Lipoxin A4 Stimulates a Cytosolic Ca2+ Increase in Human Bronchial Epithelium*

Caroline Bonnans, Brigitte Mainprice, Pascal Chanez, Jean Bousquet, and Valerie UrbachDagger

From the INSERM U454, Department of Respiratory Disease, Centre Hospitalier Universitaire Arnaud de Villeneuve, 34295 Montpellier Cedex 05, France

Received for publication, October 8, 2002, and in revised form, December 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lipoxins are biologically active eicosanoids possessing anti-inflammatory properties. Using a calcium imaging system we investigated the effect of lipoxin A4 (LXA4) on intracellular [Ca2+] ([Ca2+]i) of human bronchial epithelial cell. Exposure of the cells to LXA4 produced a dose-dependent increase in [Ca2+]i followed by a recovery to basal values in primary culture and in 16HBE14o- cells. The LXA4-induced [Ca2+]i increase was completely abolished after pre-treatment of the 16HBE14o- cells with pertussis toxin (G-protein inhibitor). The [Ca2+]i response was not affected by the removal of external [Ca2+] but completely inhibited by thapsigargin (Ca2+-ATPase inhibitor) treatment. Pre-treatment of the bronchial epithelial cells with either MDL hydrochloride (adenylate cyclase inhibitor) or (Rp)-cAMP (cAMP-dependent protein kinase inhibitor) inhibited the Ca2+ response to LXA4. However, the response was not affected by chelerytrine chloride (protein kinase C inhbitor) or montelukast (cysteinyl leukotriene receptor antagonist). The LXA4 receptor mRNA was detected, by RT-PCR, in primary culture of human bronchial epithelium and in immortalized 16HBE14o- cells. The functional consequences of the effect of LXA4 on intracellular [Ca2+]i have been investigated on Cl- secretion, measured using the short-circuit techniques on 16HBE14o- monolayers grown on permeable filters. LXA4 produced a sustained stimulation of the Cl- secretion by 16HBE14o- monolayers, which was inhibited by BAPTA-AM, a chelator of intracellular calcium. Taken together our results provided evidence for the stimulation of a [Ca2+]i increase by LXA4 through a mechanism involving its specific receptor and protein kinase A activation and resulting in a subsequent Ca2+-dependent Cl- secretion by human airway epithelial cells.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this paper, we investigated the effect of lipoxin A4 (LXA4)1 on intracellular [Ca2+] of airway epithelial cell. Lipoxins are biologically active eicosanoids, originally described to be produced by leukocytes possessing a 5-lipoxygenase activity, in the presence of 15-HETE (or 15-HPETE) (1, 2). Lipoxins also result from the conversion of LTA4 by 12-lipoxygenase activity in platelets and the 15-lipoxygenase activity in macrophages and epithelial cells (3-5). The natural isomer lipoxin A4 (5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid), which is synthesized at the inflammation site, have been shown to act as a potent anti-inflammatory mediator in different cellular systems, affecting leukocyte trafficking by modulating chemotaxis, adhesion, transmigration, and phagocytic clearance of apoptotic cells (6-9).

LXA4 has been measured in the bronchoalveolar and nasal lavage fluids of patients with respiratory diseases including asthma (4, 10). Levels of LXA4 are significantly higher in sputum of mild asthmatic patients compared with the levels measured in normal subjects or asthmatics with a severe form of the disease. Because LXA4 inhibited interleukin 8 release by peripheral blood mononuclear cells, the reduced levels of LXA4 in severe asthma might be associated with the persistence of airway inflammation (11). In addition, PMN cells from mild asthmatic patients are able to generate in vitro larger amounts of LXA4 as compared with PMN cells obtained from normal individuals (12). LXA4 causes contraction of pulmonary smooth muscle (13) although, in asthmatics, LXA4 antagonizes the broncho-constriction induced by leukotriene C4 (14). LXA4 stable analogues blocked both airway hyper-responsiveness and pulmonary inflammation in a murine model of asthma (15).

