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Published ahead of print on August 18, 2005, doi:10.1165/rcmb.2005-0181OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 601-609, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0181OC

The P2Y14 Receptor of Airway Epithelial Cells

Coupling to Intracellular Ca2+ and IL-8 Secretion

Tobias Müller, Hans Bayer, Daniel Myrtek, Davide Ferrari, Stephan Sorichter, Manfred W. Ziegenhagen, Gernot Zissel, J. Christian Virchow, Jr., Werner Luttmann, Johannes Norgauer, Francesco Di Virgilio and Marco Idzko

Department of Pneumology, University of Freiburg; Department of Pneumology, University of Rostock; Department of Dermatology, University of Jena; Wilhelm-Anton-Hospital, Goch, Germany; and Department of Experimental and Diagnostic Medicine, Section of General Pathology and Interdisciplinary Center for the Study of Inflammation (ICSI), University of Ferrara, Italy

Correspondence and requests for reprints should be addressed to Dr. Marco Idzko, Department of Pneumology, University Medical Clinic, University of Freiburg, D-79104 Freiburg i. Br., Germany. E-mail: idzko{at}med1.ukl.uni-freiburg.de


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Uridine nucleotides and UDP-glucose are endogenous molecules, which are released into the extracellular environment in a lytic manner after cell damage, as well as by regulated nonlytic mechanisms. Recently, a UDP-glucose–specific Gi protein–coupled P2Y receptor, namely P2Y14, has been cloned. In this study, we demonstrated expression of the P2Y14 mRNA in human lung epithelial cells and in the epithelial cell lines A549 and BEAS-2B. Evidence of functional expression of the P2Y14 receptor in these cell lines was provided by calcium measurements after stimulation with uridine 5'-diphosphoglucose (UDP-glc). Experiments with pertussis toxin and the Ca2+-chelator EGTA revealed participation of pertussis toxin–sensitive Gi/o-proteins in the mobilization of Ca2+-ions from intracellular stores by UDP-glc. Moreover, UDP-glc increased secretion of the potent neutrophil chemoattractant CXCL8/IL-8 in A549 and BEAS-2B cells in a pertussis toxin–sensitive manner. Moreover, reverse transcription and quantitative polymerase chain reaction revealed that UDP-glc modulated mRNA levels of IL-8/CXCL8. However, stimulation of A549 and BEAS-2B cells with UDP-glc neither modified basal nor cytokine-induced secretion of the CXC-chemokines CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. In addition, UDP-glc did not affect proliferation of the two cell lines. In summary, our data provide evidence for a distinct physiologic role of P2Y14 in the selective release of specific chemokines from human airway epithelial cells.

Key Words: airway • epithelial cells • P2Y14 • purinoceptors • UDP-glucose


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Endogenous nucleotides such as ATP and UTP are released into the extracellular space from mechanically stressed endothelial and epithelial cells, specialized compartments of nerve terminals, activated platelets, as well as lipopolysaccharide (LPS)-stimulated monocytes. In epithelial cells nucleotides such as ATP and UTP participate in cell volume control through Cl– and fluid secretion (1, 2), ciliary beat (3), mucociliary clearance (4, 5), and intracellular Ca2+ oscillations (6, 7). Moreover, nucleotides regulate cell proliferation (8) and coordinate intercellular communication between distant airway cells (9). The effects of nucleotides are mediated through interaction with two subfamilies of plasma membrane receptors named P2 receptors; the metabotropic G protein–coupled P2Y receptors and P2X receptors which are ligand-gated ion channels. Cloned human P2Y receptor subtypes include at least eight members (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) (10–12). Extensive pharmacologic studies with transfected cells revealed that P2Y1, P2Y11, P2Y12, and P2Y13 receptors selectively interacted with adenosine triphosphate (ATP) and/or adenosine diphosphate (ADP) (11, 13–15), whereas the P2Y2, P2Y4, and P2Y6 subtypes were responsive to uridine nucleotides (16–18). Moreover, detailed analyses showed that the P2Y2 receptor was activated equipotently by ATP and uridine triphosphate (UTP). UTP and uridine diphosphate (UDP) are selective agonists for the P2Y4 and P2Y6 receptor, respectively (16, 17, 19). In addition, it has been shown that the P2Y14 receptor specifically responded to UDP-Glc and related sugar-nucleotides, but and not to ATP, ADP, UTP, or UDP. Furthermore, UDP-glc does not bind to any other of the P2Y-receptor subtypes, apart from the pertussis toxin–sensitive Gi protein–coupled P2Y14 receptor (12, 20–22). Recently, expression of functional P2Y14 in dendritic cells has been revealed by UDP-glc–induced calcium transients and modulation of the expression of costimulatory molecules (22).

