ICAM-1-independent adhesion of neutrophils to phorbol ester-stimulated human airway epithelial cells

Alessandro Celi, Silvana Cianchetti, Stefano Petruzzelli, Stefano Carnevali, Filomena Baliva, and Carlo Giuntini

Laboratorio di Biologia Cellulare, Fisiopatologia Respiratoria, Dipartimento Cardiotoracico dell'Università di Pisa, 56124 Pisa, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intercellular adhesion molecule-1 (ICAM-1) is the only inducible adhesion receptor for neutrophils identified in bronchial epithelial cells. We stimulated human airway epithelial cells with various agonists to evaluate whether ICAM-1-independent adhesion mechanisms could be elicited. Phorbol 12-myristate 13-acetate (PMA) stimulation of cells of the alveolar cell line A549 caused a rapid, significant increase in neutrophil adhesion from 11 ± 3 to 49 ± 7% (SE). A significant increase from 17 ± 4 to 39 ± 6% was also observed for neutrophil adhesion to PMA-stimulated human bronchial epithelial cells in primary culture. Although ICAM-1 expression was upregulated by PMA at late time points, it was not affected at 10 min when neutrophil adhesion was already clearly enhanced. Antibodies to ICAM-1 had no effect on neutrophil adhesion. In contrast, antibodies to the leukocyte integrin beta -chain CD18 totally inhibited the adhesion of neutrophils to PMA-stimulated epithelial cells. These results demonstrate that PMA stimulation of human airway epithelial cells causes an increase in neutrophil adhesion that is not dependent on ICAM-1 upregulation.

intercellular adhesion molecule-1; phorbol 12-myristate 13-acetate; lung; inflammation; beta 2-integrins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AIRWAY INFLAMMATION is a hallmark of numerous lung diseases including chronic bronchitis (21), asthma (16), pulmonary fibrosis (17), and adult respiratory distress syndrome (9). The recruitment of circulating inflammatory cells into the airways involves both cell-cell adhesion molecules and soluble mediators. Several steps have been recognized in this process. First, circulating leukocytes adhere to the inflamed endothelium; then they migrate across the vessel wall, crawl within the interstitial space, and eventually adhere to and migrate across the airway epithelium (16).

Four superfamilies of adhesion molecules involved in the adhesion of leukocytes to endothelial cells have been identified: selectins, mucinlike selectin receptors, integrins, and immunoglobulin (Ig)-like proteins (4). Integrins are also responsible for the migration of cells within the interstitial space (16).

The molecular basis of leukocyte adhesion to epithelial cells has also been extensively investigated. A member of the Ig-like superfamily, intercellular adhesion molecule-1 (ICAM-1), originally described in endothelial cells (3), is the only inducible adhesion receptor for neutrophils identified so far that is also synthesized by airway epithelial cells. Recently, the synthesis of vascular cell adhesion molecule-1 (VCAM-1), an endothelial, inducible receptor for lymphocytes and eosinophils belonging to the Ig superfamily, has also been demonstrated in cultured, cytokine-stimulated bronchial epithelial cells (2). It is noteworthy, however, that current knowledge on the adhesion mechanisms expressed by the inflamed respiratory epithelium is largely based on studies performed with antibodies or molecular probes specific for previously known molecules, usually of endothelial origin. It is conceivable that the repertoire of adhesion mechanisms expressed by the respiratory epithelium is broader and that new, epithelium-specific molecules will be identified.

We stimulated human airway epithelial cells with different agonists and demonstrated that the phorbol ester phorbol 12-myristate 13-acetate (PMA) leads to increased neutrophil adhesion via a mechanism independent of ICAM-1 upregulation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 medium, RPMI 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, L-glutamine, trypsin, soybean trypsin inhibitor, trypan blue, formaldehyde, Ficoll-Hypaque, o-phenylenediamine (OPD), PMA, bovine serum albumin, insulin, ethanolamine, phosphorylethanolamine, transferrin, bovine pituitary extract, 3,3',5-triiodo-L-thyronine, epidermal growth factor, hexadecyltrimethylammonium bromide, dextran, anti-cytokeratin peptide-18 antibodies, FITC-conjugated anti-mouse IgG antibodies, cycloheximide, actinomycin D, and DNase were obtained from Sigma (Milan, Italy). Tumor necrosis factor-alpha (TNF-alpha ) was purchased from Genzyme (Cambridge, MA). Vitrogen 100 was from Collagen (Palo Alto, CA). LHC basal medium and trace elements were purchased from Biofluids (Rockville, MD). Hydrocortisone was from Richter Pharmaceuticals (Milan, Italy). All other chemicals were obtained from the hospital pharmacy and were of the best grade available.

