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Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulation of the alveolar
epithelium provoked the polarized apical secretion of monocyte
chemoattractant protein-1 and RANTES and up-regulation of ICAM-1
and VCAM-1 expression, accompanied by markedly enhanced transepithelial
monocyte traffic in the basal-to-apical direction. Multiple adhesive
interactions were noted to contribute to the enhanced monocyte traffic
across the TNF--stimulated alveolar epithelium: these included the
ß2 integrins CD11a, CD11b, CD11c/CD18, the
ß1 integrins very late Ag (VLA)-4, -5, and -6, and the
integrin-associated protein CD47 on monocytes, as well as ICAM-1,
VCAM-1, CD47, and matrix components on the epithelial side. In
contrast, spontaneous monocyte migration through unstimulated
epithelium depended predominantly on CD11b/CD18 and CD47, with some
additional contribution of VLA-4, -5, and -6. In summary, unlike
transendothelial monocyte traffic, for which ß1 and
ß2 integrins are alternative mechanisms, monocyte
migration across the alveolar epithelium largely depends on CD11b/CD18
and CD47 but required the additional engagement of the ß1
integrins for optimal migration. In response to inflammatory challenge,
the alveolar epithelium orchestrates enhanced monocyte traffic to the
apical side by polarized chemokine secretion and up-regulation of
ICAM-1 and VCAM-1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Much less is known about the mechanisms of monocyte migration into the alveolar compartment once the cells have traversed the vascular endothelium. In a rat model, intratracheal application of endotoxin induced a pronounced monocyte influx into the lungs, with the vast majority of monocytes being recovered from the lung parenchyma, whereas only few cells were found in the bronchoalveolar lavage (11). These findings suggested a differential regulation of transendothelial migration into the interstitial space vs emigration into the alveolar compartment in the course of the inflammatory process. Thus, alveolar epithelial cells might play an important role in regulating the expansion of the alveolar monocyte pool. In this context, it is of major interest that the epithelial cells release monocyte chemotactic activity (12, 13), such as the chemokines monocyte chemoattractant protein-1 (MCP-1) and RANTES, that may be significantly up-regulated in the presence of proinflammatory cytokines (14).
Epithelium-leukocyte interactions are partly mediated by carbohydrates (15, 16), but adhesion of lymphocytes and neutrophil granulocytes to epithelial ICAM-1 has also been reported (17, 18). ICAM-1 is expressed on type I and type II pneumocytes (19, 20, 21, 22) and is up-regulated in the presence of proinflammatory cytokines (23, 24). Other candidates for epithelial-monocyte interaction are the integrin-associated protein CD47 located at the basolateral surface of epithelial cells, which was shown to play an important role in the migratory process of neutrophils through intestinal epithelial cells (25), and the VLA-4 ligand VCAM-1, which has been detected on bronchial (17) and renal epithelium (26, 27). Epithelial cells probably regulate directional leukocyte traffic by differential expression of these adhesion molecules, compartmentalized chemokine secretion (28, 29), and deposition of extracellular matrix proteins (30, 31), which might modulate leukocyte integrin function, supporting either adhesion or migration of the cells.
In the present study, we analyzed the transmigration process of monocytes through the alveolar epithelial cell line A549 and isolated human alveolar epithelial cells (HAEpC) with type I pneumocyte characteristics. The transepithelial migration was quantified in both the apical-to-basal and the basal-to-apical direction in the absence or presence of chemoattractants and proinflammatory stimuli. Moreover, the impact of polarized chemokine secretion and the participation of monocyte and epithelial cell adhesion molecules was investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following adhesion-blocking murine mAbs against human Ags
were used: R1/1 (anti-ICAM-1 (CD54); Bender MedSystems, Vienna,
Austria), 1G11 (anti-VCAM-1 (CD106); Coulter-Immunotech, Marseille,
France), CIKM1 (anti-CD47; PharMingen, San Diego, CA), GI18
(anti-PECAM domains 1 + 2 (CD31); generously provided by S.
Santoso, Giessen, Germany), W6/32 (anti-HLA class I; a gift from A.
