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Regulation of Human Eosinophil Migration Across Lung Epithelial Monolayers by Distinct Calcium Signaling Mechanisms in the Two Cell Types¹

Department of Medical Sciences, Uppsala University, Uppsala, Sweden

	Abstract

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In asthmatic patients, eosinophils massively infiltrate the lung tissues and migrate through lung epithelium into the airways. The regulatory mechanisms involved are obscure. We studied the role of calcium in the migration of human eosinophils across monolayers of human lung epithelial H292 cell line cells induced by combined chemotactic solutions of platelet-activating factor and C5a. The transepithelial migration of eosinophils was attenuated by depletion of the external Ca²⁺ in the migration system, whereas the eosinophil migration itself was unaffected as evidenced by measuring eosinophil chemotaxis in the Boyden chamber in the absence of epithelial cells. Buffering of intracellular Ca²⁺ in eosinophils with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) inhibited both eosinophil transepithelial migration and eosinophil chemotaxis in the Boyden chamber, suggesting the importance of intracellular Ca²⁺ in eosinophil transmigration. Although loading of BAPTA/AM or addition of thapsigargin to the epithelial cells effectively changed their cytoplasmic free Ca²⁺ concentrations, neither of these treatments affected transepithelial migration of eosinophils. Interestingly, addition of La³⁺ (0.2 mM) to epithelial cells suppressed eosinophil transmigration whereas addition of La³⁺ to eosinophils did not. Taken together, these results show the importance of Ca²⁺ in eosinophil migration across lung epithelium and support a distinctive regulatory role of intracellular and extracellular Ca²⁺ for the two cell types involved in this process; i.e., the transmigration of human eosinophils across a monolayer of lung epithelial cells is regulated by the intracellular Ca²⁺ in eosinophils, whereas the ability of the lung epithelial cell monolayer to allow eosinophil passage is dependent on the extracellular Ca²⁺.

	Introduction

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Infiltration of eosinophils into the lung tissues is an important pathological feature of allergic asthmatic inflammation (1, 2). Eosinophils can be recovered from the bronchial alveolar lavage fluid in asthmatic patients, indicating that eosinophils move through lung vascular endothelium and extracellular matrix and migrate across lung epithelium into the airways (2, 3). During their migration process, eosinophils must interact with different adhesion molecules presented by other cell types and extracellular matrix, with soluble mediators such as cytokines and chemoattractants, and respond to the local signals that regulate and guide them into the inflammatory locus (3, 4). The mechanisms of the migration of these eosinophils across pulmonary endothelial and epithelial cellular layers in vivo are complex and remain incompletely understood.

Lung epithelium plays an important role in leukocyte infiltration into the inflammation sites (5, 6, 7). Lung epithelial cells are active effector cells in the initiation of inflammation through the expression of adhesion molecules (6, 7, 8, 9, 10) and the synthesis of a number of proinflammatory mediators including platelet-activating factor (PAF)³ and eotaxin (11, 12, 13, 14, 15). It has also been shown that the C5a is produced and remains biologically active as part of the inflammatory response of the lungs of asthmatic subjects (16). In vitro, we recently showed that PAF combined with another chemotaxin such as C5a induced pronounced eosinophil migration across the lung epithelium (17). In this process, PAF is a primer and a chemoattractant to eosinophils and also functions as an epithelial cell activator. Nevertheless, the signaling and regulatory mechanisms of the migration of human eosinophils across lung epithelium remain to be elucidated.

Cellular responses are controlled by receptor activation and signal transduction events. Ionized calcium (Ca²⁺) is an important and universal intracellular signaling messenger involved in a variety of cellular responses (18, 19). In granulocytes in particular, Ca²⁺ transients were found to be coupled with cell motility responses such as detachment of cell surface integrins on neutrophils from their ligands (20, 21), polarization, and chemotaxis of eosinophils (22, 23, 24). However, other reports argued that no absolute requirement of intracellular calcium responses exists for leukocyte locomotion, as was shown in neutrophils (25) and eosinophils (26). In cellular interactions, neutrophil transmigration across endothelium was regulated by the cytosolic Ca²⁺ changes in endothelial cells (27). Therefore, it is tempting to ask the question of whether Ca²⁺ plays a role during the process of eosinophil transmigration across lung epithelium.

