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Am J Physiol Lung Cell Mol Physiol 278: L139-L147, 2000;
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Vol. 278, Issue 1, L139-L147, January 2000

beta -Adrenergic agonist modulation of monocyte adhesion to airway epithelial cells in vitro

Debra J. Romberger, Peggy Heires, Stephen I. Rennard, and Todd A. Wyatt

Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, Nebraska Medical Center, Omaha, Nebraska 68198-5300


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

beta -Adrenergic agonists are commonly used in the treatment of obstructive airway diseases and are known to modulate cAMP-dependent processes of airway epithelial cells. However, little is known regarding the ability of cAMP-dependent mechanisms to influence cell-cell interactions within the airway. Thus we investigated the role of the beta -adrenergic agonist isoproterenol in modulating the ability of human bronchial epithelial cells to support the adhesion of THP-1 cells, a monocyte/macrophage cell line, in vitro. We demonstrated that pretreatment of human bronchial epithelial cells (HBECs) with 10 µM isoproterenol or 100 µM salbutamol augments the adhesion of fluorescently labeled THP-1 cells to HBEC monolayers by ~40-60%. The increase in THP-1 cell adhesion occurred with 10 min of isoproterenol pretreatment of HBECs and gradually declined but persisted with up to 24 h of isoproterenol exposure. Exposure of THP-1 cells to isoproterenol or salbutamol before the adhesion assays did not result in an increase in adhesion to HBECs, suggesting that the isoproterenol modulation was primarily via changes in epithelial cells. A specific protein kinase A inhibitor, KT-5720, inhibited subsequent isoproterenol augmentation of THP-1 cell adhesion, further supporting the role of cAMP-dependent mechanisms in modulating THP-1 cell adhesion to HBECs.

adenosine 3',5'-cyclic monophosphate-dependent protein kinase; human bronchial epithelial cells; cell adhesion; THP-1 cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MONOCYTES AND MACROPHAGES are important in immunoinflammatory processes that occur in the airways in response to infectious agents as well as noxious stimuli such as cigarette smoke. An increase in the number of cells of monocyte/macrophage phenotype is a feature of bronchial biopsies of smokers with airflow limitation (24). Mechanisms regulating interactions between monocytes and airway epithelial cells are not clearly defined, but airway epithelial cells are known to release chemotactic factors for monocytes and to support the adhesion of monocytes (19, 20, 29). Human airway epithelial cells have been shown to express several mediators capable of influencing monocyte/macrophage activity, including RANTES (regulated on activation, normal T cell expressed and presumably secreted), monocyte chemotactic protein-1, and macrophage inflammatory protein-1alpha (23). Furthermore, Robbins et al. (29) observed that stimulating bovine bronchial epithelial cells with bacterial lipopolysaccharide or cigarette smoke extract augmented the adherence of peripheral blood monocytes to epithelial cell monolayers in vitro. Thus it is likely that monocyte/macrophage interactions with epithelial cells of the airway are modulated by a variety of mediators.

In patients, the airway epithelium is frequently exposed to inhaled beta -adrenergic agonists, which are capable of modulating epithelial cell cAMP-dependent pathways. Several airway epithelial cell functions are known to be influenced by cAMP, including ciliary beat frequency, cystic fibrosis transmembrane regulation, endotoxin-induced cytotoxicity, and nitric oxide release (19, 35, 37-39). The major cellular receptor for cAMP is cAMP-dependent protein kinase A (PKA). Wyatt et al. (43) recently demonstrated PKA, as well as protein kinase G (cGMP-dependent protein kinase), in bronchial epithelial cells and correlated cyclic nucleotide kinase activation with epithelial cell function, specifically ciliary beat frequency in vitro. However, little is known regarding PKA modulation of cell-cell interactions of the airway, such as epithelial cell-monocyte interactions. cAMP-dependent processes have been shown to influence other types of cell-cell interactions, although the influence of cAMP is dependent on the cell types involved (3, 17, 25).

