Up-regulation of bradykinin receptors in a murine in-vitro model of chronic airway inflammation

doi:10.1016/j.ejphar.2004.02.033

European Journal of Pharmacology

Volume 489, Issues 1–2, 5 April 2004, Pages 117-126

https://doi.org/10.1016/j.ejphar.2004.02.033 Get rights and content

Abstract

Tumour necrosis factor-alpha (TNF-α) is a mediator with a likely role in chronic airway inflammation and airway hyperresponsiveness. In the present study, mouse tracheal segments were cultured for 1, 4 or 8 days in the absence and presence of TNF-α. Contractile response of cultured segments to des-Arg⁹-bradykinin and bradykinin was assessed in myographs and mRNA for bradykinin B₁ and B₂ receptors was quantified by real-time polymerase chain reaction. Both contraction to des-Arg⁹-bradykinin and bradykinin, mediated via bradykinin B₁ and B₂ receptors, respectively, and mRNA levels for these receptors were up-regulated following culture. These responses were markedly increased in segments treated with TNF-α. Experiments with SP600125 (anthrax(1,9-cd)pyrazol-6(2H)-one) and PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) demonstrated that both intracellular c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 pathways were implicated in this process. Thus, TNF-α causes an increase of bradykinin contractility in mouse trachea, which at least partly is due to a transcriptional increase of bradykinin receptors.

Introduction

Chronic airway inflammation is associated with the development of airway hyperresponsiveness Foresi et al., 1990, Laprise et al., 1999. The release of different mediators, such as tumour necrosis factor-alpha (TNF-α), during the inflammatory process, is known to cause both functional and structural alterations of the airways (Bousquet et al., 2000). The airway smooth muscle cells are of importance in this process Boulet et al., 1998, Halayko and Amrani, 2003. We have recently demonstrated how the long-term influence of inflammatory mediators on the isolated airway smooth muscle could induce an increased sensitivity to serotonin (5-hydroxytryptamine, 5-HT) (Adner et al., 2002).

Bradykinin and related kinins play important roles in the pathogenesis of inflammation, tissue damage and repair Dray and Perkins, 1993, Proud and Kaplan, 1988. Bradykinin is formed from the kininogen precursor after proteolytic cleavage by kallikrein, and can further be converted by carboxypeptidase N to des-Arg⁹-bradykinin. Both kinins can be degraded by kininase II that is identical to angiotensin-converting enzyme (ACE) Barnes, 1992, Kaplan et al., 2002. Bradykinin is a weak constrictor in isolated human bronchi, while, in vivo, bradykinin is a potent bronchoconstrictor in asthmatic patients. This may be due to bradykinin-induced activation of cholinergic nerves Fuller et al., 1987, Reynolds et al., 1999. Intravenous administration of bradykinin causes intense bronchoconstriction in guinea pigs (Ichinose et al., 1990). The effects of kinins are mediated by two G-protein-coupled receptors (bradykinin B₁ and B₂ receptors) (Regoli and Barabe, 1980), present in both airway smooth muscle and epithelial cells (Li et al., 1998). Both bradykinin receptor subtypes seem to contribute to allergen-induced bronchial hyperresponsiveness of rat (Huang et al., 1999). However, patients with asthma exhibit a hyperresponsiveness to aerosolized bradykinin (a bradykinin B₂ receptor agonist), but not to des-Arg⁹-bradyknin (a bradykinin B₁ receptor agonist) Polosa and Holgate, 1990, Reynolds et al., 1999.

Both TNF-α and bradykinin are synthesized and released during chronic airway inflammation and augmented TNF-α and bradykinin levels are found in airway biopsies as well as bronchoalveolar lavage fluids from asthmatic patients Cembrzynska-Nowak et al., 1993, Christiansen et al., 1992. TNF-α can be derived from mast cells and macrophages via immunoglobulin E (IgE)-dependent mechanisms (Thomas, 2001) and has been demonstrated to cause an increase of airway hyperresponsiveness (Thomas et al., 1995). In addition, recent studies have shown that cytokines such as TNF-α and interleukin-1β increase bradykinin B₁ receptor expression in human lung fibroblasts (Phagoo et al., 2000) and that bradykinin stimulates the release of TNF-α and interleukin-1β from macrophages (Tiffany and Burch, 1989). Altogether, the above-mentioned results prompted us to further investigate the possibility of a role for TNF-α and bradykinin interaction in the development of airway hyperresponsiveness in asthma and chronic airway inflammation. For this purpose, we used a newly developed organ culture assay (Adner et al., 2002), validated as a suitable in-vitro assay for evaluation of long-term effects induced by inflammatory mediators (Johnson, 2002). Special attention was given the role of the mitogen-activated protein kinase (MAPK) pathways as a possible link between TNF-α and the bradykinin B₁ and B₂ receptors.

