Accepted Manuscript Agonist-induced β2-adrenoceptor desensitization and downregulation enhance proinflammatory cytokine release in human bronchial epithelial cells Susanne Oehme , Anja Mittag , Wieland Schrödl , Attila Tarnok , Karen Nieber , Getu Abraham , PhD PII:

S1094-5539(14)00065-0

DOI:

10.1016/j.pupt.2014.05.007

Reference:

YPUPT 1383

To appear in:

Pulmonary Pharmacology & Therapeutics

Received Date: 23 January 2014 Revised Date:

26 April 2014

Accepted Date: 29 May 2014

Please cite this article as: Oehme S, Mittag A, Schrödl W, Tarnok A, Nieber K, Abraham G, Agonistinduced β2-adrenoceptor desensitization and downregulation enhance pro-inflammatory cytokine release in human bronchial epithelial cells, Pulmonary Pharmacology & Therapeutics (2014), doi: 10.1016/j.pupt.2014.05.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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β2-adrenoceptor

Agonist-induced

desensitization

and

downregulation enhance pro-inflammatory cytokine release in

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human bronchial epithelial cells

Susanne Oehmea, Anja Mittagb, Wieland Schrödlc, Attila Tarnokb, Karen Nieberd,

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Getu Abrahama*

a

Institute of Pharmacology, Pharmacy and Toxicology, University of Leipzig, Leipzig,

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Germany b

Department of Paediatric Cardiology, Heart Centre and Translational Centre Regenerative Medicine, University of Leipzig, Leipzig, Germany

c

Institute of Bacteriology and Mycology, University of Leipzig, Leipzig, Germany

d

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Institute of Pharmacy, University of Leipzig, Leipzig, Germany

*

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Running title: β2-adrenoceptor downregulation enhances cytokine release

Corresponding author:

Getu Abraham, PhD; Institute of pharmacology, pharmacy and toxicology, University of Leipzig, An den Tierkliniken 15, 04103 Leipzig, GERMANY Phone: + 49 (0) 341 97 38 134 and Fax: + 49 (0) 341 97 38 149; Email: [email protected]

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Abstract It is not clear whether increased asthma severity associated with long-term use of β2adrenoceptor (β2-AR) agonists can be attributed to receptor degradation and increased inflammation. We investigated the cross-talk between β-AR agonist-

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mediated effects on β2-AR function and expression and cytokine release in human bronchial epithelial cells. In 16HBE14o- cells grown in the presence and absence of

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β-AR agonists and/or antagonists, the β2-AR density was assessed by radioligand binding; the receptor protein and mRNA was determined using laser scanning

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cytometer and RT-PCR; cAMP generation, the cytokines IL-6 and IL-8 release were determined using AlphaScreen Assay and ELISA, respectively. Isoprenaline (ISO) and salbutamol (Salbu) induced a concentration- and time-dependent significant decrease in β2-AR density. Both Salbu and ISO reduced cAMP generation in a

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concentration-dependent manner while in same cell culture the IL-6 and IL-8 release was significantly enhanced. These effects were antagonized to a greater extent by ICI 118.551 than by propranolol, but CGP 20712A had no effect. Reduction of the β2-

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AR protein and mRNA could be seen when cells were treated with ISO for 24 h. Our findings indicate a direct link between cytokine release and altered β2-AR expression

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and function in airway epithelial cells. β2-AR desensitization and downregulation induced by long-term treatment with β2-AR agonists during asthma may account for adverse reactions also due to enhanced release of pro-inflammatory mediators and should, thus, be considered in asthma therapy.

Keywords: β-adrenoceptors; cAMP; β2-agonists; bronchial epithelial cells; asthma

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1. Introduction

β2-Adrenoceptors (β2-AR) which belong to the superfamily of seven transmembrane G-protein coupled receptors (GPCRs) and couple typically with the heterotrimeric

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stimulatory G-protein, Gs, are widely distributed in the lung, expressed in epithelial (Abraham et al. 2004; Coraux et al. 2004; Kelsen et al. 2000), smooth muscle, inflammatory and type II alveolar cells (Johnson M, 1998). Classically, β2-AR and Gs

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protein are activated by Short- or long-acting β2-AR agonists to enhance the

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adenylate cyclase activity which catalyzes the synthesis of the intracellular cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP) and activation of cAMP-dependent protein kinase (PKA) to modulate biological responses (Giembycz and Newton, 2006). A chronic activation of these receptors is said to worsen airway inflammation thus playing a crucial role in the pathogenesis of asthma

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and chronic obstructive pulmonary disease (COPD) (Broadley, 2006). In this regard, regular use of β2-AR agonists, whether short- or long-acting, that are mainstay

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bronchodilator agents in asthma and COPD and act via the β2-AR-Gsα-proteinadenylate cyclase pathway, should have deleterious pro-inflammatory responses that

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limit their application as monotherapy in patients with long-standing and severe asthma (Nguyen et al. 2009; Salpeter et al. 2006). Moreover, in the past three decades, despite the benefits of β-AR agonists afford, continuous exposure of asthmatics to bronchodilators can lead to tolerance (Svedmyr, 1990) and this has often been a serious concern and was debated in relation to the incidence of increased morbidity and mortality in asthma (Barnes and Chung, 1992; Broadley, 2006). That is, β2-AR become down-regulated and are hyporesponsive to the stimulant effects of these agents (Johnson, 2006). In late

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asthma and upon chronic usage, β2-AR agonists do not inhibit cytokine and/or mediator releases from inflammatory and airway epithelial cells but rather enhances their concentrations in airways which result in increased responsiveness to allergens, altered mucocilliary clearance and masking symptoms (Penn et al. 1998). The most

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studied causative factor has been related to receptor desensitization (Sears and Tylor, 1994). The hypothesis to be proven here is, therefore, whether distinct mechanisms of agonist-promoted regulation of these receptors, i.e. desensitization,

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internalization and downregulation, can be linked to cytokine/mediator releases.

