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Am. J. Respir. Cell Mol. Biol., Volume 17, Number 5, November 1997 571-582

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

Jun Atsuta, Sherry A. Sterbinsky, Jim Plitt, Lisa M. Schwiebert, Bruce S. Bochner, and Robert P. Schleimer

Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland


    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Airway epithelium may actively participate in inflammatory responses, such as occur in asthma. The presence and regulation of surface molecules on the airway epithelium, however, is incompletely understood. We have determined the phenotype of the human bronchial epithelial cell line BEAS-2B by flow cytometry. We confirmed previous observations that human bronchial epithelial cells constitutively express CD29, CD44, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54 (ICAM-1), CD61, and HLA class 1. BEAS-2B cells were also found to constitutively express CD9, CD13, CD15, CD15s, CD23, CD33, CD36, CD40, CD41b, CD42b, CD48, CD50, CD71, and CD102 (ICAM-2). Culture of BEAS-2B cells with tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta (1 ng/ml) was found to enhance intercellular adhesion molecule-1 (ICAM-1) expression (severalfold) and induce de novo CD106 [vascular cell adhesion molecule-1 (VCAM-1)] expression. TNF-alpha or IL-1beta did not change the expression of CD9, CD13, CD16, CD23, CD29, CD31, CD32, CD35, CD45, CD61, or CD64 in BEAS-2B cells. IL-4 (1 ng/ml) also induced expression of VCAM-1 (1.5-fold) but not ICAM-1 expression while interferon-gamma (1 ng/ml) enhanced only ICAM-1 expression (2-fold). Maximal VCAM-1 expression was obtained with the combination of TNF-alpha and IL-4 (8-fold). Using Northern blot hybridization analysis, ICAM-1 and VCAM-1 mRNA was detected in BEAS-2B cells stimulated with cytokines. VCAM-1 on stimulated BEAS-2B was functionally active as determined by adhesion of purified eosinophils and blockade with specific antibodies. Primary isolates of bronchial epithelial cells produced detectable levels of VCAM-1 protein and mRNA as detected by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. These results suggest that cytokine activation induces expression of ICAM-1 and VCAM-1 on airway epithelium, an event which may influence leukocyte infiltration and activation.


    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

It is well recognized that bronchial epithelial cells play an important role as a physical barrier protecting the underlying tissue and in maintaining the local environment in the airway. Recent findings indicate that airway epithelial cells are also able to act as immune effector cells and may actively participate in inflammatory responses, such as occur in asthma. They generate and release mediators of inflammation, such as lipid mediators, inflammatory enzymes, and a variety of cytokines (1). Airway epithelial cells, moreover, have been demonstrated to express a wide spectrum of surface molecules, including adhesion molecules (11). Some are constitutively expressed, while others are upregulated or induced during an inflammatory reaction. However, our knowledge in this area has resulted from a directed focus on particular surface molecules. Therefore we have performed a more comprehensive flow cytometric analysis of the phenotypic expression of surface molecules on human bronchial epithelial cells under basal conditions and after stimulation with cytokines. A notable finding was that cytokines induce vascular cell adhesion molecule-1 (VCAM-1) expression on human bronchial epithelial cells. We also detected soluble-VCAM-1 in culture supernatants and VCAM-1 mRNA by reverse transcription-polymerase chain reaction (RT-PCR) in primary human bronchial epithelial cells.

    Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents

The following materials were purchased: Dulbecco's modified Eagle's medium (DMEM), Ham's F12, Ca2+- and Mg2+-free Hank's balanced salt solution (HBSS), Versene (Ca2+- and Mg2+-free HBSS containing 0.02% EDTA), trace elements, phosphoethanolamine/ethanolamine, and retinoic acid (Biofluids, Inc., Rockville, MD); insulin, hydrocortisone, epidermal growth factor, and endothelial cell growth supplement (Collaborative Research, Bedford, MA); cholera toxin and triiodothyronine (Sigma Chemical Co., St. Louis, MO); fetal calf serum (FCS), penicillin/streptomycin solution, agarose, and formamide (Life Technologies, Inc., Gaithersburg, MD); fungizone (Gibco BRL, Gaithersburg, MD); RNAzol B (Tel-Test, Inc., Friendswood, TX); chloroform, isopropanol, and formaldehyde (Fisher Scientific, Fernwood, NJ); protease (Calbiochem, San Diego, CA); Genescreen membrane and [alpha -32P]-dATP (Dupont NEN, Boston, MA); human rTNF-alpha , interleukin (IL)- 1beta , IL-4, and IL-5 (R&D Systems, Minneapolis, MN); and human rIFN-gamma (Genzyme Corp., Cambridge, MA).

