Nerve growth factor is released by IL-1b and induces hyperresponsiveness of the human isolated bronchus

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1 Er Respir J 25; 26: 15 2 DOI: / CopyrightßERS Jornals Ltd 25 Nerve growth factor is released by IL-1b and indces hyperresponsiveness of the hman isolated bronchs N. Frossard, E. Naline #, C. Olgart Höglnd, O. Georges " and C. Advenier # ABSTRACT: Nerve growth factor (NGF) is a nerotrophic factor essential for the development and srvival of nerons, and is also an important mediator of inflammation. It is released by airway cells stimlated by interlekin (IL)-1b. As IL-1b indces airway hyperresponsiveness (AHR) to the tachykinin NK-1 receptor agonist [Sar 9,Met(O 2 ) 11 ]-sbstance P in hman isolated bronchi, the aim of this stdy was to determine whether IL-1b was able to indce NGF release from isolated bronchi, and whether NGF might participate into IL-1b-indced AHR. IL-1b (1 ng?ml -1 ;21 C; 15 h) increased the release of NGF from hman isolated bronchi in vitro, and, in organ bath stdies, the response of hman bronchi to [Sar 9,Met(O 2 ) 11 ]-sbstance P (.1 mm). A significant correlation was fond between these responses. AHR indced by IL-1b was abolished by a blocking anti-hman NGF antibody. Finally, NGF (1 ng?ml -1 ;37 C;.5 h) by itself indced a significant increase in [Sar 9,Met(O 2 ) 11 ]-sbstance P responsiveness. By contrast, it did not change the maximal contraction to acetylcholine. In conclsion, the present stdy clearly demonstrated that nerve growth factor may participate in the airway hyperresponsiveness indced by interlekin-1b, which spports the nero-immne cross-talk that may be active in the development of hyperresponsiveness in the hman airways, and sggests nerve growth factor is active in the airways in asthma. KEYWORDS: Asthma, bronchial hyperresponsiveness, inflammation, nerve growth factor, nerotrophin AFFILIATIONS EA 3771, Inflammation and environment in asthma, Université Lois Paster, Strasborg, and # UPRES EA22, Université de Versailles and UFR Biomédicale des Saints-Pères, and " Laboratoire d anatomo-pathologie Gigi-Georges, Paris, France. CORRESPONDENCE N. Frossard EA 3771 Inflammation and environment in asthma Faclté de pharmacie Université Lois Paster BP Illkirch Cedex France Fax: nelly.frossard@pharma.strasbg.fr Nerve growth factor (NGF) is a nerotrophic factor essential for the development and srvival of nerons [1, 2]. It has recently been sggested to fnction as an important mediator of inflammation [3 5]. In the airways in particlar, animal stdies have shown NGF to contribte to the development of airway hyperresponsiveness (AHR). First, NGF blocking antibodies abolish the AHR created in sensitised and challenged mice [6]. In addition, AHR is indced by tisse-specific overexpression of NGF in the airways [7, 8]. Also, NGF by itself indces hyperresponsiveness of the ginea pig airways in vitro [9] and in vivo [1]. However, the evidence of NGF contribting to AHR in hmans is lacking, althogh expression of NGF is increased in asthma [4, 5]. Previos stdies reported that interlekin (IL)-1b indces AHR in several animal models [11 13]. In addition, BARCHASZ et al. [14] described that IL- 1b cases AHR to the NK-1 tachykinin receptor agonist [Sar 9,Met(O 2 ) 11 ]-sbstance P (SP) in the hman isolated bronchs. The mechanism of sch hyperresponsiveness is nclear. In experiments performed in hman airway cells in cltre, IL-1b indced the release of NGF from fibroblasts [15], bronchial smooth mscle cells [16] and airway epithelial cells [17, 18]. However, whether IL-1b is able to indce release of NGF from the hman bronchs ex vivo has not yet been reported. Therefore, the aim of the present stdy was to determine whether: 1) IL-1b is able to indce release of NGF from the hman isolated bronchs, and 2) NGF participates in AHR. METHODS Preparation of hman bronchial tisse Bronchial tisses were srgically removed from 42 patients with lng cancer (27 males, 15 females; mean SD age yrs). The protocol was approved by a local ethics committee (Comité de Protection des Personnes se Prêtant