In this study, using the recently described in vitro transmigration model (17) with purified human eosinophils, monolayers of human lung H292 epithelial cells, and combined chemotactic solutions of 3PAF and C5a, we report the importance and regulatory role of Ca²⁺ during eosinophil migration across lung epithelium.

	Materials and Methods

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Reagents

L--Phosphatidylcholine, ß-acetyl--O-hexadecyl (PAF), recombinant human C5a, and thapsigargin were purchased from Sigma (St. Louis, MO). 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), fura-2/acetoxymethyl ester (fura-2/AM) were purchased from Calbiochem (La Jolla, CA). Recombinant human eotaxin was from Pepro Tech EC (London, U.K.). Lanthanum chloride was from BDH (Poole, U.K.). C5a and PAF were dissolved in PBS supplemented with 0.5 and 2% (w/v) human serum albumin (HSA, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands), respectively. Thapsigargin, BAPTA/AM, and fura-2/AM were dissolved in DMSO. LaCl₃ was dissolved in distilled water. All these reagents were stored at high concentrations at -20°C. From our preliminary experiments we found that the optimal concentrations of eosinophil chemotaxins in the transepithelial migration assay were: C5a 10 nM, PAF 1 µM, and eotaxin 10 nM. These concentrations were used in both the transmigration and chemotaxis assays. Cell culture medium and supplements used were purchased from Life Technologies (Paisley, U.K.). [³H]Inulin was purchased from Amersham (Little Chalfont, U.K.). HEPES medium used in this study contained 132 mM NaCl, 6.0 mM KCl, 1.0 mM MgSO₄, 1.2 mM KH₂PO₄, 20 mM HEPES, 5.5 mM glucose, and 0.5% (w/v) HSA (pH 7.40). HEPES + Ca²⁺ medium and HEPES Ca²⁺-free medium were also used (HEPES medium supplemented with 1.0 mM CaCl₂ or without Ca²⁺ addition but supplemented with 0.5 mM EGTA (Sigma), respectively). In experiments where La³⁺ was applied, modified HEPES + Ca²⁺ medium in which phosphates and sulfates were replaced by chlorides was used, instead of ordinary HEPES + Ca²⁺ medium. The chemotaxins used in La³⁺ experiments were prepared in modified HEPES medium without Ca²⁺ addition, because it is known that phosphates and sulfates bind La³⁺.

Epithelial cell culture

The human lung mucoepidermoid carcinoma H292 cell line cells (CRL-1848, American Type Culture Collection, Rockville, MD) (28, 29) were maintained in 25-cm² tissue culture flasks (Nunc, Roskilde, Denmark) in RPMI 1640 cell culture medium supplemented with 10% heat-inactivated FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), and 2 mM glutamine. With a split ratio of 1:6, the cells reach confluence in 3–4 days. After reaching confluence, the cells were trypsinized with 0.05% trypsin plus 0.02% EDTA (Life Technologies) and passaged. The 3rd through 30th passages of the cells reaching confluent monolayer in culture flasks were used for the transmigration and cytoplasmic free [Ca²⁺] measurement experiments.

For the transmigration assays, the epithelial cells were subcultured in an inverted position on polycarbonate membranes (3.0 µm pore size, 6.5 mm diameter) of Transwell cell culture inserts (C-93415, Corning Costar, Cambridge, MA) as described with slight modification (17, 30). Sterile polyoxymethylene polyacetal collars with an inner diameter equal to the outer diameter of the filters and with a height of 10 mm were tightly fixed to the bottoms of the inserts. Epithelial cells in aliquots were added to the inverted inserts (2 x 10⁵ cells in a volume of 200 µl for each insert) and allowed to attach at 37°C with 5% CO₂ and maximal humidity for 16 h. Thereafter, the collars were removed, the inserts were placed upright into 24-well culture dishes, and the cell culture was continued with fresh cell culture medium. In this way, "inverted" monolayers of epithelial cells hanging underneath the filters were obtained. The confluence of the epithelial cell monolayers was reached in 3 days before their use in transmigration experiments as determined by May-Grünwald/Giemsa staining and light microscopy. The confluence of the monolayers was also confirmed by [³H]inulin permeability experiments as described (31). In all experiments, the medium in both compartments was replaced with fresh culture medium 24 h before the transmigration experiment.