The alveolar surface area of the lung is covered with alveolar epithelial type I and type II cells. Type I cells function as a physical barrier and play a major role in gas exchange, whereas type II cells produce pulmonary surfactant and behave as progenitor cells to replace injured alveolar epithelial type I cells (23–26). Thus, located at the boundary between the alveolar airspace and the interstitium, alveolar epithelial type II cells (AEC-II) are ideally situated to regulate the recruitment and activation of different types of leukocytes by producing chemokines/cytokines in response to inflammatory stimuli from the alveolar space. Recent studies suggested that AEC-II secreted a variety of mediators, including proinflammatory cytokines and chemokines important for the recruitment of leukocytes into the lung interstitium and alveolar space (27–29). It has been demonstrated that primary AEC-II are important regulators of the immune function in the lung, since they express costimulatory molecules necessary for T cell activation (30). Moreover, they express NOS3 and release the CXC-chemokine CXCL8/IL-8 in response to inflammatory stimuli (31).

The cell line A549 is generally thought to resemble AEC-II (32). BEAS-2B are transformed bronchial epithelial cells, which are widely used to explore functional properties of bronchial epithelial cells (29, 33). Calcium transients are known as important intracellular signals induced by cell surface receptors in various cell types (34, 35). In alveolar epithelial cells, calcium transients influence the release of surfactant proteins and represent a way for cell-to-cell communication (36–38). The present study was undertaken to investigate the biological effects of UDP-glc in human airway epithelial cell lines. We showed that UDP-glc induced, in a pertussis toxin–sensitive manner, mobilization of intracellular Ca2+, as well as modulated the release of the proinflammatory chemokine IL-8.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Materials
A549 and BEAS-2B cells were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). Uridine 5'-diphosphoglucose (UDP-glc) was purchased from ICN Biomedicals (Aurora, ). Human recombinant IFN-{gamma} was obtained from Immuntools (Friesoythe, Germany). ELISA kits for human CXCL8/IL-8 (IL-8), CXCL9/MIG (MIG), CXCL10/IP-10 (IP-10), and CXCL11/I-TAC (I-TAC) were purchased from R&D Systems (Minneapolis, MN). Pertussis toxin was from Sigma (Deisenhofen, Germany). Dispase was from Roche (Penzberg, Germany). Buffer for AEC-II isolation (BI) contained 7.95 g/liter NaCl, 0.40 g/liter KCl, 1.11 g/liter glucose, 0.46 g/liter Na2HPO4, and 2.60 g/liter HEPES and were sterilized by filtration through a 0.22-µm filter. Buffer II (BII) is based on BI with additional 0.28 g/liter CaCl2 and 0.32 g/liter MgSO4.

Cell Culture and Stimulation
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 (39). BEAS-2B cells were cultured in the same medium (40). To favor cell adhesion, flasks were coated with a solution containing 30 µg/ml rat collagen S, 1 mg/ml human fibronectin, and 100 µg/ml BSA in MEM-medium (41). Both cell lines were grown in culture flasks. For subculture trypsin/EDTA-solution (Biochrom) was used to remove adherent cells. For subsequent experiments cells were seeded into 24-well tissue culture plates (Corning Inc., Corning, NY), at a density of 0.20 x 106 cells/well (A549) or 0.10 x 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 ELISA.