Antibodies

All antibody reagents were diluted in phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin (BSA). Fifty monoclonal antibodies were obtained as indicated in Table 1.

                              
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TABLE 1
Primary monoclonal antibodies

Isolation of Human Eosinophils

Human eosinophils were purified from normal donors or individuals with allergic rhinitis or asthma using density gradient centrifugation and a negative selection/immunomagnetic bead technique (16).

Cell Culture

BEAS-2B cells. BEAS-2B cells were a generous gift from Dr. Curtis Harris (National Cancer Institute, Bethesda, MD). This cell was derived from human bronchial epithelium transformed by an Adenovirus12-SV40 hybrid virus (17). These cells retain electron microscopic features of epithelial cells and show positive staining with antibodies to cytokeratin but do not form tight junctions ([17] and data not shown). BEAS-2B cells were cultured in 25-cm2 tissue culture flasks and maintained in F12/10X medium consisting of Ham's F12 nutrient medium containing penicillin (100 U/ml) and streptomycin (100 U/ml) and supplemented with insulin (5 µg/ml), hydrocortisone (10-7 M), triiodothyronine (3.1 × 10-9 M), cholera toxin (10 ng/ml), endothelial cell growth supplement (3.75 µg/ml), epidermal growth factor (12.5 ng/ml), phosphoethanolamine/ethanolamine (5 × 10-6 M), trace elements (1X), and retinoic acid (0.1 µg/ml). Cells were used between passage 35 and 47 and were plated on 6-well culture plates (Costar, Cambridge, MA) in F12/DMEM medium containing 5% heat-inactivated FCS, penicillin (100 U/ml), and streptomycin (100 U/ml).

IB3-1 cells. The IB3-1 cell line was derived from human bronchial epithelial cells of a cystic fibrosis patient, transformed by an adenovirus 12-SV40 (18), and was kindly provided by Dr. Pamela Zeitlin (Johns Hopkins University, Baltimore, MD). IB3-1 cells were cultured in 25-cm2 tissue culture flasks or on 6-well culture plates coated with collagen in LHC-8 medium containing 5% heat-inactivated FCS, penicillin (100 U/ml), and streptomycin (100 U/ml).

Primary normal human bronchial epithelial cells (NHBE). Primary bronchial epithelial cells were obtained using a modification of a previously described technique (19). Specimens of human bronchi were obtained from the autopsied lungs of patients without respiratory disorders. Tissues were dissected and rinsed 5 times in Ca2+- and Mg2+-free HBSS and incubated overnight at 4°C in 0.1% protease (Calbiochem) solution in Ham's F12 medium containing penicillin (100 U/ml), streptomycin (100 U/ml), and fungizone (1 µg/ml). Cells were detached by a gentle jet of 20% FCS in Ham's F12 medium. The suspension was centrifuged at 1,100 rpm for 8 min, and the cell pellet was resuspended in F12/DMEM medium containing 5% heat-inactivated FCS, penicillin (100 U/ml), and streptomycin (100 U/ml). The medium was changed at day 1 to F12/10X medium, and changed on alternate days thereafter. The purity of the cultures and identity of the cells were confirmed by light microscopy and immunocytochemical staining using specific monoclonal antibodies directed against cytokeratin.

Human umbilical vein endothelial cells (HUVEC). Endothelial cells were isolated after collagenase digestion from human umbilical cord veins and were grown to confluence in 6-well culture plates as described previously (20).

All cells were incubated at 37°C with 5% CO2 in humidified air.

Flow Cytometry Analysis

Cells were grown to 80% confluence in 6-well culture plates. In the experiments assessing the effect of cytokines, cells grown to 50% confluence were incubated with cytokines for 24 h. Cells were washed 3 times with Ca2+- and Mg2+-free HBSS and treated for 10 min with Versene (Ca2+- and Mg2+-free HBSS containing 0.02% EDTA) without trypsin, and then removed from plates by repeated pipetting. For each analysis, 1 × 106 cells were incubated in 30 µl of PBS/0.2% BSA containing saturating concentrations of each monoclonal antibody and 4 mg/ml of human IgG (to reduce nonspecific binding) on ice for 30 min, as previously described (21). Saturating concentrations were first determined on various cell types known to express each surface marker. The cells were washed, resuspended in saturating amounts of fluorescein-conjugated goat F(ab')2 antimouse IgG antibody for another 30 min, and then washed again. Fluorescence was measured using an EPICS Profile flow cytometer (Coulter Electronics, Hialeah, FL) and was expressed as percent of control mean fluorescence intensity; each mean fluorescence intensity was compared with control staining using an irrelevant class-matched mouse monoclonal antibody. For each sample, at least 5,000 events were collected, and histograms were generated. Negative staining with propidium iodide (2 µg/ml) and a combination of scatter characteristics was used to identify a uniform population of viable cells.