Cells. The alveolar epithelial cell line of alveolar origin, A549, was obtained from American Type Culture Collection (ATCC; Manassas, VA). A549 cells were maintained in DMEM supplemented with 10% (vol/vol) FBS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin (complete DMEM) in a humidified 95% air-5% CO2 atmosphere at 37°C. The murine fibroblast cell line L929 was also obtained from ATCC and cultured in complete DMEM.

Human bronchial epithelial cells (HBECs) were obtained from subjects undergoing diagnostic bronchoscopy according to the method of Kelsen et al. (13) with some modifications. Briefly, after the patient's informed consent to the procedure was received, the fiber-optic bronchoscope was positioned at the level of the carina and/or the level of second- or third-order bronchial branchings. The use of local anesthetics was kept as low as possible to minimize their effects on cell viability. Four to six brushings of grossly normal bronchial mucosa were regularly obtained. The cells were then removed from the brush by vortexing in Ham's F-12 medium-10% FBS. The cells were brought to the laboratory in ice and incubated with DNase (50 g/ml) to eliminate clumping. After a wash with ice-cold, serum-free Ham's F-12 medium, the cells were resuspended in LHC-9-RPMI 1640 medium (1:1) (15) and plated on Vitrogen 100-coated 35-mm culture dishes (Becton Dickinson, Franklin Lakes, NJ). Indirect immunofluorescence with anti-cytokeratin antibodies confirmed the epithelial origin of the cells. The cells were then cultured and passed as appropriate according to Lechner and LaVeck (15) except that LHC-9-RPMI 1640 medium was used instead of LHC-9 medium. All the experiments were performed with cells at passages 4-9.

Tests to rule out the presence of mycoplasmal contamination were not routinely performed.

Neutrophils were isolated from the peripheral blood of normal volunteers. Blood was anticoagulated with 0.1 volume of 130 mM sodium citrate and sedimented on 2% (wt/vol) dextran. The leukocyte-rich fraction was then washed and layered on Ficoll-Hypaque. After centrifugation at 400 g for 30 min, the pellet was subjected to hypotonic lysis for 30 s to remove contaminating red blood cells and resuspended in Hanks' balanced salt solution (HBSS) with 1 mM CaCl2 and 1 mM MgCl2 unless otherwise indicated. The resulting cells were consistently >95% viable as assessed by trypan blue exclusion and >95% pure.

Antibodies. The hybridoma secreting the anti-CD18 monoclonal antibody (MAb) TS1/18 was obtained from ATCC and cultured in DMEM supplemented with 10% FBS and 50 µg/ml of gentamicin. The IgG fraction was affinity purified from the supernatant on a protein A-Sepharose column (Pierce, Rockford, IL) according to the manufacturer's directions. The inhibitory MAb to ICAM-1, R6.5 (20), supplied as pure IgG, was a generous gift from Dr. R. Rothlein (Boehringer Ingelheim, Ridgefield, CT). R6.5 binds to an epitope on domain 2 of ICAM-1 (8). Another inhibitory MAb to ICAM-1, clone 84H10, directed to an epitope on domain 1 (8) was purchased as purified IgG from Immunotech (Marseille, France). The generation and characterization of rabbit antiserum to P-selectin and of the MAb to the same protein, AC1.2, have been previously described (14). The antibodies to CD11a (TS1/22; originally from ATCC), CD11b (LPM19c; originally from Dr. Karen Pulford, University of Oxford, Oxford, UK), CD11c (HC1.1; originally from Dr. Carmelo Bernabeu, Centro de Investigationes Biologicas, Madrid, Spain), and ICAM-2 (B-37) were kindly provided as pure IgG by Dr. Virgilio Evangelista (Consorzio Mario Negri Sud, S. Maria Imbaro, Chieti, Italy) (12).