In this work, we investigated the ability of two beta -adrenergic agonists, isoproterenol and salbutamol, to influence adhesion of the human monocyte/macrophage cell line THP-1 to human bronchial epithelial cells (HBECs) in vitro. We observed that HBEC exposure to beta -adrenergic agonists augments subsequent THP-1 cell adhesion to epithelial cells in a concentration- and time-dependent fashion. This augmentation of adhesion is inhibited by HBEC exposure to a specific PKA inhibitor, KT-5720, suggesting that monocyte adhesion to airway epithelial cells is, at least in part, a cAMP-dependent process.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells. THP-1 cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 (Life Technologies, Grand Island, NY) with 2-mercaptoethanol (2 × 10-5 M) and 10% fetal bovine serum (Biofluids, Rockville, MD).

HBECs were obtained from one of three sources. We utilized bronchoscopy brushings (~6-8 brushes in 3-4 locations) of patients undergoing bronchoscopy for clinical reasons after we obtained informed consent and with the approval of the Institutional Review Board of the University of Nebraska Medical Center and the Human Studies Subcommittee of the Research and Development Committee of the Omaha Veterans Affairs Medical Center. Cells were processed using the technique of Kelsen et al. (15). Cells were passaged no more than seven times before use in experiments. By use of a cytokeratin stain, cells were found to be 95-98% epithelial. We also used HBECs obtained using an explant technique as previously described (1) from an autopsy specimen of a person without lung disease. Additionally, normal HBECs (NHBE 4263, Clonetics, San Diego, CA) were examined. HBECs from all sources were maintained in culture in serum-free medium at 37°C in 5% CO2-95% air. LHC-9-RPMI 1640 medium (1:1) was used to support the growth of these cells as previously described (1, 2). LHC-9 medium contains LHC basal medium (Biofluids), 0.5 µM phosphoethanolamine or ethanolamine (Sigma, St. Louis, MO), 0.11 mM calcium (Fisher, Springfield, NJ), 50 U/ml penicillin and streptomycin (Life Technologies), 2 µg/ml amphotericin B (Fungizone, Life Technologies), trace elements, 5 µg/ml bovine insulin (Sigma), 5 ng/ml epidermal growth factor (Sigma), 10 µg/ml bovine transferrin (Sigma), 10 nM 3,3',5-triiodothyronine (Biofluids), bovine pituitary extract (50 µg protein/ml; Pel Freeze, Rogers, AR), 0.2 µM hydrocortisone (Biofluids), 0.5 µg/ml epinephrine (Sigma), and 0.1 µg/ml retinoic acid (Sigma). Epinephrine was removed from the medium immediately before HBECs were plated for use in the adhesion assays. During exposure to beta -adrenergic agonists or the PKA inhibitor, HBECs were placed in LHC-D, a growth factor-deficient medium, which contains LHC basal medium, 0.5 µM phosphoethanolamine or ethanolamine, 0.11 mM calcium, penicillin and streptomycin, amphotericin B, and trace elements.

Cell adhesion assay. HBECs were grown to confluence in 96-well tissue culture plates, black with clear bottoms (Costar, Cambridge, MA). Medium was changed to growth factor-deficient medium (1:1 LHC-D-RPMI 1640) with agents to be tested. HBEC monolayers were rinsed before initiation of the binding assay. THP-1 cells (0.6 × 106 cells/ml) that had been labeled with the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (Calbiochem, La Jolla, CA; 2 µg/ml for 15 min) were allowed to bind to the HBEC monolayers for 20 min. After incubation, wells were filled with RPMI 1640 medium, the plate was gently inverted, and nonadherent cells were sedimented. All wells were then gently washed with PBS. Attached cells were solubilized with 1% Triton X-100 in H2O. The cell lysates were evaluated by an automatic microfluorometer (Fluorolite 1000, Dynex Technologies, Chantilly, VA) at 490/530-nm excitation/emission wavelengths. In each experiment, HBECs without exposure to THP-1 cells were examined to evaluate autofluorescence of HBECs, and this value was subtracted from the fluorescence of THP-1 cells adherent to HBECs. A linear relationship existed between THP-1 cell number and amount of fluorescence measured when the number of THP-1 cells was 0.3 × 106 to 1.5 × 106 (data not shown).

To express the adhesion as percentage of THP-1 cells attached as opposed to mean fluorescence (see Fig. 5), we used the following formula: %adhesion = [fluorescence of experimental condition - background fluorescence (autofluorescence of HBECs)]/[fluorescence of THP-1 cells (same number used in the experimental conditions) in 1% Triton X-100 - background fluorescence] × 100.

