression line analysis using log/log model determining Ch1 of 0.049. This meant that an allergen extract with a concentration of 0.049 mg protein/ml contained 10 000 BU/ml, or that one ampoule of our allergen extract contained 65 300 BU/ampoule. Stock solution was diluted with sterile PBS containing 0.5% phenol to obtain provocation solutions. The weakest allergen solution contained 1.5 g of allergen extract per ml, which represents about 32.2 BU/ml, whereas the highest concentration contained 21 400 BU/ml. These concentrations meet (considering the number of breaths and total aerosol output) the Official Statement of the European Respiratory Society [37]. Allergen was administered as an aerosol generated using an Asthma Provocation System (APS dosimeter, Jaeger, Wu¨rzburg, Germany), which exposes the nebulizer to compressed air at 1.6 bar (22.8 psi.) and duration of 0.6 s from the start of each breath. Under these conditions the output for each breath was 5.02 l with the particle size mass diameter of 1.9 m. The FEVl measurements were performed in triplicate (Body Plethysmograph, Jeager) after inhalation of a diluent and each concentration of allergen. Subjects inhaled five breaths (total output 25.1 l) of diluent or an allergen solution, starting each inhalation at functional residual capacity and terminating it at approximately 70% baseline vital capacity. A 5-s breathhold was maintained at the end of each inhalation. FEV1 was measured at 3 min, and then at 15 min intervals for 45 min. If a 20% fall of FEV1 was not achieved the concentration of allergen in the nebulizer was increased and the protocol was repeated. Bronchoconstriction was reversed after the 20% fall of FEV1 by a -agonist after blood samples were drawn. During the next 24 h, the patients were observed in the hospital and PEFR was measured every 2 h. 2.4. NEP ELISA This method, fully described in Ref. [26], was used with some modifications. Briefly, polystyrene microtitre plates (A/S Nunc, Roskilde, Denmark) were coated with 100 g/
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Table 1 Subject characteristics Subject no.
NEP-IR (pg/ml) 0 min
NEP-IR (pg/ml) 30 min
NEP-IR (pg/ml) 120 min
PC20 FEV1 metacholine*
PC20 FEV1 allergen**
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
935.0 277.1 516.4 279.3 484.2 205.8 516.1 1025.1 322.4 115.4 2100.2 1940.8 782.1 727.9 571.4 330.7 1135.6 412.8 399.5 298.2 142.3
1076.5 243.0 417.5 263.5 428.3 137.6 522.6 987.4 205.1 10.2 1502.8 1364.1 753.5 702.2 631.9 365.7 1302.8 292.7 307.1 310.7 136.2
1022.5 212.7 559.3 135.1 212.9 5.3 555.6 769.1 219.2 5.3 1686.5 1126.5 7171.6 612.8 661.8 292.7 987.6 296.3 253.8 323.4 85.6
0.102 0.521 0.977 0.446 0.206 1.372 0.161 0.054 3.359 0.297 0.457 0.466 0.333 1.189 0.214 0.456 0.731 0.109 0.035 0.059 7.511
127 94 111 100 110 233 127 300 380 25 49 49 339 110 102 79 95 55 82 148 76
* PC20FEV1 metacholine in mg/ml; ** PC20FEV1 allergen in BU/ml.
well of rat-anti-NEP mAb AL 2 (Dunn Labortechnik, Asbach, Germany) at a concentration of 2 g/ml in 0.05 M carbonate-buffered saline (pH 9.6) at 4°C overnight. Nonspecific binding was blocked with 1% BSA (300 m/well) in Tris-buffered saline [TBS, 0.1 M tris-(hydroxymethyl)aminomethane, 0.15 M NaCl, 0.02% NaN3, 0.05% Tween20, pH 7.6]. Either the sample (300 l/well) or recombinant NEP (15– 4000 pg/ml) as reference (kindly provided by KHEPRI-Pharmaceutical, San Fransicso, CA, USA), was added to the plates and incubated for 1 h. Mouse mAb against human NEP, ALB-1 (100 g/well) (Dianova, Hamburg, Germany) was added to the plates at a concentration of 1 g/ml; then the plates were incubated for 60 min and washed. Alkaline phosphatase-conjugated goat-anti-mouse IgG (100 l/well) (Dianova) was added (diluted 1:5000 in TBS with 0.2% BSA) and incubated for 1 h; then 100 l/well of substrate (AMPAK, DAKO Diagnostics, Ely, Cambs., UK) was added in a timed sequence and incubated for 20 min, followed by 100 l/well of reconstituted amplifier in the same sequence. The optical density was determined at 492 nm on an automated microtitre plate reader (Multiskan MCC/340 MK II, Flow Laboratories International SA, Lugano, Switzerland). The NEP values in samples were calculated by interpolation from the reference dilution curve using the P.Fit calculator program (Biosoft, Cambridge, UK). All incubations were performed at room temperature. Because it was only possible to detect recombinant NEP in Western Blot, NEP-values are expressed as NEP-like immunoreactivity (NEP-IR). Measurements of samples were performed in a blinded fashion.