Ziegler, Berlin, Germany), clone 38 (anti-CD11a; R&D Systems,
Wiesbaden, Germany), clone 44 (anti-CD11b, R&D Systems),
CBR-p150/4G1 (anti-CD11c; Bender MedSystems), MEM 48
(anti-CD18; R&D Systems), HP2/1
(anti-4-chain of VLA-4 integrin (CD49d);
Serotec, Oxford, U.K.), SAM-1
(anti-5-chain of VLA-5 (CD49e); Serotec),
GoH3 (anti-6-chain of VLA-6 (CD49f);
Coulter-Immunotech); Dreg 56 (anti-L-selectin (CD62L); PharMingen).
Neutralizing murine Abs against human MCP-1, RANTES and
macrophage-inflammatory protein-1 (MIP-1) were obtained from R&D
Systems.
Culture of A549 cells
A total of 5 x 104 A549 cells (cell line with alveolar type II epithelial cell characteristics; CCL 185; American Type Culture Collection, Manassas, VA) were seeded on the upper or lower side of polycarbonate filter inserts (5 µm pore size; diameter, 6.5 mm; Costar, Cambridge, MA) and cultured in complete Ham’s F-12 medium (Life Technologies, Eggenstein, Germany) containing 10% FCS (Life Technologies), 4 mM L-glutamine (Life Technologies), and penicillin-streptomycin-amphotericin B solution (Life Technologies). Cells became confluent after 7 days and formed a monolayer with permeability <2% when tested by 125I-HSA (Amersham, Braunschweig, Germany) diffusion as described for endothelial cells (32).
Isolation of HAEpC
Type II HAEpC were isolated as described previously, with some modifications (19, 20, 33, 34). Human lung tissue was obtained from lobectomy specimens distal from tumors (Departments of Pathology and Surgery, Justus-Liebig-University, Giessen, Germany). This was approved by the local ethics committee of the Justus-Liebig-University. The lung tissue was minced and washed extensively in HEPES-buffered saline and was subsequently digested by the use of Dispase II (2.5 mg/ml; Boehringer Mannheim, Mannheim, Germany) in the presence of 2 mM calcium and 1.3 mM magnesium for 60 min at 37°C under continuous rotation. A cell-rich suspension was obtained by sequential filtration through sterilized 100-µm pore size, 60-µm pore size, and 20-µm pore size meshes (Millipore, Eschborn, Germany). Type II pneumocytes were separated by ficoll (Ficoll Paque, Amersham Pharmacia Biotech, Uppsala, Sweden) density centrifugation (1200 x g, 15 min, 21°C), followed by depleting the interfacial cells of contaminating leukocytes by anti-CD45 magnetic beads (Coulter-Immunotech). The isolated cells were composed of 88–95% epithelial cells (flow cytometric analysis of the epithelial cell-specific Ag HEA-125; anti-HEA-125; Camon, Wiesbaden, Germany) and 5–12% alveolar macrophages and lymphocytes (light scatter characteristics and expression of CD45; HI30; PharMingen). Because intracellular alkaline phosphatase activity is specific for type II cells in the lung (35, 36), alkaline phosphatase cytochemistry was performed on cytospin preparations and revealed 96–99% type II pneumocytes in the epithelial cell population. The freshly isolated HAEpC are termed TII-HAEpC (type II HAEpC).
Culture of HAEpC
A total of 5 x 105 human type II
pneumocytes were seeded on the upper or lower side of human type IV
collagen (Sigma, Munich, Germany)-coated (34)
polycarbonate filter inserts (5-µm pore size; diameter, 6.5 mm;
Costar) and cultured in complete Ham’s F-12 medium containing
D-valine (Life Technologies) instead of
L-valine to prevent growth of fibroblasts
(37). Medium was changed every 2 days, and when the
epithelial cells reached confluence after 6–7 days, the medium was
changed to complete Ham’s F-12 medium containing L-valine,
and cells were cultured for further 2 days. HAEpC exhibited no or only
little proliferation in tissue culture as confirmed by nuclear Ki-67
staining (<0.5%; Ref. 38), and they progressively lost
their type II cell characteristics and underwent differentiation into
type I alveolar epithelial cells (loss of intracellular alkaline
phosphatase activity; up-regulation of ICAM-1 expression and
down-regulation of HLA-DR expression as shown in Fig. 6
; Refs.