The airway epithelial barrier, besides its role in electrolyte and water balance of the body, also participates in inflammation as a target cell for inflammatory mediators and by secreting inflammatory cytokines. Intracellular processes such as Ca2+ or pHi signaling, protein kinase activities, and the airway neuro-humoral environment finely control the airway epithelial secretory function altered in respiratory diseases such as asthma and cystic fibrosis. However, the effect of LXA4 has not been investigated on the cellular process regulating the human airway epithelium secretory function, and its potential role in airway epithelium is not clear. Lipoxins, but particularly LXB4 rather than LXA4, inhibit the proliferation of the human bronchial epithelial cell line, A549 (16). In addition, the same cell line did not show appreciable levels of the LXA4 receptor (17).

Intracellular Ca2+ plays a central role in cellular functions including inflammatory processes, and interestingly LXA4 stimulates a [Ca2+]i increase in rainbow trout leukocytes (18), in human monocytes (19, 20), and in human neutrophils (21). However, the direct effect of LXA4 on [Ca2+]i has not been investigated in epithelial cells. Because [Ca2+]i is a main regulator of the secretory functions of the epithelium, the aim of our study was to investigate the effect of LXA4 on [Ca2+]i and Cl- secretion through airway epithelium. The present study is the first report of a rapid and transient [Ca2+]i increase induced by LXA4 in airway epithelial cells, the description of the cellular mechanisms generating the response, and its consequence on ionic secretion.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- The human bronchial epithelial 16HBE14o- cell line is derived from surface epithelium of mainstream, second-generation bronchi (22), forms polarized monolayers with intact tight junctions, and retains Cl- transport properties of freshly isolated surface airway epithelial cells (22). Cells were grown on a collagen/fibronectin coating, in Eagle's minimal essential medium (BioWhittaker), supplemented with 10% fetal calf serum, 1% penicillin, 1% streptomycin, and 1% L-glutamine, and incubated in a 37 °C, 5% CO2 atmosphere.

For primary culture of human bronchus epithelium, cells were obtained from surgical biopsies. The bronchial samples were taken from a normal area of bronchi removed from patients suffering from lung cancer with a normal lung function (after approval by the ethics committee). After excision, the bronchial tubes were washed and incubated overnight at 4 °C with 0.38 mg/ml hyaluronidase, 0.75 mg/ml collagenase, 1 mg/ml protease, and 0.3 mg/ml DNase in RPMI 1640 medium (Invitrogen) and then filtered through a 70-mm mesh nylon strainer. Pieces of epithelium retained on the strainer were washed with phosphate-buffered saline. After centrifugation, epithelial cells were re-suspended in small airway epithelium basal medium (Clonetics, BioWhittaker, San Diego, CA) supplemented with 0.5 µg/ml human recombinant epidermal growth factors, 7.5 mg/ml bovine pituitary extract, 0.5 mg/ml epinephrine, 10 mg/ml transferin, 5 mg/ml insulin, 0.1 µg/ml retinoic acid, 6.5 µg/ml triiodothyronine, 50 mg/ml gentamicin, 50 µg/ml amphotericin B, and 50 mg/ml bovine serum albumin- fatty acid-free.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction of LXA4 Receptor-- Total RNA was obtained by lysing airway epithelial cells using TRIzol reagent (Invitrogen) and phenol-chloroform extraction and isopropanol precipitation. After DNase treatment (Invitrogen), 1 µg of total RNA was reversed transcripted using a Moloney murine leukemia virus (M-MTV RT) reverse transcriptase (Invitrogen). A first round of PCR was performed using the sense primer 5'-CAC CAG GTG CTG CTG GCA AG-3' and the anti-sense primer 5'-AAT ATC CCT GAC CCC ATC CTC A-3' (Applied Biosystems, Foster City, CA), which were designed to amplify the human peripheral blood mononuclear cell LXA4 receptor coding region (1,095 bp) (17). In parallel, the hypoxanthine-guanine phosphoribosyltransferase gene (213 bp) was used as a housekeeping gene and amplified using the sense primer 5'-TGT AAT GAC CAG TCA ACA GGG-3' and antisense primer 5'-TGG CTT ATA TCC AAC ACT TCG-3' (23). PCRs were carried out with 2 µl of cDNA (28 cycles of 30 s at 94 °C, 45 s at 64 °C, and 80 s at 72 °C for LXA4 receptor amplification and 35 cycles of 30 s at 94 °C, 50 s at 52 °C, and 80 s at 72 °C for hypoxanthine-guanine phosphoribosyltransferase amplification) on a DNA Thermo Cycler (Thermojet; Eurogentec, Angers, France). A nested PCR was then initiated with 0.5 µl of the first-round PCR products. The primers used were as follows: 5'-TGC TTG GGG TCA CCT TTG TC-3' (forward) and 5'-TGA AGC AGA ATT GGC AGC CG-3' (reverse), corresponding to LXA4 receptor primers generating a 928-bp fragment (24). As described by Sodin-Semrl et al. (24), the second set of LXA4 receptor primers was chosen with minimal homology with other members of the formyl peptide receptor family and exclude random crossing with known genomic sequences. Samples were subjected to 28 cycles of 30 s at 94 °C, 45 s at 58 °C, and 80 s at 72 °C, followed by a final extension step at 72 °C for 5 min. PCR products were analyzed using electrophoresis in Tris-boric acid-EDTA buffer with ethidium bromide-stained (0.5 mM) 1% agarose gel. Bands were captured using an ultrasensitive photon counting imaging camera (Argus 100; Hamamatsu Photonics, Tokyo, Japan).

