Laboratorio di Biologia Cellulare, Fisiopatologia Respiratoria, Dipartimento Cardiotoracico dell'Università di Pisa, 56124 Pisa, Italy
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ABSTRACT |
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Intercellular
adhesion molecule-1 (ICAM-1) is the only inducible adhesion receptor
for neutrophils identified in bronchial epithelial cells. We stimulated
human airway epithelial cells with various agonists to evaluate whether
ICAM-1-independent adhesion mechanisms could be elicited. Phorbol
12-myristate 13-acetate (PMA) stimulation of cells of the alveolar cell
line A549 caused a rapid, significant increase in neutrophil adhesion
from 11 ± 3 to 49 ± 7% (SE). A significant increase from 17 ± 4 to 39 ± 6% was also observed for neutrophil adhesion to
PMA-stimulated human bronchial epithelial cells in primary culture.
Although ICAM-1 expression was upregulated by PMA at late time points, it was not affected at 10 min when neutrophil adhesion was already clearly enhanced. Antibodies to ICAM-1 had no effect on neutrophil adhesion. In contrast, antibodies to the leukocyte integrin -chain CD18 totally inhibited the adhesion of neutrophils to PMA-stimulated epithelial cells. These results demonstrate that PMA stimulation of
human airway epithelial cells causes an increase in neutrophil adhesion
that is not dependent on ICAM-1 upregulation.
intercellular adhesion molecule-1; phorbol 12-myristate 13-acetate; lung; inflammation; 2-integrins
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INTRODUCTION |
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AIRWAY INFLAMMATION is a hallmark of numerous lung diseases including chronic bronchitis (21), asthma (16), pulmonary fibrosis (17), and adult respiratory distress syndrome (9). The recruitment of circulating inflammatory cells into the airways involves both cell-cell adhesion molecules and soluble mediators. Several steps have been recognized in this process. First, circulating leukocytes adhere to the inflamed endothelium; then they migrate across the vessel wall, crawl within the interstitial space, and eventually adhere to and migrate across the airway epithelium (16).
Four superfamilies of adhesion molecules involved in the adhesion of leukocytes to endothelial cells have been identified: selectins, mucinlike selectin receptors, integrins, and immunoglobulin (Ig)-like proteins (4). Integrins are also responsible for the migration of cells within the interstitial space (16).
The molecular basis of leukocyte adhesion to epithelial cells has also been extensively investigated. A member of the Ig-like superfamily, intercellular adhesion molecule-1 (ICAM-1), originally described in endothelial cells (3), is the only inducible adhesion receptor for neutrophils identified so far that is also synthesized by airway epithelial cells. Recently, the synthesis of vascular cell adhesion molecule-1 (VCAM-1), an endothelial, inducible receptor for lymphocytes and eosinophils belonging to the Ig superfamily, has also been demonstrated in cultured, cytokine-stimulated bronchial epithelial cells (2). It is noteworthy, however, that current knowledge on the adhesion mechanisms expressed by the inflamed respiratory epithelium is largely based on studies performed with antibodies or molecular probes specific for previously known molecules, usually of endothelial origin. It is conceivable that the repertoire of adhesion mechanisms expressed by the respiratory epithelium is broader and that new, epithelium-specific molecules will be identified.
We stimulated human airway epithelial cells with different agonists and demonstrated that the phorbol ester phorbol 12-myristate 13-acetate (PMA) leads to increased neutrophil adhesion via a mechanism independent of ICAM-1 upregulation.