Isolation of AEC-II
Isolation of AEC-II was performed as previously described (42). Briefly, lung tissue samples were obtained from subjects with lung cancer undergoing lobectomy. 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 µm. 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% fetal calf serum and 1% penicillin/streptomycin and seeded in a density of 0.2 x 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.

Analysis of mRNA Expression
Total RNA was extracted from the cells using Trizol-Reagent (Gibco) as described by the manufacturer. To obtain cDNA, 5 µg of total RNA were primed with oligo-dT primers (Hermann GbR, Freiburg, Germany) and reverse transcribed with StrataScript reverse transcriptase (Stratagene, La Jolla, CA). Primers for the human P2Y14 receptor were designed, based on published sequence data (accession no.: NM_014879): forward 5'-TCA TTG CGG GAA TCC TAC TC-3' and reverse 5'-CCC AAA GAA CAC AAT GCT GAC-3' (PCR product, 242 bp). PCR was performed using 10 µl of iQ-Supermix (Bio-Rad, Hercules, CA), 7 µl H2O, 1 µl of each primer (final concentration 0.5 µM each), and 1 µl 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 2.5% agarose gel. PCR products were cloned and sequenced to prove their identity.

Quantification by Real-Time PCR
Total RNA was extracted using the RNeasy kit according to the manufacturer's protocol (Qiagen, Hilden, Germany). Briefly DNase I (Invitrogen, Carlsbad, CA) treatment, 1 µg of total RNA from each sample was used as template for the reverse transcription reaction. Fifty microliters of cDNA were synthesized using M-MLV reverse transcriptase and pd(N)6 primers (GIBCO BRL, Gaithersburg, MD). All samples were reverse transcribed under the same conditions and from the same reverse transcription master mix to minimize differences in reverse transcription efficiency. All oligonucleotide primers for real-time PCR were designed using Primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA, http://frodo.wi.mit.edu/cgi-bin/primer3/primer3www.cgi) and synthesized by Invitrogen.

For iCycler reaction, a master mix of the following compounds was prepared to the indicated end-concentration: 10 µl SYBR Green master mix (Bio-Rad, Herculas, CA), 6 µl water, and 1 µl sense and 1 µl antisense primers (500 nM). This master mix (18 µl) was filled in the iCycler strips and 2 µl cDNA (0.625, 2.5, 10, or 40 ng reverse transcribed total RNA) was added as PCR template. The following iCycler experimental run protocol was used: denaturation (95°C for 9 min), with 40 cycles of amplification and quantification (95°C for 30 s, 60°C for 30 s, 72°C for 30 s) with a melting curve program (60–95°C with a heating rate of 0.1°C per second). Emitted fluorescence for each reaction was measured during the extension phase. Real-time PCR efficiency (E) was calculated from the given slopes, with the iCylcer software, as previously described (43). The cycle threshold (CT)—that is, the cycle number at which the amount of the amplified gene reaches threshold fluorescence—was determined by using the iCycler software. The relative expression ratio (R) of the different target genes was calculated based on efficiency (E) and cycle threshold (CT), deviation of an unknown sample versus a control, and compared with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Intracellular Ca2+ Measurements
Ca2+ transients were measured in A549 and BEAS-2B cells loaded with the Ca2+ indicator fura-2/AM (Calbiochem) by using the digital fluorescence microscope unit Attofluor (Zeiss, Oberkochen, Germany) (34). Briefly, A549 and BEAS-2B cells were incubated with 2 µM 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 1.5 mM 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 505 nm. Curves shown are representative of the whole cell population.