à la Recherche Biomédicale, Versailles, France). After resection, segments of hman bronchi were taken and placed in oxygenated Krebs-Henseleit soltion (composition: NaCl 119 mm, KCl Received: April Accepted after revision: March SUPPORT STATEMENT C. Olgart Höglnd was spported in this stdy by the Swedish Research Concil (Stockholm, Sweden), Torsten and Ragnar Söderberg s Fondations (Sweden), Erik and Edith Fernström s Fondation (Sweden), Comité Départmental d Bas-Rhin Contre les Maladies Respiratories et la Tberclose (France), and the UCB Institte of Allergy (Belgim). This stdy received spport fnding from Inserm (Illkirch Cedex, France) and Université Lois Paster (Strasborg, France). Eropean Respiratory Jornal Print ISSN Online ISSN c EUROPEAN RESPIRATORY JOURNAL VOLUME 26 NUMBER 1 15

2 NGF INDUCES HYPERRESPONSIVENESS OF BRONCHUS N. FROSSARD ET AL. 4.7 mm, CaCl mm, KH 2 PO mm, NaHCO 3 25 mm and glcose 11.7 mm). After removal of adhering lng parenchyma and connective tisse, rings of the same bronchs were prepared (5 7 mm length6.5 1 mm internal diameter), divided into paired grops and randomised [19]. The diameter was measred with a stage micrometer at light transmission after haematoxylin, eosin and saffron staining of 5 mm paraffin sections of a sample of bronchi fixed for 24 h in 1% formyl saline. Bronchial ring segments were placed in 1 ml Krebs-Henseleit soltion at room temperatre (21 C) for 15 h in the presence or absence of IL-1b (1 ng?ml -1 ) as previosly reported [14, 15]. After incbation, the paired bronchi were taken for contractile stdies, and the spernatants kept aliqoted at -8 C ntil NGF measrement. In another set of experiments, anti-hman NGF blocking antibody or an irrelevant immnogloblin (Ig)- G antibody (2 5 ng?ml -1 ; R&D Systems Erope, Lille, France) were incbated simltaneosly to IL-1b for 15 h at 21 C, before bronchi were taken for contractile stdies. The effect of exogenos NGF (.1 1 ng?ml -1 ) was stdied on bronchial ring segments kept in Krebs-Henseleit soltion at 4 C overnight. Contractile stdies were performed after NGF pre-treatment for 3 min directly in the organ baths. Experimental procedre Bronchial ring segments were sspended in 5 ml organ baths containing Krebs-Henseleit soltion, gassed with 95% O 2 5% CO 2 and maintained at 37 C. Each preparation was connected to an isometric force displacement transdcer (UF1; Piodem, Canterbry, Kent, UK) and EMKA amplifier (EMKA Technology, Les Ulis, France). Changes in tension were recorded on a polygraph. Preparations were sspended with an initial tension of 1.5 g, washed three times every 1 min, and eqilibrated for another 3 min at g. The contractile response to the tachykinin NK-1 receptor agonist, was stdied. Bronchial ring segments were washed ot and acetylcholine was applied for maximal contraction. A.1 mm concentration of was selected for maximal NK-1 contractile response withot interference with the tachykinin NK-2 receptor, and a 3 mm acetylcholine concentration for maximal response [2]. Qantification of NGF protein by ELISA NGF was qantified in the spernatant of hman isolated bronchi pre-treated for 15 h with IL-1b (1 ng?ml -1 ) or solvent. A commercially available NGF-specific, highly sensitive, two-site ELISA kit was sed following the manfactrer s instrctions (Promega, Madison, WI, USA). Briefly, 96-well immnoplates (Maxisorp TM, Nnc, Roskilde, Denmark) were coated with a polyclonal goat anti-hman NGF antibody in a coating bffer (25 mm carbonate bffer; ph 9.7). After overnight incbation at 4 C, plates were washed (2 mm Tris-HCl, 15 mm NaCl with.5% (v/v) Tween H-2; Sigma-Aldrich, St Lois, MO, USA), and incbated in a blocking bffer for 1 h. The spernatants and the standard recombinant hman NGF diltions were incbated at 37 C for 6 h and washed. Rat monoclonal anti-hman NGF antibody (.25 mg?ml -1 ) was added for overnight incbation at 4 C, and washed. Antirat horseradish peroxidase-conjgated IgG antibodies were incbated for 2.5 h, and the sbstrate (3,39,5,59-tetramethylbenzidine.2% and hydrogen peroxidase.1%) was added. The colorimetric reaction was stopped after 