Serotoninergic Receptors on Human Airway Epithelial ... - ATS Journals

Serotoninergic Receptors on Human Airway Epithelial Cells ¨ ller*, Daniel Myrtek, Stephan Sorichter, Manfred Ziegenhagen, Johannes Norgauer, Hans Bayer*, Tobias Mu Gernot Zissel†, and Marco Idzko† Department of Pneumology, University Hospital Freiburg; Wilhelm-Anton-Hospital, Goch; and Department of Dermatology, University of Jena, Jena, Germany

There is accumulating evidence that points to a role of serotonin (5-hydroxytryptamine [5-HT]) in the pathophysiology of asthma. Therefore, we analyzed the expression of serotoninergic receptors (5-HTR), its linkage to intracellular calcium homeostasis, and its influence on the production and secretion of IL-6, prostaglandin E2, the CCL-Chemokine CCL5/Rantes, and the CXC-chemokines CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC in primary alveolar epithelial cells type II and the human lung cell lines A549 and BEAS-2B. Employing a PCR approach we were able to demonstrate mRNA expression of several 5-HTR, such as the heptahelical receptors 5-HTR1A, 5-HTR1B, 5-HTR1E, 5-HTR1F, 5-HTR2A, 5-HTR4, 5-HTR6, and 5-HTR7, as well as the ligand-gated ion channel 5-HTR3 in alveolar epithelial cells type II (AEC-II), A549, and BEAS2B cells. To verify functional expression of 5-HTR subtypes, Ca2ⴙtransients were analyzed. This enabled us to show that 5-HT induced an increase in intracellular calcium. Further experiments with isotype-selective receptor agonists allowed us to demonstrate that 5-HT induced calcium transients via activation of 5-HTR1, 5-HTR2, and 5-HTR3 in A549 and BEAS-2B cells. Moreover, we revealed that stimulation of 5-HTR1 and 5-HTR2 induced Ca2ⴙ mobilization from intracellular stores, whereas activation of 5-HTR3 induced Ca2ⴙ influx from the extracellular space. Functional studies indicated that activation of 5-HTR1B, 5-HTR1E/F, 5-HTR2, 5-HTR3, 5-HTR4, and 5-HTR7 regulated the release of the cytokine IL-6 and the CXCchemokine CXCL8/IL-8. Our study shows that 5-HT stimulates different signaling pathways and regulates cytokine release in airway epithelial cells. In summary, our data implicate a pathophysiologic role of 5-HT in the asthmatic inflammatory responses in human airway epithelial cells. Keywords: airway epithelial cells; Ca2⫹-homeostasis; cytokine release; serotonin; serotoninergic receptors

5-Hydroxytryptamine (5-HT) is a well-characterized neurotransmitter with regulative functions in multiple physiologic aspects (1). In peripheral sites 5-HT is present at high concentrations in basophils, platelets, and mast cells (2, 3). 5-HT also has immunomodulatory effects by regulating a wide variety of cell responses such as migration, phagocytosis, superoxide anion generation, and cytokine production (4–7). Furthermore, increased levels of free 5-HT are present in the plasma of symptomatic patients with asthma compared with levels in asymptomatic patients (8). Moreover, cumulating evidence points to an additional

(Received in final form April 19, 2006) * H.B. and T.M. contributed equally to this work. †

G.Z. and M.I. contributed equally to this work.

This work was supported by grants from DFG (ID7/2-1, ID7/3-1 and NO266/3-1) and IZKF/Jena (TP3.2). Correspondence and requests for reprints should be addressed to Dr. Marco Idzko, University Hospital Freiburg, Department of Pneumology, Hugstetterstraße 55, D-79106 Freiburg i. Br., Germany. E-mail: [email protected] Am J Respir Cell Mol Biol Vol 36. pp 85–93, 2007 Originally Published in Press as DOI: 10.1165/rcmb.2006-0151OC on July 27, 2006 Internet address: www.atsjournals.org