Adhesion assay. A549 cells, L929 cells, or HBECs were plated onto 96-well plates (Greiner, Nürtingen, Germany) at 5 × 104 cells/well. For HBECs, the wells were coated with Vitrogen 100. When confluent, the cells were washed with medium and stimulated with different agonists as indicated. After the appropriate time, the cells were extensively washed, and neutrophils were added at 105 cells/well. After 30 min at 37°C in a humidified 5% CO2 atmosphere, the wells were gently washed three times with HBSS containing Ca2+ and Mg2+ with a multiple pipetter to add and remove the buffer, taking great care to preserve the integrity of the monolayers. At the end of the assay, the plates were checked for monolayer integrity by phase-contrast microscopy, and the occasional wells showing any degree of monolayer disruption were removed from the analysis. When antibodies to neutrophil integrins were used, they were added to the cell suspension 30 min before the adhesion assay and maintained throughout the test. R6.5 and 84H10 were incubated with the epithelial cell monolayers for 30 min and washed three times before the assay. However, experiments in which R6.5 was maintained in the reaction mixture gave similar results. Antibodies to P-selectin and ICAM-2 were also maintained throughout the test. In experiments designed to evaluate the role of different metal ions, neutrophils were resuspended in HBSS containing either Ca2+ only or Mg2+ only, and the cell monolayers were washed throughout the test with HBSS containing the same divalent ion. In some experiments, neutrophils were resuspended in HBSS containing Mn2+. Adherent neutrophils were treated with 0.1% (wt/vol) hexadecyltrimethylammonium bromide in 0.1 M sodium acetate, pH 4, to solubilize endogenous peroxidase, and the chromogenic substrate OPD [1 mg/ml in 50 mM sodium citrate, pH 5, containing 0.15% (vol/vol) H2O2] was added. The reaction was stopped with 2 M H2SO4 (50 µl/well), and absorbance was read at 492 nm with a Titertek Multiskan MCC ELISA reader (Flow Laboratories, McLean, VA). The percentage of adherent neutrophils was calculated by comparison with a standard curve obtained by adding known numbers of neutrophils to wells that were then processed exactly as the others.

ELISA for cell-associated ICAM-1 and P-selectin. Epithelial cells were plated and stimulated as described in Adhesion assay. After an extensive wash, the cells were fixed with 3.7% (vol/vol) formaldehyde at room temperature for 15 min. Nonspecific binding sites on the solid phase were blocked with 2% (wt/vol) bovine serum albumin, and the relevant primary antibody (R6.5 at 5 µg/ml and AC1.2 ascites diluted 1:1,000 for ICAM-1 and P-selectin detection, respectively) was added. After 2 h at 37°C the wells were washed, and a peroxidase-conjugate anti-mouse IgG (Sigma) was added at the concentration indicated by the manufacturer. After a 2-h incubation at 37°C followed by an extensive wash, the chromogenic substrate OPD was added. The reaction was stopped with 2 M H2SO4, and absorbance read at 492 nm.

Data presentation and statistical analysis. Data are means ± SE unless otherwise indicated. Differences between groups were tested for significance by paired t-test. Significance was set at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PMA increases neutrophil adhesion to bronchial and alveolar epithelial cells. Under the experimental conditions employed, ~5-10% of neutrophils adhere to unstimulated A549 cells. Stimulation of A549 cells with PMA causes a significant increase in neutrophil adhesion (Fig. 1A). This effect is dose dependent and saturable, reaching a plateau at ~50 ng/ml of PMA (Fig. 1B). Figure 1C shows that the effect of PMA is rapid, being nearly maximal within 10 min. Although baseline adhesion of neutrophils to HBECs is slightly higher compared with that of A549 cells, PMA induces a further, significant increase in adhesion (Fig. 1D).


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Fig. 1.   Phorbol 12-myristate 13-acetate (PMA) stimulation of airway epithelial cells increases neutrophil adhesion. A: A549 cells were cultured in absence and presence of PMA (100 ng/ml). After 24 h, cells were extensively washed, and neutrophils were incubated for 30 min at 37°C. Adherent cells were quantitated as described in MATERIALS AND METHODS. Data are means ± SE of 7 consecutive experiments. * P < 0.05 compared with adhesion to unstimulated cells by paired t-test. B: dose-response curve of increase in neutrophil adhesion to PMA-stimulated A549 cells. A549 cells were incubated with increasing concentrations of PMA ([PMA]) for 24 h. Neutrophil adhesion was evaluated as described in MATERIALS AND METHODS. Data are from 1 experiment, representative of 3. C: kinetics of increase in neutrophil adhesion to PMA-stimulated A549 cells. A549 cells were incubated with PMA (100 ng/ml) and extensively washed at different time points before addition of neutrophils. 10' and 30', 10 and 30 min, respectively. Data are means ± SE of 3 consecutive experiments. D: human bronchial epithelial cells (HBECs) were cultured in absence and presence of PMA (100 ng/ml). After 24 h, cells were extensively washed and incubated with neutrophils for 30 min at 37°C. Adherent cells were quantitated as described in MATERIALS AND METHODS. Data are means ± SE of 6 consecutive experiments. * P < 0.05 compared with adhesion to unstimulated cells by paired t-test.

Control experiments were performed with the fibroblast cell line L929. Binding of neutrophils to L929 cells was not affected by PMA stimulation of the latter (41 ± 4 and 41 ± 7% for unstimulated and PMA-stimulated L929 cells, respectively). In contrast, PMA-stimulation of neutrophils caused a significant increase in binding (61 ± 7%; P < 0.05).