1 min by adding phosphoric acid (1 M), and the optical density was measred in dplicate at 45 nm. The detection range was pg?ml -1. Expression of reslts and statistical analysis All vales are expressed as mean SEM. Contractile responses were expressed in g and as a percentage of the maximal contraction indced by acetylcholine (3 mm). NGF protein levels were expressed as pg of NGF?mg -1 wet weight tisse. Differences between grops were analysed from raw data sing an npaired, two-tailed t-test, and a two-way ANOVA with Stdent-Newman-Kels test when more than two variables were compared. Data were considered significantly different at p,.5. The correlation between the NGF release (% increase) and contraction to (% increase) was evalated sing regression analysis. The coefficient of determination (r 2 ) was determined from the regression crve. Drgs The drgs sed were:, recombinant hman IL-1b (Bachem, Bbendorf, Switzerland), recombinant NGF, anti-hman NGF blocking antibody and an irrelevant IgG antibody (R&D Systems Erope). IL-1b and NGF were dissolved in distilled water at a concentration of.75 mm and 2 mg?ml -1, respectively, and kept aliqoted at -8 C ntil se. All drgs were dissolved in distilled water and frther dilted in Krebs-Henseleit soltion. RESULTS Effect of IL-1b on NGF release and on AHR As previosly reported, the hman isolated bronchs incbated with IL-1b became hyperresponsive to the NK-1 receptor agonist (.1 mm). Response was increased by % (p,.1; n524; fig. 1). In contrast, the response to acetylcholine (3 mm) was nchanged ( and g for IL-1b and saline, respectively; n524). Contraction g FIGURE 1. Effect of interlekin (IL)-1b (1 ng?ml -1 ;15h;21 C) on contractile responses indced by [Sar 9,Met(O 2 ) 11 ]-sbstance P (SP) (.1 mm) on the hman isolated bronchs. h: withot IL-1b; &: with IL-1b. n524. : p, VOLUME 26 NUMBER 1 EUROPEAN RESPIRATORY JOURNAL

3 N. FROSSARD ET AL. NGF INDUCES HYPERRESPONSIVENESS OF BRONCHUS 8 12 Increase of NGF release % Increased response % Increase of contraction % FIGURE 2. Correlation between the percentage of increase in contraction to [Sar 9,Met(O 2 ) 11 ]-sbstance P (SP) (.1 mm) and the percentage increase in nerve growth factor (NGF) release indced by interlekin (IL)-1b. In parallel, a significant increase in the levels of NGF, althogh slight, was measred in the incbation medim after treatment with IL-1b (3.1.5 and pg?mg -1 wet weight tisse after IL-1b and saline, respectively; n514; p,.5), i.e. a % increase in NGF release. A positive correlation between NGF release and bronchial hyperresponsiveness to IL-1b was measred (r ; p,.5; fig. 2). Effect of a NGF blocking antibody on AHR indced by IL-1b Pre-incbation of an anti-hman NGF blocking antibody (5 ng?ml -1 ) simltaneosly to IL-1b totally abolished the IL-1b-indced AHR to (p,.5 for 5 ng?ml -1 ; fig. 3), bt had no effects on the contractile response to this NK-1 receptor agonist per se. Under similar conditions, pre-incbation with an irrelevant IgG antibody had no effect on the IL-1b-indced hyperresponsiveness to [Sar 9, Met(O 2 ) 11 ]-SP responses, or on the contractile response to this agonist. FIGURE 4. Effect of nerve growth factor (NGF;.1,.1, 1 ng?ml -1 ; 3 min) on responses of hman isolated bronchi to the tachykinin NK-1 receptor agonist [Sar 9,Met(O 2 ) 11 ]-sbstance P (SP) (.1 mm). h: + NGF.1 ng?ml -1 ; &: + NGF.1 ng?ml -1 ; &: + NGF 1 ng?ml -1. Reslts are expressed as percentages of increase from control vales (mean SEM of n511 experiments). : p,.5 verss control. AHR to NGF Exogenos NGF alone had no effect on bronchial smooth mscle tone. NGF (.1 1 ng?ml -1 ) indced a concentrationdependent increase in response, with a maximal increase of % at 1 ng?ml -1 (p,.5; n511; fig. 4). In contrast, NGF (1 ng?ml -1 ) did not change the acetylcholine (3 mm)-indced contraction ( %, nonsignificant). DISCUSSION The present reslts show that IL-1b indces release of NGF from the hman isolated bronchs, and this release is correlated with the increased response to indced by IL-1b. In addition, IL-1b-indced hyperresponsiveness is abolished by pre-treatment with an