role of 5-HT in the pathophysiology of asthma (4, 9, 10). The wide variety of biological activities, which are currently not completely understood, and the complexity of pharmacologic activities mediated by 5-HT, are due to the existence of different classes of serotoninergic receptors (5-HTR). The large number of receptors combined with a highly effective re-uptake mechanism, provides nearly unlimited signaling capacity (11). The 5-HTR1 subgroup consists of at least five subtypes namely 5-HTR1A, 5-HTR1B, 5-HTR1D, 5-HTR1E, and 5-HTR1F. 5-HTR1A interacts with several G proteins, eliciting different responses (12). 5-HTR1B and 5-HTR1D are coupled to formation of inositol phosphates through interaction with pertussis toxin–sensitive Gi/o and pertussis toxin–insensitive Gq proteins (13). The G protein– coupled 5-HTR2 includes three different subtypes: 5-HTR2A, 5-HTR2B, and 5-HTR2C (14). The 5-HTR3 is a ligand-gated cation channel triggering depolarization of the plasma membrane through activation of Na⫹ and K⫹ fluxes (15). The 5-HTR4 receptor has two splice variants (5-HTR4a and 5-HTR4b) (15). The heptahelical 5-HTR5 subtype is less characterized (16). 5-HTR6 and 5-HTR7 are linked to Gs protein–mediated stimulation of adenylyl cyclase (17, 18). The alveolar surface of the lung is lined with alveolar epithelial cells type I and type II. Alveolar epithelial cells type I function as a physical barrier and provide a pathway for gas exchange, whereas alveolar epithelial cells type II (AEC-II) produce surfactant and act as progenitors to replace injured alveolar epithelial cells type I. Located on the boundary between the alveolar airspace and the lung interstitium, they are ideally situated to modulate immune responses (e.g., by producing cytokines and chemokines [19–23]). Previously it has been shown that primary AEC-II were important regulators of the immune function in the lung, since they expressed costimulatory molecules necessary for T cell activation and NOS3, and release IL-6 and the CXCchemokine CXCL8/IL-8 in response to inflammatory stimulants (24, 25). Intracellular Ca2⫹-transients are an important mechanism in the transmission of cell surface activation signals to diverse cellular processes in various cell types (26), including alveolar epithelial cells (20). The aim of this study was to characterize the biological activity of 5-HT in AEC-II, A549, and BEAS-2B cells in the context of the asthmatic inflammatory reaction. The A549 cell line is generally thought to resemble AEC-II (27). BEAS-2B are transformed bronchial epithelial cells, which are widely used to explore functional properties of bronchial epithelial cells (28). By using these cells in this fashion, we were able to show that A549, BEAS-2B, and primary AEC-II expressed different 5-HTR. 5-HT triggered calcium influx through the plasma membrane as well as Ca2⫹ mobilization from intracellular stores and participated in the regulation of the release of IL-6 and the CXCL8/ IL-8.

MATERIALS AND METHODS Reagents A549 and BEAS-2B cells were obtained from the DMSZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig,

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Germany). 5-HT, 5-methoxytryptamine (2-MHT), N-methyl-5-hydroxytryptamine (2Me5HT), ketanserin, R-(-)-DOI-hydrochloride (DOI), ketanserin, and pertussis toxin (PTX) were obtained from SigmaAldrich (Deisenhofen, Germany); 5-carboxamidotryptamine maleate (5-CT), BRL54443 (BRL), 8-hydroxy-DPAT-hydrobromide (8HDPAT), buspirone hydrochloride (BUS), and anpirtoline hydrochloride (AnHCL) were from Biotrend (Cologne, Germany).

Cell Culture A549 cells were grown in MEM-medium (Gibco, Paisley, UK) containing 5% fetal calf serum gold (PAA Laboratories, Pasching, Austria) and 1% penicillin/streptomycin (Biochrom, Berlin, Germany) in 175 cm2 culture flasks (BD Falcon, Bedford, MA) at 37⬚C, 5% CO2 and 100% humidity, as described previously (20). BEAS-2B cells were cultured in the same medium, under similar conditions as previously described (20). To favor cell adhesion, flasks were coated with a solution containing 30 ␮g/ml rat collagen S, 1 mg/ml human fibronectin, and 100 ␮g/ml BSA in MEM-medium. Both kinds of cells were grown in culture flasks; cells were then removed using trypsin/EDTA solution (Biochrom) and seeded into 24well tissue culture plates (Corning Inc., Corning, NY), at a density of 0.2 ⫻ 106 cells/well (A549) or 0.1 ⫻ 106 cells/well (BEAS-2B), respectively. After 24 h, medium was changed and cells were stimulated. After additional 24 h, cell supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA).

Isolation of AEC-II Isolation of AEC-II was performed as previously described (20). Briefly, lung tissue samples were obtained from subjects with lung cancer undergoing lobotomy. Tissue was cut into pipettable pieces and washed with BI. The washed pieces were incubated in a solution of BII and dispase (2.5 g/liter) at 37⬚C for 1 h. After dispase, digestion tissue was cut again. Crude tissue and cell suspensions were filtered through nylon gauze with meshes of 50 and 20 ␮m. The resulting single-cell suspension was placed on Ficoll separating solution (Biochrom) and centrifuged at 1,990 rpm for 25 min. The AEC-II-enriched cells from the interphase were washed and resuspended in medium and incubated in 100-mm plastic dishes at 37⬚C in humidified air containing 5% CO2 for 15, 20, and 30 min to remove adherent cells (alveolar macrophages, dendritic cells, fibroblasts, and endothelial cells). Nonadherent cells were seeded onto fresh dishes after each adherence step. To remove remaining leukocytes, cells were incubated with anti-CD45 antibodies conjugated beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and depleted using LD-columns. Cells were routinely checked by modified Papanicolaou staining and flow cytometric analysis. CD45-negative cells were used for RT-PCR. Cells were then resuspended in MEM medium containing 5% FCS and 1% penicillin/streptomycin and seeded in a density of 0.2 ⫻ 106 cells in 24-well tissue culture plates. After 24 h, medium was changed and cells were stimulated. After an additional 24 h, cell supernatants were collected and analyzed by ELISA.