Eosinophil purification

Granulocytes were purified from a buffy coat of 450 ml blood obtained from healthy, nonallergic volunteers by density gradient centrifugation at room temperature over 67% isotonic Percoll (Pharmacia, Uppsala, Sweden) as described (32). After centrifugation, the pellet was handled on ice or at 4°C and with ice-cold buffers unless otherwise stated. The erythrocytes were removed by hypotonic lysis of the pellet, and the granulocytes were washed twice in PBS and resuspended in PBS with 2% heat-inactivated newborn calf serum (Life Technologies).

Human eosinophils were purified via removal of neutrophils (CD16 positive) according to a negative immunomagnetic selection technique (33). In brief, isolated granulocytes were incubated for 1 h at 4°C with anti-CD16 mAb-coated magnetic microbeads (at a proportion of 10⁷ granulocytes in 30 µl PBS with 2% newborn calf serum to 15 µl microbeads, Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were subsequently allowed to pass through a steel matrix column in a magnetic field. Thereafter, the eosinophils that passed through were collected, washed, and suspended in HEPES medium. The purity and the viability of the eosinophils were >96 and 99%, respectively.

Eosinophil transepithelial migration

In the transmigration experiments, HEPES + Ca²⁺ medium or modified HEPES + Ca²⁺ medium was used except for the experiments described in Fig. 2. Identical medium was used for both chemotaxins and eosinophils. The upper and lower compartments of the cell culture chamber inserts with confluent epithelial cell monolayers were washed twice with 37°C-prewarmed medium. The purified eosinophils were washed, resuspended, adjusted to 10⁶ cells/ml, and prewarmed at 37°C before being added into the upper compartment. The chemotactic solutions of PAF, C5a, or a combination (PAF/C5a) in the lower compartment were prewarmed to 37°C before the start of the assay. Volumes of the upper and lower chambers were 0.1 and 0.6 ml, respectively. The 24-well culture plates containing these inserts were then incubated at 37°C with 5% CO₂ and maximal humidity for 2 h. Kinetic measurements showed that in the presence of 1 mM Ca²⁺ the eosinophil transepithelial migration toward PAF/C5a reached its plateau after 1.5 h (data not shown). We thus performed the transmigration assay in a 2-h incubation time. After incubation, the cell suspensions from both chambers were collected separately. Volumes of 0.1 and 0.6 ml of HEPES + Ca²⁺ medium and sample buffer were used to wash upper and lower chambers, respectively, and were also collected. These suspensions and the membranes cut from the inserts were dissolved in sample buffer, containing 0.1% Tween 20, 0.2% N-cetyl-N,N,N-trimethylammonium bromide (Merck, Darmstadt, Germany), 0.2% BSA, and 15 mM EDTA (Merck) in PBS, and stored at -20°C for eosinophil cationic protein (ECP) quantification. After transmigration assay, the epithelial monolayers remained intact as confirmed by fixation, staining, and examination of the filter membranes under light microscopy as described (17, 30).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Effect of extracellular Ca2+ on eosinophil transmigration across H292 epithelial cell monolayers (A) and eosinophil migration in the Boyden chamber chemotaxis assay in the absence of epithelial cells (B). Data are means ± SEM of four independent experiments, each performed with duplicate monolayers/membranes. Both assays were conducted with eosinophils and/or epithelial monolayers in HEPES + Ca2+ medium () or in HEPES Ca2+-free medium () in the migration systems. Eosinophil migration in the Boyden chamber chemotaxis assay (B) toward PAF, C5a, or PAF/C5a without external Ca2+ in the medium did not differ significantly from that in HEPES + Ca2+ medium (p > 0.05). *, p < 0.05 compared with transmigration in HEPES + Ca2+ medium.

Samples from eosinophil transmigration assays were quantitated for ECP content using ECP fluoroimmunoassay via the Pharmacia CAP System, as described by the manufacturer (Pharmacia-Upjohn Diagnostics, Uppsala, Sweden). The percentage of eosinophil transmigration was calculated from the ECP content detected in the lower compartment compared with the total ECP added to the insert. The recovery of ECP content was > 90% for all experiments.

