Increased Th2 Cytokine Secretion, Eosinophilic Airway Inflammation, and Airway Hyperresponsiveness in Neurturin-Deficient Mice This information is current as of September 26, 2021. Tatiana Michel, Maud Thérésine, Aurélie Poli, Olivia Domingues, Wim Ammerlaan, Nicolaas H. C. Brons, François Hentges and Jacques Zimmer J Immunol 2011; 186:6497-6504; Prepublished online 20 April 2011; Downloaded from doi: 10.4049/jimmunol.1001673 http://www.jimmunol.org/content/186/11/6497 http://www.jimmunol.org/ References This article cites 34 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/186/11/6497.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Increased Th2 Cytokine Secretion, Eosinophilic Airway Inflammation, and Airway Hyperresponsiveness in Neurturin-Deficient Mice Tatiana Michel,* Maud The´re´sine,* Aure´lie Poli,* Olivia Domingues,* Wim Ammerlaan,† Nicolaas H. C. Brons,† Franc¸ois Hentges,* and Jacques Zimmer* Neurotrophins such as nerve growth factor and brain-derived neurotrophic factor have been described to be involved in the path- ogenesis of asthma. Neurturin (NTN), another neurotrophin from the glial cell line-derived neurotrophic factor family, was shown to be produced by human immune cells: monocytes, B cells, and T cells. Furthermore, it was previously described that the secretion of inflammatory cytokines was dramatically stimulated in NTN knockout (NTN2/2) mice. NTN is structurally similar to TGF-b, a protective cytokine in airway inflammation. This study investigates the implication of NTN in a model of allergic airway Downloaded from inflammation using NTN2/2 mice. The bronchial inflammatory response of OVA-sensitized NTN2/2 mice was compared with wild-type mice. Airway inflammation, Th2 cytokines, and airway hyperresponsiveness (AHR) were examined. NTN2/2 mice showed an increase of OVA-specific serum IgE and a pronounced worsening of inflammatory features. Eosinophil number and IL-4 and IL-5 concentration in the bronchoalveolar lavage fluid and lung tissue were increased. In parallel, Th2 cytokine secretion of lung draining lymph node cells was also augmented when stimulated by OVA in vitro. Furthermore, AHR was markedly 2/2 enhanced in NTN mice after sensitization and challenge when compared with wild-type mice. Administration of NTN before http://www.jimmunol.org/ challenge with OVA partially rescues the phenotype of NTN2/2 mice. These findings provide evidence for a dampening role of NTN on allergic inflammation and AHR in a murine model of asthma. The Journal of Immunology, 2011, 186: 6497–6504. llergic asthma is characterized by an inflammatory air- nergic noncholinergic nervous system, through the release of way obstruction triggered by inhalation of specific neuropeptides such as substance P, contributes to airway smooth A allergens. A hallmark of allergic asthma is an inap- muscle contraction and to airway hyperresponsiveness (AHR) and propriate regulation of the Th1/Th2 balance in favor of a Th2 modulates neurotrophin synthesis (2). Neurotrophins are mole- response. Asthmatic airways are characterized by infiltrates of eo- cules originally described for their effects on neuronal cells. They sinophils and T lymphocytes secreting Th2 cytokines: IL-4, IL-5, control differentiation, development, and survival of neurons (3, by guest on September 26, 2021 and IL-13 (1). The production of IL-5 results in maturation and 4). Factors with a neurotrophic activity can be divided into three enhanced recruitment of eosinophils into airways mucosa. Asthma structurally different families: 1) the nerve growth factor (NGF) may result in acute life-threatening reactions but more commonly family including NGF, brain-derived neurotrophic factor (BDNF), results in a chronic disabling disease. Many efforts have been neurotrophin-3, and neurotrophin-4; 2) the ciliary neurotrophic spent in the last 10 years to characterize the regulatory events factor/IL-6 family; and 3) the glial cell line-derived neurotrophic involved in the balance of Th1/Th2 cytokines, to identify signif- factor (GDNF) family, including GDNF, neurturin (NTN), arte- icant therapeutic targets. An effective therapy for asthma would be min, and persephin. Neurotrophins such as NGF and BDNF are to inhibit the development of such allergen-specific Th2 cells, but expressed in a variety of nonneural tissues, including the pulmo- the factors responsible for the initial induction of the Th2-like nary system (5). Recent studies suggest that NGF and BDNF response in vivo remain incompletely understood. participate in the pathogenesis of many features and symptoms of Neural regulation of the airways consists