Cytokine Assays
CXCL8/IL-8 (IL-8), CXCL9/MIG (MIG), CXCL10/IP-10 (IP-10), and CXCL11/I-TAC (I-TAC) were measured by ELISA (R&D Systems) and performed according to the manufacturer's recommendations. Samples were assayed in triplicate for each condition.

Determination of Cell Proliferation
Cell proliferation was measured by BrdU-incorporation during DNA-synthesis detected with an anti–BrdU-POD antibody (Roche) (8, 44). Briefly, cells were seeded at a density of 2.5 x 103/well in MEM-medium containing 0.5% fetal calf serum gold. After 24 h, cells were incubated with different concentrations of UDP-glc. After the indicated time period of incubation (1–72 h), 20 µl of BrdU-labeling solution was added to each well and cells were labeled for 16 h. Thereafter, cells were washed three times with buffer and fixed with 70% ethanol. To improve the accessibility of DNA to the detection antibody, cells were incubated with 1.5 N of hydrochloric acid for 20 min at room temperature. Plates were washed again and incubated with peroxidase-conjugated anti-BrdU antibodies. Unbound antibodies were washed off the samples and 100 µl of the peroxidase substrate (tetra-methyl-benzidine solution) were added. The reaction was stopped using sulphuric acid and the optic density (OD) was quantified using an ELISA-reader (µquant; Bio-Tek Instruments, Bad Friedrichshall, Germany). Proliferation was determined by measuring the OD of BrdU-labeled cellular DNA, in each well. Proliferation of stimulated cells is expressed as stimulation index (SI = ODtreated cells/ODbaseline) indicating a multiple of the baseline proliferation. Each experiment was run in eight independent wells.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Human Airway Epithelial Cells Express the mRNA for the P2Y14 Receptor
Expression of the P2Y14 receptor mRNA in human airway epithelial cells was analyzed using RT-PCR. Figure 1 shows that A549, BEAS-2B, and AEC-II express the mRNA for the P2Y14 subtype. In each cell type we yielded the expected product of 242 bp. Cloning and sequencing confirmed the identity of the PCR products. No products were obtained omitting reverse transcription. In addition, relative mRNA quantification by real-time PCR was performed, to investigate whether P2Y14 receptor mRNA expression in human airway epithelial cells can be modulated by inflammatory cytokines like TNF-{alpha}, IFN-{gamma}, or a combination of both. However, these experiments showed that expression of P2Y14 mRNA was not influenced by these agents (data not shown).



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  		Figure 1. A549, BEAS-2B, and human airway epithelial cells express the mRNA for the P2Y14 receptor. RT-PCR analysis was performed with mRNA isolated from A549, BEAS-2B, and purified AEC-II cells (see MATERIALS AND METHODS). For each kind of cell we performed control experiments omitting reverse transcription (negative), leading to no PCR products. One representative experiment of four is shown (n = 4).

 
Effect of UDP-glc on the Intracellular Free Ca2+ Concentration of A549 and BEAS-2B Cells
It has been reported previously that the P2Y14 receptor is involved in the regulation of intracellular Ca2+ homeostasis (22). Stimulation with UDP-glc (as indicated by arrows) of A549 (Figure 2A) or BEAS-2B cells (Figure 2B) induced a rapid and concentration-dependent Ca2+ increase. In both cell lines, half maximum and maximum effects were observed at an UDP-glc concentration of 10–6 M and 10–4 M, respectively. Compared with 10–4 M no significant changes were seen after stimulation with 10–3 M UDP-glc in A549 (1.16 ± 0.03 versus 1.18 ± 0.05, means ± SEM; n = 4, P > 0.05) or BEAS-2B cells (1.18 ± 0.03 versus 1.21 ± 0.05, means ± SEM; n = 4, P > 0.05). Preincubation of the cells with apyrase (4 U/ml) did not affect the UDP-glc induced Ca2+ transient in A549 and BEAS-2B, suggesting that the observed effect was not due to other nucleotides like ATP, ADP, or UTP (data not shown).