Calcium Imaging-- Intracellular [Ca2+] was determined in 16HBE14o- cells, in primary culture of human bronchial epithelial cells, or in A549 cells according to a method previously described (25). Because A549 cells do not form tight junctions, the calcium experiments were performed on isolated A549 cells. The 16HBE14o- cells and primary culture of human bronchial epithelial cells grown on permeable support reached confluency and their highest electrical resistance, on average, 4 days after plating. Therefore, we arbitrarily distinguished three types of cell preparations on glass: isolated cells, non-confluent monolayers (2 days after plating), and confluent monolayers (4 days after plating). For isolated cells the glass was treated with poly L-lysine and for monolayers with a fibronectin collagen solution. The cells were loaded with 5 µM the Ca2+-sensitive fluorescent probe fura-2 acetoxy-methyl ester for 30 min, in the dark, at room temperature (22 °C) and were then washed twice in HEPES-buffered Krebs-Hensleit solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4, 280-290 mOsmol). The coverslip covered with cell monolayer or isolated bronchial cells was mounted on the stage of an inverted microscope equipped for epi-fluorescence (TE-300, Nikon, Badhoeve Dorp, Netherlands). Calcium cellular imaging was performed using the Metafluor Imaging System (Universal Imaging Corporation). The cell preparation was excited alternatively at 340 and 380 nm using the Optoscan monochromator (Cairn Research Ltd, Kent, UK). The emission fluorescence produced after fura-2 excitation was filtered at 510 nm. The transmitted light image was detected using a Photometrics CoolSNAP-fx video camera (Roper Scientific, Evry, France) coupled to the microscope. The fluorescence obtained at each excitation wavelength (F340 and F380) depends upon the level of Ca2+ binding to fura-2, according to an in vivo calibration performed using a range of EGTA-buffered Ca2+ solutions of the fura-2-free acid. The [Ca2+]i was calculated automatically by a computer program using the Grynkiewicz equation (26).