39, 40, 41). Based on these findings, HAEpC that were cultured
for 7–9 days are termed TI-HAEpC (type I HAEpC). TI-HAEpC formed tight
monolayers, with a 125I-human serum albumin
permeability < 0.5%.
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Human monocytes (from buffy coats of healthy blood donors, approved by the local ethics committee) were isolated using a combination of ficoll density gradient centrifugation (800 x g, 30 min, 21°C) and counterflow centrifugal elutriation (Beckmann J2-21 M/E centrifuge with JE-B6 elutriator rotor, standard 5-ml elutriation chamber; Beckman Instruments, Palo Alto, CA). Cell counts were determined by hemocytometer counts of trypan blue-stained aliquots. The monocyte fraction consisted of 93–97% monocytes, 3–7% lymphocytes, 0–1% granulocytes, and essentially no platelets, and cell viability always ranged >95% as determined by Pappenheim-stained cytospin preparations and trypan blue dye exclusion, respectively. The isolated monocytes were radiolabeled with 5 µCi 111In (10 mCi/ml 111InCl; Amersham) tropolon (Fluka, Neu-Ulm, Germany) as previously described (42). Labeled monocytes were suspended at a density of 1 x 107 cells/ml in RPMI 1640 (Life Technologies) containing 10% heat-inactivated FCS (RPMI-FCS).
Monocyte migration across A549 and human alveolar epithelium barriers
A549 or TI-HAEpC monolayers on the inserts and the lower
compartments of the transwell chambers were washed twice with complete
HAM’s F12 medium and incubated for 24 h in fresh medium or were
stimulated for 4, 12, or 24 h by the addition of 10 ng/ml TNF-
(sp. act. 1.1 x 106 U/mg protein; R&D
Systems) to the medium. After this incubation, the filters were washed
trice on the lower and upper surfaces with RPMI-FCS and transferred to
new, clean wells of 24-well low-cluster plates (Costar). To these
wells, 500 µl of RPMI-FCS were added, and before immersion of the
inserts, 100 µl of the monocyte suspension containing 1 x
106 111In-labeled cells were added above the
filter units. Monocytes were allowed to transmigrate the epithelium
barrier for 120 min at 37°C and 5% CO2 in
absence or presence of 1, 10, or 100 ng/ml MCP-1 (sp. act. 2.5 x
105 U/mg protein; R&D Systems) in the lower
compartment of the transwell chamber. The migration was stopped by
washing the upper compartment with RPMI-FCS, and the undersurface was
rinsed into the lower compartment and was swabbed with a cotton swab
soaked in ice-cold PBS/EDTA solution (43). The monocytes
in the lower compartment were lysed by the addition of 0.5% Triton
X-100 (Sigma), and lysed cells combined with the respective cotton swab
were counted in a gamma counter to determine the number of migrated
cells. The number of monocytes that migrated through the epithelial
barrier was expressed as percentage of lysed cells plus swab counts in
relation to counts initially added to the upper compartment.
In some experiments, saturating amounts of neutralizing murine Abs
against human MCP-1, RANTES, or MIP-1 were added to the epithelial
cells before the addition of monocytes. To investigate the role of
adhesion molecules in monocyte transepithelial migration,
111In-labeled monocytes were treated with
saturating amounts of adhesion blocking mAbs (30–50 µg/ml) for 30
min at room temperature and epithelial cells were incubated with mAbs
(40–80 µg/ml) for 60 min at 37°C. FcIgG
receptors were blocked by preincubation with human Igs (10 mg/ml;
Octagam; Octapharma, Langenfeld, Germany). Monocytes and epithelial
cells were washed twice in RPMI-FCS to remove unbound mAbs before the
migration assay.
Monocyte-specific chemokine secretion by alveolar epithelial cells
A total of 5 x 104 A549 or 5 x
105 HAEpC were seeded on the upper (for
quantification of basolateral chemokine secretion) or the lower side
(for quantification of apical chemokine secretion) of polycarbonate
filter inserts and cultured for 7 days. Epithelial cell monolayers on
the inserts of the transwell chambers were washed twice with complete
Ham’s F-12 medium, and 100 µl of fresh medium was added to the upper
and 500 µl to the lower compartment of the transwell chamber,
respectively. Cells were incubated for 24 h or stimulated for 4,
12, and 24 h by the addition of 10 ng/ml TNF- to the medium.