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  		Figure 2. UDP-glc triggers Ca2+ transients A549 and BEAS-2B cells. A549 (A) or BEAS-2B cells (B) were loaded with the Ca2+ indicator fura-2/AM and stimulated with the indicated concentration of UDP-glc. Traces of single cell measurements were averaged and representative traces are shown. Experiments were repeated four times with similar results. A549 (C) or BEAS-2B (D) were labeled with fura-2/AM and stimulated in the absence (solid lines) or presence (dashed lines) of 4 mmol/ml EGTA with 10–4 M UDP-glc. Representative data of one experiment out of three are shown.

 
Intracellular Ca2+ transients are either caused by Ca2+ influx from the extracellular milieu or by mobilization from intracellular stores (35). Preincubation of A549 (Figure 2C) and BEAS-2B (Figure 2D) cells with 4 mM of EGTA, a known Ca2+ chelator, did not significantly affect the UDP-glc response during the observed time period. Therefore these findings show that UDP-glc at early time points induced mobilization of Ca2+ from intracellular stores. To investigate involvement of Gi/o-proteins in UDP-glc response, A549 (Figure 3A) and BEAS-2B cells (Figure 3B), were incubated for 2 h with pertussis toxin. As assessed by Trypan blue exclusion, no changes in cell viability of were seen upon pertussis toxin treatment. Pertussis toxin catalyzes ADP-ribosylation of heterotrimeric Gi/o-proteins thereby leading to their inactivation. As shown in Figure 3, pertussis toxin pretreatment completely inhibited Ca2+-transients evoked by UDP-glc.




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  		Figure 3. Pertussis toxin inhibits Ca2+ transients induced by UDP-glc in A549 and BEAS-2B cells. A549 (A) and BEAS-2B (B) were incubated with or without 50 ng/ml pertussis toxin for 4 h, loaded with fura-2/AM, and then stimulated with 10–4 M UDP-glc. Ca2+ transients were analyzed, and the ratio after stimulation for 10 s is given. Data are means ± SEM (n = 3).

 
UDP-glc Induces IL-8 Secretion from AEC-II, A549, and BEAS-2B Cells
IL-8 is an important chemotactic factor for different leukocyte subtypes (45, 46). Stimulation of A549 (Figure 4A), BEAS-2B (Figure 4B), and AEC-II (Figure 4C) with the P2Y14 agonist UDP-glc enhanced the production of IL-8 in a concentration-dependent manner. The highest release of IL-8 was seen at an UDP-glc concentration of 10–3 M, whereas a half maximal response was observed at an UDP-glc concentration of 10–5 M. In addition, relative mRNA quantification by real-time PCR indicated that UDP-glc enhanced IL-8 mRNA levels in A549 (Figure 4D).






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  		Figure 4. UDP-glc increases IL-8 protein release and mRNA expression in A549, BEAS-2B cells, and AEC-II. A549 (A), BEAS-2B cells (B), and AEC-II (C) were stimulated with the indicated concentration of UDP-glc. Supernatants were collected after 24 h, and IL-8 content determined by ELISA. Data are expressed as mean ± SEM (n = 5). Total RNA was isolated from A549 (1 x 106) stimulated in absence or present of 10–3 M UDP-glc for 24 h (D). IL-8 mRNA expression was quantified as described in MATERIALS AND METHODS. Number of transcripts is normalized to the number of copies of GAPDH ones. Data are means ± SEM (n = 3).

 
To investigate whether Gi/o-proteins are also involved in UDP-glc–induced IL-8 production, A549 (Figure 5A) and BEAS-2B cells (Figure 5B) were incubated for 4 h with pertussis toxin. Pertussis toxin pretreatment completely inhibited IL-8 secretion evoked by UDP-glc.