Short-circuit Current Measurement-- For transepithelial transport study, the 16HBE14o- cells were grown on permeable filters (Costar Snapwell-Clear 3801, 12-mm diameter, 0.4 mm pore size). Monolayers of 1.2 cm2 exposed surface area were mounted in a horizontal Ussing chamber between 0 and 3 days after the electrical resistance reached a maximum value (800-1200 ohms) as previously described (27). To create a favorable electrochemical gradient for Cl- secretion, the basolateral side of the monolayer was bathed in HEPES-buffered Krebs-Hensleit solution, and the apical was bathed in a low Cl- solution (125 mM sodium gluconate, 15 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4, 280-290 mOsmol). The spontaneous trans-membrane potential was measured and clamped to 0 mV by application of a short-circuit current (Isc) using a voltage clamp model amplifier (EVC 4000, World Precision Instrument). Frequency of acquisition was every 10 s. Under these conditions the Isc is a measure of electrogenic transepithelial ion transfer. The output from the amplifier was digitized using a PowerLab system (Chart for Windows v4.0, ADInstrument).

Lipoxin-- The LXA4 used in this study was provided by Calbiochem. The absence of transformation into all-trans lipoxin isomers was verified using reverse phase-high pressure liquid chromatography analysis according to the methods previously described (12). Aliquots of LXA4 solution in ethanol were stored at -80 °C to avoid any damage of the molecule.

Data Analysis-- The mean [Ca2+]i variations correspond to the [Ca2+]i variations between the mean [Ca2+]i measured during the 2 min prior to exposure to LXA4 and the [Ca2+]i measured at the peak. In each experiment, the mean [Ca2+]i was obtained from all cells in the microscope field. Data are shown as the mean ± S.E. of n experiments. Measures of statistical significance were obtained using the Kruskal-Wallis non-parametric test or the Student's t test for paired data. A p value <0.05 was deemed to be significant. All statistical operations were performed using Excel software (Microsoft).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

LXA4 Effect on Intracellular [Ca2+]-- Exposure of human bronchial epithelial cells to LXA4 produced a large and transient increase in [Ca2+]i (Fig. 1, A and B). In primary culture of human bronchial (PCHB) epithelial non-confluent cell monolayer (2 days after plating), apical LXA4 (10-8 M) produced an increase in [Ca2+]i of 526 ± 59 nM (n = 3, p < 0.001) from a basal value of 129 ± 35 nM (n = 3). In 16HBE14o- non-confluent cell monolayers or isolated cells exposure to LXA4 produced a [Ca2+]i increase of 407 ± 57 nM (n = 6, p < 0.001) from a basal value of 135 ± 30 nM (n = 6) and an increase of 510 ± 50 nM (n = 7, p < 0.001) from a basal value of 135 ± 50 nM (n = 7), respectively. In 16HBE14o- monolayers having reached confluency for more than 4 days, apical exposure to LXA4 did not affect [Ca2+]i. In A549-isolated cells, LXA4 did not affect the [Ca2+]i. However, as a positive control we verified that a further exposure of A549 cells to apical ATP (10-4 M), known to stimulate a [Ca2+]i increase, produced the expected response (Fig. 1C). The dose dependence of the Ca2+ response to LXA4 in 16HBE14o- cells is shown in Fig. 1D. The larger increase in [Ca2+]i was obtained using LXA4 at 0.1 µM. One nM of LXA4 did not produce any detectable change in [Ca2+]i. Ethanol used as a LXA4 solvent did not affect [Ca2+]i.


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Fig. 1.   LXA4 effect on [Ca2+]i. Typical intracellular [Ca2+]i recording upon exposure to LXA4 (10-8 M) in non-confluent PCHB monolayers (A), in non-confluent 16HBE14o- monolayers (B), and in A549-isolated cells (C) loaded with fura-2 acetoxy-methyl ester (5 µM). D, effect of increasing doses of LXA4 on [Ca2+]i variations in non-confluent 16HBE14o- cells (D). For each LXA4 concentration, 4 independent experiments have been performed. LXA4 10-9 M and ethanol used as the LXA4 solvent did not produce any significant [Ca2+]i changes. All the other LXA4 concentrations used produced a significant [Ca2+]i increase. (**, p < 0.01; *, p < 0.05, and NS, non-significant, p > 0.05).