2.5.Statistical analysis Significance of differences between NEP values at 0, 30, and 120 min after the challenge was determined using the Wilcoxon signed rank test. Correlation coefficients were calculated by use of the Spearman rank test. Regression analysis was performed with the software package of SPSS. The difference between groups was considered significant at P ⬍ 0.05. 3. Results NEP-like immunoreactivity (NEP-IR) was found in the serum of all studied persons (Table 1). Marked interindividual serum NEP-IR (pg/ml) differences were found. In the group of asthmatics, the serum NEP levels measured 30 and 120 min after allergen inhalation challenge were significantly lower (P ⫽ 0.058 and P ⫽ 0.0017, respectively), whereas in healthy volunteers, no significant change was seen. Comparing the serum NEP-IR levels of asthmatics with healthy volunteers at basal conditions, no significant difference was observed (Table 2). Furthermore, no correlation nor regression was found between the decrease of the serum NEP-IR level and PC20 for metacholine and/or allergen. 4. Discussion The results of the present study indicate that allergen inhalation challenge induces, in asthmatic patients, a signif-
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Table 2 The serum level of NEP-IR* in asthmatics and controls
Asthmatics Controls
NEP-IR (0 min) MED (25%–75%)
NEP-IR (30 min) MED (25%–75%)
NEP-IR (120 min) MED (25%–75%)
484.2 (298.2–782.5) 496.2 (321.5–758.8) NS
412.3 (263.8–753.4) 530.5 (339.2–796.3) P ⬍ 0.05
323.4 (212.9–717.7) 508.4 (349.2–769.7) P ⬍ 0.05
P ⬍ 0.0017 NS
* NEP-IR (pg/ml) is expressed as median value (MED) and 25th and 75th percentile (values in parentheses).
icant decrease of the serum NEP-IR level. Based on evidence that NEP is the major enzyme responsible for tachykinin degradation [9,38,29,7], this finding favors the hypothesis that tachykinins are involved in the pathophysiological mechanisms of bronchial asthma in humans. NEP, also known as CALLA (common acute lymphocytic leukemia antigen), has been shown to be a membranebound molecule on cells of common acute lymphoblastic leukemia [22], neutrophil granulocytes [33], and many other organs including the kidney and brain [16,21,35]. More recently, studies described the role of peptidases in modulating peptide-induced responses in airways, and enkephalinase activity has been found in crude lung homogenates [23], causing more interest in the location and distribution of NEP in airways. The enzyme has been shown by enzymological methods to be present in the airway epithelium of the rat and ferret [5,6] and in the submucosa and airway smooth muscle of the ferret [6,36]. Human studies showed a significantly different distribution of NEP in the human respiratory tract. Using immunohistochemical staining of lung biopsy specimens, Johnson et al. [19] determined NEP predominantly associated with alveolar surface cells, possibly type I or type II epithelial cells, whereas Haver et al. found NEP associated with alveolar walls and the subepithelial cell layer [18]. In cultured lung cells, the same authors determined NEP in lung fibroblasts and in filamentous material that might be beneath the cells [19]. Our unpublished data corroborate these findings, as specific NEP immunoreactivity was found in lung biopsy specimens predominantly localized in subepithelial, filamentous connective tissue and elastic elements, alveolar epithelium cells, and intercellular connective tissue. These findings also agree with a report from Niehus et al. [31], who found specific NEP positive immunostaining in the cell layer under the basement membrane and in the connective tissue surrounding the submucosal glands of the human nasal mucosa. The authors believe that these cells may correspond to subepithelial myofibroblasts described in the lower airways by Brewster et al. [8]. In addition, the airway and lung distribution of NEP described in the cited studies [18,19], which appears to be similar to that in the upper airways [31], corresponds adequately to the distribution of sensory neuropeptides (substance P and calcitonin generelated peptide) in human airways [27].