anti- NGF blocking antibody. Finally, NGF by itself indces a) 6 b) NS NS Contraction % 4 2 FIGURE 3. a) Effect of an anti-hman NGF blocking antibody (anti-ngf; 2 5 ng?ml -1 ). h: control; &: anti-ngf (5 ng?ml -1 ); &: interlekin (IL)-1b; p: IL-1b + anti-ngf (2 ng?ml -1 ); : IL-1b + anti-ngf (5 ng?ml -1 ). b) Effect of an irrelevant immnogloblin(ig)-g (5 ng?ml -1,15h,21 C) on IL-1b-indced hyperresponsiveness to [Sar 9,Met(O 2 ) 11 ]-sbstance P (SP) (.1 mm) in hman isolated bronchi. h: control; &:IL-1b;&: IgG (5 ng?ml -1 ); q: IL-1b + IgG (5 ng?ml -1 ). Reslts are expressed as percentage of contraction verss acetylcholine (3 mm). Data are mean SEM of n58 experiments. NS: nonsignificant; : p,.5. c EUROPEAN RESPIRATORY JOURNAL VOLUME 26 NUMBER 1 17

4 NGF INDUCES HYPERRESPONSIVENESS OF BRONCHUS N. FROSSARD ET AL. hyperresponsiveness of the hman isolated bronchs to the tachykinin NK-1 receptor agonist in vitro. This stdy was designed to investigate new mechanisms involved in previosly reported AHR observed in inflammatory conditions created in vitro by the pro-inflammatory cytokine IL-1b [14]. In hman airway cells in cltre, IL-1b indced release of NGF from airway smooth mscle cells [16], epithelial cells [17, 18] and lng fibroblasts [15]. The crrent stdy demonstrated that IL-1b is also able to stimlate release of NGF from isolated ring segments from the hman bronchs. Ths, overexpression of NGF may be de to increased release of NGF from bronchial strctral cells, smooth mscle, epithelial cells and/or fibroblasts, pon stimlation by IL-1b. Acqisition of a secretory phenotype by the cells of hman bronchi has been previosly sbmitted as a new characteristic of airway cells actively participating in inflammation [15, 18, 2]. Additionally, increased release of NGF may also be derived from immne/inflammatory cells, sch as lymphocytes, macrophages or mast cells [4] since these cells are also reported to secrete NGF [4, 21 23]. This sggests that increased NGF levels might occr in inflammatory conditions in vivo. Indeed, overexpression of NGF has been reported in hmans in vivo, in sitations where IL-1b is increased, particlarly in asthma [24 27]. The present stdy also demonstrated that an anti-ngf blocking antibody abolishes the hyperresponsiveness to indced by IL-1b of the hman bronchs in vitro, sggesting contribtion of NGF to the development of AHR to IL-1b. Concordant sggestion has recently been reported in animal models, when administration of a blocking anti-ngf antibody prevents development of allergen-indced AHR to electrical field stimlation in mice [6] or attenates the allergen-specific early airway response to ovalbmin in sensitised and challenged rats [28]. In addition, exogenos NGF has been reported to indce AHR by itself in the gineapig in vivo [9, 29], and tracheal hyperresponsiveness in the ginea-pig in vitro [1]. The present findings show that NGF also indces a dose-dependent AHR in the hman isolated bronchs in vitro. These reslts provide a potential mechanism to explain the findings of an preglation of NGF protein in the airways of asthmatic patients, which has been associated with AHR, althogh no casal relationship between these events has been demonstrated. Indeed, enhanced NGF levels are reported in serm from patients with asthma, with the highest NGF levels in patients with more severe allergic asthma displaying a high degree of AHR [24]. Also, local preglation of NGF protein was detected in bronchoalveolar lavage flid from asthmatics, displaying AHR, as compared with control sbjects [25]. These NGF levels have been frther enhanced following allergen challenge in mild asthmatics [26]. In addition, previos data from KASSEL et al. [27] spport an preglation of NGF in the bronchi as an early event in response to allergen in asthma, in association with an increased AHR. Indeed, increased NGF transcripts and AHR were detected in the airway tisse from mild asthmatics following exposre to allergen at a low sbclinical dose [27]. All these findings sggest a close association between increased NGF levels and AHR. The present reslts bring spport