Generally, an intense and long-lasting stimulation of the β2-AR by β-AR agonists

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leads often to rapid desensitization due to uncoupling of the receptor from Gs protein and adenylate cyclase. Prolonged activation leads to internalization of the receptor into an intracellular endosomal pool, thereby effectively removing the receptor from the cell surface (Johnson, 2001; Zetterlund et al. 2001, Inglese et al. 1993), and

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eventually to degradation and permanent receptor downregulation defined by an overall decrease in receptor number and the corresponding intracellular cAMP accumulation. Also, in asthmatic airways, reduced relaxant responses to β2-AR

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agonists have been related to desensitization of the Gs coupled β2-AR pathway

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(Barnes, 1999). This means: several inflammatory mediators and cytokines in the asthmatic airways have the potential to induce decreased β2-AR responsiveness and desensitization concomitant to β2-AR agonist usage (Heijink et al. 2004). This scenario has been also shown after strong and repeated exposure of β2-ARs to specific β2-AR agonists in vivo or in vitro (Abraham et al. 2002; Kelsen et al. 1997). The airway epithelium represents physical barrier against external pathogen or nonpathogen agents (Diamond et al. 2000) and plays a crucial role in orchestrating airway inflammatory response by interacting with several environmental factors which

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disrupt epithelial tight junction to result in chronic wound scenario and airway remodelling (Holgate, 2007). Airway epithelial cells are capable of synthesizing and secreting mediators (van der Velden et al. 1998) which influence chemotaxis (Interleukin 8; IL-8) (Gordon et al. 2003) and immune response inflammation as well

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as acute phase reactions (Interleukin 6; IL-6) (Barnes et al. 1998; Sehgal, 1990). Expression and production of these mediators is controlled by catecholamines via the β2-AR-Gs-protein-cAMP-system (von Patay et al. 1998), but can also be regulated in

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an autocrine manner (Allen-Gipson et al. 2004). Previous studies have shown that increased IL-6 and IL-8 release from bronchial epithelial cells is accompanied by β-

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AR agonist-induced increase in intracellular cAMP accumulation (Edwards et al 2007; Holden et al. 2010; Lindén, 1996; Strandberg et al. 2008); however, the influence of receptor number on cAMP response in link to cytokine release has not been examined. Moreover, secretion of a range of mediators from the airway

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epithelium is also strongly implicated to interact with the underlying mesenchyme, smooth muscle cells, inflammatory cells and vasculature to maintain asthmatic inflammation (Holgate, 2007).

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The purpose of this study was, therefore, to examine how the density of β2-AR in the

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human bronchial epithelial cell line, 16HBE14o-, affects a) the β-AR agonist-induced (ISO and salbu) cAMP accumulation and b) β-AR agonist-induced IL-6 and IL-8 secretion in relation to cAMP production in same cell culture. We used 16HBE14o- as cell models since they express exclusively β2-AR subtypes and are capable to release cytokines after stimulation by β-agonists.

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2. Material and methods

2.1. Chemicals

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Media and plastic wares: All culture media and reagents were from PAA Laboratories GmbH (Pasching, Germany). Cell culture plastics were from Greiner Biosciences (Frickenhausen, Germany).

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Ligands: (±)-CGP 12177, CGP 20712A, ICI 118.551, (-)-propranolol, (-)-isoprenaline, (-)-salbutamol and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma

Life Sciences (Zaventem, Belgium).

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Aldrich (Deisenhofen, Germany). (-)-[125I]-iodocyanopindolol (ICYP) from PerkinElmer

Kits: cAMP-AlphaScreen Assay Kit was obtained from PerkinElmer Life Sciences (Zaventem, Belgium). IL-6 and IL-8 enzyme-linked immunosorbent assay (ELISA)

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kits were obtained from BD Bioscience (Heidelberg, Germany). Antibodies: The monoclonal mouse-anti-human β2-AR-antibody (clone I3D6), coupled to phycoerythrine (PE), was purchased from Santa Cruz Biotechnology (Heidelberg, Fluoromount-GTM-media

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Germany).

were

obtained

from

Southern

Biotech

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(Birmingham, USA).

Other substances were obtained at per analysis (p.A.) quality from commercial sources.

2.2. Cell Culture

16HBE14o- cells were cultured to confluence in culture flasks or in 24-well plates in Eagle’s minimum essential medium (EMEM) containing 10% fetal calf serum (FCS) supplemented with non-essential amino acids (1:100) and L-glutamine (2 mM) as

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previously described (Abraham et al. 2004). All cultures were maintained in a humidified atmosphere at 37°C in 5% CO 2. For passaging and where necessary, cells were harvested by treatment with trypsin/EDTA (0.05% in PBS). After

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harvesting, cell viability was tested by trypan blue exclusion test.

2.3. Cell Stimulation

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Confluent 16HBE14o- cells were washed twice with PBS before adding drugs and cultured in a fresh serum-free medium, to avoid nonspecific effects of FCS.