Calcium Source for LXA4 Response-- To identify the source of the [Ca2+]i increase induced by LXA4 we investigated the effect of thapsigargin, a Ca2+-ATPase pump inhibitor, and removal of external Ca2+ on 16HBE14o- non-confluent cell monolayers. As shown in Fig. 2A, thapsigargin (1 µM) produced a slow [Ca2+]i increase by 165 ± 47 (n = 5) followed by a return to a plateau value. After 15 min of thapsigargin treatment, the effect of LXA4 (10-8 M) on [Ca2+]i was completely abolished. After thapsigargin, exposure of the 16HBE14o- cells to LXA4 (10-8 M) did not produce any significant change in [Ca2+]i (Delta [Ca2+]i = 2.5 ± 5 nM, n = 5, p > 0.1) (Fig. 2, A and C). Exposure of 16HBE14o- cells to external Ca2+-free medium did not affect the response to LXA4. In Ca2+-free solution (0 mM CaCl2 and 5 mM EGTA), LXA4 (10-8 M) produced a [Ca2+]i increase by 348 ± 105 nM (n = 5, p < 0.001), which was not significantly different from the LXA4-induced Ca2+ increase produced in control conditions (p > 0.05, Fig. 2, B and C). Taken together, these results indicate that the response of human bronchial epithelial cells to LXA4 mainly involvs the Ca2+ release from thapsigargin-sensitive intracellular stores rather than Ca2+ entry from the extracellular compartment.


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Fig. 2.   Calcium source of the LXA4 effect. Intracellular [Ca2+]i recording upon exposure to LXA4 (10-8 M) in non-confluent 16HBE14o- monolayers after thapsigargin (1 µM) treatment (A) or after removal of Ca2+ (+ 5 mM EGTA) from the external bathing solution (B). C, statistical representation of the [Ca2+]i changes upon the two experimental protocols described above. (**, p < 0.01 and NS, p > 0.05).

Effect of (Rp)-cAMP, MDL Hydrochloride, and Chelerythrine Chloride-- The role of either protein kinase A or protein kinase C signaling pathways in the mediation of the response have been investigated using (Rp)-cAMP (inhibitor of protein kinase A activity), MDL hydrochloride (inhibitor of adenylate cyclase), and chelerytrine chloride (inhibitor of protein kinase C activity) on 16HBE14o- non-confluent cell monolayers. Neither (Rp)-cAMP (200 µM), MDL hydrochloride (50 µM), nor chelerythrine chloride (10-7 M) significantly affected the basal level of [Ca2+]i (Delta [Ca2+]i = 6 ± 8 nM, n = 4; Delta [Ca2+]i = -5.6 ± 6.1 nM, n = 3; Delta [Ca2+]i = -1.4 ± 4 nM, n = 3, respectively). As shown in Fig. 3, A, B, and D, (Rp)-cAMP (200 µM) and MDL (50 µM), largely antagonized the response to 10-8 M of LXA4 (Delta [Ca2+]i = 26 ± 26 nM, n = 4; Delta [Ca2+]i = -142 ± 13 nM, n = 3, respectively). After chelerythrine chloride (10-7 M), the Ca2+i response to LXA4 (10-8 M) was not significantly different from control conditions (Delta [Ca2+]i = 495 ± 21 nM, n = 3, p > 0.05) (Fig. 3, C and D).


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Fig. 3.   (Rp)-cAMP, MDL hydrochloride, and chelerythrine chloride effects. A, intracellular [Ca2+]i recording upon exposure to LXA4 in non-confluent 16HBE14o- monolayers during (Rp)-cAMP (200 µM) treatment. MDL hydrochloride (50 µM) treatment (B) or chelerythrine chloride (10-7 M) exposure (C). D, statistical representation of the [Ca2+]i changes upon the three experimental protocols described above.