Determination of cyclic nucleotide-dependent kinase activity. PKA activity was determined in diethylaminoethyl fractions as well as in crude whole cell fractions of bronchial epithelial cells. The assay is a modification of procedures previously described by Jiang et al. (12) using 130 µM PKA substrate heptapeptide (LRRASLG), 10 µM cAMP, 0.2 mM 3-isobutyl-1-methylxanthine, 20 mM magnesium acetate, and 0.2 mM [gamma -32P]ATP in a 40 mM Tris · HCl buffer (pH 7.5). Negative controls consisted of similar assay samples without the appropriate substrate peptide or cyclic nucleotide. A positive control of 0.4 ng/ml purified catalytic subunit from type I bovine PKA (Promega) was included as a sample. Kinase activity was expressed in relationship to total cellular protein assayed and calculated in picomoles per minute per milligram. The absolute kinase activity of HBECs from different sources and at different passages is somewhat variable, as has been demonstrated with other HBEC components (22a, 42). Therefore, we have expressed the PKA data as magnitude activation over baseline (unstimulated HBECs).

Reagents. Isoproterenol, salbutamol, and dibutyryl cAMP (DBcAMP) were obtained from Sigma, and KT-5720 was obtained from Calbiochem.

Statistical evaluation. Values are means ± SE. Experimental values were compared using a one-way ANOVA for repeated measures.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THP-1 adherence to HBEC monolayers is augmented by HBEC exposure to beta -adrenergic agonists and DBcAMP. To examine whether exposure of airway epithelial cells to beta -adrenergic agonists influences subsequent monocyte binding to epithelial cells, HBECs were initially pretreated with various concentrations of isoproterenol (1 µM-1 mM) or salbutamol (10-100 µM) for 24 h before assessment of THP-1 cell adhesion to confluent HBEC monolayers. Media containing beta -adrenergic agonists were removed, and HBECs were rinsed before fluorescently labeled THP-1 cells were allowed to adhere to the HBECs for 20 min. As shown in Fig. 1, pretreatment with isoproterenol (10 µM) or salbutamol (100 µM) for 24 h increased the subsequent binding of THP-1 cells to HBECs by ~40% (percent increase in adhesion compared with THP-1 adhesion to unstimulated control HBECs = 100%: 137 ± 6.5% for 10 µM isoproterenol, 117 ± 6.5% for 100 µM isoproterenol, 127 ± 4.6% for 10 µM salbutamol, and 137 ± 2.5% for 100 µM salbutamol, means ± SE, n = 6, P < 0.005, by ANOVA, for 10 µM isoproterenol and 100 µM salbutamol). In repeat experiments with various HBECs, 10 µM isoproterenol and 100 µM salbutamol consistently demonstrated a maximal effect in terms of augmenting THP-1 cell adhesion to HBECs.


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  		Fig. 1.   THP-1 cell adhesion to cultured human bronchial epithelial cells (HBECs) pretreated with various concentrations of isoproterenol (Iso) or salbutamol (Sal). HBECs were plated on type I collagen in serum-free medium without epinephrine on 96-well plates. Confluent monolayers of HBECs were established by the following day. HBECs were then pretreated with 10 or 100 µM isoproterenol or salbutamol for 24 h in serum-free, growth factor-deficient medium. Medium was removed, and HBEC monolayers were rinsed. Labeled THP-1 cells were allowed to adhere to HBECs for 20 min. Nonadherent cells were removed, monolayers were rinsed with PBS, and attached cells were solubilized in 1% Triton X-100. Fluorescence was evaluated by automatic microfluorometer at 490/530-nm excitation/emission wavelength. Results are from a single experiment, representative of triplicate experiments; n = 6 for each condition. Vertical axis, percent change in adhesion, as measured by fluorescence (mean ± SE), compared with unstimulated control HBECs (THP-1 adhesion to unstimulated control HBECs = 100%); horizontal axis, experimental conditions. * P < 0.005 by ANOVA.