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Radioligand binding studies were used to assess concentration- (1 nM – 10 µM) and time- dependent (0.5 - 24h) effects of ISO or Salbu on β2-AR density. Moreover, cells were incubated with 10 - 100 nM ISO or Salbu, to quantify IL-6 and IL-8 release in cell supernatants by ELISA. In parallel, cell lysates were harvested from same

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cultures to detect intracellular cAMP formation by AlphaScreen assay. To test β2-AR subtype mediated effects on cAMP formation and cytokine release, the β2-selective antagonist ICI 118.551 (100 nM), β1-selective antagonist CGP 20712A (300 nM) or

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the non-selective β-AR-antagonist propranolol (300 nM) were added alone or

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concomitantly with β-AR agonists ISO and Salbu. Furthermore, the β2-AR protein content was detected with laser scanning cytometry after 24 h treatment with 10 µM ISO. As well, cells were stimulated time-dependently (1 – 48 h) with 10 µM ISO to assess the β2-AR mRNA expression by RT-PCR.

2.4. Radioligand Binding Studies

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Radioligand binding studies were carried out in intact and viable (>98%; trypan blue exclusion

test)

16HBE14-

cells

by

radioligand

binding

using

(-)-[125I]-

iodocyanopindolol (ICYP) as previously described (Abraham et al. 2004). Cells treated with ISO or Salbu in a time- and concentration-dependent manner were

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trypsinized, resuspended in EMEM and washed twice in PBS by centrifugation (500 x g, 10 min and 4°C). Pellets were resuspended in the incubation buffer (10 mM TrisBase, 154 mM NaCl, 0.55 mM ascorbic acid, pH 7.4 at 25°C) and counted. 10 5 cells

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(in 150 µl) were incubated with six increasing ICYP concentrations (concentrations 10 to 150 pM) for 90 min at 37°C. Non-specific bind ing was determined in the

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presence of 1 µM (±)-CGP 12177. The reaction was terminated by adding ice-cold Tris-buffer following rapid filtration over GF/B Whatman glass fibre filters (Whatman Inc. Clifton, NJ). The radioactivity was measured in Wizard automatic gamma counter (PerkinElmer Life Sciences). The specific binding of ICYP was determined as

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difference between total binding and non-specific binding.

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2.5. Analysis of β-ARs by Laser Scanning Cytometry

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To quantify possible alterations of β2-AR protein level by β-agonists, cells were treated merely with ISO for 24 h, and adherent cells were immunocytochemically stained for β2-AR protein and analysed using laser scanning cytometry “iCysTM” (CompuCyte Cooperation, Westwood, USA) as recently shown (Mittag et al. 2011). In brief, 104 cells/m were seeded on glass cover slips in 24-well plates and cultured for 7 days. Sub-confluent monolayers were then exposed to 10 µM ISO for 24h. Thereafter, plates were placed on ice and each well was washed twice for 10 min with HBSS (Hank’s Balanced Salt Solution). Subsequently, cells were fixed with icecold 95% ethanol and 5% acetic acid for 1 min. The reaction was stopped by adding

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100 mM glycine in HBSS on ice for 3 min. Cells were subsequently double stained overnight (dark, 4°C) with the PE–conjugated monocl onal antibody to β2-ARs (PE; λEx = 488 nm; λEm = 578 nm) (dilution: 1:50) and the DNA-staining fluorescence dye 4,6diamidino-2-phenylindole (DAPI; λEx = 405 nm; λEm = 461 nm) (dilution: 1:2000)

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diluted in HBSS, containing 50 mM glucose and 0.1 % BSA. Thereafter, each well was washed four times each for 5 min with HBSS. Cells on cover slips were embedded using Fluoromount-GTM-media for analysis. Cells with DAPI staining are

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used as negative control. The fluorescence intensity of bound PE-conjugated anti-β2AR antibody which was associated with the β2-AR protein level was quantified using

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the corresponding iCys analysis software (version 3.4.0).

2.6. β2-AR RNA Extraction and RT-PCR Analysis

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To investigate the influence of β-agonists on β2-AR mRNA, cells were treated timedependently with 10 µM ISO for 1 - 48h. Total RNA was prepared from 5x106

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16HBE14o- cells using the Qiagen RNeasy mRNA Mini-Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. On-column DNase I digestion was

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applied to reduce contamination with genomic DNA. The quality of the isolated RNA was controlled by agarose gel electrophoresis and the RNA concentration was determined spectrophotometrically at 260 nm. Using the First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) equal amounts of total RNA (1 µg) were transcribed into cDNA. The presence of β2-AR cDNA sequences were analyzed by PCR using sense (5’-TCGTCATGTCTCTCATCGTC-3’) and anti-sense primers (5’AATGGCATAGGCTTGGTTCG-3’) as previously described (Abraham et al. 2004). Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) detected by sense (5’-

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and

anti-sense

primers

(5’-

AGCTCAGGGATGACCTTGCCCA-3’) was used as positive control. For PCR, 1 µl per cDNA sample was amplified in the GeneAmp 9700 thermal cycler (Applied Biosystems, Foster City, USA) in reaction volumes of 50 µl containing 1.5 U Dream

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Taq DNA polymerase, 0.2 mM dNTPs, 1 mM MgCl2, 1x Dream-Taq-buffer (Fermentas, St.Leon-Rot, Germany) and 0.5 µM of each primer (Eurofins MWG Operon, Ebersberg, Germany) diluted in nuclease-free water. Initial denaturation at

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95°C for 3 min, amplification procedure of 35 cycle s at 95°C for 30s, 56°C for 30s, 72°C for 1 min and subsequently annealing at 72°C f or 1 min. To determine the size

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of the PCR products for β2-AR (~480 bp) and for GAPDH (~430 bp) we used a 100 bp-ladder from New England BioLabs GmbH (Frankfurt, Germany).