To the cited differences in NEP localization, a simple extrapolation of the hypothesis regarding the role of tachykinins and NEP in neurogenic inflammation [2,3,24 –26] from animal studies to the pathophysiology of human bronchial asthma, is not allowed; however there are several intriguing reports. Thus, both the number and length of substance P-containing nerves were reported as being increased in asthmatic airways [32]. Furthermore, a specific NEP inhibitor was shown as having the ability to increase the bronchoconstrictive activity of LTD4, which suggests that this potent inflammatory mediator of asthma exerts its effect partly by secondary release of endogenous tachykinins [14]. A significantly higher level of substance P (SP) was found in BAL fluid obtained from atopics compared to normal controls [30], with increase of SP after allergen provocation only in asthmatics. More recently, the low plasma concentration of VIP and elevated levels of other neuropeptides during exacerbation of asthma were documented [10]. The findings, which clearly documented the uniform distribution of NEP in the human lungs and airways [18,19,31], together with the cited reports, make the hypothesis about the regulatory role of NEP in human asthma more likely. This hypothesis is supported by the findings that endogenous NEP is capable of degrading exogenously administered tachykinins [12,13,42]. We believe that the present finding corroborates this hypothesis. The decreased serum level of NEP may indicate its mobilization to the lungs as the site of increased neuropeptide activity. The important data regarding the functional state of the NEP determined are still not available. However, in cultured human endothelial cells, NEP-IR as well as its functional activity, measured as a specific inhibition by phosphoramidon, has been determined [17]. The present finding offers new details for the hypothesis of the regulatory role of tachykinins in allergic reactions. Based on early animal studies [15,27,28,40] and reports that inhibition of endogenous NEP increases respiratory responses to exogenously administered tachykinins [11–13], the existence of NEP in the human airway epithelium, smooth muscle, and blood vessel endothelium was presumed. Thus, released tachykinins might be degraded instantly by the NEP located in their close proximity. This hypothesis has been supported by immunohistological findings of NEP and NEP mRNA in human bronchi [1]. On the
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contrary, immunohistological studies of human lung periphery have shown no NEP at the mentioned locations [18,19]. If this is so, NEP soluble in tissue fluids or associated with cellular membranes of neutrophils [13] and macrophages [19] may be the mechanism of a greater importance via leakage and/or cell migration. Acknowledgment This work was supported by a research grant from the Deutscher Akademischer Austauschdienst. References [1] Baraniuk JN, Ohkubo K, Kwon OJ, Max J, Ali M, Davies R, Twort C, Kaliner M, Letarte M, Barnes PJ. Localization of neutral endopeptidase (NEP) mRNA in human bronchi. Eur Respir J 1995;9: 1458 – 64. [2] Barnes PJ, Baraniuk JN, Belvisi MG. Neuropeptides in the respiratory tract. Part I. Am Rev Respir Dis 1991;144:1187–98. [3] Barnes PJ, Baraniuk JN, Belvisi MG. Neuropeptides in the respiratory tract. Part II. Am Rev Respir Dis 1991;144:1391–9. [4] Bertrand C, Geppetti P, Graf PD, Foresi A, Nadel JA. Involvement of neurogenic inflammation in antigen-induced bronchoconstriction in guinea pigs. Am J Physiol 1993;265:L507–11. [5] Borson DB, Brokaw JJ, Sekizawa K, McDonald D, Nadel JA. Neutral endopeptidase and neurogenic inflammation in rats with respiratory infections. J Appl Physiol 1989;66:2653– 8. [6] Borson DB, Malfroy B, Gold M, Ramachandran J, Nadel JA. Tachykinins inhibit enkephalinase activity from tracheas and lungs of ferrets. Physiologist 1986;29:174. [7] Borson DB. Roles of neutral endopeptidase in airways. Am J Physiol 1991;260:L212–25. [8] Brewster CEP, Howarth PH, Djukanovic R, Wilson J, Holgate ST, Oche WR. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am Resp Cell Mol Biol 1990;3:507–11. [9] Bunnett NW. Postsecretory metabolism of peptides. Am Rev Respir Dis 1987;136:S27–34. [10] Cardel LO, Uddman R, Edvinsson L. Low plasma concentration of VIP and elevated levels of other neuropeptides during exacerbations of asthma. Eur Respir J 1994;7:2169 –73. [11] Cheung D, Bel EH, Den Hartigh J, Dijkman JH, Sterk PJ. The effects of an inhaled neutral endopeptidase inhibitor, thiorphan, on airway response to neurokinin A in normal humans in vivo. Am Rev Respir Dis 1992;145:1275– 80. [12] Cheung D, Timmers MC, Zwinderman AH, Den Hartigh J, Dijkman JH, Sterk PJ. Neutral endopeptidase activity and airway hyperresponsiveness to neurokinin A in asthmatic subjects in vivo. Am Rev Respir Dis 1993;148:1467–73. [13] Crimi N, Palermo F, Oliveri R, Polosa R, Magri S, Mistretta A. Inhibition of neutral endopeptidase potentiates bronchoconstriction induced by neurokinin A in asthmatic patients. Clin Exp Allergy 1994;24:115–20. [14] Diamant Z, Timmers MC, Van der Veen H, Booms P, Sont JK, Sterk PJ. Effect of an inhaled neutral endopeptidase inhibitor, thiorphan, on airway responsiveness to leukotriene D4, in normal and asthmatic subjects. Eur Respir J 1994;7:459 – 66. [15] Ellis JL, Undem BJ. Antigen-induced enhancement of noncholinergic contractile response to vagus nerve and electrical field stimulation in guinea pig isolated trachea. J Pharmacol Exp Ther 1992;262:646 –53. [16] Gafford J, Skidgel RA, Erdo¨s EG, Hersh LB. Human kidney “enkephalinase,” a neutral metalloendopeptidase that cleaves active peptides. Biochemistry 1983;22:3265–71.
363
[17] Graf K, Kunkel G, Zhang M. Activation of adenylate cyclase and phosphodiesterase inhibition enhance NEP activity in human endothelial cells. Peptides 1995;16:1273– 8. [18] Haver K, Kobzik L, Shore SA. Neutral endopeptidase activity and localization in human lungs and airways. Am Rev Respir Dis 1993; 147: A15 (Abstr). [19] Johnson AR, Ashton J, Schulz WW, Erdo¨s EG. Neutral metalloendopeptidase in human lung tissue and culture cells. Am Rev Respir Dis 1985;132:564 – 8. [20] Kawano O, Kohrogi H, Yamaguchi T, Araki S, Ando M. Neutral endopeptidase inhibitor potentiates allergic bronchoconstriction in guinea pigs in vivo. J Appl Physiol 1993;75:185–90. [21] La Bien TW, McCormack RT. The common acute lymphoblastic leukemia antigen (CD10)— emancipation from a functional enigma. Blood 1989;73:625–35. [22] Letarte M, Vera S, Tan R, et al. Common acute lymphocytic leukemia antigen is identical to neutral endopeptidase. J Exp Med 1988;168: 1247–51. [23] Llorens C, Schwartz JC. Enkephalinase activity in rat peripheral organs. Eur J Pharmacol 1981;69:113– 6. [24] Lundberg JM, Brodin E, Saria A. Effects of distribution of vagal capsaicin-sensitive substance P neurons with special reference to the trachea and lungs. Acta Physiol Scand 1983;119:243–52. [25] Lundberg JM, Hoekfelt T, Martling C-R, Saria A, Cuello C. Sensory substance P-immunoreactive nerves in the lower respiratory tract of various mammals including man. Cell Tissue Res 1984;235:251– 