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T-3⬘, antisense 5⬘-GCA AAG TAG CCA GGC GAT TCT CT-3⬘; 5-HTR4 (411 bp)—sense 5⬘-GGC CTT CTA CAT CCC ATT TCT CCT-3⬘, antisense 5⬘-CTT CGG TAG CGC TCA TCA TCA CA-3⬘; 5-HTR6 (342 bp)—sense 5⬘-CCG CCG GCC ATG CTG AAC G-3⬘, antisense 5⬘-GCC CGA CGC CAC AAG GAC AAA AG-3⬘; 5-HTR7 (436 bp)—sense 5⬘-GCG CTG GCC GAC CTC TC-3⬘, antisense 5⬘-TCT TCC TGG CAG CCT TGT AAA TCT-3⬘. PCR was performed using 10 ␮l of iQ-Supermix (BioRad, Hercules, CA), 7 ␮l H2O, 1 ␮l of each primer (final concentration 0.5 ␮M each) and 1 ␮l of cDNA. Amplification conditions were: 9 min initial denaturation at 95⬚C, then 40 cycles of 94⬚C for 15 s, 57⬚C for 30 s, and 72⬚C for 30 s. PCR products were resolved by electrophoresis on a 2% agarose gel. PCR products were cloned and sequenced to prove their identity.

Quantification by Real-Time PCR Total RNA was extracted and cDNA was synthesized as described above. All oligonucleotide primers for real-time PCR were designed using Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3www. cgi) and synthesized by MWG (Ebersberg, Germany). For iCycler reaction, a master mix of the following compounds was prepared to the indicated end concentration: 10 ␮l SYBR Green master mix (BioRad), 6 ␮l water, and 1 ␮l each of sense and antisense primers (500 nM). The target gene mRNA was indexed to the housekeeping gene glyceraldehyde-3phosphate dehydrogenase (GAPDH) using the formula: 10,000 ⫻ 2deltaCT (deltaCT ⫽ CTGADPH ⫺ CTtarget gene).

Intracellular Ca2ⴙ Measurement Ca2⫹ transients were measured in A549 and BEAS-2B cells loaded with the Ca2⫹ indicator Fura-2/AM (Calbiochem, La Jolla, CA) by using the digital fluorescence microscope unit Attofluor (Zeiss, Oberkochen, Germany) as previously described (20). Briefly, A549 and BEAS-2B were incubated with 2 ␮M Fura-2/AM for 30 min at 37⬚ C in a Ca2⫹and Mg2⫹-free Hanks’ BSA solution. Cells were then washed twice and finally resuspended in the same buffer containing 1.5 mM CaCl2 and MgCl2. Traces were followed spectrofluorometrically and Ca2⫹ transients were determined by multiple cell acquisitions with the 340/380 wavelength excitation ratio at an emission wavelength of 505nm.

Cytokine and Prostaglandin E2 Assays 5-HT and 5-HTR agonists were added to cells for the indicated time points. Thereafter, supernatants of A549, BEAS-2B, and AEC-II cells were collected. IL-6, RANTES (regulated on activation, normal T cells expressed and secreted), IL-8, monokine induced by gamma interferon (MIG), IP-10, I-TAC, and prostaglandin (PG)E2 were measured by ELISA or EIA (R&D Systems, Minneapolis, MN). Samples were assayed in triplicate for each condition.

Statistical Analyses Detection of 5-HTR mRNA by RT-PCR Analysis Total RNA was extracted from the cells using Trizol-Reagent (Gibco). To obtain cDNA, 5 ␮g of total RNA were primed with oligo-dT primers (Gibco) and reverse-transcribed with StrataScript reverse transcriptase (Stratagene, La Jolla, CA). All oligonucleotides used as primers in PCR were designed to recognize sequences specific for each target cDNA. Primer sequences were as follows: 5-HTR1A (411-bp product)— sense 5⬘-GCC GCG TGC GCT CAT CTC G-3⬘, antisense 5⬘-GCG GCG CCA TCG TCA CCT T-3⬘; 5-HTR1B (460-bp product)—sense 5⬘-CAG CGC CAA GGA CTA CAT TTA CCA-3⬘, antisense 5⬘GAA GAA GGG CGG CAG CGA GAT AGA-3⬘; 5-HTR1E (461bp product)—sense 5⬘-CAA GAG GGC CGC GCT GAT GAT-3⬘, antisense 5⬘-CTG CCT TCC GTT CCC TGG TGG TGC TA-3⬘; 5-HTR2A (359-bp product)—sense 5⬘-ACT CGC CGA TGA TAA CTT TGT CCT-3⬘, antisense 5⬘-TGA CGG CCA TGA TGT TTG TGA T-3⬘; 5-HTR2B (416-bp product)—sense 5⬘-GGC CCC TCC CAC TTG TTC T-3⬘, antisense 5⬘-TAG GCG TTG AGG TGG CTT GTT-3⬘; 5-HTR2C (449-bp product)—sense 5⬘-TGT GCC CCG TCT GGA TTT CTT TAG-3⬘, antisense 5⬘-CTC TTC CTC GGC CGT ATT CCT CTT-3⬘; 5-HTR3 (448/352 bp)—sense 5⬘-CCG GCG GCC CCT CTT CTA