Eosinophil chemotaxis in a Boyden chamber assay

A modified Boyden chamber chemotaxis assay was performed using purified human eosinophils, Boyden microchambers, and 135-µm-thick Micropore filter membranes with 5 µm pore size (Millipore, Bedford, MA). The incubation was at 37°C with maximal humidity for 1 h. Eosinophil migration was measured according to the leading front technique as described (34).

Cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) measurement

For [Ca²⁺]_i measurement, H292 epithelial cells or purified eosinophils (10 x 10⁶ cells/ml in HEPES + Ca²⁺ medium) in suspension were loaded with 2 µM fura-2/AM for 40 min at 37°C in the dark under gentle agitation. In some experiments, BAPTA/AM was added to the cells after the cells had been incubated with fura-2/AM for 10 min, and the incubation was continued for another 30 min. The cells were then washed twice, resuspended in HEPES medium to the previous concentration, and kept in the dark at room temperature. Before being transferred to a cuvette (containing 2 x 10⁶ cells in 2 ml), the fura-2-loaded cells were diluted 10 times in HEPES + Ca²⁺ medium and prewarmed for 5 min at 37°C. The concentration of Ca²⁺ in cell suspension was adjusted to 1 mM. Fluorescence changes of the cells, magnetically stirred and kept at 37°C, were monitored with a dual-excitation wavelength spectrofluorometer (Model F-2000, Hitachi, Tokyo, Japan), with 340 and 380 nm as excitation wavelengths and emission at 490 nm. To calibrate the fura-2 fluorescence as a function of [Ca²⁺]_i, maximal fluorescence was achieved by adding Triton X-100 (0.05%) and minimal fluorescence by 10 mM EGTA/150 mM Tris (pH > 8.3). Absolute [Ca²⁺]_i was calculated according to the formula described by Grynkiewicz et al. (35) with 224 nM as the dissociation constant for Ca²⁺-fura-2 complex at 37°C.

Statistical analysis

Results were expressed as the mean ± SEM of the number of different experiments mentioned in the legends. Data were analyzed with a paired Student t test. Probability values were calculated, and p values less than 0.05 were considered significant.

	Results

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Role of extracellular Ca²⁺ in eosinophil transepithelial migration and chemotaxis

To investigate the role of extracellular Ca²⁺ in eosinophil transepithelial migration, we performed eosinophil transmigration assays in the presence and absence of Ca²⁺ in the medium. To eliminate Ca²⁺ from the transmigration system, we washed both freshly purified eosinophils and the Transwell inserts containing confluent monolayers of H292 lung epithelial cells with HEPES Ca²⁺-free medium (containing 0.5 mM EGTA). The control Transwell inserts and eosinophils were washed with HEPES + Ca²⁺ medium. Because Ca²⁺ is important for maintaining the integrity of epithelium (10, 36, 37), we examined the epithelial cell monolayers after they had been washed and incubated in HEPES Ca²⁺-free medium. After a 2-h incubation in HEPES Ca²⁺-free medium, the epithelial monolayers were still intact as judged by light microscopy, although 10–20% of the epithelial cells showed a slightly rounded and slightly shrunken appearance at the apical surface, which was not the case for monolayers kept in HEPES + Ca²⁺ medium. The basal side of the cells adhered well to the filter membrane. No hole was observed in the cell monolayers under these conditions. We also tested the permeability of the epithelial monolayers after removal of external Ca²⁺ in the transmigration system. As is shown in Fig. 1, without extracellular Ca²⁺ in the medium (in the presence of 0.5 mM EGTA), the permeability of the monolayers of the epithelial cells, as judged by [³H]inulin permeability measurement (31), did not change within a 2-h incubation, compared with that with 1 mM Ca²⁺ control medium. These results suggested that the epithelial cells were functioning normally in a short term incubation with HEPES Ca²⁺-free medium.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. [3H]Inulin permeability of monolayers of H292 epithelial cells. The effect of depletion of Ca2+ in the medium on the permeability of epithelial cell monolayers was monitored using HEPES + Ca2+ medium and HEPES Ca2+-free medium, with 10 mM EDTA in HEPES medium as a positive control. The epithelial monolayers were washed with respective media, and [3H]inulin was added to the upper chamber. Incubation was at 37°C with 5% CO2 and maximal humidity for 2 h with the identical medium in both upper and lower chambers. [3H]Inulin permeability was determined as described (31 ). Data are mean values of percentage leakage per hour of two independent experiments, each performed with duplicate monolayers. , buffer controls; , PAF/C5a combination.