61. [26] Lundberg JM, Saria A, Theodorsson–Norheim E, Brodin E, Hua X, Martling C-R, Gamse R, Hoekfelt T. Multiple tachykinins in capsaicin-sensitive afferents: occurrence, release and biological effects with special reference to irritation of the airways. In: Håkanson R, Sundler F, editors. Tachykinin antagonists. Amsterdam: Elsevier, 1985. pp. 159 – 69. [27] Luts A, Uddman R, Alm P, Basterra J, Sundler F. Peptide-containing nerve fibers in human airways: distribution and coexistence pattern. Int Arch Allergy Immunol 1993;101:52– 60. [28] Martins MA, Shore SA, Drazen JM. Release of tachykinins by histamine, metacholine, PAF, LTD4 and substance P from guinea pig lungs. Am J Physiol 1991;261:449 –55. [29] Nadel JA. Neutral endopeptidase modulates neurogenic inflammation. Eur Respir J 1991;4:745–54. [30] Nieber K, Baumgarten C, Witzel A, Rathsack R, Oehme P, Barunnee T, Kleine-Tebbe J, Kunkel G. The possible role of substance P in the allergic reaction, based on two different provocation models. Int Arch Allergy Immunol 1991;94:334 – 8. [31] Niehus J, Schnellbacher T, Mu¨ller S, De Baey C, Mir–Salim P, Kunkel G. Neutral endopeptidase (NEP) 3.4.24.11. in human nasal mucosa—an immunohistological study. Allergy Clin Immunol News 1994;6:85– 8. [32] Ollerenswah SL, Jarvis D, Sullivan CE, Woolcock AJ. Substance P immunoreactive nerves in airways from asthmatics and nonasthmatics. Eur Respir J 1991;4:673– 82. [33] Painter RG, Dukes R, Sullivan J, Carter R, Erdo¨s EG, Johnson AR. Function of neutral endopeptidase on the cell membrane of human neutrophils. J Biol Chem 1988;263:9456 – 61. [34] Saria A, Martling CR, Yan Z, Theodorsson–Norheim E, Gamse R, Lundberg JM. Release of multiple tachykinins from capsaicin-sensitive sensory nerves in the lung by bradykinin, histamine, dimethylphenyl piperazinium, and vagal nerve stimulation. Am Rev Respir Dis 1988;137:1330 –5. [35] Schwartz JC, Malfroy B, De La Baume S. Biological inactivation of enkephalins and the role of enkephalin-dipeptidyl-carboxypeptidase (“enkephalinase”) as neuropeptidase. Life Sci 1981;29:1715. [36] Sekizawa K, Tamaoki J, Graf PD, Basbaum CB, Borson DB, Nadel JA. Enkephalinase inhibitor potentiated mammalian tachykinin-induced contraction in ferret trachea. J Pharmacol Exp Ther 1987;243: 1211–7.
364
N. Tudoric et al. / Peptides 21 (2000) 359 –364
[37] Sterk PJ, Fabbri LM, Quanjer PH, Cockcroft DW, O’Byrne PM, Anderson SD, Juniper EF, Malo J-L. Airway responsiveness. Standardized challenge testing with pharmacological, physical, and sensitizing stimuli in adults. Eur Respir J 1993;4(suppl. 16):53– 83. [38] Stimler–Gerard NP. Neutral endopeptidase-like enzyme controls the contractile activity of substance P in guinea pig lung. J Clin Invest 1987;79:1819 –25. [39] Trescec A, Kolevska T, Cvoriscec B, Krnic B, Stipic–Markovic A, Tudman Z, Dekaris D. Characterization and partial purification of the Croatian national standard Dermatophagoides pteronyssinus allergen extract. Allergy 1993;48:454 –9.
[40] Tudoric N, Coon RL, Bosnjak ZJ. Inhibition of neutral endopeptidase potentiates compound 48/80-induced constriction of guinea-pig tracheal smooth muscle. Pulm Pharmacol 1994;7:343–7. [41] Tudoric N, Coon RL, Bosnjak ZJ. Phosphoramidon augments contraction of guinea pig tracheal smooth muscle induced by histamine and leukotriene-D4. Int Arch Allergy Immunol 1994; 103:286 –92. [42] Zhang M, Niehus J, Schnellbacher T, Mu¨ller S, Graf K, Schultz KD, Baumgarten CR, Lucas C, Kunkel G. ELISA for the neuropeptide degrading endopeptidase 3.4.24.11. in human serum and leukocytes. Peptides 1994;15:843– 8.