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  		Figure 5. Pertussis toxin inhibits UDP-glc induced IL-8 release in A549 and BEAS-2B cells. A549 (A) and BEAS-2B cells (B) were incubated with or without 50 ng/ml pertussis toxin for 4 h, and then stimulated with the indicated concentration of UDP-glc. Supernatants were collected after 24 h, and IL-8 content determined by ELISA. Data are expressed as mean ± SEM (n = 3).

 
UDP-glc Does Not Modify Production of the Chemokines IP-10, MIG, and I-TAC
The respiratory epithelium is known to be an important source for the CXC-chemokines IP-10, MIG, and I-TAC, preferably attracting Th1-lymphocytes. As described previously, no basal secretion of these chemokines was seen but they were inducible by IFN-{gamma}/TNF-{alpha} (100 IU/ml) in A549 and BEAS-2B cells (29). Here we studied whether secretion of IP-10, MIG, and I-TAC is influenced by stimulation with UDP-glc in A549 and BEAS-2B cells. However, UDP-glc neither induced secretion of these chemokines (data not shown) nor influenced TNF-{alpha}/IFN-{gamma}–induced release of IP-10, MIG, or I-TAC in A549 (Figure 6A) and BEAS-2B cells (Figure 6B). To exclude unresponsiveness of cells primed with 100 IU/ml IFN-{gamma}/TNF-{alpha}, we performed experiments with cells only primed with IFN-{gamma} and then stimulated increasing concentration of TNF-{alpha} or UDP-glc. Again, UDP-glc had no influence on the secretion of MIG, I-TAC, or IP-10, whereas TNF-{alpha} increased production of these chemokines in IFN-{gamma}–pretreated A549 (Figures 6C–6E) and BEAS-2B (data not shown).







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  		Figure 6. UDP-glc does not influence secretion of IP-10, MIG, or I-TAC in A549 or BEAS-2B cells. A549 (A) and BEAS-2B cells (B), were stimulated with a combination of TNF-{alpha}/IFN-{gamma} (100 IU each) and the indicated concentration of UDP-glc. Supernatants were collected after 24 h, and MIG, ITAC, and IP-10 content determined by ELISA. Data are expressed as mean ± SEM (n = 4). A549 were pretreated with 100 IU IFN-{gamma} and stimulated with the increasing concentrations of TNF-{alpha} or 10–3 M UDP-glc. Supernatants were collected after 24 h, and MIG (C), ITAC (D), and IP-10 (E) content determined by ELISA. Data are expressed as mean ± SEM (n = 3).

 
UDP-glc Does Not Increase the Proliferation Rate of A549 and BEAS-2B Cells
It has been reported that ATP and UTP can stimulate proliferation of A549 and BEAS-2B cells (8). Here we show that UDP-glc did not increase BrdU uptake and DNA synthesis of both A549 (Figure 7A) and BEAS-2B cells (Figure 7B) at any concentration and time point tested. Here, high concentrations of FCS were used as positive controls (Figure 7).








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  		Figure 7. UDP-glc does not increase the proliferation rate of A549 or BEAS-2B cells. A549 (A) or BEAS-2B (B) cells were stimulated with different concentrations or for different time periods with UDP-glc. One out of three independent experiment is shown. Data are means ± SD (n = 8).

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Previous reports have shown that human airway epithelial cells express uridine nucleotide receptors (8, 47). In epithelial cells, nucleotides regulate the cell volume through Cl– and fluid secretion (1, 2), ciliary beat (3), and mucociliary clearance (4, 5). Moreover, UTP or UDP stimulated proliferation of both A549 and BEAS-2B cells (8, 47). Recently, a novel Gi protein–coupled purinoreceptor, P2Y14, has been cloned and characterized. Reconstitution experiments revealed that P2Y14 specifically responds to UDP-glucose (UDP-Glc) and related sugar-nucleotides (12), but does not interfere with ATP, ADP, UTP, or UDP. Expression of functional P2Y14 receptor expression has been revealed by UDP-glc–induced calcium transients and modulatory effects on the expression of costimulatory molecules in monocyte-derived dendritic cells (22). Since expression and function of this receptor in lung biology is not known, we performed experiments in primary human airway epithelial cells and derived cell lines.