Unless stated otherwise, data are expressed as mean ⫾ SEM. ANOVA was used to compare experimental groups to control values. When the global test of differences was significant at the 5% level, pairwise tests of differences between groups were applied (Tukey’s comparison test). For PCR bands, statistical analysis was performed by the Dunnet comparison test (ANOVA).

RESULTS Human AEC Express the mRNA of Different 5-HTR Subtypes

Expression of mRNA for the different 5-HTR subtypes was analyzed by RT-PCR in A549 (Figure 1A) and BEAS-2B (Figure 1B) cells, as well as in primary human AEC-II cells (Figure 1C). These figures indicate that the analyzed cells expressed mRNA of the G protein–coupled 5-HTR1A, 5-HTR1B, 5-HTR1E, 5-HTR1F, 5-HTR2A, 5-HTR4, 5-HTR6, and 5-HTR7. Moreover, transcripts of the ligand-gated cation channel 5-HTR3 were revealed. However, we found no transcripts for 5-HTR1D, 5-HTR2B, or 5-HTR2C in these cells (data not shown).

Bayer, Mu¨ller, Myrtek, et al.: Serotoninergic Receptors on Airway Cells

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Figure 1. A549 (A ), BEAS-2B (B ), and alveolar epithelial type II cells (C ) express the mRNA for several 5-HTR subtypes. (A ) 5-HTR1A; (B ) 5-HTR1B; (C ) 5-HTR1E; (D ) 5-HTR1F; (E ) 5-HTR2A; (F ) 5-HTR3; (G ) 5-HTR4; (H ) 5-HTR6; (I ) 5-HTR7. RT-PCR analysis was performed with mRNA isolated from A549, BEAS-2B, and purified AEC-II cells (see MATERIALS AND METHODS). One representative experiment of four is shown (n ⫽ 4).

5-HT Stimulates Ca2ⴙ-Transients in A549 and BEAS-2B Cells

In order to reveal the functional expression of 5-HTR, Ca2⫹transients after stimulation with 5-HT were followed. Stimulation of A549 (Figure 2A) and BEAS-2B cells (Figure 2B) with 5HT induced rapid and concentration-dependent Ca2⫹-transients in both cell lines. In both cells half-maximal and maximal responses were seen with 10⫺4 and 10⫺6 M 5-HT, respectively. To study involvement of different 5-HTR subtypes in Ca2⫹ transients, A549 and BEAS-2B were stimulated with different selective 5-HTR agonists. Therefore, BEAS-2B were stimulated with 5-CT, an agonist for 5-HTR1, 5-HTR4, and 5-HTR7. This compound induced a spiking Ca2⫹ rise followed by a slow declining phase in BEAS-2B (Figure 3A). Since the 5-HTR4 agonist 2MHT as well as the 5-HTR7 agonist 8HDPAT did not trigger any Ca2⫹ response in these cells (data not shown), experiments with isotype-selective 5-HTR1 agonists were performed. Thereby, Ca2⫹ transients in BEAS-2B were seen after stimulation with the 5-HTR1A agonist BUS (Figure 3B), the 5-HTR1B agonist AnHCL (Figure 3C), and the 5-HTR1E/F agonist BRL54443 (Figure 3D). Moreover, the 5-HTR2 agonist DOI was able to induce Ca2⫹-transients (Figure 3E). Identical conclusions about functional expression of 5-HTR1A, 5-HTR1B, 5-HTR1E, and 5-HTR2 could be drawn from experiments with A549 (Table 1). In order to corroborate participation of different 5-HTR in this signal pathway, cell studies with the selective 5-HTR2 antagonist ketanserin were performed. Ketanserin completely abolished the Ca2⫹ increase induced by 5-HTR2 agonist DOI, while it failed to block the cell responses induced with the 5-HTR1 agonists CT, BUS, AnHCL, and BRL54443 in A549 (data not shown).

Figure 2. 5-HT triggers Ca2⫹ transients in A549 (A ) and BEAS-2B cells (B ). Cells were loaded with the Ca2⫹ indicator Fura-2/AM and stimulated with the indicated concentrations of 5-HT. Representative traces are shown. Experiments were repeated four times with similar results.