Northern Blot Hybridization Analysis

BEAS-2B cells grown to 80% confluence were incubated with cytokines for 2 h. Total RNA was extracted from BEAS-2B cells using the RNAzol B extraction technique (22). Samples of RNA (10 µg) were denatured with formaldehyde/formamide and subjected to electrophoresis on 1% agarose formaldehyde gels. Gels were run for 1.5 h at 85 V and the RNA was transferred onto a GeneScreen nylon membrane (New England Nuclear, Boston, MA). Membranes were crosslinked by exposure to ultraviolet radiation, then were subjected to prehybridization treatment for 20 h at 37°C in 2× PIPES, 50% formamide buffer containing 0.5% sodium dodecyl sulfate (SDS), and 100 µg/ml sonicated salmon sperm DNA. Blotted membranes were hybridized with 32P-labeled human intercellular adhesion molecule-1 (ICAM-1) and VCAM-1 cDNA probes generated by the random hexamer priming method (0.5 × 106 count per min/ml). A 1.6-kB ICAM-1 cDNA probe excised from Bluescript® II SK vector was prepared by purifying plasmid DNA and digesting with restriction endonucleases Xba I and Xho I. A 2.2-kB VCAM-1 cDNA probe excised from pcDNA3 vector was prepared by purifying plasmid DNA and digesting with restriction endonuclease Not I. Human ICAM-1 plasmid was a gift from Dr. Richard Hampton (Johns Hopkins University). Human VCAM-1 plasmid was a gift from Dr. Venkot Gopal (Clonexpress, Inc., Gaithersburg, MD). Membranes were washed twice at room temperature in 2× SSC, twice at 60°C in 2× SSC/0.1% SDS, again at room temperature in 0.1× SSC and then subjected to autoradiography at -80°C using an intensifying screen. Quantification of RNA was done by densitometry. For the densitometric analysis, autoradiographs were carried out using a scanning densitometer attached to a computer with Image 1.53 software (National Institutes of Health Public Software, Bethesda, MD). Loading of lanes and integrity of total RNA were confirmed by ethidium bromide staining.

Eosinophil-Epithelial Cell Monolayer Adhesion Assay

Eosinophil adherence to BEAS-2B cells was measured using a 51Cr-radiolabeled leukocyte collection assay (23). BEAS-2B cells in 24-well culture plates were preincubated for 24 h with or without 1 ng/ml of tumor necrosis factor (TNF)-alpha . Purified eosinophils were radiolabeled (51Cr, 0.2 uCi, 37°C, 30 min) and washed before adhesion assays. Eosinophils (2.5 × 105 cells) and BEAS-2B cells were coincubated in 5% CO2/95% air for 10 min at 37°C before removal of nonadherent eosinophils by rinsing. Adherent cells were lysed in ammonium hydroxide and radioactivity measured using a Beckman 5500 gamma counter (Beckman Instruments, Inc., Fullerton, CA). Results were expressed as a percentage of total added counts bound to the monolayers. For anti-adhesion molecule blocking experiments, duplicate wells containing cell monolayers were incubated with saturating concentrations of F(ab')2 preparations of mAb to HLA class I, ICAM-1, or VCAM-1 (Caltag Laboratories, South San Francisco, CA) in 5% CO2/95% air for 30 min at 37°C prior to the addition of eosinophils.

Measurement of Soluble VCAM-1

Culture supernatants from primary normal human bronchial epithelial cell cultures were analyzed for the presence of soluble VCAM-1 by specific enzyme-linked immunosorbent assay (ELISA) (R&D Systems Ltd., Oxford, UK) according to the manufacturer's instructions. With this assay, the minimum detectable concentration is 40 pg/ml.

RT-PCR Analysis of VCAM-1 mRNA in Primary Normal Human Bronchial Epithelial Cells

Total RNA extracted from normal human bronchial epithelial cells was reverse transcribed using oligo(dT) as a primer and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Paisley, Scotland). The VCAM-1 primers used were: forward primer, 5'-ATTGGGAAAAACAGAAAAGAG-3'; reverse primer, 5'-GGCAACATTGACATAAAGTG-3'; these primers produced a 642-bp product. Thermocycler settings were an initial step at 94°C for 5 min to denature and linearize cDNA followed by 30 cycles of 94°C for 30 s for denaturing, 50°C for 30 s for annealing, and 72°C for 1 min for polymerization. Amplified products were electrophoresed on a 1.5% agarose gel which was then stained with ethidium bromide.