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MATERIALS AND METHODS |
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Reagents. Dulbecco's modified
Eagle's medium (DMEM), Ham's F-12 medium, RPMI 1640 medium, fetal
bovine serum (FBS), penicillin, streptomycin,
L-glutamine, trypsin, soybean
trypsin inhibitor, trypan blue, formaldehyde, Ficoll-Hypaque,
o-phenylenediamine (OPD), PMA, bovine serum albumin,
insulin, ethanolamine, phosphorylethanolamine, transferrin, bovine
pituitary extract,
3,3',5-triiodo-L-thyronine, epidermal growth factor, hexadecyltrimethylammonium bromide,
dextran, anti-cytokeratin peptide-18 antibodies, FITC-conjugated
anti-mouse IgG antibodies, cycloheximide, actinomycin D, and DNase were
obtained from Sigma (Milan, Italy). Tumor necrosis factor- (TNF-
)
was purchased from Genzyme (Cambridge, MA). Vitrogen 100 was from Collagen (Palo Alto, CA). LHC basal medium and trace elements were
purchased from Biofluids (Rockville, MD). Hydrocortisone was from
Richter Pharmaceuticals (Milan, Italy). All other chemicals were
obtained from the hospital pharmacy and were of the best grade available.
Cells. The alveolar epithelial cell line of alveolar origin, A549, was obtained from American Type Culture Collection (ATCC; Manassas, VA). A549 cells were maintained in DMEM supplemented with 10% (vol/vol) FBS, 100 U/ml of penicillin, and 100 µg/ml of streptomycin (complete DMEM) in a humidified 95% air-5% CO2 atmosphere at 37°C. The murine fibroblast cell line L929 was also obtained from ATCC and cultured in complete DMEM.
Human bronchial epithelial cells (HBECs) were obtained from subjects undergoing diagnostic bronchoscopy according to the method of Kelsen et al. (13) with some modifications. Briefly, after the patient's informed consent to the procedure was received, the fiber-optic bronchoscope was positioned at the level of the carina and/or the level of second- or third-order bronchial branchings. The use of local anesthetics was kept as low as possible to minimize their effects on cell viability. Four to six brushings of grossly normal bronchial mucosa were regularly obtained. The cells were then removed from the brush by vortexing in Ham's F-12 medium-10% FBS. The cells were brought to the laboratory in ice and incubated with DNase (50 g/ml) to eliminate clumping. After a wash with ice-cold, serum-free Ham's F-12 medium, the cells were resuspended in LHC-9-RPMI 1640 medium (1:1) (15) and plated on Vitrogen 100-coated 35-mm culture dishes (Becton Dickinson, Franklin Lakes, NJ). Indirect immunofluorescence with anti-cytokeratin antibodies confirmed the epithelial origin of the cells. The cells were then cultured and passed as appropriate according to Lechner and LaVeck (15) except that LHC-9-RPMI 1640 medium was used instead of LHC-9 medium. All the experiments were performed with cells at passages 4-9.
Tests to rule out the presence of mycoplasmal contamination were not routinely performed.
Neutrophils were isolated from the peripheral blood of normal volunteers. Blood was anticoagulated with 0.1 volume of 130 mM sodium citrate and sedimented on 2% (wt/vol) dextran. The leukocyte-rich fraction was then washed and layered on Ficoll-Hypaque. After centrifugation at 400 g for 30 min, the pellet was subjected to hypotonic lysis for 30 s to remove contaminating red blood cells and resuspended in Hanks' balanced salt solution (HBSS) with 1 mM CaCl2 and 1 mM MgCl2 unless otherwise indicated. The resulting cells were consistently >95% viable as assessed by trypan blue exclusion and >95% pure.
Antibodies. The hybridoma secreting the anti-CD18 monoclonal antibody (MAb) TS1/18 was obtained from ATCC and cultured in DMEM supplemented with 10% FBS and 50 µg/ml of gentamicin. The IgG fraction was affinity purified from the supernatant on a protein A-Sepharose column (Pierce, Rockford, IL) according to the manufacturer's directions. The inhibitory MAb to ICAM-1, R6.5 (20), supplied as pure IgG, was a generous gift from Dr. R. Rothlein (Boehringer Ingelheim, Ridgefield, CT). R6.5 binds to an epitope on domain 2 of ICAM-1 (8). Another inhibitory MAb to ICAM-1, clone 84H10, directed to an epitope on domain 1 (8) was purchased as purified IgG from Immunotech (Marseille, France). The generation and characterization of rabbit antiserum to P-selectin and of the MAb to the same protein, AC1.2, have been previously described (14). The antibodies to CD11a (TS1/22; originally from ATCC), CD11b (LPM19c; originally from Dr. Karen Pulford, University of Oxford, Oxford, UK), CD11c (HC1.1; originally from Dr. Carmelo Bernabeu, Centro de Investigationes Biologicas, Madrid, Spain), and ICAM-2 (B-37) were kindly provided as pure IgG by Dr. Virgilio Evangelista (Consorzio Mario Negri Sud, S. Maria Imbaro, Chieti, Italy) (12).