Role of known adhesion molecules. Airway epithelial cells of different origin express ICAM-1 on stimulation with various agonists (3, 20). We therefore investigated whether ICAM-1 was responsible for the increased adhesion induced by PMA. As expected (3), PMA upregulated ICAM-1 expression by A549 cells at 24 h as assessed by ELISA. However, ICAM-1 expression at 10 min was not affected by PMA (Fig. 2A). Furthermore, increased expression of ICAM-1 induced by TNF-alpha was not paralleled by increased neutrophil adhesion under the experimental conditions described (Fig. 2B). Similar results were obtained with interferon-gamma (data not shown). These data suggest that ICAM-1, although upregulated by PMA under appropriate conditions, is not responsible for the increased adhesion induced by PMA. To investigate further the role of ICAM-1 in this phenomenon, we used the inhibitory MAb to ICAM-1, R6.5. As shown in Fig. 3, R6.5 at saturating concentrations does not inhibit neutrophil adhesion to PMA-stimulated A549 cells (A) and HBECs (B). In control experiments, R6.5 at similar concentrations inhibited, as expected, neutrophil adhesion to interleukin-1-stimulated human umbilical vein endothelial cells (data not shown). To confirm the results obtained with R6.5, we repeated the experiments with another inhibitory anti-ICAM-1 antibody, 84H10. In a series of four experiments, binding of neutrophils to A549 cells was 10 ± 4 and 32 ± 9% for unstimulated and PMA-stimulated cells, respectively (P < 0.05). Binding to PMA-stimulated cells in the presence of 84H10 (20 µg/ml) was 41 ± 13%.


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Fig. 2.   A: intercellular adhesion molecule (ICAM)-1 expression by A549 cells cultured under different conditions. A549 cells were stimulated with PMA (100 ng/ml) or tumor necrosis factor (TNF)-alpha (500 U/ml). ICAM-1 expression was assessed at indicated time points by ELISA and is expressed as absorbance at 492 nm (A492). * P < 0.05 compared with expression by unstimulated cells by paired t-test. B: neutrophil adhesion to A549 cells under different conditions. A549 cells were stimulated as in A, and neutrophil adhesion was assessed as described in MATERIALS AND METHODS. Data are means ± SE of 3 consecutive experiments. * P < 0.05 compared with adhesion to unstimulated cells by paired t-test.



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Fig. 3.   Effect of anti-ICAM-1 antibody (R6.5) on neutrophil adhesion to PMA-stimulated airway epithelial cells. A549 cells (A) and HBECs (B) were stimulated for 24 h with PMA (100 ng/ml) and preincubated for 30 min with inhibitory anti-ICAM-1 antibody R6.5 (30 µg/ml) before adhesion assay. Data are means ± SE of 3 consecutive experiments. * P < 0.05 compared with adhesion to unstimulated cells by paired t-test.

P-selectin is a member of the selectin family of cell-cell adhesion molecules stored in the alpha -granules of platelets and the Weibel-Palade bodies of endothelial cells. On cell stimulation, P-selectin is rapidly translocated to the cell membrane where it functions as a receptor for leukocytes (4). Although the presence of P-selectin has never been shown in other cell types, we investigated whether P-selectin could be involved in the rapid increase in neutrophil adhesion on stimulation of epithelial cells with PMA. As shown in Fig. 4, a blocking antiserum to P-selectin did not inhibit neutrophil adhesion to PMA-stimulated epithelial cells. Furthermore, P-selectin was not detectable in A549 cells, either resting or PMA stimulated, by ELISA with AC1.2 as the detection antibody (data not shown).


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Fig. 4.   Effect of anti-P-selectin antibodies on neutrophil adhesion to PMA-stimulated airway epithelial cells. A549 cells were stimulated with PMA (100 ng/ml) for 15 min and preincubated with a polyclonal antiserum to P-selectin (1:100) 30 min before adhesion assay. Data are means ± SD from 1 experiment representative of 2.

The leukocyte integrins CD11a/CD18 (lymphocyte function-associated antigen-1) and CD11b/CD18 (Mac-1) function as ligands for adhesion molecules, including ICAM-1, and have been implicated in adhesive interactions between leukocytes and both endothelial and epithelial cells (16, 19). We used an MAb (TS1/18) directed against the common beta -chain CD18 to investigate the role of these integrins in the adhesion of neutrophils to PMA-stimulated airway epithelial cells. Figure 5A shows that TS1/18 totally inhibits the adhesion of neutrophils to PMA-stimulated A549 cells. Similar results were obtained with HBECs (Fig. 5B). We further characterized the role of beta 2-integrins by adding inhibitory antibodies to CD11a, CD11b, and CD11c to the neutrophil suspension. The results of these experiments for A549 cells and HBECs are shown in Fig. 6, A and B, respectively. Antibodies to CD11b totally blocked the adhesion to both PMA-stimulated A549 cells and HBECs. Antibodies to CD11a and CD11c inhibited adhesion to a much smaller degree.