The 5-HT–Induced Ca2ⴙ-Transients Are Partially Dependent on Extracellular Calcium and Gi Proteins

Besides Ca2⫹ influx through the plasma membrane, Ca2⫹ transients can be due to mobilization of the ion from the intracellular stores. To better discriminate between the two pathways, BEAS2B and A549 were stimulated with 5-HT in the absence of extracellular Ca2⫹. The presence of the Ca2⫹ chelator ethylene-glycotetraacetic-acid (EGTA) partially reduced 5-HT-induced Ca2⫹ transients in BEAS-2B and A549. However, chelation of extracellular Ca2⫹ did not affect the 5-HTR1– and 5-HTR2–mediated Ca2⫹-transients induced by 5-CT, BUS, AnHCL, BRL 54443, and DOI (Table 2). 5-HTR3 is a ligand-gated cation channel triggering Ca2⫹ influx from the extracellular medium (15). For a better understanding of the above-reported effects, studies with the 5-HTR3 agonist 2Me5HT were performed. This ligand also induced Ca2⫹-transients in BEAS-2B in a concentration-dependent manner. Half-maximal and maximal responses were seen with 10⫺5 and 10⫺3 M 2Me5HT, respectively (Figure 4). In contrast to 5-HTR1 and 5-HTR2 agonist, the 5-HTR3–mediated response was totally blocked by the absence of extracellular Ca2⫹ in BEAS-2B (Table 2). Similar data were obtained in experiments with A549. Mobilization of Ca2⫹ from intracellular stores by heptahelical receptors is often mediated via pertussis toxin–sensitive Gi/o proteins (29). To study participation of Gi/o proteins in 5-HTR1– and 5-HTR2–mediated signaling, BEAS-2B were preincubated with pertussis toxin. This toxin uncouples Gi/o proteins from

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Figure 3. 5-HTR1 and 5-HTR2 agonists trigger Ca2⫹ transients in BEAS-2B. Cells were loaded with Fura-2/AM and stimulated with increasing concentrations of the reported agonists. Representative curves are shown. Experiments were repeated four times with similar results.

5-HTR1–stimulating 5-CT, and Ca2⫹-transients were monitored (Figure 6A). After recovery to initial values, cells were stimulated a second time with an optimal concentration of the 5-HTR2 agonist DOI. This agent was able to provoke a second cell response. After a further recovery to initial values, the 5-HTR3 agonist 2Me5HT was added to the cells. Again, a Ca2⫹response could be observed. Similar observations were seen with the stimulation cascades DOI/5-CT/2Me5HT (Figure 6B), 2Me5HT/DOI/5-CT (Figure 6C), or 2Me5HT/5-CT/DOI (Figure 6D). In addition, BEAS-2B cells were initially stimulated with optimal concentration of 5-CT (Figure 6E), DOI (Figure 6F), or 2-Me5HT (Figure 6G) and calcium transients were analyzed. After

heptahelical receptors by ADP-ribosylation (30). Pertussis toxin reduced the 5-HT response and almost completely abolished Ca2⫹-transients induced by 5-CT, BUS, AnHCL, and BRL54443 in A549 and BEAS-2B (Figures 5A and 5B). To exclude the possibility that lack of response of A549 and BEAS-2B upon treatment with pertussis toxin was due to cytotoxic side effects of the toxin, cells were also stimulated with the 5-HTR3 agonist 2-Me5HT. Pertussis toxin did not affect the Ca2⫹ response triggered by these agonists (data not shown). To gain further insight into the 5-HTR agonist stimulation mechanism, desensitization studies were performed. Therefore, BEAS-2B were exposed to optimal concentration of the

TABLE 1. EFFECTS OF SELECTIVE 5-HT RECEPTOR AGONISTS ON INTRACELLULAR Ca2⫹ IN A549 CELLS 1 mM 5-CT BUS AnHCL BRL 54443 DOI 2Me5HT

1.29 ⫾ 0.06 — 1.25 ⫾ 0.05 1.23 ⫾ 0.04 1.24 ⫾ 0.05 1.18 ⫾ 0.02

0.1 mM 1.22 1.19 1.18 1.21 1.19 1.15

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.05 0.05 0.04 0.03 0.05 0.03

0.01 mM 1.18 1.11 1.11 1.10 1.12 1.07

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.04 0.03 0.05 0.04 0.04 0.04

0.001 mM

0.0001 mM

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

— — — — — 0.96 ⫾ 0.05

1.04 1.04 1.04 1.03 1.07 0.99

0.05 0.04 0.03 0.04 0.05 0.04

0 mM 0.93 0.92 0.91 0.90 0.91 0.84

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.05 0.03 0.06 0.05 0.03 0.04

Definition of abbreviations: 2Me5HT, N-methyl-5-hydroxytryptamine; 5-CT, 5-carboxamidotryptamine maleate; AnHCL, anpirtoline hydrochloride; BUS, buspirone hydrochloride; DOI, R-(-)-DOI-hydrochloride. A549 were loaded with Fura-2/AM and then stimulated with the indicated concentrations of the different agonists. The Ca2⫹ transients were analyzed, and the ratio after stimulation for 10 s is given. Data are means ⫾ SEM (n ⫽ 5).