After this incubation, the medium from the lower compartment was
collected, centrifuged, and stored at -80°C until chemokine
measurement.
The apical and basolateral secretion of the chemokines MCP-1 and RANTES was quantified by ELISA technique. Maxisorp microtiter plates (Nunc, Wiebaden, Germany) were coated overnight at 4°C with polyclonal goat Abs to human MCP-1 and RANTES (R&D Systems) followed by three washing steps with PBS containing 0.05% Tween 20 (Sigma). Then, 50 µl of cell culture samples was dispensed into the wells and incubated for 2 h at room temperature. After washing, application of a monoclonal mouse Ab directed against MCP-1 or RANTES (R&D Systems) was followed by sequential incubation with a biotinylated donkey anti-mouse Ig Ab (Dianova, Hamburg, Germany), avidin and biotinylated HRP (Dianova), and the substrate 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (Dianova). Serial dilutions of human recombinant MCP-1 or RANTES (R&D Systems) provided a standard curve for each individual ELISA.
Expression of adhesion molecules by A549 and HAEpC
A total of 5 x 105 A549 cells were
cultured in six-well tissue culture plates for 7 days, and cells were
washed twice and incubated for 24 h in fresh medium or were
stimulated by the addition of 10 ng/ml TNF-. HAEpC were cultured for
7 days on type IV collagen-coated six-well tissue culture plates before
TNF- stimulation. The medium was removed and cells were detached by
a short incubation in trypsin solution (Life Technologies).
Immunofluorescence labeling of cultured A549 and TI-HAEpC as well as
freshly isolated TII-HAEpC was performed by incubation with mouse mAbs
directed against VCAM-1, ICAM-1, MHC class II (HLA-DR; Becton
Dickinson, Heidelberg, Germany), CD47, PECAM-1, E-selectin (BBIG-E1
(CD62E); R&D Systems); P-selectin 9E1 (CD62P); R&D Systems), or isotype
controls (Dianova), and PE-labeled F(ab')2 of an
anti-mouse Igs Ab (Dianova).
For analysis of VCAM-1 gene expression by TII-HAEpC, TI-HAEpC, as well
as TNF- stimulated HUVEC and TI-HAEpC, total cellular RNA of these
cells was isolated using the acid guanidinium
thiocyanate-phenol-chloroform method (44). The mRNA was
reverse transcribed in a GeneAmp PCR System 2400 (Perkin-Elmer,
Norwalk, CA), and the PCR was performed with first-strand DNA using
intron-spanning specific primers for ß-actin
(5'-AAAGAACCTGTACGCCAACACAGTGCTGTCT-3',
5'-CGTCATACTCCTGCTTGCTGATCCACATCTG-3'; Stratagene, Heidelberg, Germany)
and VCAM-1 (5'-GAAGATGGTCGTGATCCTTG-3', 5'-GGACTTCCTGTCTGCATCCT-3';
Stratagene). Aliquots of RT-PCR products were electrophoresed through
1.8% (w/v) Nusieve/agarose gels and stained with ethidium bromide for
2 h at 75V.
Statistitcal analysis
Data were expressed as mean ± SEM. For analyzing statistical difference, two-tailed Student’s t test for unpaired samples was performed. After Bonferroni’s correction, statistically significant differences were defined as values of p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A significant spontaneous migration of monocytes through A549 and
TI-HAEpC monolayers from the apical to the basal (epithelium seeded on
the upper side of transwell filter inserts) as well as from the
basolateral to the apical surface (cells seeded on the lower side of
filter inserts) of epithelial cells was noted. The migratory response
was markedly and dose-dependently enhanced in the presence of MCP-1
admixed beneath the epithelium barrier (Fig. 1). Notably, spontaneous transepithelial
migration through TI-HAEpC was significantly higher when monocytes
migrated from the basolateral to the apical compartment of the
epithelium, whereas the enhanced monocyte migration in the presence of
MCP-1 displayed no significant differences when both directions were
compared (Fig. 1). Stimulation of epithelial monolayers with TNF-
time-dependently up-regulated monocyte transepithelial migration (Fig. 1), with significant predominance of the basal-to-apical direction
(Fig. 1). Monocyte migration across the epithelial barrier was
confirmed by cell counting in the lower compartments of transwell
chambers and by flow cytometric analysis of migrated leukocytes (data
not given in detail).