Adhesion assay. A549 cells, L929 cells, or HBECs were plated onto 96-well plates (Greiner, Nürtingen, Germany) at 5 × 104 cells/well. For HBECs, the wells were coated with Vitrogen 100. When confluent, the cells were washed with medium and stimulated with different agonists as indicated. After the appropriate time, the cells were extensively washed, and neutrophils were added at 105 cells/well. After 30 min at 37°C in a humidified 5% CO2 atmosphere, the wells were gently washed three times with HBSS containing Ca2+ and Mg2+ with a multiple pipetter to add and remove the buffer, taking great care to preserve the integrity of the monolayers. At the end of the assay, the plates were checked for monolayer integrity by phase-contrast microscopy, and the occasional wells showing any degree of monolayer disruption were removed from the analysis. When antibodies to neutrophil integrins were used, they were added to the cell suspension 30 min before the adhesion assay and maintained throughout the test. R6.5 and 84H10 were incubated with the epithelial cell monolayers for 30 min and washed three times before the assay. However, experiments in which R6.5 was maintained in the reaction mixture gave similar results. Antibodies to P-selectin and ICAM-2 were also maintained throughout the test. In experiments designed to evaluate the role of different metal ions, neutrophils were resuspended in HBSS containing either Ca2+ only or Mg2+ only, and the cell monolayers were washed throughout the test with HBSS containing the same divalent ion. In some experiments, neutrophils were resuspended in HBSS containing Mn2+. Adherent neutrophils were treated with 0.1% (wt/vol) hexadecyltrimethylammonium bromide in 0.1 M sodium acetate, pH 4, to solubilize endogenous peroxidase, and the chromogenic substrate OPD [1 mg/ml in 50 mM sodium citrate, pH 5, containing 0.15% (vol/vol) H2O2] was added. The reaction was stopped with 2 M H2SO4 (50 µl/well), and absorbance was read at 492 nm with a Titertek Multiskan MCC ELISA reader (Flow Laboratories, McLean, VA). The percentage of adherent neutrophils was calculated by comparison with a standard curve obtained by adding known numbers of neutrophils to wells that were then processed exactly as the others.
ELISA for cell-associated ICAM-1 and P-selectin. Epithelial cells were plated and stimulated as described in Adhesion assay. After an extensive wash, the cells were fixed with 3.7% (vol/vol) formaldehyde at room temperature for 15 min. Nonspecific binding sites on the solid phase were blocked with 2% (wt/vol) bovine serum albumin, and the relevant primary antibody (R6.5 at 5 µg/ml and AC1.2 ascites diluted 1:1,000 for ICAM-1 and P-selectin detection, respectively) was added. After 2 h at 37°C the wells were washed, and a peroxidase-conjugate anti-mouse IgG (Sigma) was added at the concentration indicated by the manufacturer. After a 2-h incubation at 37°C followed by an extensive wash, the chromogenic substrate OPD was added. The reaction was stopped with 2 M H2SO4, and absorbance read at 492 nm.
Data presentation and statistical analysis. Data are means ± SE unless otherwise indicated. Differences between groups were tested for significance by paired t-test. Significance was set at P < 0.05.
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RESULTS |
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PMA increases neutrophil adhesion to bronchial and
alveolar epithelial cells. Under the experimental
conditions employed, ~5-10% of neutrophils adhere to
unstimulated A549 cells. Stimulation of A549 cells with PMA causes a
significant increase in neutrophil adhesion (Fig.
1A).