Effects of Pertussis Toxin and Montelukast-- Because A549 cells do not express detectable levels of LXA4 receptor, the absence of the effect of LXA4 on [Ca2+]i in A549 cells suggested that in 16HBE14o- cells the Ca2+ response to LXA4 is mediated by the LXA4 receptor. Because the LXA4 receptor have been identified to be coupled to pertussis toxin-sensitive G-protein, we tested the effect of the toxin on the Ca2+ response induced by LXA4 in 16HBE14o--isolated cells. As shown in Fig. 4, A and C, in 16HBE14o- cells pertussis toxin treatment completely abolished the response to LXA4 at either 10-8 or 10-7 M. LXA4 produced a non-significant [Ca2+]i variation of -11 ± 15 nM (n = 5, p > 0.1) at 10-8 M and of -2.5 ± 3 nM (n = 5, p > 0.1) at 10-7 M. We also investigated the eventual role of the cysteinyl leukotriene receptor, which can be recognized by LXA4 and is also sensitive to pertussis toxin in the [Ca2+]i response to LXA4. As shown in Fig. 4, B and C, montelukast (10-6 M) used as a specific leukotriene receptor antagonist did not affect the Ca2+ response to LXA4 (10-8 M).


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Fig. 4.   Pertussis toxin and montelukast effects. Intracellular [Ca2+]i recording upon exposure to LXA4 of non-confluent 16HBE14o- monolayers treated during 1 h with 2.5 µg/ml of pertussis toxin (A) or after montelukast (10-6 M) treatment (B). C, statistical representation of the [Ca2+]i changes during LXA4 (10-8 M) exposure in control conditions, after pertussis toxin or montelukast treatment as described above. (**, p < 0.01 and NS, p > 0.05).

LXA4 Receptor mRNA-- The LXA4 receptor mRNA expression was investigated in three different human bronchial epithelial cells (the PCHB, the 16HBE14o-, and the A549 cells) by RT-PCR using specific primers for LXA4 receptor cDNA. Expression of the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase was determined to provide an internal control for RT and PCR efficiencies. Peripheral blood mononuclear cells, from which the LXA4 receptor has been cloned, were used as positive control (20). Representative electrophoretic analysis of the PCR products obtained is shown in Fig. 5. LXA4 receptor mRNA expression was not detected after a first round of RT-PCR amplification of any of the A549, 16HBE14o-, and PCHB cell mRNAs. Detection of LXA4 receptor transcripts in human bronchial epithelial cells required the use of nested PCR, as shown in the representative electrophoretic analysis in Fig. 5. We demonstrated that 16HBE14o- and PCHB expressed LXA4 receptor mRNA, whereas A549 cells did not.


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Fig. 5.   LXA4 receptor mRNA expression in human bronchial epithelial cells. RT-PCR amplification of a 1095-bp fragment from 16HBE14o-, A549 and PCHB cells was performed using specific primers for LXA4 receptor cDNA. Peripheral blood mononuclear cells were used as positive control. After 28 cycles of PCR amplification (top), 0.5 µl of the resulting PCR product were used for a nested PCR step (28 cycles) using a second set of internal LXA4 receptor primers amplifying a 928-bp fragment (middle PCR). PCR products were resolved in 1% agarose gel.

