Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, Nebraska Medical Center, Omaha, Nebraska 68198-5300
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ABSTRACT |
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-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
-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
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INTRODUCTION |
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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-1 (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
-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 -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
-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.
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MATERIALS AND METHODS |
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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 × 105 M) and 10% fetal
bovine serum (Biofluids, Rockville, MD).
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 conditionDetermination 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
[-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.
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RESULTS |
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THP-1 adherence to HBEC monolayers is augmented by HBEC
exposure to -adrenergic agonists and
DBcAMP.
To examine whether exposure of airway epithelial cells to
-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
-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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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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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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DISCUSSION |
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-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
-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
-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
-adrenergic receptors, which modulate intracellular cAMP (16, 36).
The regulation of airway epithelial cell
-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
-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-1
(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-1 (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.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the secretarial assistance of Sheryl Latenser and the technical assistance of Tara Wish.
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FOOTNOTES |
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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.
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