This effect is dose dependent and saturable, reaching a plateau at
~50 ng/ml of PMA (Fig. 1B). Figure
1C shows that the effect of PMA is
rapid, being nearly maximal within 10 min. Although baseline adhesion
of neutrophils to HBECs is slightly higher compared with that of A549
cells, PMA induces a further, significant increase in adhesion (Fig.
1D).
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Control experiments were performed with the fibroblast cell line L929. Binding of neutrophils to L929 cells was not affected by PMA stimulation of the latter (41 ± 4 and 41 ± 7% for unstimulated and PMA-stimulated L929 cells, respectively). In contrast, PMA-stimulation of neutrophils caused a significant increase in binding (61 ± 7%; P < 0.05).
Role of known adhesion molecules.
Airway epithelial cells of different origin express ICAM-1 on
stimulation with various agonists (3, 20). We therefore investigated
whether ICAM-1 was responsible for the increased adhesion induced by
PMA. As expected (3), PMA upregulated ICAM-1 expression by A549 cells
at 24 h as assessed by ELISA. However, ICAM-1 expression at 10 min was
not affected by PMA (Fig.
2A).
Furthermore, increased expression of ICAM-1 induced by TNF- was not
paralleled by increased neutrophil adhesion under the experimental
conditions described (Fig. 2B).
Similar results were obtained with interferon-
(data not shown).
These data suggest that ICAM-1, although upregulated by PMA under
appropriate conditions, is not responsible for the increased adhesion
induced by PMA. To investigate further the role of ICAM-1 in this
phenomenon, we used the inhibitory MAb to ICAM-1, R6.5. As shown in
Fig. 3, R6.5 at saturating concentrations
does not inhibit neutrophil adhesion to PMA-stimulated A549 cells
(A) and HBECs
(B). In control experiments, R6.5 at
similar concentrations inhibited, as expected, neutrophil adhesion to
interleukin-1-stimulated human umbilical vein endothelial cells (data
not shown). To confirm the results obtained with R6.5, we repeated the
experiments with another inhibitory anti-ICAM-1 antibody, 84H10. In a
series of four experiments, binding of neutrophils to A549 cells was 10 ± 4 and 32 ± 9% for unstimulated and PMA-stimulated
cells, respectively (P < 0.05). Binding to
PMA-stimulated cells in the presence of 84H10 (20 µg/ml) was 41 ± 13%.
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P-selectin is a member of the selectin family of cell-cell adhesion
molecules stored in the -granules of platelets and the Weibel-Palade
bodies of endothelial cells. On cell stimulation, P-selectin is rapidly
translocated to the cell membrane where it functions as a receptor for
leukocytes (4). Although the presence of P-selectin has never been
shown in other cell types, we investigated whether P-selectin could be
involved in the rapid increase in neutrophil adhesion on stimulation of
epithelial cells with PMA. As shown in Fig.
4, a blocking antiserum to P-selectin did
not inhibit neutrophil adhesion to PMA-stimulated epithelial cells.
Furthermore, P-selectin was not detectable in A549 cells, either
resting or PMA stimulated, by ELISA with AC1.2 as the detection antibody (data not shown).
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The leukocyte integrins CD11a/CD18 (lymphocyte function-associated
antigen-1) and CD11b/CD18 (Mac-1) function as ligands for adhesion
molecules, including ICAM-1, and have been implicated in adhesive
interactions between leukocytes and both endothelial and epithelial
cells (16, 19). We used an MAb (TS1/18) directed against the common
-chain CD18 to investigate the role of these integrins in the
adhesion of neutrophils to PMA-stimulated airway epithelial cells.
Figure 5A
shows that TS1/18 totally inhibits the adhesion of neutrophils to
PMA-stimulated A549 cells. Similar results were obtained with HBECs
(Fig. 5B). We further characterized the role of
2-integrins by adding
inhibitory antibodies to CD11a, CD11b, and CD11c to the neutrophil
suspension. The results of these experiments for A549 cells and HBECs
are shown in Fig. 6, A and
B, respectively. Antibodies to CD11b
totally blocked the adhesion to both PMA-stimulated A549 cells and
HBECs. Antibodies to CD11a and CD11c inhibited adhesion to a much
smaller degree.