Effect of LXA4 on Transepithelial Ion Transport-- Because intracellular [Ca2+]i is a major regulator of epithelial secretory function we investigated a possible role for LXA4 on a Ca2+-dependent Cl- secretion. LXA4 (10-8 M) applied on the basolateral side of the monolayer produced a sustained increase by 6 ± 1 µA/cm2, (p < 0.01, n = 6) of the transepithelial short-circuit current (Isc). The Isc increase started between 0 and 5 min after the beginning of LXA4 exposure and reached a plateau value within 10 min (Fig. 6A). In absence of external Cl- ions on both side of the epithelium, the response to LXA4 was completely abolished, indicating that the LXA4 effect on the Isc was mainly due to Cl- secretion (Fig. 6B). The role of intracellular [Ca2+]i on the Isc increase was investigated using the intracellular calcium chelator, the BAPTA-AM. As shown in Fig. 6C, BAPTA-AM (10 µM) produced a low decrease (-0.9 ± 0.2 µA/cm2, n = 3) of the initial Isc, indicating a weak contribution of intracellular [Ca2+] to the basal trans-epithelial ion transport. However, after 60 min of treatment with BAPTA-AM, the LXA4 effect on short-circuit current was completely inhibited. These results indicated that the LXA4-induced short-circuit current increase is due to the stimulation of a Ca2+-dependent secretory pathway.


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Fig. 6.   LXA4 effect on short-circuit current. Basolateral effect of LXA4 on the transepithelial short-circuit current of 16HBE14o- grown on a permeable filter. The short-circuit current was measured in control condition (basolateral Krebs-Hensleit solution and apical low chloride solution) (A) or in Cl--free solution (B) or in presence of BAPTA-AM (10 µM) on both sides of the monolayer.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The present study is the first description of the rapid [Ca2+]i increase induced by LXA4 in human airway epithelial cells including the description of the cellular mechanism involved in the response. The stimulation of a [Ca2+]i increase by LXA4 has been reported in rainbow trout leukocytes (18), in human monocytes (19, 20), and in human neutrophils (21). In airway epithelial cell, identification of the intracellular calcium stores as the main source of the LXA4-induced [Ca2+]i increase is consistent with the previous report of an intracellular calcium mobilization induced by LXA4 in other cell types (19, 20). In addition, the murine LXA4 receptor recently identified and cloned was also shown to induce the production of inositol triphosphate, a cellular event related to Ca2+ mobilization (28). There is no previous report of a [Ca2+]i increase induced by LXA4 in epithelial cells. In T84 cells, an intestinal epithelial cell line, LXA4 inhibited the transmigration of PMN by decreasing the Ca2+ response to the pathogen-elicited epithelial chemo-attractant, but the direct effect of LXA4 on [Ca2+]i was not reported (29).

The [Ca2+]i response to LXA4 was dose-dependent with a maximal response measured at 10-7 M, which is consistent with the [Ca2+]i response to LXA4 in other cell types. A maximal effect was obtained at 10-6 M in human neutrophils and human monocytes and at 10-8 M in rainbow leukocytes (18-21). In human monocytes the [Ca2+]i response was mediated by stimulation of the LXA4 receptor (20).

The LXA4 receptor has been cloned in human leukocytes (30) and in intestinal epithelial cell lines (17). We found mRNA expression of the LXA4 receptor in the 16HBE14o- cell line and in human bronchial epithelial cells in culture, but not in A549 cells. The detection of the LXA4 receptor mRNA in human airway epithelial cells is consistent with the detection of LXA4 receptor cDNA in murine lung (28). Our results are in accordance with the previous publication (31) reporting that the human bronchial epithelial cell line A549 did not show appreciable levels of LXA4 receptor cDNA, which we interpreted as a cell line characteristic rather than a tissue specificity of the LXA4 receptor expression. Because there is not detectable LXA4 receptor expression in A549 cells, we considered this cell line as a negative control. Therefore, the relation between the LXA4 receptor mRNA detection and the [Ca2+]i response strongly suggests that the LXA4 receptor was involved in the Ca2+ response. Moreover, because the LXA4 receptor activation has been shown to be associated to pertusssis toxin-sensitive G-protein, the sensitivity of the LXA4-induced [Ca2+]i response to pertussis toxin is also consistent with a role for LXA4 receptor in the response. It has been reported that in rat glomerular mesengial cells, LXA4 binds the LTD4 receptor, which is also coupled to a G-protein (32). However, we have shown that the LTD4 antagonist montelukast did not affect the [Ca2+]i response to LXA4 in airway epithelium, which indicates that the [Ca2+]i response to LXA4 is not mediated by the LTD4 receptor (34). This last result constitutes further evidence for the involvement of the LXA4 receptor in the Ca2+ response to LXA4 in airway epithelial cell.