Effect of buffering intracellular Ca²⁺ in eosinophils on transepithelial migration and chemotaxis

The inhibition of eosinophil transmigration across epithelial cells toward PAF and PAF/C5a by depletion of Ca²⁺ in the medium could have been the result of an effect on either the eosinophils or the epithelial cells. Because depletion of extracellular Ca²⁺ had no inhibitory effect on eosinophil chemotaxis toward PAF and/or C5a (Fig. 2B), we tested the role of eosinophil intracellular Ca²⁺ in the transmigration process using the intracellular Ca²⁺ chelator BAPTA/AM. By measuring [Ca²⁺]_i, we found that the intracellular Ca²⁺ responses of eosinophils preloaded with 25 µM BAPTA/AM to the addition of 1 µM PAF were completely abrogated (data not shown). Both the transmigration across H292 epithelial cell monolayers and chemotaxis in the absence of epithelial cells in the Boyden chamber of 25 µM BAPTA-loaded eosinophils toward combined solutions of PAF and C5a were reduced by 66.1 and 63.1%, respectively, compared with control eosinophils (Table I, p < 0.05). A similar inhibition was also observed in the eotaxin-induced transepithelial migration of 25 µM BAPTA-loaded eosinophils (data not shown). These results indicated the importance of eosinophil intracellular Ca²⁺ for the migratory responses of human eosinophils.

To investigate the role of intracellular Ca²⁺ of epithelial cells in the eosinophil transmigration process, we quantitated eosinophil transmigration across monolayers of epithelial cells loaded with different doses of BAPTA/AM. As is shown in Fig. 3, loading of epithelial cells with 1 to 25 µM BAPTA/AM did not change the eosinophil transmigration (p > 0.05). Eosinophil transepithelial migration still remained unchanged when the loading dose of BAPTA/AM was increased to 50 µM (data not shown). Because chelation of epithelial cell intracellular Ca²⁺ with BAPTA/AM had no effect on eosinophil transmigration, we investigated the effects of thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca²⁺-ATPase (38) that irreversibly depletes intracellular Ca²⁺ stores and thus elevates the [Ca²⁺]_i in cells, and of LaCl_3, an inorganic, nonspecific calcium channel blocker. Pretreatment of epithelial cell monolayers with thapsigargin (0.2 or 0.02 µM) for 5 min did not change the subsequent eosinophil transmigration (Fig. 4, p > 0.05). In a separate dose-response experiment with thapsigargin, pretreatment of epithelial cells with thapsigargin at concentrations ranging from 1 nM to 1 µM did not affect the subsequent eosinophil transmigration (data not shown). Addition of 0.2 mM La³⁺ to epithelial cells in the lower chamber potently inhibited eosinophil transmigration (Fig. 4, p < 0.01). Combined pretreatment of epithelial cells with 0.2 µM thapsigargin and 0.2 mM La³⁺ also resulted in potent inhibition of eosinophil transepithelial migration (p < 0.01), but there was no significant difference between the treatment with 0.2 mM La³⁺ alone and the combined treatment with 0.2 mM La³⁺ and 0.2 µM thapsigargin (Fig. 4, p > 0.05). To exclude a possible inhibitory effect of La³⁺ on eosinophils, we incubated eosinophils with 0.2 or 0.02 mM LaCl₃ at 37°C for 3–5 min and then applied these cells to the upper chamber in the transmigration assay with untreated epithelial cell monolayers. On addition of 0.2 or 0.02 mM La³⁺ and incubation, eosinophils did not form aggregates, and the transepithelial migration of these cells was not decreased (data not shown). Therefore, La³⁺ inhibits eosinophil transepithelial migration through an effect only on epithelial cells.