To ascertain that the beta -agonist stimulation of THP-1 adhesion was due primarily to an effect on the epithelial cells as opposed to the THP-1 cells, THP-1 cells (before labeling) were placed in medium containing 10 µM isoproterenol or 100 µM salbutamol for 30 min before adhesion to unstimulated HBECs and compared with HBECs pretreated with isoproterenol or salbutamol for 1 h before the adhesion assay. There was again an ~35-40% increase in THP-1 binding when HBECs were pretreated with beta -agonists (Fig. 2). However, there was no change in binding when only the THP-1 cells were exposed to the beta -agonists (percent increase in adhesion compared with unstimulated control HBECs = 100%: 134 ± 3.6% for 10 µM isoproterenol on HBECs, 134 ± 4.9% for 100 µM salbutamol on HBECs, 86 ± 1.9% for 10 µM isoproterenol with THP-1 cells, and 94 ± 4.0% for 100 µM salbutamol with THP-1 cells, n = 6, P < 0.0001, by ANOVA, for 10 µM isoproterenol and 100 µM salbutamol on HBECs).


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  		Fig. 2.   Pretreatment of HBECs or THP-1 cells with isoproterenol or salbutamol before THP-1 cell adhesion to cultured HBECs. After confluent monolayers of HBECs were established, HBECs only (for 1 h) or THP-1 cells only (for 30 min) were pretreated with 10 µM isoproterenol in serum-free, growth factor-deficient medium. Medium was removed, and HBEC monolayers were rinsed. Labeled THP-1 cells were allowed to adhere to HBECs for 20 min. Nonadherent cells were removed, monolayers were rinsed with PBS, and attached cells were solubilized in 1% Triton X-100. Fluorescence was evaluated by automatic microfluorometer. Results are from a single experiment, representative of duplicate experiments; n = 6 for each condition. Vertical axis, percent change in adhesion, as measured by fluorescence (mean ± SE) compared with unstimulated HBECs (control); horizontal axis, experimental conditions. * P < 0.0001 by ANOVA.

Because beta -adrenergic agonists are known to influence bronchial epithelial intracellular signals such as cAMP within a short time period, we determined the time course of isoproterenol pretreatment of HBECs on subsequent THP-1 cell adhesion. Pretreatment of HBECs with 10 µM isoproterenol for only 10 min resulted in a maximal increase in THP-1 cell adhesion to HBECs (Fig. 3A). There was a 56% increase in adhesion at 10 min, which declined with longer pretreatment time (percent increase in adhesion compared with unstimulated control HBECs = 100%: 157 ± 7.5, 142 ± 10.3, and 119 ± 3.0% for 10, 30, and 60 min of isoproterenol pretreatment, respectively, n = 6, P < 0.008, by ANOVA, for 10 and 30 min of isoproterenol pretreatment). Thus HBEC pretreatment with 10 µM isoproterenol for as little as 10 min enhances THP-1 adhesion. This effect diminishes with time, although a statistically significant augmentation of adhesion was still observed after 24 h of isoproterenol HBEC pretreatment (Fig. 1).


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  		Fig. 3.   THP-1 cell adhesion to HBECs pretreated with isoproterenol for various time periods. Confluent monolayers of HBECs were pretreated with 10 µM isoproterenol for various time periods in serum-free, growth factor-deficient medium. Medium was removed, and HBEC monolayers were rinsed. Labeled THP-1 cells were allowed to adhere to HBECs for 20 min. Nonadherent cells were removed, monolayers were rinsed with PBS, and attached cells were solubilized in 1% Triton X-100. Fluorescence was evaluated by automatic microfluorometer. Results are from a single experiment, representative of triplicate experiments; n = 6 for each condition. A: percent change in adhesion, as measured by fluorescence, compared with adhesion to unstimulated control HBECs plotted against time of isoproterenol pretreatment of HBECs before adhesion assay. Values are means ± SE. * P < 0.008 by ANOVA. B: protein kinase A (PKA) activity in HBECs cultured as in adhesion assay and exposed to 10 µM isoproterenol for same time periods. Kinase activity was measured as described in MATERIALS AND METHODS and expressed as multiple of increase in PKA activation compared with PKA activation in unstimulated HBECs (vertical axis). Values are means ± SE. P equal 0.001, isoproterenol-treated vs. unstimulated cells.