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Fig. 5.   Effect of anti-CD18 antibody (TS1/18) on neutrophil adhesion to PMA-stimulated airway epithelial cells. A549 cells (A) and HBECs (B) were stimulated for 24 h with PMA (100 ng/ml). Neutrophils were preincubated for 30 min with inhibitory TS1/18 before adhesion assay. Data are means ± SE of 3 consecutive experiments. * P < 0.05 compared with adhesion to unstimulated cells by paired t-test. ** P < 0.05 compared with adhesion in absence of antibody.



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Fig. 6.   Effect of anti-CD11a (TS1/22), anti-CD11b (LPM19c), and anti-CD11c (HC1.1) antibodies on neutrophil adhesion to PMA-stimulated airway epithelial cells. A549 cells (A) and HBECs (B) were stimulated for 30 min with PMA (100 ng/ml). Neutrophils were preincubated for 30 min with inhibitory TS1/22, LPM19c, or HC1.1 before adhesion assay. Data are means ± SE of 3 consecutive experiments. * P < 0.05 compared with adhesion to unstimulated cells by paired t-test. ** P < 0.05 compared with adhesion in absence of antibodies.

ICAM-2 is an adhesion receptor for beta 2-integrins expressed by bronchial epithelial cells (2). We investigated the role of ICAM-2 in the increased adhesion of neutrophils to PMA-stimulated cells. As shown in Fig. 7, an inhibitory monoclonal antibody to ICAM-2 (B-37) did not inhibit neutrophil adhesion in our system.


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Fig. 7.   Role of ICAM-2 on neutrophil adhesion to PMA-stimulated airway epithelial cells. A549 cells were cultured in either presence or absence of PMA (100 ng/ml) for 15 min and incubated with inhibitory anti-ICAM-2 antibody (B-37) for 30 min before adhesion assay. Data are means ± SD of 1 experiment, representative of 2.

Metal ions, metabolic energy, and protein synthesis dependence of neutrophil adhesion to PMA-stimulated epithelial cells. To further characterize the interaction between neutrophils and PMA-stimulated epithelial cells at the molecular level, we investigated the role of different divalent cations and the requirement for metabolic energy and de novo protein synthesis. All adhesion experiments shown so far were performed in the presence of both Ca2+ and Mg2+. Figure 8A shows the binding of neutrophils to PMA-stimulated A549 cells in the presence of Ca2+ and Mg2+, Ca2+ only, and Mg2+ only. Because metal ions are required for A549 cells to bind to the plastic growth surface, we could not perform experiments in the absence of metal ions or in the presence of EDTA. The binding of neutrophils to PMA-stimulated A549 cells was optimal in the presence of Mg2+ regardless of the availability of Ca2+. Ca2+ alone was not sufficient to support the binding. In contrast, binding of neutrophils to the plastic growth surface in the absence of epithelial cells was optimal in the presence of either metal ion (data not shown). Mn2+ upregulates CD18-dependent adhesive interactions by increasing the binding affinity of beta 2-integrins (10). We performed experiments in the presence of Mn2+ to confirm the role of beta 2-integrins in our system. As shown in Fig. 8B, Mn2+ greatly enhances binding of neutrophils to alveolar cells both in resting conditions and upon stimulation with PMA.


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Fig. 8.   Requirement for divalent metal ions on neutrophil adhesion to PMA-stimulated airway epithelial cells. A: A549 cells were cultured for 24 h in absence and presence of PMA (100 ng/ml). Neutrophil adhesion was assessed as described in MATERIALS AND METHODS. Experiments were carried out in presence of 1 mM CaCl2 and 1 mM MgCl2 (open bars), 1 mM CaCl2 (solid bars), or 1 mM MgCl2 (hatched bars). See MATERIALS AND METHODS for further details. B: experiments were carried out as in A except that neutrophils were suspended in 1 mM MnCl2. Data are means ± SE of 3 consecutive experiments. * P < 0.05 compared with adhesion to unstimulated cells by paired t-test.

The requirement for metabolic energy was investigated both by lowering the binding temperature to 4°C and by adding NaN3 to the neutrophil suspension during the incubation. As shown in Fig. 9A, the binding of neutrophils to PMA-stimulated A549 cells at 4°C was low. The binding was also blocked by the metabolic inhibitor NaN3 (Fig. 9B). To confirm that de novo protein synthesis is not required for increased adhesion, experiments were performed in the presence of cycloheximide and actinomycin D. Figure 9C shows that neither cycloheximide nor actinomycin D blocked the increased binding of neutrophils to A549 cells stimulated with PMA for 24 h. Similar results were obtained with cells stimulated for 15 min (data not shown). In parallel experiments, both reagents inhibited, as expected, the synthesis of ICAM-1 by A549 cells stimulated with TNF-alpha as assessed by ELISA (data not shown).