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Effect of buffering intracellular Ca2+ in H292 epithelial cells on eosinophil transepithelial migration. Epithelial cells in confluent monolayers were loaded with different concentrations (in µM) of BAPTA/AM in HEPES + Ca2+ medium for 30 min at 37°C with 5% CO2 and maximal humidity and were subsequently washed twice. Control epithelial cells were treated with same amount of DMSO. Purified human eosinophils were untreated. The transmigration assay was conducted in HEPES + Ca2+ medium. Data are means ± SEM of three independent experiments, each performed with duplicate monolayers. No significant difference was found between eosinophil transmigration across monolayers of control H292 cells and that of BAPTA-loaded cells (p > 0.05).

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Effect of treatment of epithelial cells with thapsigargin and La3+ on eosinophil transepithelial migration toward PAF/C5a. Epithelial cell monolayers were washed twice with medium and then incubated with thapsigargin (TG, 0.2 or 0.02 µM) or control medium for 5 min at 37°C and subsequently washed. Epithelial cell monolayers were then incubated with or without LaCl3 (La, 0.2 or 0.02 mM) in the lower compartment for 3–5 min at 37°C before the start of the transmigration assay. Modified HEPES + Ca2+ medium was used for washing and for incubation. La3+ was present in the lower compartment throughout the 2-h transmigration assay. Data are means ± SEM of four different experiments each performed with duplicate monolayers. **, p < 0.01 compared with PAF/C5a control.

Treatment of epithelial cells with BAPTA/AM or thapsigargin did not affect eosinophil transmigration across lung epithelial cell monolayers. This suggested that changes of [Ca²⁺]_i in epithelial cells may not be crucial for the eosinophil transmigration. To confirm that these treatments effectively changed the [Ca²⁺]_i in epithelial cells, we treated H292 epithelial cells in suspension under conditions similar to when these cells were treated with BAPTA/AM or thapsigargin in monolayers in the transmigration assay (Figs. 3 and 4). Loading epithelial cells in suspension with BAPTA/AM dose dependently decreased both the basal [Ca²⁺]_i and the elevation of [Ca²⁺]_i in these cells on addition of PAF (1 µM; Fig. 5). Elevation of [Ca²⁺]_i on PAF activation was completely or partially prevented when these cells were preloaded with 50 or 25 µM BAPTA/AM, respectively. As has been shown in many other cell types (38, 39, 40, 41, 42, 43), thapsigargin (0.2 µM) raised [Ca²⁺]_i in H292 lung epithelial cells and, after an initial increase in [Ca²⁺]_i, the [Ca²⁺]_i elevation sustained for >8 min (Fig. 6). After >5 min thapsigargin addition, LaCl₃ (0.2 mM) was added, and the elevated [Ca²⁺]_i was rapidly lowered to the basal level. Prior addition of LaCl₃ (0.2 mM) did not change either the basal [Ca²⁺]_i or the thapsigargin-induced initial increase of [Ca²⁺]_i in H292 epithelial cells, but it abrogated the subsequent sustained [Ca²⁺]_i elevation observed in Fig. 6 (data not shown). This suggested that La³⁺ itself does not have any activating effect on the [Ca²⁺]_i response in epithelial cells but that it effectively blocks the Ca²⁺ influx across the cell membrane. In addition, La³⁺ (0.2 mM) or elevation of external Ca²⁺ from 1 to 4 mM did not change the basal [Ca²⁺]_i in H292 epithelial cells.

View larger version (11K): [in this window] [in a new window]   FIGURE 5. Effect of buffering intracellular Ca2+ on [Ca2+]i changes of epithelial cells in suspension. H292 epithelial cells suspended in HEPES + Ca2+ medium were incubated with DMSO control (trace A) or loaded with BAPTA/AM (trace B, 25 µM; trace C, 50 µM) and subsequently washed twice. [Ca2+]i was measured as described in Materials and Methods. Arrow, PAF addition (1 µM). Curves show one representative experiment of four.