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-induced migration through TI-HAEpC was
significantly inhibited in the presence of neutralizing Abs against the
epithelium-derived monocyte-specific chemokines MCP-1 and RANTES (Fig. 2), whereas a neutralizing Ab against
MIP-1 had no effect (Fig. 2). Monocyte migration across unstimulated
A549 cells was slightly but not significantly inhibited by
anti-RANTES (14.4 ± 8.6% inhibition; n = 5),
and anti-MCP-1 and anti-MIP-1 had no effect. Like with
TI-HAEpC, the migration across TNF--stimulated A549 cells was
markedly inhibited by anti-RANTES (38.6 ± 9.1% inhibition;
n = 5, p < 0.01) and anti-MCP-1
(53.5 ± 5.9% inhibition; n = 5,
p < 0.005), and anti-MIP-1 had no effect.
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Because larger numbers of monocytes transmigrated the
TNF--stimulated epithelium in the basal-to-apical compared with the
apical-to-basal direction, and migration was inhibited in the presence
of neutralizing Abs against MCP-1 and RANTES, we hypothesized a
polarized secretion of MCP-1 and RANTES by alveolar epithelial cells.
Therefore, the release of both chemokines was analyzed separately in
the apical and the basolateral compartment of the epithelial barrier.
TI-HAEpC secreted RANTES and MCP-1 under baseline conditions (Fig. 2
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whereas resting A549 cells released small amounts of RANTES in both
directions without significant difference (apical 65 ± 17 pg/ml,
basolateral 43 ± 29 pg/ml; p = 0.18,
n = 5) but no MCP-1 (apical and basolateral <5 pg/ml;
n = 5). After stimulation with TNF-, chemokine
secretion was time-dependently increased in both cell types (shown for
HAEpC in Fig. 2). The release of MCP-1 and RANTES into the apical
compartment of TI-HAEpC markedly surpassed the basolateral secretion
(Fig. 2) under both baseline (MCP-1; Fig. 2) and inflammatory
conditions (MCP-1 and RANTES; Fig. 2). Within 24 h,
TNF--stimulated A549 cells secreted 892 ± 175 pg/ml MCP-1 into
the apical and 225 ± 89 pg/ml into the basolateral compartment
(p < 0.01, n = 5), and they
secreted 592 ± 108 pg/ml RANTES into the apical and 236 ±
71 pg/ml into the basolateral compartment (p <
0.01, n = 5). In addition, the apical and basolateral
chemokine secretion of A549 cells seeded on the upper side of transwell
filter inserts was analyzed in the same culture. Native A549 cells did
not secrete MCP-1 (<5 pg, n = 5) within 24 h, but
they released 41 ± 13 pg RANTES into the apical and 21 ± 12
pg into the basolateral compartment without statistical significant
difference (absolute amounts; p = 0.29,
n = 5). TNF--stimulated A549 cells released 247
± 29 pg RANTES into the upper and 118 ± 31 pg RANTES into the
lower compartment (p < 0.01, n
= 5), and they secreted 388 ± 68 pg MCP-1 into the apical and
113 ± 48 pg MCP-1 into the basolateral compartment
(p < 0.01, n = 5).
Expression of adhesion molecules by alveolar epithelial cells
It has been reported previously, that ICAM-1 is constitutively
expressed on type II pneumocytes and expression increases upon
differentiation into type I cells (19, 20, 21). Indeed, ICAM-1
was noted to be expressed at low levels on unstimulated A549 cells and
freshly isolated TII-HAEpC, and expression increased on cultured
TI-HAEpC. TNF- stimulation of A549 cells, and to a minor extent of
cultured TI-HAEpC, increased the expression of ICAM-1 (Fig. 3). The integrin-associated protein CD47
was expressed on unstimuated and TNF--stimulated A549 and TI-HAEpC
with no significant differences, as well as on freshly isolated type II
HAEpC (Fig. 3). HLA-DR was expressed at high levels on freshly isolated
TII-HAEpC and expression decreased during cell culture and
differentiation into TI-HAEpC, with no difference in absence or
presence of TNF- (Fig. 3). The expression of VCAM-1 by epithelial
cells was reported for bronchial and renal epithelium (17, 26, 27), whereas contradictory results were reported for alveolar
epithelial cells (19, 20). Our results indicated
expression of VCAM-1 on A549 and cultured TI-HAEpC, but VCAM-1
expression was not detected on freshly isolated TII-HAEpC (Fig. 3).