Section snippets

Chemicals

Recombinant murine TNF-α was obtained from R&D Systems (Abingdon), UK. des-Arg⁹-bradykinin and bradykinin were purchased from Neosystem (Strasbourg, France). HOE140 (d-Arg-[Hyp³, Thi⁵, d-Tic⁷, Oic⁸]bradykinin), [Des-Arg¹⁰]HOE140 (DesArg⁹-d-Arg[Hyp³,Thi⁵,d-Tic⁷,Oic⁸]bradykinin), indomethacin, carbachol, PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one), atropine, N-monomethyl-l-arginine (l-NMMA), captopril, dimethyl sulfoxide (DMSO), Dulbecco's modified Eagle's medium (DMEM; 4500 mg/l d

Contractile effects of des-Arg⁹-bradykinin and bradykinin on freshly isolated mouse tracheal segments

Basal contractile responses of des-Arg⁹-bradykinin and bradykinin on isolated mouse trachea were studied in freshly isolated segments. The segments elicited a negligible contractile effect by des-Arg⁹-bradykinin, whereas bradykinin induced a weak contraction (Table 1).

Organ culture, with and without TNF-α: effects on des-Arg⁹-bradykinin-induced contractions

Tracheal segments were cultured for 1, 4 or 8 days. Subsequent exposure to des-Arg⁹-bradykinin elicited a minor contractile response in the tested segments. The responses were small both in mN and expressed as a in percentage of

Discussion

We have recently developed a novel in-vitro assay for the evaluation of inflammatory-mediators effects on mouse airways Adner et al., 2002, Johnson, 2002. Using this culture model, we demonstrated that long-term exposure to TNF-α enhanced 5-HT_2A receptor-mediated airway contractions (Adner et al., 2002). In the present study, the effect of long-term exposure to TNF-α on des-Arg⁹-bradykinin- and bradykinin-induced airway contractions were investigated using the same model. Both the functional

Acknowledgements

The present work was supported by the Swedish Medical Research Council, the Swedish Heart Lung Foundation, the Swedish Association for Allergology, the Swedish Foundation for Health Care Science and Allergic Research and the Royal Physiographic Society.