The PKA activity of HBECs pretreated with 10 µM isoproterenol was also measured to ascertain that HBEC PKA activity was enhanced by isoproterenol exposure (Fig. 3B). Pretreatment with 10 µM isoproterenol resulted in a threefold increase in HBEC PKA activity at 10, 30, and 60 min (P equal 0.001 for each time point compared with unstimulated HBECs, by Student's t-test).

beta -Adrenergic agonists such as isoproterenol and salbutamol are known to influence a variety of intracellular signals including cAMP. To examine the role of cAMP, HBECs were pretreated with DBcAMP (10 pM-10 µM) for 10 min before THP-1 cells were allowed to adhere to HBEC monolayers. As shown in Fig. 4A, there was an approximately threefold increase in THP-1 adhesion to HBECs when HBECs were pretreated with 100 nM DBcAMP for 10 min (percent increase in adhesion compared with unstimulated control HBECs = 100%: 138 ± 3.5, 288 ± 10.5, 295 ± 10, 332 ± 7.6, and 279 ± 5.7% for 10 pM, 100 pM, 1 nM, 100 nM, and 10 µM DBcAMP pretreatment, respectively, n = 6, P < 0.0001, by ANOVA, for 100 pM, 1 nM, 100 nM, and 10 µM DBcAMP). Thus direct augmentation of HBEC cAMP activity with DBcAMP pretreatment was associated with a significant augmentation of THP-1 cell adhesion to bronchial epithelial cells. The fluorescence of THP-1 cells adherent to unstimulated HBEC monolayers was found to represent ~10-30% THP-1 cell adhesion depending on the various HBECs utilized. An example of actual percentage of THP-1 cells adherent to HBECs exposed to DBcAMP is shown in Fig. 5,with a twofold increase in percent adherent THP-1 cells to HBECs pretreated with 100 nM DBcAMP for 10 min (10.7 ± 0.85% THP-1 cell adhesion for unstimulated control HBECs and 14.5 ± 0.76 and 21.7 ± 2.0% THP-1 cell adhesion for HBECs pretreated with 10 pM and 100 nM DBcAMP, respectively, P equal 0.002, by ANOVA).


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  		Fig. 4.   THP-1 cell adhesion to HBECs pretreated with dibutyryl cAMP (DBcAMP). Confluent monolayers of HBECs were pretreated with various concentrations of cAMP analog DBcAMP for 10 min before adhesion assay with fluorescently labeled THP-1 cells. Results are from a single experiment, representative of triplicate experiments; n = 6 for each condition. A: percent change in adhesion, as measured by fluorescence, compared with adhesion to unstimulated control HBECs plotted against log molar concentrations of DBcAMP used in pretreatment of HBECs before adhesion assay. Values are means ± SE. * P < 0.0001 by ANOVA. B: PKA activity in HBECs cultured as in adhesion assay and exposed to various concentrations of DBcAMP. Kinase activity is expressed as multiple of increase in PKA activation compared with PKA activation in unstimulated HBECs (vertical axis). Values are means ± SE.



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  		Fig. 5.   THP-1 cell adhesion to HBECs pretreated with DBcAMP. Experiment was performed as described in Fig. 4A legend, with confluent monolayers of HBECs pretreated with 2 concentrations of DBcAMP for 10 min before adhesion assay with fluorescently labeled THP-1 cells. Results are from a single experiment, representative of duplicate experiments; n = 6 for each condition. Vertical axis, actual percentage of THP-1 cells adherent to HBEC monolayers, either unstimulated (0 DBcAMP) or exposed to 10-11 or 10-7 M DBcAMP. * P equal 0.002 by ANOVA.

Assay of the PKA activity of HBECs pretreated with DBcAMP demonstrated that the PKA activity increased with increasing concentrations of DBcAMP (Fig. 4B). Small increases in PKA activation (<2-fold) were observed between 10 pM and 1 nM, and maximal PKA activity levels were observed at 100 nM to 10 µM pretreatment.