Because LXA4 is a lipophylic molecule, the side of epithelium exposure to LXA4 should not affect the response. However, the absence of Ca2+ response to apical exposure to LXA4 of highly confluent 165HBE14o- monolayers compared with the response obtained in isolated cells or non-confluent cell monolayers could be explained by the lack of accessibility of the extracellular side of the receptor. This would suggest a basolateral location of the LXA4 receptor. In addition this observation is consistent with the recent report (35) of a preferential lateral location of the LXA4 receptor in intestinal epithelial cells. Therefore, we tested the modulation of the Isc by LXA4 applied on the basolateral side.

Because the role of LXA4 in protein kinase C activation have been largely reported, we investigated the involvement of protein kinase C activity in the generation of the Ca2+ response (36-39). The ineffectiveness of chelerythrine chloride, a protein kinase C inhibitor, in the Ca2+ response to LXA4 suggests that protein kinase C activity is not involved in the generation of the [Ca2+]i increase. However, the stimulation of protein kinase C activity by LXA4 might involve an initial Ca2+ increase, as described in the present study. The role of protein kinase A activity has also been investigated. The inhibitory effect of (Rp)-cAMP and MDL hydrochloride suggested that, in airway epithelium, the Ca2+ response to LXA4 involved stimulation of adenylate cyclase and protein kinase A activities. In contrast, the LXA4 effect on actin rearrangement in monocytes and macrophages is mimicked by the protein kinase A inhibitor and inhibited by the cell permeant cAMP analogue 8-Br-cAMP (40).

Intracellular [Ca2+] plays a central role in epithelial cellular function such as ion or protein secretion. The LXA4-induced [Ca2+]i response might be involved in eicosanoid production by epithelium, which is known to depend on [Ca2+]i (41-43). More specifically, a [Ca2+]i increase is necessary for lipoxygenase activity in the production LXA4 from its substrate (12, 42, 43). Therefore, the LXA4-induced [Ca2+]i increase might play a central role in an autocrine production of LXA4 and constitute an amplificatory mechanism for the biosynthesis of LXA4 itself.

In addition, our results suggested that the Cl- secretion induced by LXA4 is one of the functional consequences of the LXA4-induced [Ca2+]i increase. This would indicate that, in airway epithelial cells, beside its anti-inflammatory role LXA4 is involved in ionic transport regulation. This might have important clinical implications, in particular in the treatment of respiratory diseases associated with ion transport dysfunction, and necessitating anti-inflammatory treatment such as cystic fibrosis and asthma.

    ACKNOWLEDGEMENTS

We thank D.C. Gruenert (Gene Therapy Core Center, University of California, San Francisco, CA) for the gift of the 16HBE14o- cell line.

    FOOTNOTES

* This work was funded by The Wellcome Trust (055695/Z/98/Z/CH/TG/JF), Convention Industrielle de Formation par la Recherche research Grant 99679, the Fondation Institut de France, and the Regional Council of Languedoc, Roussillon, France.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: INSERM U454, CHU Arnaud de Villeneuve, 34295 Montpellier Cedex 05, France. Tel.: 33-4-67-33-59-31; Fax: 33-4-67-63-28-55; E-mail: urbach@montp.inserm.fr.

Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M210294200

    ABBREVIATIONS

The abbreviations used are: LX, lipoxin; LT, leukotriene; PMN, polymorphonuclear leukocytes; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; RT, reverse transcriptase; PCHB, primary culture of human bronchial epithelium; MDL, MDL-12, 330A, hydrochloride; BAPTA-AM, bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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