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In addition, clinical studies confirmed the role of corticosteroids in decreasing bronchial hyperresponsiveness to bradykinin and the bradykinin receptor expression in eosinophils/neutrophils from asthmatics in association with the improvement of respiratory symptoms (Bertram et al., 2007, 2009; Reynolds et al., 2002) indicating that the bradykinin-induced effects on the asthmatic airways seem to be corticosteroid sensitive. In line with the latter studies, dexamethasone, a well-known anti-inflammatory corticosteroid drug, inhibited the kinin receptor upregulation and bronchial hyperresponsiveness to kinins in murine airways in vitro and on cigarette smoke-induced bronchial hyperresponsiveness in vivo (Zhang et al., 2004; Lei et al., 2008) confirming a steroid sensitive modulation of kinin-induced airway responses. In a nice review, Zhang et al. pointed out that exposure to allergen, cigarette smoke or viral infection stimulated MAPK/NF-κB pathway promoting the gene upregulation of bradykinin receptors and subsequently increased bronchial responsiveness.
Bradykinin, a pro-inflammatory molecule, and its related peptides have been studied for their effects on acute reactions in upper and lower airways, where they can be synthesised and metabolized after exposure to different stimuli including allergens and viral infection. Bradykinin B₁ and B₂ receptors are constitutively expressed in the airways on several residential and/or immune cells. Their expression can also be induced by inflammatory mediators, usually associated with eosinophil and neutrophil recruitment, such as IL-4, IL-13, TNF-α, IL-6 and IL-8, via intracellular MAPK and NF-κB signalling. In turn, the latters up-regulate both bradykinin receptors. Bradykinin activates epithelial/endothelial and immune cells, neurons and mesenchymal cells (such as fibroblasts, myofibroblasts and smooth muscle cells), which are implicated in the development of airway chronic inflammation, responsiveness and remodelling (a major feature of severe asthma). This review highlights the role of bradykinin and its receptors in respect to chronic inflammatory response involving eosinophils/neutrophils and to vascular/matrix-related airway remodelling in asthmatic airways. This scenario is especially important for understanding the mechanisms involved in the pathogenesis of eosinophilic and/or neutrophilic asthma and hence their therapeutic approach.
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Bradykinin-induced asthmatic fibroblast/myofibroblast activities via bradykinin B<inf>2</inf> receptor and different MAPK pathways
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Constitutive bradykinin B₂ receptor and α-SMA expression was higher in HBAFb as compared to HNBFb. Bradykinin increased bradykinin B₂ receptor expression in HBAFb. Bradykinin, via bradykinin B₂ receptor, significantly increased fibroblast proliferation at lower concentration (10⁻¹¹ M) and α-SMA expression/polymerization at higher concentration (10⁻⁶ M) in both cells. Bradykinin increased ERK1/2 and p38 phosphorylation via bradykinin B₂ receptor; EGF receptor inhibitor AG1478 and panmetalloproteinase inhibitor GM6001 blocked bradykinin-induced ERK1/2 activation but not p38 phosphorylation. Bradykinin, via bradykinin B₂ receptor, induced EGF receptor phosphorylation that was suppressed by AG1478. In HBAFb AG1478, GM6001, the ERK1/2-inhibitor U0126 and the p38 inhibitor SB203580 suppressed bradykinin-induced cell proliferation, but only SB203580 reduced myofibroblast differentiation.
These data indicate that bradykinin is actively involved in asthmatic bronchial fibroblast proliferation and differentiation, through MAPK pathways and EGF receptor transactivation, by which bradykinin may contribute to airway remodeling in asthma, opening new horizons for potential therapeutic implications in asthmatic patients.
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More interestingly, there is increased expression of kinin receptors in eosinophils and human monocyte-derived dendritic cells (hMo-DCs) from asthmatic patients [41,115], and the kinin B1 receptor upregulation is also seen in airways during allergic inflammation [36] and following stimulation by the inflammatory mediator IL-4 [116]. We have demonstrated that exposure of murine airways to LPS/polyinosinic polycytidylic acid (Poly I:C) [39], TNF-α [70], IL-1β [8] or nicotine [43] induce upregulation of kinin B1 and B2 receptors in airway smooth muscle cells, which mediates AHR to kinins. In an asthma model in rats [73] and in a mouse in vitro model of chronic airway inflammation [8], kinin receptor upregulation mediates AHR to kinins and airway inflammation, which can be attenuated by administration of infliximab (an anti-TNF-α antibody).
Airway hyperreactivity (AHR) is a major feature of asthmatic and inflammatory airways. Cigarette smoke exposure, and bacterial and viral infections are well-known environmental risk factors for AHR, but knowledge about the underlying molecular mechanisms on how these risk factors lead to the development of AHR is limited. Activation of intracellular mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB) and their related signal pathways including protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and protein kinase A (PKA) signaling pathways may result in airway kinin receptor upregulation, which is suggested to play an important role in the development of AHR. Environmental risk factors trigger the production of pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukins (ILs) that activate intracellular MAPK- and NF-κB-dependent inflammatory pathways, which subsequently lead to AHR via kinin receptor upregulation. Blockage of intracellular MAPK/NF-κB signaling prevents kinin B₁ and B₂ receptor expression in the airways, resulting in a decrease in the response to bradykinin (kinin B₂ receptor agonist) and des-Arg⁹-bradykinin (kinin B₁ receptor agonist). This suggests that MAPK- and NF-κB-dependent kinin receptor upregulation can provide a novel option for treatment of AHR in asthmatic as well as in other inflammatory airway diseases.
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The hyperactivity of smooth muscle cells may not only be due to an increase in the secretion of contractile mediators but also inflammation-induced alterations in the airway smooth muscle contractile phenotype [8]. In mice, studies have shown that long-term inflammatory stimulation with TNFα, IL-1β and IL-4, alters the responses to contractile agonists such as 5-hydroxytryptamine (5-HT) and bradykinin [9–13]. Moreover, smooth muscle cells can be activated directly by stimuli of microbial origin through Toll-Like receptors (TLRs) [14,15].
Different mouse strains display different degrees of inflammation-induced airway hyperresponsiveness in vivo. It is not known whether these variations are attributable to distinct properties of the airway smooth muscle. Therefore, tracheal ring segments from C57BL/6 and BALB/c mice were exposed to three different pro-inflammatory stimuli for 4 days while maintained under tissue-culture conditions: tumour necrosis factor α (100 ng/ml), the Toll-like receptor (TLR) 3 agonist polyI:C (10 μg/ml), and the TLR4 agonist LPS (10 μg/ml). The contractile responses to carbachol, 5-hydroxytryptamine (5-HT) and bradykinin were assessed after culture. In addition, gene expression of TLR1–TLR9, pivotal inflammatory signal transduction proteins (jun-kinase, p38 and p65) and critical negative regulators of inflammation (A20, Itch, Tax1bp1 and RNF11) were studied in tracheal smooth muscle strips, fresh and following treatment for 4 days with LPS, from both strains. No differences between the strains were detected regarding the response of freshly isolated preparations to carbachol, 5-HT and bradykinin. After stimulation with pro-inflammatory mediators, contractions in response to 5-HT and bradykinin, but not to carbachol, were up-regulated. This up-regulation was markedly larger in BALB/c than in C57BL/6 segments and depended on the type of inflammatory stimulus. Expression of the genes investigated did not differ between the two strains. These findings indicate that strain differences in airway hyperresponsiveness can be linked to differences in the responsiveness of airway smooth muscle to pro-inflammatory mediators per se. The differences do not appear to be due to differential expression of TLR or common inflammatory transduction and repressor proteins.
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Although we had no evidence for the presence of B1R in DRG neurons under our culturing conditions in previous studies (Petersen et al., 1998; Segond von Banchet et al., 2000) we cannot exclude that under the conditions of IL-1β stimulation some B1R become expressed. Because under inflammatory conditions B2R is upregulated (Segond von Banchet et al., 2000; Banik et al., 2001; Zhang et al., 2004) it could be that IL-1β upregulates the expression of the B2R in primary cultures of DRG neurons and thereby increases responses to BK (or reduces internalization). However, the exposure to IL-1β did neither alter the proportion of neurons expressing the B2R nor the proportion of neurons showing the B2 receptor in the membrane.
In dorsal root ganglion sections numerous small-to medium-sized neurons were found to exhibit extensive colocalization of the bradykinin receptor 2, the interleukin-1 receptor 1 and G protein-coupled receptor kinase 2. Application of bradykinin to cultured DRG neurons caused substantial internalization of the bradykinin 2 receptor which significantly reduced the responsiveness of DRG neurons to a second application of bradykinin. Such an internalization was not observed in DRG neurons which were exposed to long-term pretreatment with interleukin-1β. The long-term incubation with interleukin-1β on its own did neither change the proportion of neurons which expressed the bradykinin 2 receptor in the cytoplasma nor the proportion of neurons expressing the bradykinin 2 receptor in the membrane but it reduced the proportion of neurons expressing G protein-coupled receptor kinase 2, an enzyme which facilitates the internalization of G protein-coupled receptors. These results show that interleukin-1β maintains the responsiveness of DRG neurons to bradykinin in the long-term range, and they suggest that the downregulation of G protein-coupled receptor kinase 2 could be a cellular mechanism involved in this interleukin-1β effect.