Inhibition of isoproterenol-stimulated THP-1 adhesion to HBECs by PKA inhibitor KT-5720. To evaluate the role of bronchial epithelial cell cAMP-dependent PKA activity in modulating the ability of HBECs to support THP-1 adhesion, HBECs were pretreated with a selective and potent inhibitor of PKA, KT-5720, before adhesion studies were performed. Confluent monolayers of HBECs were pretreated with 10 µM KT-5720 for 2 h. HBECs were then exposed to 10 µM isoproterenol for 1 h. Media were removed, HBECs were rinsed, and adhesion assay was performed with fluorescently labeled THP-1 cells. As seen in Fig. 6A, HBECs treated with 10 µM isoproterenol alone (no KT-5270 pretreatment) supported THP-1 adhesion that was 170 ± 6% compared with that in unstimulated HBECs (THP-1 adhesion to unstimulated HBECs = 100%). Pretreatment of HBECs with 10 µM KT-5720 for 2 h before isoproterenol stimulation resulted in a reduction of THP-1 adhesion to 53 ± 9% of unstimulated cells. THP-1 adhesion to HBECs pretreated with 10 µM KT-5720 and then exposed to 10 µM isoproterenol was 71 ± 4% compared with adhesion to unstimulated HBECs (P < 0.0001 for both comparisons by ANOVA). Thus HBEC exposure to the PKA inhibitor KT-5720 reduced THP-1 cell adhesion to isoproterenol-stimulated bronchial epithelial cells. In addition, measurement of PKA activity of HBECs confirmed that exposure to 10 µM KT-5270 inhibits intracellular HBEC PKA activity (Fig. 6B).


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  		Fig. 6.   Effect of PKA inhibitor KT-5720 on HBECs. A: inhibition of THP-1 cell adhesion to isoproterenol-pretreated HBECs by PKA inhibitor KT-5720. Confluent monolayer cultures of HBECs were pretreated with 10 µM KT-5720 for 2 h. Medium was removed, and HBECs were exposed to 10 µM isoproterenol for 1 h. Adhesion assay with fluorescently labeled THP-1 cells was performed. Vertical axis, percentage of control THP-1 adhesion to HBECs (not exposed to KT-5720 or isoproterenol); horizontal axis, various conditions. * P < 0. 0001 by ANOVA. B: inhibition of PKA activity in HBECs pretreated with KT-5720. Confluent monolayer cultures of HBECs maintained in serum-free medium were pretreated with various concentrations of KT-5720 and subsequently exposed to 10 µM isoproterenol or no isoproterenol for 1 h. Kinase activity is expressed as multiple of increase in PKA activation compared with unstimulated control HBECs.

HBEC isoproterenol exposure influences soluble mediators of subsequent THP-1 adhesion to bronchial epithelial cell monolayers. Exposure of HBECs to isoproterenol may influence a variety of epithelial cell processes, such as mediator release or cell surface changes, that could result in augmented THP-1 cell adhesion to bronchial epithelial cells. To assess whether isoproterenol-stimulated mediator release was involved, we used conditioned media from HBECs exposed to 10 µM isoproterenol for 10 min or 1 h to stimulate additional HBEC monolayers or THP-1 cells for 20 min before assessment of THP-1 adhesion to epithelial cells. As seen in Table 1, HBEC exposure to conditioned media from isoproterenol-stimulated HBECs resulted in statistically significant THP-1 cell adhesion only when conditioned medium from HBECs exposed to isoproterenol for 1 h was utilized. Exposure of THP-1 cells only to conditioned medium from isoproterenol-stimulated HBECs did not augment THP-1 cell adhesion. Similar to our results in Fig. 2, exposure of THP-1 cells only to 10 µM isoproterenol for 10 min or 1 h did not influence THP-1 adhesion. This suggests that isoproterenol stimulation of HBECs for 1 h may influence soluble mediator(s) release capable of influencing subsequent THP-1 cell adhesion, whereas the shorter isoproterenol exposure of 10 min did not cause enough change in the conditioned media and possible mediator release to influence THP-1 binding. However, 10 min of isoproterenol exposure directly to HBECs is sufficient to augment THP-1 adhesion (Fig. 3).