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ICAM-2 is an adhesion receptor for
2-integrins expressed by
bronchial epithelial cells (2). We investigated the role of ICAM-2 in
the increased adhesion of neutrophils to PMA-stimulated cells. As shown
in Fig. 7, an inhibitory monoclonal
antibody to ICAM-2 (B-37) did not inhibit neutrophil adhesion in our
system.
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Metal ions, metabolic energy, and protein synthesis
dependence of neutrophil adhesion to PMA-stimulated epithelial
cells. To further characterize the interaction between
neutrophils and PMA-stimulated epithelial cells at the molecular level,
we investigated the role of different divalent cations and the
requirement for metabolic energy and de novo protein synthesis. All
adhesion experiments shown so far were performed in the presence of
both Ca2+ and
Mg2+. Figure
8A shows
the binding of neutrophils to PMA-stimulated A549 cells in the presence
of Ca2+ and
Mg2+,
Ca2+ only, and
Mg2+ only. Because metal ions are
required for A549 cells to bind to the plastic growth surface, we could
not perform experiments in the absence of metal ions or in the presence
of EDTA. The binding of neutrophils to PMA-stimulated A549 cells was
optimal in the presence of Mg2+
regardless of the availability of
Ca2+.
Ca2+ alone was not sufficient to
support the binding. In contrast, binding of neutrophils to the plastic
growth surface in the absence of epithelial cells was optimal in the
presence of either metal ion (data not shown).
Mn2+ upregulates CD18-dependent
adhesive interactions by increasing the binding affinity of
2-integrins (10). We performed
experiments in the presence of
Mn2+ to confirm the role of
2-integrins in our system. As
shown in Fig. 8B,
Mn2+ greatly enhances binding of
neutrophils to alveolar cells both in resting conditions and upon
stimulation with PMA.
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The requirement for metabolic energy was investigated both by lowering
the binding temperature to 4°C and by adding
NaN3 to the neutrophil suspension
during the incubation. As shown in Fig. 9A, the
binding of neutrophils to PMA-stimulated A549 cells at 4°C was low.
The binding was also blocked by the metabolic inhibitor NaN3 (Fig.
9B). To confirm that de novo protein
synthesis is not required for increased adhesion, experiments were
performed in the presence of cycloheximide and actinomycin D. Figure
9C shows that neither cycloheximide
nor actinomycin D blocked the increased binding of neutrophils to A549
cells stimulated with PMA for 24 h. Similar results were obtained with
cells stimulated for 15 min (data not shown). In parallel experiments,
both reagents inhibited, as expected, the synthesis of ICAM-1 by A549
cells stimulated with TNF- as assessed by ELISA (data not shown).
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DISCUSSION |
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The molecular mechanisms of leukocyte recruitment into the airways have been extensively studied in the past decade. A number of cell-cell adhesion molecules and soluble mediators that are involved in the process have been identified (4, 19). However, although several adhesion receptors of endothelial origin have been recognized, ICAM-1 is currently the only known inducible receptor for neutrophils expressed by airway epithelia. The aim of this work was to investigate whether stimulation of such cells with different agonists would cause an increase in neutrophil adhesion independent of ICAM-1 upregulation.
Several lines of evidence suggest that, indeed, increased adhesion of
neutrophils to human airway epithelial cells on incubation with the
phorbol ester PMA is not dependent on ICAM-1 upregulation. First, the
phenomenon is rapid, being nearly complete after 10 min. ICAM-1
expression requires de novo protein synthesis and therefore takes
several hours (20). Accordingly, on PMA stimulation, ICAM-1 expression
is increased at 24 h, but it is unaffected at early time points (e.g.,
10 min). Furthermore, two inhibitory MAbs to ICAM-1 have no effect on
adhesion. Finally, we showed that other agonists capable of
upregulating ICAM-1 expression, such as TNF-, do not affect
neutrophil adhesion to alveolar cells. Although this observation, which
has been preliminarily reported (5), lends support to the hypothesis
that ICAM-1 upregulation is not responsible for increased adhesion in
the presence of PMA, at present we have no explanation for it. The
integrin receptor CD11/CD18 must be activated and undergo a
conformational change to bind ICAM-1. It is possible that in the
absence of such activation, leukocytes cannot bind to ICAM-1-bearing
cells due to the absence of a functional receptor. In vivo this would
be irrelevant because leukocytes must be exposed to chemotactic factors
and therefore be activated before they reach the epithelial barrier.