View larger version (10K): [in this window] [in a new window]   FIGURE 6. Changes in [Ca2+]i of epithelial cells in suspension after thapsigargin and subsequent LaCl3 addition. H292 epithelial cells were washed and suspended in modified HEPES + Ca2+ medium, and [Ca2+]i was measured as described in Materials and Methods. After thapsigargin (0.2 µM, first vertical arrow) was added for >5 min, LaCl3 addition (0.2 mM, second vertical arrow) was followed. A representative curve for five independent experiments is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Using this model, we determined the importance of external Ca²⁺ for eosinophil transepithelial migration. Our results showed that H292 epithelial monolayers maintained functionally intact in a short term incubation after depletion of Ca²⁺ from the transmigration system. This was indicated by testing the inulin permeability through H292 epithelial monolayers. Moreover, eosinophils displayed a decreased transepithelial migration toward chemotaxins and did not transmigrate toward medium control after Ca²⁺ depletion (Fig. 2A). The inhibition of eosinophil transepithelial migration by depletion of extracellular Ca²⁺ can be due to the changes of Ca²⁺ signaling in either epithelial cells or eosinophils.

We found in this study that eosinophil migration in both transepithelial migration assay and the Boyden chamber chemotaxis assay was inhibited by buffering intracellular Ca²⁺ in eosinophils with BAPTA/AM and that eosinophil migration in the Boyden chamber without epithelial cells was independent of extracellular Ca²⁺. Our results suggest that the appropriate basal [Ca²⁺]_i and [Ca²⁺]_i responses to chemoattractants are vital for eosinophil migratory responses across epithelial cells or in matrix and support the importance and a regulatory role of intracellular Ca²⁺ in eosinophil migration. In a recent study on chemotaxis of newt eosinophils (23), the intracellular Ca²⁺ changes were found pivotal to the polarization and locomotion responses of these cells, but newt eosinophil chemotaxis was inhibited by depletion of Ca²⁺ in the buffer. This latter finding is in contrast to the absence of a role of external Ca²⁺ found in our present eosinophil chemotaxis study. Obviously, newt and human eosinophils present very large species differences. In the newt eosinophil chemotaxis study, incubation with 5 mM EGTA could effectively interfere with the Ca²⁺ homeostasis in eosinophils (23). In our chemotaxis assay, the effect of extracellular Ca²⁺ depletion on intracellular Ca²⁺ homeostasis was kept minimal, and human eosinophils were incubated for only 1 h with 0.5 mM EGTA. Our results are consistent with the findings by Zigmond et al. and Schweizer et al. about the role of external Ca²⁺ in human neutrophil (25) and eosinophil (26) chemotaxis responses.

In contrast to the importance of intracellular Ca²⁺ in eosinophils, lung epithelial cells do not depend on intracellular Ca²⁺ changes to regulate eosinophil transmigration. Neither the buffering of intracellular Ca²⁺ in epithelial cells in monolayers with BAPTA/AM (Figs. 3 and 5), nor the elevation of [Ca²⁺]_i in epithelial cells by thapsigargin treatment (Figs. 4 and 6) had any effect on eosinophil transepithelial migration. Additionally, we observed that eotaxin at 10 nM was as effective as PAF and C5a in combination in inducing eosinophil transepithelial migration, yet H292 epithelial cells showed no measurable Ca²⁺ response to eotaxin (our unpublished data). These results suggest that a global change of [Ca²⁺]_i in epithelial cells is not crucial for the regulation of eosinophil transepithelial migration.

In this study, we applied LaCl₃ (0.2 mM) to either human eosinophils or H292 epithelial cells. La³⁺ inhibited eosinophil transepithelial migration only when La³⁺ was added to the epithelial cell monolayers. La³⁺ activates the calcium receptors expressed in parathyroid cells, including an elevation of [Ca²⁺]_i (44). However, such an action is unlikely in H292 cells because addition of La³⁺ (or of a high concentration Ca²⁺) per se does not cause any intracellular calcium signals. Thapsigargin depletes intracellular Ca²⁺ stores and induces Ca²⁺ influx over the plasma membrane in cells (18, 41, 42, 43). La³⁺ reversed thapsigargin-induced sustained elevation of [Ca²⁺]_i to basal level (Fig. 6). This indicates that La³⁺ blocks the Ca²⁺ influx across the plasma membrane. We observed that addition of different doses of verapamil, nifedipine or diltiazem to epithelial cells did not inhibit eosinophil transmigration (unpublished observations) indicating that L-type voltage-operated Ca²⁺ channels are not involved in eosinophil transepithelial migration. Based on our present data, it is not clear which type of Ca²⁺ channels in epithelial cells is responsible for the Ca²⁺ influx involved in eosinophil transmigration.