to an nderlying casal mechanism since they demonstrate that exogenos NGF is responsible for an increased responsiveness of the hman bronchs, and that hyperresponsiveness indced by IL-1b is abolished by anti- NGF antibodies. In contrast, no increased contraction was observed in response to a maximal acetylcholine response. This is in agreement with previos findings in hman isolated bronchi that IL-1b neither modifies acetylcholine maximal response nor displaced the acetylcholine concentration-response crve [14]. This may be linked to the reported heterogeneity of AHR to contractile agonists in asthma, which is now clearly established, bt remains nexplained [3]. The mechanism by which NGF indces AHR is not known. NGF is reported to activate immne/inflammatory cells, sch as mast cells, lymphocytes, basophils or macrophages, as well as strctral resident cells, sch as fibroblasts or airway smooth mscle cells [5]. NGF is also able to sensitise nerons, and it indces an enhanced prodction of tachykinins in sensory nerons. This was shown by HUNTER et al. [31], who demonstrated in the ginea-pig that NGF indces a phenotypic switch of airway sensory nerons 24 h after intratracheal administration, and that NGF increases SP content in neronal cell bodies. Frthermore, HOYLE et al. [7] showed that transgenic mice overexpressing NGF in the airways develop a hyperinnervation of the airways, and an increase in SP content. However, these effects have been observed hors to days after NGF administration, involving transcriptional mechanisms that wold probably not accont for the rapid effect of NGF in the present experimental conditions. This evidence is frther spported by the absence of sensory cell bodies within the preparation, indicating that preglation of SP content cannot be a mechanism of NGF action in the hman bronchs in these conditions in vitro. However, an early local mechanism may be proposed, as also observed in the hyperalgesia mechanism in the skin. Indeed, NGF, when applied directly to nociceptive afferents, lowers the threshold of thermal stimlation in an isolated skin-nerve preparation [32]. Similarly, NGF applied for 1 min on dorsal root ganglion nerons was able to abolish the tachyphylaxis observed in response to capsaicin or, even, to potentiate these responses [33]. Interestingly, NGF has also recently been shown in a model of inflammatory pain in the rat to have short-term effects on nociceptor sensitisation of sensory nerves of the dorsal root ganglia [34]. NGF might, therefore, contribte to an increased neronal hyperexcitability, possibly throgh vanilloid receptor-1 activation withot transcriptional events inclding their increased expression [35, 36]. These reslts, together with the present data, sggest that NGF is able to indce early post-transcriptional changes in nerons and cells, which may be involved in the tisse sensitisation [1, 37 39], and sggest parallel mechanisms in the generation of AHR and of hyperalgesia. In conclsion, the crrent reslts clearly show that nerve growth factor released by interlekin-1b is involved in the airway hyperresponsiveness indced by this cytokine, and spport the evidence of nero-immne cross-talk in the hyperresponsiveness of the hman bronchs. 18 VOLUME 26 NUMBER 1 EUROPEAN RESPIRATORY JOURNAL

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6 NGF INDUCES HYPERRESPONSIVENESS OF BRONCHUS N. FROSSARD ET AL. capsaicin sensitivity of rat dorsal root ganglion sensory nerones. Nerosci Lett 1996; 19,23: Szallasi A, Blmberg PM. Vanilloid (capsaicin) receptors and mechanisms. Pharmacological Rev 1999; 51: Lewin GR, Reff A, Mendell LM. Peripheral and central mechanisms of NGF-indced hyperalgesia. Er J Nerosci 1994; 6: Nicholas RS, Winter J, Wren P, Bergmann R, Woolf CJ. Peripheral inflammation increases the capsaicin sensitivity of dorsal root ganglion nerons in a nerve growth factordependent manner. Nerosci 1999; 91: Woolf CJ, Costigan M. Transcriptional and posttranslational plasticity and the generation of inflammatory pain. Proc Nat Acad Sci USA 1999; 96: VOLUME 26 NUMBER 1 EUROPEAN RESPIRATORY JOURNAL