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TABLE 2. EFFECT OF EXTRACELLULAR Ca2⫹ CHELATION ON INTRACELLULAR Ca2⫹ TRANSIENTS INDUCED BY DIFFERENT 5-HT RECEPTOR AGONISTS A549 AND BEAS-2B Control A549 Medium EGTA BEAS-2B Medium EGTA

5-HT

5-CT

BUS

AnHCL

BRL 54443

DOI

2Me5HT

0.87 ⫾ 0.06 1.17 ⫾ 0.04 1.28 ⫾ 0.04 1.18 ⫾ 0.07 1.20 ⫾ 0.04 1.19 ⫾ 0.05 1.21 ⫾ 0.03 1.15 ⫾ 0.04 0.85 ⫾ 0.03 1.06 ⫾ 0.03 1.25 ⫾ 0.05 1.17 ⫾ 0.05 1.18 ⫾ 0.03 1.18 ⫾ 0.04 1.19 ⫾ 0.04 0.89 ⫾ 0.06 0.95 ⫾ 0.04 1.27 ⫾ 0.04 1.34 ⫾ 0.06 1.21 ⫾ 0.06 1.26 ⫾ 0.05 1.23 ⫾ 0.05 1.23 ⫾ 0.03 1.19 ⫾ 0.04 0.92 ⫾ 0.03 1.14 ⫾ 0.03 1.29 ⫾ 0.08 1.19 ⫾ 0.05 1.24 ⫾ 0.03 1.22 ⫾ 0.04 1.19 ⫾ 0.04 0.98 ⫾ 0.06

Definition of abbreviations: 5-HT, 5- hydroxytryptamine; EGTA, ethylene-glyco-tetraacetic-acid. A549 and BEAS-2B were loaded with Fura-2/AM as reported in MATERIALS AND METHODS and stimulated with 10⫺3 M of the indicated agonists or 10⫺5 M for BP554 respectively, in the absence or presence of 4 mM EGTA. Results are expressed as ratios (⫾ SD; n ⫽ 3).

recovery to initial values at the end of the first cell response, the cells were this time stimulates a second time with the same agonist, respectively. Under this protocol, none of the tested compounds were able to prove a second cell response (Figures 6E–6G). Identical results could be seen for A549 cells (data not shown). 5-HT Stimulates IL-6 and CXCL8/IL-8 Release from A549 and BEAS-2B Cells

There is evidence suggesting that Ca2⫹-transients regulate the production of cytokines and chemokines in different kinds of cells (31, 32). Therefore, we analyzed the influence of 5-HT on CXCL8/ IL-8 and IL-6 production in A549, BEAS-2B, and AEC-II. These experiments showed that 5-HT concentration-dependently increased the secretion of CXCL8/IL-8 in A549 (Figure 7A), BEAS-2B (Figure 7B), and AEC-II (Figure 7C). Half-maximal and maximal effects were seen at concentration of 10⫺5 and 10⫺4 M 5-HT, respectively. Using selective receptor agonists at optimal concentrations, 5-HTR1A agonist BUS, 5-HTR1B agonist AnHCL, the 5-HTR1E/F agonist BRL54443, the 5-HTR2 agonist DOI, the 5-HTR3 agonist 2Me5HT, 5-HTR4 agonist Me5HT, and the 5-HTR7 agonist 8HDPAT revealed that all 5-HTR subtypes are involved in the 5-HT–induced CXCL8/IL-8 production (Figures 7D–7F). However, the most potent analyzed activators of CXCL8 release were the 5-HTR3 agonist 2Me5HT and the 5-HTR4 agonist Me5HT. To prove the selectivity of the used agonists, the cells were pretreated with the 5-HTR3 antagonists AP and Y-25130 and the 5-HTR4 antagonist RS 39604. This led to a block of the 2Me5-HT– and the Me-5HT–induced response (data not shown).

Figure 4. Stimulation of the 5-HTR3 subtype triggers Ca2⫹ influx BEAS2B. Fura-2/AM loaded BEAS-2B were stimulated with the indicated concentrations of the 5-HTR3 agonist 2-Methyl-5-HT. Representative curves are shown. Experiments were repeated four times with similar results.