                              
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  		Table 1.   Effect of conditioned media from HBECs exposed to isoproterenol on subsequent THP-1 cell adhesion to HBECs


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

beta -Adrenergic agonists are widely used in the treatment of obstructive lung diseases because of their ability to rapidly enhance bronchodilation. Airway epithelial cells are among the first cells of the airway to encounter inhaled beta -adrenergic agonists, and it is increasingly recognized that a wide variety of epithelial cell functions may be modulated by these agents (8, 18, 30, 35, 37-39, 44). In this report, we have demonstrated that exposure to beta -adrenergic agonists, specifically isoproterenol and salbutamol, augments the capacity of HBECs in vitro to support the adhesion of THP-1 cells, a monocyte/macrophage cell line. Furthermore, inhibition of HBEC PKA activity with KT-5720 blocked the isoproterenol augmentation of THP-1 cell adhesion, supporting the role of cAMP-dependent protein kinase activity in modulating monocyte/macrophage cell adhesion to airway epithelial cells.

Human airway epithelial cells in vitro and in vivo express beta -adrenergic receptors, which modulate intracellular cAMP (16, 36). The regulation of airway epithelial cell beta -adrenergic receptors has been described (26). Utilizing a transformed human airway epithelial cell line, Kelsen et al. (14) recently observed that maintained exposure (24 h) to isoproterenol, forskolin, and DBcAMP results in desensitization of the cAMP response to isoproterenol, whereas only isoproterenol causes significant beta -adrenergic receptor downregulation. In addition, HBECs obtained via a variety of techniques have been shown to synthesize cAMP in vitro, and this synthesis is modulated by inflammatory mediators such as interleukin-1beta (4, 13, 27). Thus regulation of airway epithelial cell cAMP is complex, and cAMP-dependent cellular processes are modulated by a variety of endogenous as well as exogenous substances.

Using bronchial epithelial cells obtained from different sources, we observed an increase in THP-1 cell adhesion to isoproterenol-stimulated HBECs. Specifically, we utilized HBECs that we prepared from an autopsy specimen as well as cells obtained from brushings at bronchoscopies, as previously published (1, 30). In addition, we utilized commercially available normal primary HBECs. HBECs from all sources were passaged to obtain the number of cells required to perform multiple experiments. Despite different patient sources, techniques in obtaining cells, and number of passages, we routinely observed a 40-60% augmentation of THP-1 cell adhesion to isoproterenol-exposed HBECs.

Cells of the monocyte/macrophage phenotype are associated with airway inflammation and disease. Recently, O'Shaughnessy et al. (24) examined bronchial biopsies of normal nonsmoking subjects as well as patients with chronic bronchitis with and without airflow limitation and noted an increase in CD68+ (monocyte/macrophage phenotype)-staining cells in smokers with airflow limitation. Similarly, increases in the number of macrophages have also been observed in the bronchioles of smokers with airflow limitation (6). Saetta et al. (31) reported an increase in the number of macrophages in the bronchial glands of airway tissue from smokers with chronic bronchitis but not in the epithelium and submucosa. These observations support the importance of macrophages within the airway wall in participating in inflammatory processes that appear to lead to airway remodeling and clinically significant airflow obstruction (10, 11). Macrophages within the airway epithelium may be derived directly from blood monocytes migrating into airway tissue or from monocytes migrating into alveolar tissue, which differentiate into alveolar macrophages and then migrate from the alveolar space to the airways. We utilized the monocyte THP-1 cell line in our studies because it provided a uniform population of monocyte/macrophage cells in which we could examine the ability to adhere to bronchial epithelial cells. THP-1 cells and peripheral blood monocytes have demonstrated several similar properties (22, 34).

We have demonstrated that epithelial cell PKA modulates the adhesion of THP-1 cells to HBECs in that stimulation of HBECs with isoproterenol and DBcAMP is associated with an augmentation of adhesion, and this augmentation is inhibited by HBEC pretreatment with a specific PKA inhibitor, KT-5720. In addition, pretreatment of THP-1 cells only with isoproterenol did not modulate subsequent THP-1 adhesion to HBECs (Fig. 2, Table 1), although exposure to isoproterenol did increase PKA activity of THP-1 cells (data not shown). This also supports the importance of increases in epithelial cell PKA activity in modulating THP-1 cell adhesion to HBECs. We have not yet identified the mechanism(s) by which PKA mediates monocyte adhesion to bronchial epithelial cells. Our data suggest that PKA may influence more than one aspect of the adhesion process. Isoproterenol treatment of HBECs for 1 h, but not for 10 min, appears to alter soluble mediator(s) release capable of enhancing subsequent THP-1 adhesion in that exposure of HBEC supernatants to isoproterenol for 1 h augmented subsequent THP-1 adhesion (Table 1). Such mediator(s) release may be responsible for the augmentation of THP-1 adhesion that we observed after >= 1 h of HBEC pretreatment with isoproterenol (Figs. 1 and 3). The absence of an effect of the supernatants from 10 min of isoproterenol HBEC pretreatment on subsequent THP-1 adhesion (Table 1) suggests that putative soluble mediator(s) release does not occur instantaneously or at least not at a concentration great enough to influence adhesion appreciably. Thus the increase in adhesion we observed after only 10 min of isoproterenol or DBcAMP exposure of HBECs (Figs. 3 and 4) may be due to a targeted effect of PKA activation on receptors and/or ligands mediating THP-1 adhesion to the epithelial cells.