However, preliminary data indicate that activation of neutrophils with the chemotactic peptide
N-formyl-methionyl-leucyl-phenylalanine does not increase adhesion to ICAM-1-bearing alveolar epithelial cells
(Celi, unpublished data). Interestingly, Cunningham and Kirby (7) have shown that stimulation of human alveolar
cells with interferon-
, although upregulating ICAM-1 expression, did not influence the adhesion of phytohemoagglutinin-activated lymphocytes.
Experiments with inhibitory MAbs have shown that whatever adhesion molecule is expressed by PMA-stimulated airway epithelial cells, its interaction with neutrophils is mediated by the integrin CD11/CD18. Beside ICAM-1, CD11/CD18 binds to other receptors expressed in different conditions. The first Mac-1 ligand to be identified was iC3b, a complement C3 fragment (1). A role for iC3b in binding of neutrophils to tracheal epithelial cells pretreated with human serum has been previously demonstrated (24). However, metal ion requirements for the binding of Mac-1 to iC3b appear to be different from those for the adhesion of neutrophils to PMA-stimulated epithelial cells because the latter is strictly dependent on the presence of Mg2+, whereas either Ca2+ or Mg2+ is sufficient for iC3b binding to Mac-1 (11, 24). Other ligands for CD11/CD18 include coagulation factor X and fibrinogen. Binding to these receptors has been shown to require Ca2+ but not Mg2+ (1). CD11/CD18 also bears recognition sites for polysaccharides (1). Interestingly, a role for cell surface carbohydrates in neutrophil adhesion to alveolar epithelial cells of different origin, including A549 cells, has been demonstrated (6), although the exact molecular species involved has never been fully characterized. It is possible that carbohydrate residues are involved in the increased binding of neutrophils to airway epithelial cells in our conditions.
Finally, another member of the Ig-like superfamily of adhesion
molecules, ICAM-2, serves as a receptor for
2-integrins (4). ICAM-2 is
expressed by both endothelial (4) and epithelial (2) cells. However,
ICAM-2 is constitutively synthesized, and its expression is not
upregulated by cell activation (4). Accordingly, an inhibitory antibody
to ICAM-2 did not affect neutrophil adhesion to PMA-activated cells.
Robbins et al. (18) have previously shown that PMA, as well as other agonists, upregulate neutrophil and mononuclear cell adhesion to bovine bronchial epithelial cells. However, the molecular bases of adhesion of leukocytes to PMA-stimulated epithelial cells of bovine origin are not necessarily identical to those described for human cells in the present report. First, the authors did not specifically address the issue of the dependence of the phenomenon on ICAM-1. Furthermore, an MAb to CD18 did not affect adhesion in their conditions (18).
Exposure of human airway epithelial cells to ozone has been shown to increase neutrophil adhesion in a CD11/CD18-dependent, ICAM-1-independent manner (22). Although the adhesion receptor expressed under these conditions has not been characterized, time-course experiments show that increased adhesion takes several hours and is maximal 24 h after ozone exposure, suggesting that different mechanisms, likely to require de novo protein synthesis, are involved. Similar considerations apply to the increased adhesion of leukocytes to parainfluenza virus-infected airway epithelial cells reported by the same group (23).
PMA activation of neutrophils increases their adhesion to airway epithelial cells (18). Thus it could be conceivable that small amounts of PMA that remain bound to the cell membranes are responsible for the direct activation of neutrophils and therefore for their increased binding. The observation that PMA stimulation of L929 cells has no effect on neutrophil adhesion rules out this possibility.