Statistical Analysis

Data are expressed as mean ± SEM. Statistical significance was assessed with a paired two-group t test, an unpaired two-group t test, or the ANOVA test followed by Bonferroni/Dunn analysis; P < 0.05 was considered significant. Statistical analysis of data by flow cytometry was performed using percent of control mean fluorescence intensity.

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Phenotyping of Bronchial Epithelial Cells

Table 2 lists the expression of cluster of differentiation structures, HLA class 1, and HLA-DR on unstimulated BEAS-2B human bronchial epithelial cells. We confirmed previous observations (12) that human bronchial epithelial cells constitutively expressed (i.e., statistical differences at least P < 0.05 from control mean fluorescence intensity) CD29, CD44, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54 (ICAM-1), CD61, and HLA class 1. A number of surface molecules, previously not studied on epithelial cells, were found to be expressed on BEAS-2B cells; CD9, CD13, CD15, CD15s, CD23, CD33, CD36, CD40, CD41b, CD42b, CD48, CD50 (ICAM-3), CD71, and CD102 (ICAM-2).

                              
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TABLE 2
Constitutive expression of CD structures and other surface markers on unstimulated BEAS-2B

Effect of Cytokines on the Expression of Surface Molecules on Bronchial Epithelial Cells

We tested a selected panel of markers, primarily not expressed on resting BEAS-2B cells, to determine whether cytokine stimulation induced them. Expression of CD9, CD13, CD16, CD23, CD29, CD31, CD32, CD35, CD45, CD61, and CD64 was not changed after stimulation with TNF-alpha , IL-1beta , IL-4, IL-5, or interferon-gamma (IFN-gamma ) (1 ng/ml, 24 h) (data not shown). Expression of CD71 did not change with IFN-gamma (1 ng/ml, 24 h) (data not shown). Expression of ICAM-1 was significantly enhanced with 1 ng/ ml of TNF-alpha (3.5-fold of ICAM-1 expression without stimulus, P < 0.05), IL-1beta (2.5-fold, P < 0.05), or IFN-gamma (2-fold, P < 0.05) (Figure 1). IL-4 and IL-5 had no effect on ICAM-1 expression. Expression of VCAM-1 was significantly induced with 1 ng/ml of TNF-alpha (6-fold of VCAM-1 expression without stimulus, which was not different from control, P < 0.05), IL-1beta (2.5-fold, P < 0.05), or IL-4 (1.5-fold, P < 0.05). IL-5 and IFN-gamma had no effect on VCAM-1 expression.


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  		Figure 1.   Induction of ICAM-1 and VCAM-1 expression by cytokines on BEAS-2B cells. Cells were incubated with 1 ng/ml of TNF-alpha , IL-1beta , IL-4, IL-5, or IFN-gamma for 24 h. Values are expressed as mean ± SEM (n = 4, except for IL-5 where n = 1). Significant differences are *P < 0.05 from values obtained without stimulus, as assessed with a paired two-group t test.

Effect of Cytokines on ICAM-1 and VCAM-1 Expression on Bronchial Epithelial Cells

Data in Figure 2 show the concentration dependence of induction of ICAM-1 and VCAM-1 expression by cytokines on BEAS-2B cells. A significant (P < 0.05) increase in ICAM-1 expression over basal levels was observed with concentrations of TNF-alpha as low as 0.1 ng/ml or IL-1beta at 0.01 ng/ml. Induction reached a plateau at 1.0 ng/ml of TNF-alpha or 0.1 ng/ml of IL-1beta . A significant (P < 0.05) increase in VCAM-1 expression over basal levels was observed with 1.0 ng/ml of TNF-alpha , 0.1 ng/ml of IL-1beta , or 10 ng/ml of IL-4. IL-4 did not enhance ICAM-1 expression. A representative fluorescence histogram of VCAM-1 expression induced with TNF-alpha is shown in Figure 3. Staining patterns of VCAM-1 were bimodal with a progressively larger proportion of the cells staining positively as the concentration of TNF-alpha was increased.


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  		Figure 2.   Induction of ICAM-1 (a) and VCAM-1 (b) expression by cytokines on BEAS-2B cells. Cells were incubated with the indicated concentrations of TNF-alpha (closed circles), IL-1beta (open circles), or IL-4 (closed squares) for 24 h. Values are expressed as mean ± SEM (n = 4). Significant differences are *P < 0.05 and **P < 0.01 from values obtained without stimulus, as assessed with Bonferroni/Dunn analysis.


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  		Figure 3.   Representative fluorescence histograms showing VCAM-1 expression on BEAS-2B cells. Cells were incubated with or without TNF-alpha for 24 h. IgG1 control was unchanged even after TNF stimulation. +ve = positive.