of cholinergic, ad- asthma (2, 6–8). renergic, and nonadrenergic noncholinergic nerves. The nonadre- Intensive research has focused on NGF, which is the prominent member of the neurotrophin family; however, several recent reports suggest that GDNF family members may also modulate airway *Laboratoire d9Immunoge´ne´tique et d9Allergologie, Centre de Recherche Public de inflammation. It was described that GDNF is expressed in mouse la Sante´, L-1526 Luxembourg, Luxembourg; and †Platforme de Cytometrie en Flux, lung, in particular in airway smooth muscle cells. After allergen Centre de Recherche Public de la Sante´, L-1526 Luxembourg, Luxembourg challenge in OVA-sensitized mice, the number of GDNF+ cells was Received for publication May 19, 2010. Accepted for publication March 14, 2011. shown to increase within the inflammatory infiltrate (9). Another This work was supported by a grant from the Fonds National de la Recherche, member of this family, NTN, exerts its effects through a multi- Luxembourg. component receptor system consisting of the GDNF family re- Address correspondence and reprint requests to Dr. Tatiana Michel and Dr. Jacques Zimmer, Laboratoire d9Immunoge´ne´tique et d9Allergologie, Centre de Recherche ceptor a 2 (GFRa-2) and the proto-oncogene RET (10, 11). It was Public CRP-Sante´, 84 Val Fleuri, L-1526 Luxembourg, Luxembourg. E-mail previously shown that NTN is produced by human immune cells: addresses: [email protected] and [email protected] monocytes, B cells, and T cells (12). NTN is also structurally Abbreviations used in this article: AHR, airway hyperresponsiveness; BALF, bron- b b choalveolar lavage fluid; BDNF, brain-derived neurotrophic factor; CBA, Cytometric similar to TGF- , and it was demonstrated that TGF- 1 is a Bead Array; Cdyn, dynamic compliance; GDNF, glial cell line-derived neurotrophic protective cytokine in airway inflammation (13). Furthermore, it factor; GFRa-2, GDNF family receptor a 2; LN, lymph node; NGF, nerve growth was described that the production of inflammatory cytokines was factor; NTN, neurturin; PAS, periodic acid-Schiff; WT, wild-type. dramatically stimulated in the corneal epithelia of the NTN 2/2 Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 knockout (NTN ) mice. These mice also have an immune www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001673 6498 NEURTURIN INVOLVED IN ALLERGIC ASTHMA methacholine. Before testing, mice were anesthetized by s.c. injection of 80 mg/kg ketamine, 10 mg/kg xylazine, and 2.5 mg/kg midazolam. Then mice were tracheotomized, mechanically ventilated, and lung function was an- alyzed by Flexivent equipment (Emka Technologies, Paris, France). Bronchoalveolar lavage fluid, lung, and lymph node cell isolation and analysis FIGURE 1. Expression of GFRa-2 mRNA by RT-PCR. Tissues from Immediately after assessment of AHR, lungs were washed via a tracheal 3 WT brain, lung, and lung draining LN were prepared. The experiments tube with PBS and 5% BSA (700 ml) followed by PBS (2 500 ml). Lung cells were isolated using collagenase digestion, followed by staining for were performed by using primers for GFRa-2 and GAPDH as a house- FACS analysis. Lung draining lymph nodes (LNs) were excised from mice, keeping gene. There were three mice in each group. B, brain; L, lung; W, and cells were isolated, adjusted to 5 3 106 cells/ml, and cultured in 48- water. well plates in DMEM supplemented with 10% FBS, 100 U/ml penicillin/ streptomycin, and IL-2 (1000 U/ml) for 1 or 3 d with or without OVA (200 activation of the conjunctival epithelium and an abnormal gut mg/ml). Bronchoalveolar lavage fluid (BALF), lung, and lung draining LN 2 motility caused by a reduction in substance P secretion by gut supernatants were frozen at 80˚C for later cytokine analysis. All cell culture reagents were purchased from Invitrogen (Paisley, U.K.). muscle cells (14, 15). In this study, we assessed the influence of 2/2 NTN on lung function. NTN mice were investigated for airway Measurement of OVA-specific Abs inflammation and for development of AHR after sensitization and Before performing BALF, blood was removed from the heart of mice using challenge with OVA. a syringe for measurement of OVA-specific IgG1, IgG2a, and IgE. Serum levels of anti-OVA IgG1, IgG2a, and IgE were measured by ELISA. Ninety- Downloaded from Materials and Methods six–well plates were coated with either OVA or purified anti-IgE (BD Mice Biosciences, Erembodegem, Belgium). After addition of serum samples, IgE standard (BD Biosciences), biotin rat anti-mouse IgE Ab (BD Biosciences), C57BL/6 mice were obtained from Harlan (Horst, The Netherlands).