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Fig. 9.   Temperature, metabolic energy, and protein synthesis dependence of neutrophil adhesion to PMA-stimulated airway epithelial cells. A: A549 cells were cultured in absence and presence of PMA (100 ng/ml) for 24 h. Adhesion assay was then carried out at 37 (solid bars) or 4°C (open bars). B: A549 cells were cultured as in A; adhesion assay was then carried out in absence and presence of NaN3. Data are means ± SE of 3 consecutive experiments. * P < 0.05 compared with adehsion to unstimulated cells by paired t-test. C: A549 cells were stimulated with PMA for 24 h in absence and presence of cycloheximide (cyclo; 50 µg/ml) or actinomycin D (ActD; 5 µg/ml). Data are means ± SD from 1 experiment, representative of 2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The molecular mechanisms of leukocyte recruitment into the airways have been extensively studied in the past decade. A number of cell-cell adhesion molecules and soluble mediators that are involved in the process have been identified (4, 19). However, although several adhesion receptors of endothelial origin have been recognized, ICAM-1 is currently the only known inducible receptor for neutrophils expressed by airway epithelia. The aim of this work was to investigate whether stimulation of such cells with different agonists would cause an increase in neutrophil adhesion independent of ICAM-1 upregulation.

Several lines of evidence suggest that, indeed, increased adhesion of neutrophils to human airway epithelial cells on incubation with the phorbol ester PMA is not dependent on ICAM-1 upregulation. First, the phenomenon is rapid, being nearly complete after 10 min. ICAM-1 expression requires de novo protein synthesis and therefore takes several hours (20). Accordingly, on PMA stimulation, ICAM-1 expression is increased at 24 h, but it is unaffected at early time points (e.g., 10 min). Furthermore, two inhibitory MAbs to ICAM-1 have no effect on adhesion. Finally, we showed that other agonists capable of upregulating ICAM-1 expression, such as TNF-alpha , do not affect neutrophil adhesion to alveolar cells. Although this observation, which has been preliminarily reported (5), lends support to the hypothesis that ICAM-1 upregulation is not responsible for increased adhesion in the presence of PMA, at present we have no explanation for it. The integrin receptor CD11/CD18 must be activated and undergo a conformational change to bind ICAM-1. It is possible that in the absence of such activation, leukocytes cannot bind to ICAM-1-bearing cells due to the absence of a functional receptor. In vivo this would be irrelevant because leukocytes must be exposed to chemotactic factors and therefore be activated before they reach the epithelial barrier. However, preliminary data indicate that activation of neutrophils with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine does not increase adhesion to ICAM-1-bearing alveolar epithelial cells (Celi, unpublished data). Interestingly, Cunningham and Kirby (7) have shown that stimulation of human alveolar cells with interferon-gamma , although upregulating ICAM-1 expression, did not influence the adhesion of phytohemoagglutinin-activated lymphocytes.

Experiments with inhibitory MAbs have shown that whatever adhesion molecule is expressed by PMA-stimulated airway epithelial cells, its interaction with neutrophils is mediated by the integrin CD11/CD18. Beside ICAM-1, CD11/CD18 binds to other receptors expressed in different conditions. The first Mac-1 ligand to be identified was iC3b, a complement C3 fragment (1). A role for iC3b in binding of neutrophils to tracheal epithelial cells pretreated with human serum has been previously demonstrated (24). However, metal ion requirements for the binding of Mac-1 to iC3b appear to be different from those for the adhesion of neutrophils to PMA-stimulated epithelial cells because the latter is strictly dependent on the presence of Mg2+, whereas either Ca2+ or Mg2+ is sufficient for iC3b binding to Mac-1 (11, 24). Other ligands for CD11/CD18 include coagulation factor X and fibrinogen. Binding to these receptors has been shown to require Ca2+ but not Mg2+ (1). CD11/CD18 also bears recognition sites for polysaccharides (1). Interestingly, a role for cell surface carbohydrates in neutrophil adhesion to alveolar epithelial cells of different origin, including A549 cells, has been demonstrated (6), although the exact molecular species involved has never been fully characterized. It is possible that carbohydrate residues are involved in the increased binding of neutrophils to airway epithelial cells in our conditions.

Finally, another member of the Ig-like superfamily of adhesion molecules, ICAM-2, serves as a receptor for beta 2-integrins (4). ICAM-2 is expressed by both endothelial (4) and epithelial (2) cells. However, ICAM-2 is constitutively synthesized, and its expression is not upregulated by cell activation (4). Accordingly, an inhibitory antibody to ICAM-2 did not affect neutrophil adhesion to PMA-activated cells.