The kinetics of enhancement of ICAM-1 expression with 1 ng/ml of TNF-alpha are shown in Figure 4a. The response reached a plateau by 8 h and persisted to 48 h. As shown in Figure 4b, VCAM-1 expression induced by 1 ng/ ml of TNF-alpha reached a maximum after 8 h and declined slightly thereafter, reaching a plateau at 24-48 h. Induction of VCAM-1 expression exceeded the additive values when IL-4 was combined with either IL-1beta or TNF-alpha (Figure 5). Incubation with TNF-alpha and IL-4 together resulted in the greatest enhancement of VCAM-1 expression (8-fold of VCAM-1 expression without stimulus); the combination of TNF-alpha and IL-1beta did not result in a response greater than that induced by TNF-alpha alone. The effect of TNF-alpha and IL-4 together on VCAM-1 expression was confirmed using a second human bronchial epithelial cell line, IB3-1. The combination of 100 ng/ml of TNF-alpha and IL-4 induced a 2.5-fold increase of VCAM-1 expression after 24 h of incubation (n = 2, data not shown). Although TNF-alpha (100 ng/ ml) or the combination of TNF-alpha (100 ng/ml) and IL-4 (10 ng/ml) did not induce VCAM-1 expression by flow cytometry on primary human bronchial epithelial cells (data not shown), both mRNA and protein were detected by more sensitive assays (see below).


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  		Figure 4.   Kinetics of induction of ICAM-1 (a) and VCAM-1 (b) expression by TNF-alpha on BEAS-2B cells. Cells were incubated with 1 ng/ml of TNF-alpha for up to 48 h. Values are expressed as mean ± SEM (n = 3).


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  		Figure 5.   Effects of combinations of cytokines on VCAM-1 expression on BEAS-2B cells. Cells were incubated with 1 ng/ml of TNF-alpha , IL-1beta , and/or IL-4 for 24 h. Values are expressed as mean ± SEM (n = 3).

Effect of Cytokines on ICAM-1 and VCAM-1 mRNA Expression

Northern blot analysis was used to confirm flow cytometry results and assess cytokine effects on mRNA expression in BEAS-2B cells (Figure 6). Neither ICAM-1 or VCAM-1 mRNA was detected under basal conditions but both were expressed after stimulation for 2 h with 1 ng/ml of TNF-alpha . Expression of VCAM-1 mRNA was further enhanced with the combination of TNF-alpha and IL-4 (1.5-fold of VCAM-1 mRNA expression induced by TNF-alpha alone).


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  		Figure 6.   Northern blot analysis of ICAM-1 and VCAM-1 mRNA induced in BEAS-2B cells. Cells were incubated with 1 ng/ml of TNF-alpha with or without 1 ng/ml of IL-4 for 2 h. (I) Representative autoradiography of ICAM-1 (a) and VCAM-1 (b) mRNA expression. (II) Densitometric analysis of the autoradiography. Values are expressed as mean ± SEM (n = 5). (III) Ethidium-bromide staining of gel showing loading of lanes and integrity of mRNA.

Eosinophil Adhesion to BEAS-2B Cell Monolayers

To test the functional status of VCAM-1 expressed on BEAS-2B cells, we assessed the ability of purified eosinophils to adhere to stimulated epithelial cells. Baseline values of eosinophil adhesion to unstimulated BEAS-2B cell monolayers were 5.7 ± 0.3%, and were significantly increased by the stimulation of BEAS-2B cells with 1 ng/ml of TNF-alpha for 24 h (15.7 ± 3.0%, P < 0.02, Figure 7). To determine the surface molecules responsible for eosinophil adhesion to BEAS-2B cell monolayers, eosinophil adhesion was assessed in the presence of monoclonal antibodies to ICAM-1 and VCAM-1. Eosinophil adhesion to TNF-alpha -stimulated BEAS-2B cell monolayers was not decreased by pre-incubation with anti-ICAM-1 F(ab')2 antibody. In contrast, anti-VCAM-1 F(ab')2 antibody completely inhibited eosinophil adhesion to TNF-alpha -stimulated BEAS-2B cell monolayers (5.9 ± 0.6%, P < 0.02).


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  		Figure 7.   Eosinophil adhesion to BEAS-2B cell monolayers. BEAS-2B cells were incubated with or without 1 ng/ml of TNF-alpha for 24 h. Pre-treatment of TNF-alpha -stimulated BEAS-2B monolayers with anti-VCAM-1 F(ab')2 antibody resulted in a significant decrease of eosinophil adhesion compared with values obtained with TNF-alpha stimulation alone (*P < 0.05). Values are presented as mean ± SEM (n = 3). Statistical significance was assessed with the Bonferroni/Dunn analysis.