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2.7. Cyclic AMP Assay

To determine prolonged effects of β-agonists on cAMP formation , confluent 16HBE14o- cells were treated with ISO or Salbu concentration-dependently for 24h

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in serum-free medium in 24-well culture plates in the presence of 1 mM IBMX.

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Thereafter, cells were washed twice with PBS in the presence of 1 mM IBMX and incubated with 1 µM ISO for 15 min at 37°C to immed iately stimulate cAMP formation. The reaction was stopped on ice for 75 min by adding a lysis buffer containing 1 mM IBMX, 5 mM HEPES (pH=7.4), 0.1% BSA and 0.3%Tween-20. Cell lysates were collected and kept at -20°C until analysis. Aliquots of cell lysates were thawed and cAMP was determined in triplicates in 384-well white opaque microplates at room temperature at a total volume of 25 µl/well using AlphaScreen®cAMP assay kit as recently described (Abraham et al. 2011). The standard cAMP dilutions (0–5 µM) were prepared with PBS buffer

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containing 100 µM IBMX. Then, 5 µl sample or 5 µl standard cAMP were added to each well, followed by the addition of 0.1 µl anti-cAMP acceptor beads in 10 µl assay buffer (5 mM HEPES, 1 x HBSS, 0.1% BSA, pH 7.4). Microplates were incubated in the dark for 30 min at room temperature. A master mix, containing

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0.1 µl of streptavidin donor beads and 0.0075 µl of biotinylated-cAMP in 10 µl of lysis buffer (5 mM HEPES, 0.1% BSA, 0.3% Tween-20, pH 7.4) per well, was prepared and incubated also for 30 min in the dark. Subsequently, the donor bead

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solution was added to each well and incubated with the other substances for 1h at RT in the dark. Finally, the AlphaScreen signals were measured by Fusion-Alpha®

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Multilabel Reader (PerkinElmer, Boston, MA, USA).

To correlate β2-AR function with cytokine production, both the β-agonist-induced cAMP accumulation and cytokine releases were assessed in same 16HBE14o-

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cultures.

2.8. Measurement of IL-6 and IL-8 Levels

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Cells cultured to assess cAMP in the presence of IBMX were used at the same time to detect cytokine releases. After stimulation of cells concentration-dependently with

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ISO or Salbu for 24h in serum-free medium, also, in the presence or absence of β-AR antagonists (ICI 118.551, CGP 20712A, propranolol), cell supernatants (~1 mL) were collected, centrifuged (2000 rpm, 15 min, RT) to remove debris, and aliquots were then stored at -80 °C until analysis. Specific ELIS A kits for IL-6 and IL-8 were used to measure IL-6 and IL-8 according to the manufacturer’s instructions (BD Bioscience, Heidelberg, Germany). Here, we used PBS containing 3% BSA as an assay and blocking buffer to inhibit non-specific binding of the antibodies, and samples from stimulated 16HBE14o- cells were diluted in same buffer.

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2.9. Statistical Analysis

The maximum number of binding sites (Bmax) and the equilibrium dissociation

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constants (KD) for ICYP were calculated from non-linear regression curve fittings for one-site binding model using the software GraphPad Prism (GraphPad Software, San Diego, CA). Dose-response increases in cAMP formation to ISO/Salbu were

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analyzed by fitting the sigmoidal curves to the experimental data by equating Y= Bottom + (Top-Bottom)/1 + 10^((LogEC50-X)*Hill Slope).

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Data that follow the Gaussian distribution, proved by D’Agostino and Pearson omnibus normality test, were presented as mean ± S.E.M from n experiments in duplicates. Two-tailed paired Student’s t test was used to determine significant differences between ISO-induced cAMP production vs. control as well as cytokine

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production in absence and presence of β-antagonists. Given the low number of some samples, the Gaussian distribution could not be assumed. Here values are expressed as median and the Wilcoxon match-paired signed rank test was used for

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comparison between two matched groups (time-dependent and ISO-concentration-

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dependent differences in β2-AR density, β2-AR protein levels as well in β2-ARmediated cAMP production and cytokine release after 24 h treatment with Salbu in absence and presence of β-antagonists.

3. Results

3.1. Time- and Concentration-Dependent Isoprenaline- and Salbutamol-Induced β2AR Downregulation

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ISO and Salbu were tested initially for their effect on β-AR density and expression in 16HBE14o- cells which exclusively contain the β2-AR subtype (Abraham et al. 2004). Cells were treated with either ISO (1 nM to 10 µM) or Salbu (1 nM, 100 nM, 10 µM)

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for 30 min to 24 h. Fig. 1 shows time- and concentration-dependent decrease in β2AR density (Bmax) in 16HBE14o- cells by ISO (Fig. 1A) and Salbu (Fig. 1B). While lower ISO concentrations reduced gradually the β2-AR density, ISO concentrations 1

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- 10 µM caused a rapid and significant β2-AR downregulation. 24 h treatment with all

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tested ISO concentrations resulted in depletion of the β2-AR number even to a nondetectable level (Fig. 1A). Here, there was no difference between total and nonspecific ICYP binding. Similarly, 24 h treatment of 16HBE14o- cells with Salbu resulted in non-detectable β2-AR concentration at all tested concentrations (Fig. 1B). Salbu had a less pronounced effect on β2-AR density at time points less than 6 h. But significant

β2-AR

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statistically

downregulation

could

be

achieved

at

Salbu

concentrations >100 nM and time points >12 h (38% median of control) of cell pre-

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treatment (p < 0.001) as similar as the data obtained for ISO.