Robbins et al. (18) have previously shown that PMA, as well as other agonists, upregulate neutrophil and mononuclear cell adhesion to bovine bronchial epithelial cells. However, the molecular bases of adhesion of leukocytes to PMA-stimulated epithelial cells of bovine origin are not necessarily identical to those described for human cells in the present report. First, the authors did not specifically address the issue of the dependence of the phenomenon on ICAM-1. Furthermore, an MAb to CD18 did not affect adhesion in their conditions (18).

Exposure of human airway epithelial cells to ozone has been shown to increase neutrophil adhesion in a CD11/CD18-dependent, ICAM-1-independent manner (22). Although the adhesion receptor expressed under these conditions has not been characterized, time-course experiments show that increased adhesion takes several hours and is maximal 24 h after ozone exposure, suggesting that different mechanisms, likely to require de novo protein synthesis, are involved. Similar considerations apply to the increased adhesion of leukocytes to parainfluenza virus-infected airway epithelial cells reported by the same group (23).

PMA activation of neutrophils increases their adhesion to airway epithelial cells (18). Thus it could be conceivable that small amounts of PMA that remain bound to the cell membranes are responsible for the direct activation of neutrophils and therefore for their increased binding. The observation that PMA stimulation of L929 cells has no effect on neutrophil adhesion rules out this possibility.

Taken together, our data demonstrate that PMA activation of airway epithelial cells causes a rapid increase in neutrophil adhesion through an as yet unknown CD11/CD18 receptor. The interaction is dependent on Mg2+ and requires metabolic energy. A better characterization of this phenomenon at the molecular level, currently underway, should shed new light into the basic mechanisms of lung inflammation.


    ACKNOWLEDGEMENTS

We thank Dr. R. Rothlein for the generous gift of the anti-intercellular adhesion molecule (ICAM)-1 antibody R6.5 and Dr. G. Evangelista for kindly providing purified antibodies to CD11a, CD11b, CD11c, and ICAM-2.


    FOOTNOTES

This work was supported in part with grants from the Italian Ministry of the University and of Scientific and Technologic Research.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. Celi, U. O. Fisiopatologia Respiratoria, Dipartimento Cardiotoracico, Via Paradisa 2, 56124 Pisa, Italy (E-mail: aceli{at}dcap.med.unipi.it).

Received 23 November 1998; accepted in final form 17 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arnaout, M. A. Structure and function of the leukocyte adhesion molecule CD11/CD18. Blood 75: 1037-1050, 1990[Medline].

2.   Atsuta, J., S. A. Sterbinsky, J. Plitt, L. M. Schwiebert, B. B. Bochner, and R. P. Schleimer. Phenotyping and cytokine regulation of the BEAS-2B human bronchial epithelial cell: demonstration of inducible expression of the adhesion molecules VCAM-1 and ICAM-1. Am. J. Respir. Cell Mol. Biol. 17: 571-582, 1997[Abstract/Free Full Text].

3.   Bloemen, P. G. M., M. C. van den Tweel, P. A. J. Henricks, F. Engels, S. S. Wagenaar, A. A. J. J. L. Rutten, and F. P. Nijkamp. Expression and modulation of adhesion molecules on human bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 9: 586-593, 1993[Medline].

4.   Celi, A., R. Lorenzet, B. Furie, and B. C. Furie. Platelet-leukocyte-endothelial cell interaction on the blood vessel wall. Semin. Hematol. 4: 1-10, 1997.

5.   Celi, A., S. Petruzzelli, S. Cianchetti, L. Valdisserri, and C. Giuntini. Interleukin-1beta (IL-1beta ) and interferon-gamma (IFN-gamma ) upregulate ICAM-1 expression but not adhesiveness for neutrophils in the human alveolar type II cell line, A5F9 (Abstract). Eur. Respir. J. 8, Suppl. 19: 548s, 1995.

6.   Crestani, B., C. Rolland, A. Petiet, N. Colas-Linhart, and M. Aubier. Cell surface carbohydrates modulate neutrophil adherence to alveolar type II cells in vitro. Am. J. Physiol. 264 (Lung Cell. Mol. Physiol. 8): L391-L400, 1993[Abstract/Free Full Text].

7.   Cunningham, A. C., and J. A. Kirby. Regulation and function of adhesion molecule expression by human alveolar epithelial cells. Immunology 86: 279-286, 1995[Medline].

8.   Diamond, M. S., D. E. Staunton, S. D. Marlin, and T. Springer. Binding of the integrin Mac-1 (CD11b/CD18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosilation. Cell 65: 961-971, 1991[Medline].