Soluble-VCAM-1 in Cultures of Primary Human Bronchial Epithelial Cells

To determine whether VCAM-1 expression also occurs in primary bronchial epithelial cells, 10 ng/ml of the combination of TNF-alpha and IL-4 were added to subconfluent cultures of primary human bronchial epithelial cells, and the supernatants were harvested after 24-48 h for assay of immunoreactive soluble-VCAM-1 content. As shown in Figure 8, stimulated bronchial epithelial cells released significant detectable amounts of immunoreactive soluble VCAM-1 compared with unstimulated cells (from 0.39 ± 0.05 ng/ml in control to 0.69 ± 0.10 ng/ml in stimulated cultures, P < 0.01). As would be expected based upon the Northern blot and flow cytometry results, BEAS-2B produced significantly greater levels of soluble-VCAM-1 (from 0.9 ng/ml in control to 70.3 ng/ml in stimulated cultures).


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  		Figure 8.   ELISA of soluble-VCAM-1 in culture supernatants of primary normal human bronchial epithelial cells. Cells were incubated with or without the combination of TNF-alpha and IL-4 (10 ng/ml of each cytokine) for 24-48 h. Statistical significance was assessed with a paired two-group t test (n = 7, P < 0.01).

VCAM-1 mRNA Expression in Primary Human Bronchial Epithelial Cells

The expression of VCAM-1 mRNA by primary bronchial epithelial cells was examined by RT-PCR analysis. As shown in Figure 9 (I, II), stimulation with the combination of 10 ng/ml of TNF-alpha and IL-4 for 24 h resulted in detectable levels of amplified VCAM-1 cDNA (results are from one of three similar experiments). The combination of TNF-alpha and IL-4 enhanced VCAM-1 mRNA expression in primary human bronchial epithelial cells (6-fold of VCAM-1 mRNA expression without stimulus). To rule out a small contamination with endothelial cells, the expression of E-selectin by HUVEC or primary human bronchial epithelial cells was examined [Figure 9 (III)]. E-selectin mRNA was induced in HUVEC stimulated with 100 ng/ ml of TNF-alpha for 2 or 4 h, however, E-selectin mRNA was not detected in primary human bronchial epithelial cells stimulated with 10 ng/ml of TNF-alpha for 3 h, suggesting that contamination with endothelial cells is not responsible for the detection of VCAM-1 in the primary epithelial cell cultures which we have used.


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  		Figure 9.   VCAM-1 and E-selectin mRNA analysis of primary NHBE by RT-PCR. (I ) Representative VCAM-1 mRNA induced in NHBE. Cells were incubated with the combination of TNF-alpha and IL-4 (10 ng/ml of each cytokine) for 24 h. (II) Densitometric analysis of VCAM-1 mRNA in NHBE. Values represent mean ± SEM (n = 3). (III) E-selectin mRNA analysis of NHBE and HUVEC by RT-PCR. E-selectin mRNA was induced in HUVEC, not in NHBE. HUVEC were incubated with 100 ng/ml of TNF-alpha for 2 or 4 h. NHBE were incubated with 10 ng/ml of TNF-alpha for 3 h. Results are from one of three similar experiments.

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The initial goal of this study was to characterize the cell surface phenotype of resting and activated airway epithelial cells, using the BEAS-2B cell line. The BEAS-2B cell line was originally established from healthy bronchial epithelium. They have been shown to maintain typical epithelial morphology and many epithelial functional characteristics (17, 24). Cultured BEAS-2B cells were found to express numerous surface molecules, including several previously found on epithelial cells; CD29, CD44, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54 (ICAM-1), CD61, and HLA class 1 (12, 25). Of these, CD29, CD49a, CD49b, CD49c, CD49d, CD49e, and CD49f are members of the beta 1 subfamily of integrins, known to bind to matrix proteins including collagen, laminin, and fibronectin. CD51 and CD61 form a beta 3 integrin heterodimer termed the "vitronectin receptor." CD44 is known to be a receptor for hyaluronic acid, an important interstitial component. The integrins may play important roles in maintaining contact between bronchial epithelial cells and their underlying substratum. ICAM-1 is one of the ligands of leukocyte function-associated antigen-1 and Mac-1, beta 2 integrins commonly expressed on leukocytes (26), and is probably important in leukocyte-epithelial interaction.