Taken together, our data demonstrate that PMA activation of airway epithelial cells causes a rapid increase in neutrophil adhesion through an as yet unknown CD11/CD18 receptor. The interaction is dependent on Mg2+ and requires metabolic energy. A better characterization of this phenomenon at the molecular level, currently underway, should shed new light into the basic mechanisms of lung inflammation.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. Rothlein for the generous gift of the anti-intercellular adhesion molecule (ICAM)-1 antibody R6.5 and Dr. G. Evangelista for kindly providing purified antibodies to CD11a, CD11b, CD11c, and ICAM-2.
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FOOTNOTES |
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This work was supported in part with grants from the Italian Ministry of the University and of Scientific and Technologic Research.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Celi, U. O. Fisiopatologia Respiratoria, Dipartimento Cardiotoracico, Via Paradisa 2, 56124 Pisa, Italy (E-mail: aceli{at}dcap.med.unipi.it).
Received 23 November 1998; accepted in final form 17 May 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arnaout, M. A.
Structure and function of the leukocyte adhesion molecule CD11/CD18.
Blood
75:
1037-1050,
1990[Medline].
2.
Atsuta, J.,
S. A. Sterbinsky,
J. Plitt,
L. M. Schwiebert,
B. B. Bochner,
and
R. P. Schleimer.
Phenotyping and cytokine regulation of the BEAS-2B human bronchial epithelial cell: demonstration of inducible expression of the adhesion molecules VCAM-1 and ICAM-1.
Am. J. Respir. Cell Mol. Biol.
17:
571-582,
1997
3.
Bloemen, P. G. M.,
M. C. van den Tweel,
P. A. J. Henricks,
F. Engels,
S. S. Wagenaar,
A. A. J. J. L. Rutten,
and
F. P. Nijkamp.
Expression and modulation of adhesion molecules on human bronchial epithelial cells.
Am. J. Respir. Cell Mol. Biol.
9:
586-593,
1993[Medline].
4.
Celi, A.,
R. Lorenzet,
B. Furie,
and
B. C. Furie.
Platelet-leukocyte-endothelial cell interaction on the blood vessel wall.
Semin. Hematol.
4:
1-10,
1997.
5.
Celi, A.,
S. Petruzzelli,
S. Cianchetti,
L. Valdisserri,
and
C. Giuntini.
Interleukin-1 (IL-1
) and interferon-
(IFN-
) upregulate ICAM-1 expression but not adhesiveness for neutrophils in the human alveolar type II cell line, A5F9 (Abstract).
Eur. Respir. J.
8, Suppl. 19:
548s,
1995.
6.
Crestani, B.,
C. Rolland,
A. Petiet,
N. Colas-Linhart,
and
M. Aubier.
Cell surface carbohydrates modulate neutrophil adherence to alveolar type II cells in vitro.
Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8):
L391-L400,
1993
7.
Cunningham, A. C.,
and
J. A. Kirby.
Regulation and function of adhesion molecule expression by human alveolar epithelial cells.
Immunology
86:
279-286,
1995[Medline].
8.
Diamond, M. S.,
D. E. Staunton,
S. D. Marlin,
and
T. Springer.
Binding of the integrin Mac-1 (CD11b/CD18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosilation.
Cell
65:
961-971,
1991[Medline].
9.
Donnelly, S. C.,
C. Haslett,
I. Dransfield,
C. E. Robertson,
D. C. Carter,
J. A. Ross,
I. S. Grant,
and
T. F. Tedder.
Role of selectins in development of adult respiratory distress syndrome.
Lancet
344:
215-219,
1994[Medline].
10.
Dransfield, I.,
C. Cabanas,
A. Craig,
and
N. Hogg.
Divalent cation regulation of the function of the leukocyte integrin LFA-1.
J. Cell Biol.
116:
219-226,
1992[Abstract].
11.
Dransfield, I.,
and
N. Hogg.
Regulated expression of Mg2+ binding epitope on leukocyte integrin subunit.
EMBO J.
8:
3759-3765,
1989[Abstract].
12.