In addition, relative quantification of mRNA in iCycler realtime PCR indicated that stimulation with 5-HT also enhanced CXCL8 mRNA level in A549 (Figure 7G). Furthermore, 5-HT stimulates the production of IL-6 in A549, BEAS-2B, and AEC-II (Figures 8A–8C). Again, this response was mediated by the different 5-HTR1 subtypes, the 5-HTR2, 5-HTR3, the 5-HTR4, and 5-HTR7 (Figures 8D–8F). The most potent receptor agonists inducing the secretion of IL-6 were the 5-HTR3 agonist 2Me5HT and the 5-HTR4 agonist Me-5HT, as well as the 5-HTR7 agonist 8HDPAT, while the other 5-HTR agonist showed a weaker potency as for IL-6 secretion (Figures 8D–8F). Again, pretreatment of the cells with the selective antagonists Pimozide (5-HTR7 antagonist), SB 269970 (5-HTR7 antagonist), AP (5-HTR3 antagonist), Y-25130 (5-HTR3 antagonist), and

Figure 5. Pertussis toxin (PTX) inhibits Ca2⫹ transients elicited by 5-HTR1 and 5-HTR2 agonists in A549 (A ) and BEAS-2B cells (B ). Cells were incubated in the presence or absence of 10 ␮g/ml PTX for 2 h, loaded with Fura-2/AM, and then stimulated with optimal concentrations of 5-HTR1 and 5-HTR2 agonists. Data are means ⫾ SEM (n ⫽ 3).

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Figure 6. Heterologous and homologous desensitization studies of 5-HTR agonists in BEAS-2B. BEAS-2B were labeled with Fura-2 and stimulated for the indicated time with the first stimulus, followed by a second and third stimulation with the indicated agonists. The serial stimulation was performed with (A ) 10⫺4 M 5-CT, 10⫺4 M DOI, and 10⫺4 M 2Me5HT; (B ) 10⫺4 M DOI, 10⫺4 M 5-CT, and 10⫺4 M 2Me5HT; (C ) 10⫺4 M 2Me5HT, 10⫺4 M DOI, and 10⫺4 M 5-CT; (D ) 10⫺4 M 2Me5HT, 10⫺4 M 5-CT, and 10⫺4 M DOI; (E ) 10⫺4 M 5-CT and 10⫺4 M 5-CT; (F ) 10⫺4 M 5-CT and 10⫺4 M 5-CT; (G ) 10⫺4 M 2Me5HT and 10⫺4 M 2Me5HT. The experiment was repeated four times with identical results.

RS 39604 (5-HTR4 antagonist) led to a complete abolishment of the 8HDPAT-, 2Me5HT-, and Me-5HT–induced response (data not shown). In addition, relative quantification of mRNA in iCycler realtime PCR revealed that the 5-HT–increased IL-6 secretion was paralleled by enhanced IL-6 mRNA levels in A549 (Figure 8G). Similar data were observed by experiments in BEAS-2B and AEC-II (data not shown). In contrast to the situation regarding IL-6 and IL-8/CXCL8, parallel-performed experiments could not show any significant effect of 5-HT on the production of CCL5/Rantes, CXCL9/

MIG, CXCL 10/IP-10, or CXCL11/I-TAC and the cycloxygenase product PGE2 (data not shown).

DISCUSSION The role of 5-HT in the pathogenesis of bronchial asthma has been shown in various studies (33, 34). 5-HT has complex actions on the human lung. Symptomatic patients with asthma have increased plasma levels of free serotonin compared with asymptomatic patients with asthma, correlating positively with their clinical status and negatively with pulmonary function (8).


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Figure 7. 5-HT and 5-HT agonists stimulate CXCL8/ IL-8 release from (A, D ) A549 cells, (B, E ) BEAS2B cells, and (C, F ) AECII. Cells were stimulated with the indicated concentration of 5-HT (A–C) or 10⫺4 M of the indicated 5-HTR agonists (D– F). Supernatants were collected after 24 h, and the CXCL8/IL-8 content was determined by ELISA. Data are expressed as mean ⫾ SEM (n ⫽ 5). (G ) Cells were stimulated in absence or present of 10⫺4 M 5-HT for 24 h. Total RNA was isolated from A549. CXCL8/IL-8 mRNA expression was quantified as described in MATERIALS AND METHODS. Number of transcripts is normalized to the number of copies of GAPDH. Data are means ⫾ SEM (n ⫽ 3).