These studies identified several surface markers not previously shown to be expressed by epithelial cells; CD9, CD13, CD15, CD15s, CD23, CD33, CD36, CD40, CD41b, CD42b, CD48, CD50 (ICAM-3), CD71, and CD102 (ICAM-2). Among these, CD9, CD13, and CD71 were strongly expressed. CD9 is a cell-surface glycoprotein expressed at high levels on the surface of developing B-lymphocytes, platelets, eosinophils, basophils, stimulated T-lymphocytes, and a neuroblastoma cell line (27). The functional role of CD9 on bronchial epithelial cells is presently unclear. However, studies with platelets or pre-B cells showed that anti-CD9 monoclonal antibody induced activation and aggregation, leading to what is termed an adherent phenotype (31, 32). Rather than being directly involved in adhesion, it is thought that CD9 may function in the initiation of signals leading to cell adhesion, together with other proteins, such as the integrin GPIIb/IIIa or VLA beta 1 (33, 34). CD13 has been detected in many tissues, including the brush border membrane of the small intestine, the epithelium of renal proximal tubules, synaptic membranes of the central nervous system, and on the surface of granulocytes and macrophages (35). CD13 is aminopeptidase-N, a protease shown to inactivate neuropeptides. CD13 expression on human bronchial epithelial cells may play a role in modulating neurogenic inflammation by neuropeptides such as substance P in asthmatic lungs. It has been reported that IL-4 enhances CD13 expression on monocytes (39); however, CD13 expression on BEAS-2B cells was not altered by IL-4 in our study (data not shown). CD71 is the transferrin receptor that is expressed ubiquitously on proliferating cells. Several findings suggest that the interaction between transferrin and its receptor may play an important role in the control of cell growth (40). It has been demonstrated that IFN-gamma inhibits the expression of transferrin receptor on monocytes (41), however, IFN-gamma did not inhibit CD71 expression on BEAS-2B cells (data not shown).

We studied the effects of cytokines on the expression of several surface molecules on BEAS-2B cells, and observed that TNF-alpha and IL-1beta both enhanced ICAM-1 expression and induced VCAM-1 expression. IL-4 induced VCAM-1 expression but not ICAM-1 expression, while IFN-gamma only enhanced ICAM-1 expression among the markers we studied. The increase in adhesion molecule expression may influence the recruitment of leukocytes to the surface of epithelium. Alternatively, the expression of adhesion molecules may influence leukocyte survival or facilitate degranulation and release of cytotoxic products that damage the epithelium during the process of airway inflammation, as is often observed in patients with asthma.

Results of the study of ICAM-1 expression on bronchial epithelial cells induced by cytokines are conflicting. Look and coworkers found that IFN-gamma , but not TNF-alpha or IL-1beta , enhanced ICAM-1 expression on human tracheal epithelial cells in primary culture (42). Bloemen and coworkers also found that IFN-gamma , but not TNF-alpha , enhanced ICAM-1 expression on the NCI-H292 cell line (11). In contrast, both TNF-alpha and IL-1beta were found by Wegner and coworkers to enhance ICAM-1 expression in a dose-dependent manner on cultured monkey bronchus epithelial cells (43). It is likely that ICAM-1 expression under basal conditions or upon stimulation with cytokines plays an important role in the adhesion of leukocytes. In some studies (42, 44), the expression of ICAM-1 on airway epithelium was shown to be functionally relevant by the inhibition of adhesion of polymorphonuclear leukocytes to airway epithelium with anti-ICAM-1 monoclonal antibody. Leukocyte adhesion, however, was not completely inhibited with the anti-ICAM-1 monoclonal antibody in those studies, indicating that other cell adhesion molecules must be present on airway epithelial cells.

Our finding that activated human bronchial epithelial cells express VCAM-1 is in contrast to previous studies (11) and has at least two implications: (1) it is the first report, to the best of our knowledge, demonstrating that VCAM-1 expression can be induced on bronchial epithelial cells, although VCAM-1 expression has been demonstrated on renal tubular epithelial cells (45), tonsil epithelial cells (46), or skin keratinocytes (47); (2) it suggests the existence of a previously unrecognized mechanism for the selective recruitment of VLA-4-positive cells, such as eosinophils, into airways. Anwar and coworkers have shown that culture of eosinophils on fibronectin prolongs their survival by engagement of VLA-4 and perhaps induction of autocrine synthesis of survival-promoting cytokines such as granulocyte macrophage colony-stimulating factor (48). It is conceivable that epithelial VCAM-1 could prolong eosinophil survival by a similar VLA-4-dependent effect. The inability of Bloemen and coworkers to detect the induction of VCAM-1 expression on BEAS-2B cells may be due to their use of vitrogen- and fibronectin-coated flasks to maintain cells while we used uncoated culture flasks (11). Alternatively, Bloemen and coworkers used a serum-free keratinocyte medium, whereas we used F12/ DMEM medium containing FCS to culture cells. Finally, we found that early passage BEAS-2B responded significantly better than late passage cells. In studies not shown, cells older than passage 50 failed to respond. Studies utilizing biopsy of upper and lower airways have not identified epithelial expression of VCAM-1 (49). This could result from the reduced sensitivity of immunohistochemical techniques compared with flow cytometry, the potential transient nature of expression of VCAM-1 in vivo, or the accessibility of epithelial VCAM-1 to antibody probes in situ. Alternatively, it is possible that this response either occurs weakly in vivo or is restricted to special circumstances. In fact, VCAM-1 expression was observed with primary normal human bronchial epithelial cells, although the signal was weaker than in BEAS-2B cells. Our flow cytometry studies could not demonstrate VCAM-1 expression on stimulated primary human bronchial epithelial cells; however, small amounts of soluble-VCAM-1 were found in culture supernatants and PCR-detectable VCAM-1 mRNA was detected. We have not performed functional studies on primary epithelial cells which express VCAM-1 and do not know if the amount expressed is adequete to serve either as a substrate for firm adhesion or to activate VLA-4-positive cells. These studies do, however, establish that primary epithelial cells have the capacity to activate expression of the VCAM-1 gene. Clearly, the present results warrant a careful analysis specifically directed at assessing whether epithelium can express VCAM-1 in vivo.