9.   Donnelly, S. C., C. Haslett, I. Dransfield, C. E. Robertson, D. C. Carter, J. A. Ross, I. S. Grant, and T. F. Tedder. Role of selectins in development of adult respiratory distress syndrome. Lancet 344: 215-219, 1994[Medline].

10.   Dransfield, I., C. Cabanas, A. Craig, and N. Hogg. Divalent cation regulation of the function of the leukocyte integrin LFA-1. J. Cell Biol. 116: 219-226, 1992[Abstract].

11.   Dransfield, I., and N. Hogg. Regulated expression of Mg2+ binding epitope on leukocyte integrin alpha  subunit. EMBO J. 8: 3759-3765, 1989[Abstract].

12.   Evangelista, V., S. Manarini, R. Sideri, S. Rotondo, N. Martelli, A. Piccoli, L. Totani, P. Piccardoni, D. Vestweber, G. de Gaetano, and C. Cerletti. Platelet/polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorilation-dependent CD11b/CD18 adhesion: role of PSGL-1 as a signaling molecule. Blood 93: 876-885, 1999[Abstract/Free Full Text].

13.   Kelsen, S. J., I. A. Mardini, S. Zhou, J. L. Benovic, and N. C. Higgins. A technique to harvest viable tracheobronchial epithelial cells from living human donors. Am. J. Respir. Cell Mol. Biol. 7: 66-72, 1992[Medline].

14.   Larsen, E., A. Celi, G. E. Gilbert, B. C. Furie, J. K. Erban, R. Bonfanti, D. D. Wagner, and B. Furie. PADGEM: a receptor that mediates the interaction of activated platelets with neutrophils and monocytes. Cell 59: 305-312, 1989[Medline].

15.   Lechner, J., and M. A. LaVeck. A serum-free method for culturing normal human bronchial epithelial cells at clonal density. J. Tissue Cult. Methods 9: 43-48, 1985.

16.   Leff, A. R., K. J. Hamann, and C. D. Wegner. Inflammation and cell-cell interactions in airway hyperresponsiveness. Am. J. Physiol. 260 (Lung Cell. Mol. Physiol. 4): L189-L206, 1991[Abstract/Free Full Text].

17.   Lynch, J. P., III, T. J. Standiford, M. W. Rolfe, S. L. Kunkel, and R. Strieter. Neutrophilic alveolitis in idiopathic pulmonary fibrosis. Am. Rev. Respir. Dis. 145: 1433-1439, 1991.

18.   Robbins, R. A., S. Koyama, J. J. R. Spurzem, K. A. Richard, K. J. Nelson, G. L. Gossman, G. M. Thiele, and S. I. Rennard. Modulation of neutrophil and mononuclear cell adherence to bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 7: 19-29, 1992[Medline].

19.   Roche, W., S. Montefort, J. Baker, and S. Holgate. Cell adhesion molecules and the bronchial epithelium. Am. Rev. Respir. Dis. 148, Suppl.: S79-S82, 1993[Medline].

20.   Rothlein, R., M. C. Czajkowski, M. M. O'Neill, S. D. Marlin, E. Mainolfi, and V. J. Merluzzi. Induction of intercellular adhesion molecule 1 on primary and continuous cell lines by pro-inflammatory cytokines. J. Immunol. 141: 665-1669, 1988.

21.   Thompson, A. B., D. Daughton, R. A. Robbins, M. A. Ghafouri, M. Oehlerking, and S. I. Rennard. Intraluminal airway inflammation in chronic bronchitis. Characterization and correlation with clinical parameters. Am. Rev. Respir. Dis. 140: 1527-1537, 1989[Medline].

22.   Tosi, M. F., A. Hamedani, J. Brosovich, and S. E. Alpert. ICAM-1 independent, CD18-dependent adhesion between neutrophils and human airway epithelial cells exposed in vitro to ozone. J. Immunol. 152: 1935-1942, 1994[Abstract/Free Full Text].

23.   Tosi, M. F., J. M. Stark, A. Hamedani, C. Wayne-Smith, D. C. Gruenert, and Y. T. Hang. Intercellular adhesion molecule-1 (ICAM-1) dependent and ICAM-1 independent adhesive interactions between polymorphonuclear leukocytes and human airway epithelial cells infected with parainfluenza virus type 2. J. Immunol. 149: 3345-3349, 1992[Abstract/Free Full Text].

24.   Varsano, S., N. Joseph-Lerner, T. Reshef, and I. Frolkis. Normal serum increases adhesion of neutrophils to tracheal epithelial cells by a CD11b/CD18-dependent mechanism. Am. J. Respir. Cell Mol. Biol. 10: 298-305, 1994[Abstract].


Am J Physiol Lung Cell Mol Physiol 277(3):L465-L471
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