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3.2. Isoprenaline-Induced Reduction of Total β2-AR Protein

As shown in Fig. 2A, treatment of cells with ISO (10 µM) for 24 h resulted in decreased PE-fluorescence intensity of bound anti-β2-AR antibody compared to untreated cells. The corresponding PE-fluorescence intensity of ISO-treated cells was also reduced (Fig. 2B) indicated as leftward shift compared to untreated cells. The median values of the fluorescence intensity peaks (PE-MFI) from untreated and ISO-treated cells of all nine experiments were highly variable as depicted in the box

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plot in Fig. 2C. Here, we obtained 851 ± 102.9 PE-MFI for untreated cells and 657 ± 66.6 PE-MFI after 24 h treatment of cells with 10 µM ISO, i.e., β2-AR protein levels of 16HBE14o- were significantly reduced by 23% (p = 0.004) after long-term ISO treatment.

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To evaluate the reproducibility of decreased β2-AR protein levels with regard to differentiation state and density of 16HBE14o- cell cultures, we assessed cell growth and cycle. Accordingly, the DAPI fluorescence integral, i.e., the DNA content, was

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used to assign the cells to different cell cycle stages (G1/ G0-, S-, G2-phase) and to detect cells before and after mitosis. The analysis of PE-MFI values in this population

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yielded information on cell cycle dependent β2-AR protein levels. After 24 h treatment with ISO the β2-AR protein level was reduced by about 23% in all cell cycle phases. There was no correlation between cell culture density and β-AR protein level.

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3.3. Time-Dependent Effect of Isoprenaline on β2-AR Gene Expression

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β2-AR mRNA expression in 16HBE14o- cells was measured by RT-PCR following 1, 12, 24 and 48 h cell stimulation with ISO. Fig. 3 shows that 10 µM ISO-induced no

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reduction of the β2-AR mRNA (480 bp) up to 24h (1, 12 or 24 h). Then, cells were stimulated for 48 h with ISO, and here, the β2-AR mRNA level was prominently decreased. In contrast, the intensity of the GAPDH mRNA (430 bp) signal was similar in both control and treated 16HBE14o-.

3.4. Isoprenaline- and Salbutamol-Induced β2-AR Desensitization in 16HBE14o-

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As shown in Fig. 4A, pre-treatment of 16HBE14o- cells with increasing concentrations of ISO caused a concentration-dependent reduction in β-AR-mediated cAMP formation (EC50 = 1 - 2 µM). Treatment of cells with Salbu had led to a much faster and larger decrease in subsequent β-AR-mediated cAMP accumulation (Fig.

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4B) than with ISO; even at low concentration, i.e., 10 nM, Salbu induced >70% reduction in cAMP levels, indicating much greater desensitization than ISO-induced cAMP decrease at concentrations below 1 µM. Higher concentrations of Salbu (>1

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µM) induced a total loss of the β2-AR-mediated cAMP formation. In both β-AR agonist treatments, the basal cAMP accumulation remained unchanged. The application of

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ICI 118.551 (100 nM) and propranolol (300 nM) inhibited the concentrationdependent ISO- and Salbu-induced inhibition of cAMP production after 24 h stimulation of cells, suggesting the β2-receptor subtype role in this mechanism, while

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CGP 207.12A (300 nM) did not have any influence on such effects (Fig. 4A and 4B).

3.5. Effects of Isoprenaline- and Salbutamol-Induced β2-AR Stimulation on IL-6- and

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IL-8 Release

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Fig. 5 and 6 show the concentration-dependent (10 nM - 100 µM) effect of ISO and Salbu on IL-6 and IL-8 levels in 16HBE14o- cell cultures, respectively, which were at the same time used for cAMP determination. Incubation with either ICI 118.551 (100 nM), CGP 207.12A (300 nM) or propranolol (300 nM) was examined to determine whether the observed enhancement by ISO or Salbu of cytokine release from 16HBE14o- cells was receptor-dependent. Fig. 5A shows the concentrationdependent (10 nM to 100 µM) effect of ISO and Fig. 5B Salbu on IL-6 levels in the absence or presence of β-AR antagonists.

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ISO induced a sharp and statistically significant concentration-dependent (6-fold) increase in IL-6 production versus control (EC50 = 2 µM). ICI 118.551 inhibited significantly the ISO-induced IL-6 release in ISO concentration ranges 10 nM to 10 µM (Fig. 5A). Interestingly, propranolol increased basal (control) IL-6 secretion (in the

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absence of ISO). Propranolol did show differential effects in the presence of ISO: at lower ISO concentrations (10-100 nM) propranolol did not inhibit IL-6 secretion, while at ISO concentrations (1-10 µM) propranolol was able to inhibit this cytokine release

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(Fig. 5A). However, CGP 207.12A was not able to block the cytokine release.