VCAM-1 seems to have a selective role in adhesion of lymphocytes and eosinophils but not neutrophils (12, 21). Similar to findings in vascular endothelial cells (21, 56), IL-4 selectively induced VCAM-1 expression but not ICAM-1 expression on BEAS-2B cells, and the combination of TNF-alpha and IL-4 was a potent stimulus of VCAM-1 expression. One possible explanation for this effect is modulation of expression of one cytokine receptor by the other cytokine. Another possible explanation is at the level of transcriptional regulation. The combination of TNF-alpha and IL-4 synergistically increases the level of VCAM-1 mRNA in endothelial cells (59).

In order to confirm the observations with flow cytometry and to achieve a greater appreciation of the mechanism of cytokine-induced enhancement, RNA extracts were subjected to Northern blot hybridization analysis. Both ICAM-1 and VCAM-1 mRNA expression were induced in BEAS-2B cells after TNF-alpha treatment for 2 h. Moreover, strong induction of VCAM-1 mRNA expression was observed in response to the combination of TNF-alpha and IL-4, indicating that they may have a greater-than-additive effect on expression of VCAM-1 mRNA. Even though basal ICAM-1 mRNA was not detected under the conditions utilized, basal expression of ICAM-1 was observed by flow cytometry, which may suggest the reduced sensitivity of Northern blot analysis compared with flow cytometry analysis. Induction of ICAM-1 and VCAM-1 mRNA parallelled that observed with the expression of the protein, suggesting that ICAM-1 and VCAM-1 expression is due to either transcriptional activation or cytokine-mediated stabilization of ICAM-1 and VCAM-1 transcripts.

VCAM-1 expressed on activated BEAS-2B cells was found to be functionally active in mediating eosinophil adhesion. We did not find evidence for ICAM-1 mediated eosinophil adhesion. This data indicate that VCAM-1 may be important for the interaction of eosinophil with airway epithelium. Gogging and coworkers demonstrated that ICAM-1 did not mediate eosinophil adhesion to respiratory epithelial cells (60). The expression of VCAM-1 which we observed in primary human bronchial epithelial cells was quite small. It remains to be established whether these low levels of VCAM-1 can act as a signal to activate leukocytes, especially eosinophils, or can serve as a sufficient substrate for adhesion. The contribution of VCAM-1 activation in vivo to leukocyte recruitment is unknown and awaits future studies.

In conclusion, this study has provided evidence for the constitutive expression of many surface molecules on human bronchial epithelial cells and upregulation of ICAM-1 and VCAM-1 with several cytokines. In aggregate, these findings suggest a potential role of epithelial surface molecules in inflammatory reactions, such as asthma, by facilitating leukocyte recruitment and activation.

    Footnotes

Address correspondence to: Dr. Robert P. Schleimer, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-6801. E-mail: rschleim{at}welchlink.welch.jhu.edu

(Received in original form June 24, 1996 and in revised form February 10, 1997).

Acknowledgments: The authors thank Dr. Cristiana Stellato and Dr. Masafumi Arima for kind help in cell culture, Carol A. Bickel for general assistance, and Dr. Satsuki Atsuta for helpful discussions.

Abbreviations BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; HBSS, Hank's balanced salt solution; HUVEC, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule-1 (CD54); IFN-gamma , interferon-gamma; IL, interleukin; NHBE, normal human bronchial epithelial cells; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction; SDS, sodium dodecyl sulfate; TNF, tumor necrosis factor; VCAM-1, vascular cell adhesion molecule-1 (CD106).

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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