PKA activation clearly regulates a number of responses in the airway epithelium. For example, in the differentiated cell, the upregulation of the ciliary beating response is mediated via PKA activation (43). Because the role of protein kinases in the cell is potentially multifaceted, it is commonly accepted that protein kinase activation is orchestrated in a highly compartmentalized manner (5, 9, 28). Indeed, PKA has been shown to be targeted to specific anchoring proteins (AKAP) localized in distinct regions of the cell (7, 32). Recently, an AKAP has been reported to exist in the apical membrane surface for type II PKA in the human airway epithelial cell (33). Although we have demonstrated a significant amount of soluble type I PKA in the airway epithelial cell, the presence of a particulate membrane-associated type II PKA might explain the ability of a localized, albeit lower, PKA activation to produce significant epithelial cell-monocyte binding. This cell-cell binding might even be signaled by a compartmentalized PKA that is fully active before total cellular PKA activity levels as measured by cyclic nucleotide concentration or kinase activity. This would explain our observations that PKA remains activated longer than the observed increase in cell-cell binding (Fig. 3) and that levels of DBcAMP that appear to stimulate monocyte binding fail to fully activate PKA (Fig. 4). Inhibition of all PKA activity results in the abrogation of cell-cell binding in our model (Fig. 6). Although it would be advantageous to selectively activate type I or type II PKA, all known analogs of cAMP activate both isozymes, while some analogs, such as benzoyl-cAMP, are only partially (not fully) specific for type II PKA.

HBECs are known to release and express a variety of molecules that may influence monocyte/macrophage adhesion to epithelial cells as soluble mediators or as receptors/ligands. However, relatively little is known regarding how PKA modulates airway epithelial cell expression of such molecules. For example, bronchial epithelial cells in vitro have been shown to release monocyte chemotactic activity, which is, at least in part, due to leukotriene B4 (19, 20). More recently, human airway epithelial cells have been shown to express RANTES, monocyte chemotactic protein-1, and macrophage inflammatory protein-1alpha (23). Localized release of such chemokines may influence monocyte recruitment and subsequent adhesion to epithelial cells. In addition, airway epithelial cells express molecules such as intercellular adhesion molecule-1, which participates in epithelial cell adhesion to mononuclear cells (21, 40, 41). Depending on the type of cell involved, cAMP-dependent pathways have been shown to enhance and inhibit intercellular adhesion molecule-1 expression (3, 17, 25). We have not yet defined specific mediators or receptors/ligands that may be responsible for the PKA-mediated changes in THP-1 cell adhesion to HBECs.

In summary, we have demonstrated that monocyte adhesion to human airway epithelial cells in vitro is augmented via a cAMP-dependent pathway. This suggests that agents that target PKA within the airway epithelium may have potential utility in modulating monocyte/macrophage inflammatory responses, which may contribute to airway diseases such as chronic bronchitis.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the secretarial assistance of Sheryl Latenser and the technical assistance of Tara Wish.


    FOOTNOTES

Funding for this work was provided by the Department of Veterans Affairs Merit Review research program (D. J. Romberger).

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: D. J. Romberger, Dept. of Internal Medicine, Pulmonary and Critical Care Medicine, Box 985300 Nebraska Medical Center, Omaha, NE 68198-5300 (E-mail: dromberg{at}unmc.edu).

Received 21 December 1998; accepted in final form 12 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 278(1):L139-L147
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