In this study we showed expression of P2Y14 receptor mRNA in A549, BEAS-2B, and AEC-II cells. These findings are surprising, since a previous study could not detect P2Y14-specific mRNA in lung tissue (12). However, the presence of the mRNA for the P2Y14 subtype in primary AEC-II demonstrates that expression of this mRNA by A549 or BEAS-2B cells was not just due to neoplastic or viral transformation. Moreover, measurement of intracellular Ca2+ transients induced by the P2Y14 agonist UDP-glc implicate functional expression of this receptor subtype. The Ca2+ response was not influenced by the absence of external Ca2+ (i.e., incubation of cells in a Ca2+-free, EGTA-containing solution), but abrogated by pertussis toxin. Therefore, these findings implicate that the P2Y14 receptor at early time points regulates mobilization of Ca2+ from intracellular stores in both A549 and BEAS-2B cells. Pertussis toxin ADP-ribosylates Gi/o-proteins (48). This post-translational modification interrupts coupling of the modified Gi/o-proteins with seven membrane-spanning serpentine receptors. Based on these observations it can be assumed that in airway epithelial cells the P2Y14 receptor activates Gi/o-proteins, which in turn activate phospholipase C. This enzyme cleaves phosphatidylinositol-4,5-bisphosphate into diacylglycerol and inositoltrisphosphate causing Ca2+ mobilization from intracellular stores. Similar conclusions were drawn on P2Y14 receptor signaling in immature human dendritic cells (22).

Recently constitutive release of UDP-glc has been detected from multiple cellular sources, including highly differentiated polarized airway epithelial cells and tumor cells (49, 50). The detected extracellular concentrations were in similar (nanomolar) range to the well established extracellular signaling molecules ATP and UTP (50). However, it is known that after mechanical stress the release of nucleotides like ATP or UTP via lytic and nonlytic pathways is elevated (51). Thus it is possible that tissue concentration of UDP-glc is much higher after cell activation. Purinoceptors are known to influence proliferation rate of different cell types (52, 53). It has been demonstrated that ATP and UTP stimulate BrdU incorporation into DNA of A549 and BEAS-2B cells via activation of P2Y2 and P2Y6 receptors (8). However, here performed cell studies provide no evidence for proliferation-promoting activity of UDP-glc in these cells.

The airway epithelium is also a well-established source of various proinflammatory molecules, including chemokines (29, 31, 41). Several studies have shown that allergen challenge in humans as well as in animal models causes a CXCL8-mediated increase of neutrophils in the lung (54). It has been reported that extracellular nucleotides like ATP regulate secretion of IL-8 and IP-10 in dendritic cells or astrocytes (55, 56). Here we show that UDP-glc increased secretion of IL-8 in A549, BEAS-2B cells and primary AEC-II. Since UDP-glc did not influence release of chemoattractants for Th1-cells such as IP-10, MIG, and I-TAC in A549 and BEAS-2B, this activity seems to be specific for CXCL8. Therefore, one might assume that UDP-glc modulates expression of a rather distinct proinflammatory activity profile in airway epithelial cells.

In summary, here we provide evidence for the expression of the Gi/o protein–coupled P2Y14-receptor in human airway epithelial cells. We also showed that UDP-glc stimulates Ca2+-transients and release of proinflammatory chemokine IL-8. These data further support to the hypothesis that extracellular nucleotides play an important role in activation of airway epithelial cells and regulation of immune responses.


   Footnotes
 
Originally Published in Press as DOI: 10.1165/rcmb.2005-0181OC on August 18, 2005

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.

Received in original form May 13, 2005

Accepted in final form July 22, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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