As shown in Fig. 5A (inset), the ISO-induced IL-6 release negatively correlated (IL-6:

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r = -0.84 (p < 0.0001) with ISO-mediated cAMP production after 24 h exposure of the same 16HBE14o- cultures. By squaring the correlation coefficient r, we could show that the increase of IL-6 release was influenced by 70% decrease in β2-AR-induced cAMP formation.

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Fig. 5B demonstrates the concentration-dependent (10 nM – 100 µM) effect of the selective β2-AR agonist Salbu on IL-6 release in the absence or presence of β-AR antagonists after 24 h of cell treatment. Salbu strongly enhanced IL-6 release at all

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tested concentrations, except at 100 µM Salbu. The cytokine release was abolished

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by ICI 118.551 (100 nM). Also, propranolol increased basal (control) IL-6 secretion (in the absence of Salbu). Salbu-induced IL-6 secretion could also be significantly inhibited by propranolol (Fig. 5B), except at 100 µM Salbu. Again, CGP 207.12A (300 nM) did not influence this effect. Fig. 6 shows the concentration-dependent (10 nM to 100 µM) effect of ISO (Fig. 6A) and Salbu (Fig. 6B) in the absence or presence of β-AR antagonists on IL-8 supernatant levels of 16HBE14o- cultures. ISO increased IL-8 release with statistical significance at concentrations above 10 nM (Fig. 6A). Propranolol increased basal (control) IL-8 secretion (in the absence of ISO). ICI 118.551 diminished significantly

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IL-8 secretion at ISO concentrations 1 and 10 µM. Also, propranolol reduced ISOinduced IL-8 release at ISO concentrations between 10 nM – 1 µM, the latter with statistical significance, whereas CGP 207.12A did not affect IL-8 secretion. As shown in Fig. 6A, the ISO-induced IL-8 production correlated negatively (r = -0.54; p =

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0.0003) with β-AR-mediated cAMP production after 24 h exposure of cells with ISO. The ISO-induced IL-8 release seemed to be caused by loss of cAMP production to 29%.

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Furthermore, Fig. 6B depicts the concentration-dependent effect of Salbu on IL-8 release in absence or presence of β-AR antagonists. Salbu strongly enhanced IL-8

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release at all tested concentrations. The cytokine release was strongly abolished by ICI 118.551 at all Salbu concentrations, and even if to a lesser extent, by propranolol. CGP 207.12A (300 nM) did not have any influence on this effect. Because Salbu increased IL-6 and IL-8 releases at all tested concentrations, correlation analysis with

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4. Discussion

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β-AR-induced cAMP production could not be assessed.

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Main findings our study were: First, ISO and Salbu induced a time- and concentration-dependent reduction in β2-AR number and concomitantly in ISOevoked intracellular cAMP accumulation. Second, in same cell cultures, both agonists enhanced IL-6 and IL-8 releases in a concentration-dependent manner. Third, ICI 118.551, prevented markedly the ISO- and Salbu-induced cAMP production and IL-6 and IL-8 release, and propranolol partially inhibited the stimulatory effects of ISO and Salbu, whereas CGP 207.12A did not alter these effects, suggesting the effects of ISO and Salbu are β2-AR subtype mediated. This

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study may provide a mechanistic explanation for the paradoxical findings that β2-AR given as monotherapy alone are pro-inflammatory in asthma. As airway epithelial cells are considered as an essential controller of inflammation, immunity (innate or adaptive) and airway remodelling by synthesizing and releasing

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several inflammatory mediators and signal molecules, they are important targets in therapeutic interventions (for review see (Lambrecht and Hammad, 2012) and references therein). In the airways, β-AR agonists mediate their effects not only by

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primarily activating β2-ARs on airway smooth muscle cells but also on airway epithelial cells. The stimulatory effects of increasing concentrations of Salbu (except

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the highest concentration) on both IL-6 and IL-8 were largely prevented by ICI 118.551 (with inverse agonistic activity) and less prominently by propranolol that are considered specific for interactions at β2-AR subtype (Hall et al. 1992), whereby ICI 118.551 had greater efficacy than propranolol. The reason for this effect is unclear; it

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seems likely that both ISO and propranolol are classified as non-selective β1- and β2AR agonist and antagonist, respectively. Interestingly, propranolol did not reduce the basal (not β-agonist-induced) IL-6 and IL-8 secretion, and rather increased IL-6

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production. This effect seems independent of its β2-antagonistic effect. It has been

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proposed that propranolol acts as inverse agonist so that reduce basal cAMP accumulation but at the same time increase basal cytokine release which may result from stimulating a CRE-mediated gene transcription response (Azzi et al. 2001; Chidiac et al. 1994). Moreover, this paradoxical response to propranolol might be explained by the fact that propranolol caused the β2-adrenoceptor to couple to more than one G-protein or switch coupling from Gs to another G-protein. A role for β1-AR in this response can, however, entirely be excluded because the β1-AR-selective antagonist CGP 207.12A did not exhibit any inhibitory effect. The fact that Salbu

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responses to IL-6 and IL-8 releases as well as to cAMP content are almost completely blocked by ICI 118.551, it can be concluded that β2-AR are responsible for these effects, in agreement with studies shown in airway smooth muscle cells (Hallsworth et al. 2001). Moreover, we have previously shown that 16HBE14o- cells

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express a single population of the adrenoceptors of the β2-subtype (Abraham et al. 2004).