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Up-regulation of bradykinin receptors in a murine in-vitro model of chronic airway inflammation

Abstract

Introduction

Section snippets

Chemicals

Contractile effects of des-Arg9-bradykinin and bradykinin on freshly isolated mouse tracheal segments

Organ culture, with and without TNF-α: effects on des-Arg9-bradykinin-induced contractions

Discussion

Acknowledgements

J. Biol. Chem.

Trends. Neurosci.

Chest

Res. Physiol. Neurobiol.

J. Allergy Clin. Immunol.

Matrix Biol.

Exp. Cell Res.

Eur. J. Pharmacol.

Peptides

FEBS Lett.

An assay to evaluate the long-term effects of inflammatory mediators on murine airway smooth muscle: evidence that TNF-α up-regulates 5-HT2A-mediated contraction

Br. J. Pharmacol.

Bradykinin and asthma

Thorax

Further analysis of anomalous pKB values for histamine H2-receptor antagonists on the mouse isolated stomach assay

Br. J. Pharmacol.

Airway inflammation and structural changes in airway hyper-responsiveness and asthma: an overview

Can. Respir. J.

Asthma. From bronchoconstriction to airways inflammation and remodeling

Am. J. Respir. Crit. Care Med.

Elevated release of tumor necrosis factor-alpha and interferon-gamma by bronchoalveolar leukocytes from patients with bronchial asthma

Am. Rev. Respir. Dis.

Elevation of tissue kallikrein and kinin in the airways of asthmatic subjects after endobronchial allergen challenge

Am. Rev. Respir. Dis.

Bradykinin-induced bronchoconstriction in humans. Mode of action

Am. Rev. Respir. Dis.

Contractile effects of des-Arg⁹-bradykinin and bradykinin on freshly isolated mouse tracheal segments

Organ culture, with and without TNF-α: effects on des-Arg⁹-bradykinin-induced contractions

An assay to evaluate the long-term effects of inflammatory mediators on murine airway smooth muscle: evidence that TNF-α up-regulates 5-HT_2A-mediated contraction

Further analysis of anomalous pK_B values for histamine H₂-receptor antagonists on the mouse isolated stomach assay