Evangelista, V.,
S. Manarini,
R. Sideri,
S. Rotondo,
N. Martelli,
A. Piccoli,
L. Totani,
P. Piccardoni,
D. Vestweber,
G. de Gaetano,
and
C. Cerletti.
Platelet/polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorilation-dependent CD11b/CD18 adhesion: role of PSGL-1 as a signaling molecule.
Blood
93:
876-885,
1999
13.
Kelsen, S. J.,
I. A. Mardini,
S. Zhou,
J. L. Benovic,
and
N. C. Higgins.
A technique to harvest viable tracheobronchial epithelial cells from living human donors.
Am. J. Respir. Cell Mol. Biol.
7:
66-72,
1992[Medline].
14.
Larsen, E.,
A. Celi,
G. E. Gilbert,
B. C. Furie,
J. K. Erban,
R. Bonfanti,
D. D. Wagner,
and
B. Furie.
PADGEM: a receptor that mediates the interaction of activated platelets with neutrophils and monocytes.
Cell
59:
305-312,
1989[Medline].
15.
Lechner, J.,
and
M. A. LaVeck.
A serum-free method for culturing normal human bronchial epithelial cells at clonal density.
J. Tissue Cult. Methods
9:
43-48,
1985.
16.
Leff, A. R.,
K. J. Hamann,
and
C. D. Wegner.
Inflammation and cell-cell interactions in airway hyperresponsiveness.
Am. J. Physiol.
260 (Lung Cell. Mol. Physiol. 4):
L189-L206,
1991
17.
Lynch, J. P., III,
T. J. Standiford,
M. W. Rolfe,
S. L. Kunkel,
and
R. Strieter.
Neutrophilic alveolitis in idiopathic pulmonary fibrosis.
Am. Rev. Respir. Dis.
145:
1433-1439,
1991.
18.
Robbins, R. A.,
S. Koyama,
J. J. R. Spurzem,
K. A. Richard,
K. J. Nelson,
G. L. Gossman,
G. M. Thiele,
and
S. I. Rennard.
Modulation of neutrophil and mononuclear cell adherence to bronchial epithelial cells.
Am. J. Respir. Cell Mol. Biol.
7:
19-29,
1992[Medline].
19.
Roche, W.,
S. Montefort,
J. Baker,
and
S. Holgate.
Cell adhesion molecules and the bronchial epithelium.
Am. Rev. Respir. Dis.
148, Suppl.:
S79-S82,
1993[Medline].
20.
Rothlein, R.,
M. C. Czajkowski,
M. M. O'Neill,
S. D. Marlin,
E. Mainolfi,
and
V. J. Merluzzi.
Induction of intercellular adhesion molecule 1 on primary and continuous cell lines by pro-inflammatory cytokines.
J. Immunol.
141:
665-1669,
1988.
21.
Thompson, A. B.,
D. Daughton,
R. A. Robbins,
M. A. Ghafouri,
M. Oehlerking,
and
S. I. Rennard.
Intraluminal airway inflammation in chronic bronchitis. Characterization and correlation with clinical parameters.
Am. Rev. Respir. Dis.
140:
1527-1537,
1989[Medline].
22.
Tosi, M. F.,
A. Hamedani,
J. Brosovich,
and
S. E. Alpert.
ICAM-1 independent, CD18-dependent adhesion between neutrophils and human airway epithelial cells exposed in vitro to ozone.
J. Immunol.
152:
1935-1942,
1994
23.
Tosi, M. F.,
J. M. Stark,
A. Hamedani,
C. Wayne-Smith,
D. C. Gruenert,
and
Y. T. Hang.
Intercellular adhesion molecule-1 (ICAM-1) dependent and ICAM-1 independent adhesive interactions between polymorphonuclear leukocytes and human airway epithelial cells infected with parainfluenza virus type 2.
J. Immunol.
149:
3345-3349,
1992
24.
Varsano, S.,
N. Joseph-Lerner,
T. Reshef,
and
I. Frolkis.
Normal serum increases adhesion of neutrophils to tracheal epithelial cells by a CD11b/CD18-dependent mechanism.
Am. J. Respir. Cell Mol. Biol.
10:
298-305,
1994[Abstract].