It has been shown that β-AR agonists regulate β2-AR number and function and

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resulted in enhanced, for example, ISO-mediated intracellular cyclic AMP production which could selectively be inhibited by ICI 118.551 (McDonnell et al. 1998). Such

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agonist concentration-response-relationships (Whaley et al. 1994) indicate the increased signal amplification, and consequent saturation of intracellular biological signalling processes resulting from high receptor density even with higher basal cAMP accumulation (Abraham et al. 2003; Samama et al. 1994). Accordingly, it can

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strongly be assumed that in tissues/cells with intact β2-AR number, receptor stimulation by an agonist leads to activation of the adenylate cyclase resulting in enhanced intracellular cAMP levels (Benovic, 2002). Hence, in asthma and COPD,

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not surprisingly, full inhibitory responses (airway smooth muscle relaxation,

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mucociliary clearance) as a result of activation of the β2-AR/cAMP pathway are aims of β-AR agonist therapy. Certainly, on the contrary, many other studies in humans and animals in vivo, also in vitro, in different cell types, have shown that persistent administration and stimulation of β-AR agonists causes rather impaired β2-AR/cAMP response from the cell (Abraham et al. 2004; Chong et al. 2003; Tittelbach et al. 1998; Brodde et al. 1985) which can hamper their therapeutic implication. Consequently, we have examined time- and concentration-dependent effects of ISO (5 concentrations) and Salbu (3 concentrations) on β2-AR density/expression and

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cAMP content in 16HBE14o- cells. Both agonists induced successive decrease in β2AR density accompanied by the concomitant decrease in ISO-evoked cAMP formation, paralleled by ISO-induced decrease in receptor protein and mRNA. Treatment of 16HBE14o- cells with all ISO or Salbu concentrations for 24h resulted in

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almost no detectable β2-AR (cf. Fig. 1). The fact that CGP 12177, a hydrophilic ligand, was used to displace ICYP binding, it can be argued that loss of receptors from the cell membrane resulted from receptor internalization into the cytoplasm as

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described in vivo (Abraham et al. 2002). On the other hand, considerable level of β2AR protein was still expressed in 16HBE14o-, even if 10 µM ISO were added to the

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cells for 24h, as measured by flow cytometric analysis using specific β2-AR antibody (cf. Fig. 2). We assume that not or not all receptors internalized into the cytoplasm were degraded by cytoplasmic enzymes, and since the β2-AR antibody cannot differentiate the receptor localization, the fluorescence detected was, thus, not

receptors.

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apparently confined to the cell membrane but shows the intercellular presence of the

Even if both ISO and Salbu have similar intrinsic activity, incubation of 16HBE14o-

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with Salbu for 24h substantially attenuated cAMP formation than ISO, presumably

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due to its β2-AR selectivity. Our data, indeed, don’t support the observation that longterm treatment of airway epithelial cells with β2-AR agonists increases cAMP formation upon β2-AR activation (Düringer et al. 2009; Lindén, 1995). The decrease in β2-AR number (cf. Fig. 1) and cAMP content by ISO or Salbu after long-term 16HBE14o- treatment appears to correlate well with receptor changes that occur during β2-AR agonist therapy as described above, suggesting functional receptor desensitization and downregulation in vivo and in vitro which blunt drug

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and

worsen

disease

pathology,

presumably,

by

inducing

inflammatory mediator releases. Interestingly, a consistent finding with this regard was that β-AR agonist-mediated decrease in β-AR number and intracellular cAMP production was inversely linked to

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enhanced IL-6 and IL-8 release measured in same 16HBE14o- cultures, indicating the first time the role of receptor status in modulating cytokine releases in bronchial epithelial cells. The receptor density was completely reduced 24h after the addition of

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both ISO and Salbu to 16HBE14o-, while the concentration-dependent decrease in cAMP formation was rather remarkabley induced by Salbu than ISO, suggesting the

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β2-AR selectivity of Salbu. ISO dramatically reduced cAMP at concentrations above 1 µM which correlated well but negatively to the successive enhancement of IL-6 and IL-8 release (cf. Fig. 5A and 6A, inserts). However, previous in-vitro studies have shown, in part contradictory to our data, that the use of β-agonists produced

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diverging effects on IL-6, IL-8 and other cytokine releases with human airway epithelial, airway smooth muscle cells and mast cells. In human bronchial epithelial

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cell line, basal and TNF-α-stimulated IL-8 release was enhanced by β2-AR activation (leading to increased cAMP content) (Lindén, 1995). In alveolar epithelial cells

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(A549) and primary human bronchial epithelial cells, formoterol and salmeterol stimulated highly IL-6 and IL-8 releases in the presence of organic dust (Strandberg et al. 2007). In other bronchial epithelial cell line, BEAS-2B, as well in primary human bronchial epithelial cells, pre-incubation with β2-AR agonists augmented the release and mRNA expression of IL-6 and IL-8 induced by IL-1β, TNF-α plus histamine (Holden et al. 2010). On the other hand, in airway smooth muscle cells, the IL-1β- or TNF-α-mediated release and expression of eosinophil-activating cytokines such as GM-CSF, RANTES and eotaxin but not IL-8 were attenuated by ISO and Salbu

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(Hallsworth et al. 2001; Johnson, 1998). In human mast cells which express exclusively a population of the β2-AR subtype, decrease in receptor number and cAMP generation by long-term (24h) treatment with β2-AR agonists enhanced ISOstimulated histamine release, suggesting that, in good agreement with our data,

cellular responses (Scola et al. 2004; Chong et al. 2003).