Previously it has been shown that the serotonin-uptake accelerator “Tianeptine” led to a clinical improvement of asthmatic symptoms (34–36). Recently we could show that 5-HT modulated, in a maturation-dependent manner, the function of human monocyte–derived dendritic cells and monocytes, leading to an induction and/or sustaining of the Th2-dominated immunity in patients with allergic asthma (4, 9). Therefore, we investigated the effects of 5-HT on primary human alveolar epithelial cells type II and the human epithelial lung cell lines A549 and BEAS-2B. Here we show for the first time the expression of functional 5-HTR in A549, BEAS-2B, and AEC-II, by analyzing mRNA expression and Ca2⫹-transients, as well as cytokine release, after stimulation with selective 5-HTR agonists. Thereby we demonstrated that BEAS-2B and A549 expressed different G protein–coupled 5-HTR such as 5-HTR1A, 5-HTR1B, 5-HTR1E, 5-HTR1F, and 5-HTR2A. Experiments with EGTA in the extracellular medium indicated that stimulation of these receptors apparently activated mobilization of Ca2⫹ from the

intracellular stores. Therefore it can be assumed that these receptors activate the phospholipase C. This enzyme cleaves phosphoinositides into diacylglycerol and the inositol 1,4,5-trisphosphate, which mobilizes Ca2⫹ from the intracellular stores. 5-HTR1 and 5-HTR2 are able to couple Gi/o protein as well as to Gq proteins (29). Experiments with pertussis toxin indicated that the detected 5-HTR1 and 5-HTR2 apparently only couple to Gi/o proteins in A549 and BEAS-2B. In addition to the Gi protein–coupled 5HTR1 and 5-HTR2, we detected functional expression of the cation channel 5-HTR3 in BEAS-2B and A549 leading to a calcium influx from the extracellular space (4, 11, 15). In order to understand the influence between the different activation pathways of 5-HT in epithelial cells, desensitization studies were performed. Thereby we found no evidence that one agonist diminished the responsiveness of another 5-HTR agonist. Taken together, these findings suggest that in epithelial cells 5-HT independently regulates intracellular Ca2⫹ homeostasis by different mechanisms: 5-HTR1– and 5-HTR2–mediated

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Figure 8. 5-HTR stimulation induces IL-6 production in (A, D ) A549, (B, E ) BEAS-2B cells, and (C, F ) AEC-II. Cells were stimulated with the indicated concentration of 5-HT (A–C) or 10⫺4 M of indicated 5-HTR agonists (D–F). Supernatants were collected after 24 h, and IL-6 content was determined by ELISA. Data are expressed as mean ⫾ SEM (n ⫽ 5). (G ) Cells were stimulated in absence or present of 10⫺4 M 5-HT for 24 h. Total RNA was isolated from A549. IL-6 mRNA expression was quantified as described in MATERIALS AND METHODS. Number of transcripts is normalized to the number of copies of GAPDH. Data are means ⫾ SEM (n ⫽ 3).

ion mobilization from the intracellular stores, and 5-HTR3– mediated Ca2⫹ influx through the plasma membrane. To get insight into the physiologic significance of 5-HT in epithelial cells, cytokine secretion was analyzed. We found that stimulation of 5-HTR1A, 5-HTR1B, 5-HTR1E/F, 5-HTR2, 5-HTR3, 5-HTR4, and 5-HTR7 mediated the release of IL-6 and CXCL8/ IL-8 in A549, BEAS-2B, and AEC-II, but not of MIG, IP-10, I-TAC, and PGE2. Recently, a connection between 5-HT and CXCL8/IL-8 and IL-6 has been described in human dendritic cells (4). Moreover, mRNA analyses suggested that 5-HT effects IL-6 and CXCL8/IL-8 secretion by a similar mechanism, involving transcriptional regulation. These findings are interesting as on the one side, 5-HT stimulates the secretion of IL-6 and IL-8, while it does not influence the secretion of MIG, IP-10, or I-TAC, which are well-known TH1 mediators. Several studies have confirmed the pathophysiologic role of IL-6 and IL-8 in asthma. In this context, it has been shown that allergen challenge in humans causes a CXCL8/IL8–mediated increase of neutrophils in the lung (37). Moreover,

IL-6 plays a key role in “airway remodeling,” which represents another classical pathophysiologic feature of the asthmatic lung. In this case, IL-6 seems to be responsible for the subepithelial fibrosis as well as the dyscrinie, since the regulation of the mucin genes expression is mediated through an IL-6–dependent autocrine/paracrine loop (38). Therefore, it can be hypothesized that in patients with asthma, 5-HT activates epithelial cells leading to a release of IL-6 and IL-8, which could cause a TH2-dominated reaction. Enhanced secretion of IL-6 may then exacerbate the pro-asthmatic changes due to mucus hypersecretion. Furthermore, CXCL8/IL-8 and IL-6 concentration in BAL fluids from patients with asthma is significantly increased compared with that of healthy subjects (38–40). Since CXL8/IL-8 is a chemotaxin for eosinophils of atopic individuals, one can suggest the involvement of 5-HT on IL-8 mediated recruitment of eosinophils and thus in the development and maintenance of allergic diseases (41). Taken together, our study shows that 5-HT stimulates different Ca2⫹-signaling pathways in epithelial cells. Moreover, this


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study implicates control of cytokine and chemokine release in human airways epithelial cells by 5-HTR with linkage to the intracellular Ca2⫹ homeostasis.

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Conflict of Interest Statement : None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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