From our results, we conclude that lung epithelial cells depend on external Ca²⁺ for the changes to allow eosinophils to migrate through. The mechanisms of how lung epithelial cells sense extracellular Ca²⁺ and transmit the signal(s) into the cells to affect eosinophil transmigration are still unclear. Apparently, a proper external Ca²⁺ concentration supports the normal functions of epithelial cell adhesion molecules, such as cadherins and certain types of integrins (10, 36, 37). Which types of adhesion molecules are involved in eosinophil transepithelial migration is yet to be determined. Because La³⁺ is restricted to the extracellular space (45), it is possible that the results obtained in the transmigration assay with omission of extracellular Ca²⁺ and addition of La³⁺ to the epithelial cells are due to similar mechanisms. Accordingly, La³⁺ may bind to the Ca²⁺-binding sites of adhesion molecules and may also via these molecules affect the epithelial cytoskeleton system, thus inhibiting eosinophil penetration. Ca²⁺ influx plays an important role in integrin functions. In Madin-Darby canine kidney epithelial cells, integrin-mediated adhesion was shown to be predominantly regulated by Ca²⁺ influx instead of inositol 1,4,5-trisphosphate-mediated intracellular Ca²⁺ release (41, 46). It was suggested that plasma membrane proximal calcium influx is required for integrin-mediated adhesion (47). Lung epithelial cells express several types of integrins (10). It is thus possible that lung epithelial cells may rely on a proximal calcium influx over the plasma membrane for the functional changes of integrins to allow eosinophils to move through. Another possibility is that the interference of proximal Ca²⁺ influx on the functional changes of the epithelial cells for eosinophil transmigration is mediated via the local cytoskeleton system. The local cytoskeleton system involved in cellular adhesion and cell movement is closely linked with integrins and proximal to the plasma membrane (48, 49). When granulocytes migrate through the paracellular space of epithelial cell layers, the cellular junctions between epithelial cells that are coupled to the cytoskeleton network open and reseal reversibly (48, 50). One could thus speculate that the epithelial cell membrane proximal cytoskeleton system can be easily affected by a very localized Ca²⁺ signal.

We show here the importance and the regulatory role of Ca²⁺ for the migration of human eosinophils across a monolayer of lung epithelial cells. We demonstrate that a Ca²⁺ signal is utilized distinctively in the two types of cells involved in this integral transmigration process. Eosinophil transepithelial migration needs external Ca²⁺ in the surrounding environment, but the two cell types rely on different Ca²⁺ signaling mechanisms to regulate eosinophil migration across the epithelial cell monolayer. The intracellular Ca²⁺ in eosinophils is crucial whereas the epithelial cells rely on extracellular Ca²⁺, probably through a very localized Ca²⁺ signaling and/or a proximal Ca²⁺ influx across the plasma membrane, for the functional changes to allow eosinophil passage. The implication and importance of this distinctive regulation of calcium signals are unclear. This may be a mechanism for a very accurate regulation at different cellular levels for the eosinophil efflux to the airways, an important feature in vivo in asthmatic patients.

	Acknowledgments

 
We thank Lena Moberg and Kerstin Lindblad for skillful technical assistance and Drs. Rodolfo Garcia and Yi-Jia Liu for helpful discussions.

	Footnotes

 
¹ This study was supported by grants from the Swedish Medical Research Council, the Swedish Foundation for Health Care Sciences and Allergy Research, the Faculty of Medicine, Uppsala University, and Bror Hjerpstedts Stiftelse of Uppsala University Hospital.

² Address correspondence and reprint requests to Dr. Lixin Liu, Department of Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden. E-mail address:

³ Abbreviations used in this paper: PAF, platelet-activating factor; BAPTA/AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; HSA, human serum albumin; ECP, eosinophil cationic protein; [Ca²⁺]_i, cytoplasmic free Ca²⁺ concentration; fura-2/AM, fura-2/acetoxymethyl ester.

Received for publication June 29, 1999. Accepted for publication September 7, 1999.

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