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extensive levels of receptor desensitization and downregulation mediate this adaptive

A suppressive signal in inflammation and cytokine release is generally provided by

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the second messenger cyclic AMP, and increased cAMP is generally thought to inhibit cytokine release. Our data suggest that the mechanism by which long-term

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treatments with ISO and Salbu attenuate the inhibition of IL-6 and IL-8 release by βAR agonists in human bronchial epithelial cells involves effects at the receptor level itself. From our data, it can strongly be suggested that pro-inflammatory responses by β-AR agonists involve desensitization processes including phosphorylation,

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uncoupling, internalization and downregulation (Barak et al. 1997; Lefkowitz, 1998; Ferguson et al. 1998). Prolonged high receptor occupancy by β2-AR agonists drives

EP

often internalization of β2-ARs with subsequent receptor downregulation, so that receptor mediated activation of adenylate cyclase and hence cAMP production is

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hampered. It is interesting to note that, in contrary to our study, many other studies have shown that the extent of IL-6 and IL-8 releases caused by ISO and Salbu correlate well with the degree of β2-AR-mediated cAMP formation, perhaps indicating that processes leading to receptor downregulation as a result of lysosomal degradation are important. There is a number of evidence that the enhancement of cAMP accumulation in cells in vitro may decrease inflammatory gene expression possibly via a repressive effect on the transcription factor NF-κB (Ye, 2000); and accordingly, the marked IL-6 and

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IL-8 release in relation to attenuated cAMP formation observed in our study may presumably result from activation of NF-κB; indeed, it needs to be proven in 16HBE14o- cells. In BEAS-2B cells, β2-AR agonists and other cAMP-elevating agents (forskolin,

cAMP

analogues)

decreased

NF-κB-dependent

transcription

but

(Coraux et al. 2004; Holden et al. 2010).

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enhancement of cytokine releases could not be explained by this mechanism

Enhanced IL-6 and IL-8 releases upon long-term β-AR agonist treatments in the

SC

present study could be the result of decreased PKA activation, since the level of

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cAMP that results from receptor desensitization is low to activate this enzyme. In agreement, the direct activation of the cAMP-PKA cascade by forskolin, 8-bromocAMP and prostaglandin E2 in airway smooth muscle cells repressed IL-8 release (Kaur et al. 2008). Even if we did not test substances that activate adenylate cyclase, the fact that the marked β-AR agonist-mediated β2-AR downregulation ultimately

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hampers receptor-induced and ISO-evoked cAMP generation, it is logical to assume that the enhanced cytokine release was due to low PKA activation. Our results are

EP

most consistent with the interpretation that ISO and Salbu do in fact provoke rather GRK-mediated phosphorylation and downregulation; indeed, even if the ISO-evoked

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increase in cAMP as a result of receptor activation was not fully suppressed by low βAR agonist concentrations, PKA might have been activated but might not trigger enhanced cytokine releases. Even if a complete receptor downregulation has occurred, an explanation for the little amount of cAMP produced after treatment of cells with ISO and Salbu for 24h, is that internalized β2-ARs activate presumably persistent intracellular cAMP formation, but this may not play a role in cytokine synthesis (Calebiro et al. 2010). Possibly, internalization or downregulation of the β2AR by increasing concentrations of β-agonists could cause switching to cAMP-

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independent signalling to activate IL-6- and IL-8-production (for review see Giembycz et al. 2006). In the clinical context, our data suggest that β2-AR agonists likely to prevent cytokine releases at early treatment period of airway diseases than after development of drug-

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induced tolerance. Some studies have shown that β2-AR agonists, when given for long time on their own alone, are pro-inflammatory (Cockcroft et al. 1993; Vathenen et al. 1988), and our finding with IL-6 and IL-8 production are consistent with the pro-

SC

inflammatory effect. The fact that IL-8 is released from 16HBE14o- implies the association with significant neutrophilic inflammation since IL-8 is attractant for these

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cells (Gordon et al. 2003). IL-6 is involved in inflammatory and acute phase reactions and also mediates Th2-directed inflammation in the airways (Barnes et al. 1998; Sehgal, 1990).

In conclusion, IL-6 and IL-8 production is regulated by the β2-AR/cAMP pathway with

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regard to long-term treatment with β2-AR agonists. Thus, these drugs have upregulated inflammatory signals (IL6, IL-8) as a result of blunted signal transduction pathways through β2-ARs. Indeed, molecular mechanisms that prevent receptor

EP

downregulation and as a result β2-AR agonist induced airway inflammation which is

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relevant for clinical outcomes and therapeutic intervention should be evaluated in further studies.

Acknowledgements

We would like to thank the technical assistant Ina Hochheim for her support in radioligand binding studies. The SV40-transformed human bronchial epithelial cell line 16HBE14o- was kindly donated by Dr. Ulrich Sydlik (Institute of Environmental-

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Medical Research, Department of Toxicology, Heinrich-Heine-university, Düsseldorf,

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Germany).

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ACCEPTED MANUSCRIPT Conflict of interest statement

None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the

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paper.

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Figures Legends Figure 1: Time courses of the β2-AR downregulation after concentration-dependent treatment

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of 16HBE14o- cells with ISO (A) and Salbu (B). Box and Whiskers show range of data of three separate experiments performed in duplicate. Concentration-dependent significance vs. control (100%) (∗p < 0.05;

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Wilcoxon Test); Time-dependent significance vs. 0.5 h within one concentration (#p