Stimulation of A549 and TI-HAEpC with TNF- up-regulated the
expression of VCAM-1 by both cell types (Fig. 3). In line with flow
cytometric results, RT-PCR revealed basal VCAM-1 gene expression by
cultured TI-HAEpC, which was markedly up-regulated by TNF- (Fig. 4). VCAM-1 gene expression was not
detected in freshly isolated TII-HAEpC (Fig. 4). Native TII-HAEpC as
well as native or TNF--stimulated TI-HAEpC and A549 cells did not
express PECAM-1, E-selectin, or P-selectin.
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Under baseline conditions, the transepithelial migration of
monocytes through A549 monolayers from the basolateral to the apical
surface depended mainly on the ß2 integrin
CD11b/CD18 and the integrin-associated protein CD47, whereas mAbs
against CD11a and CD11c only marginally inhibited migration (Fig. 5). CD47 is expressed on monocytes as
well as on epithelial cells (Fig. 4), and both blocking CD47 on
monocytes (M-CD47; Fig. 5) or epithelial cells (Ep-CD47; Fig. 5)
virtually completely inhibited the migration response. However,
blocking the ß2 integrin ligand ICAM-1, which
is constitutively expressed on epithelial cells (Fig. 4) did not
inhibit monocyte migration (Fig. 5), suggesting the presence of
alternative ligands for CD11b/CD18 on the basolateral surface of
alveolar epithelium. The ß1-integrins VLA-4,
-5, and -6 on monocytes were also involved in the process of monocyte
transepithelial migration, as suggested by the inhibitory effect of the
corresponding Abs. Extracellular matrix proteins such as fibronectin
and laminin, secreted by epithelial cells in culture (30, 31), are known to act as ligands for these monocyte
ß1 integrins (45). Anti-VLA-4
inhibited monocyte migration by 26%, and anti-VLA-5 and VLA-6
inhibited migration by 50%. The combination of anti-VLA-4, -5,
and -6 resulted in 75% inhibition (Fig. 5). Although the VLA-4
ligand VCAM-1 was expressed by A549 cells, incubation of unstimulated
A549 with a blocking mAb against VCAM-1 did not significantly inhibit
the transepithelial migration of monocytes (Fig. 5). Incubation of
epithelial cells with a mAb against MHC class I also had no effect on
monocyte migration. Blocking L-selectin (mAb Dreg 56) or domains 1 and
2 of PECAM-1 (mAb GI18), which reportedly mediated monocyte
transendothelial migration (46), also did not influence
the monocyte migration process through the A549 barrier (Fig. 5).
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). Moreover, blocking ICAM-1 or
VCAM-1 under these conditions markedly reduced the transepithelial
migration of monocytes (Fig. 6). Anti-PECAM-1 and anti-L-selectin
did not inhibit monocyte migration in the presence of MCP-1 (Fig. 6).
TNF- stimulation of A549 cells further increased ICAM-1 and VCAM-1
dependency of the transepithelial monocyte migration (Fig. 7). In line with this finding, the
engagement of the ICAM-1 ligand CD11a and the VCAM-1 ligand VLA-4 was
also enhanced. Anti-CD11c also markedly inhibited TNF--induced
monocyte recruitment, but the participation of CD11c did not
significantly differ from MCP-1-induced monocyte migration through
unstimulated epithelium. The inhibitory capacity of anti-VLA-6 was
also increased when epithelial cells were stimulated with TNF-,
whereas VLA-5-dependent migration was not significantly different from
migration through unstimulated A549 in absence or presence of MCP-1
(Fig. 7). Anti-PECAM-1 and anti-L-selectin did not inhibit monocyte
migration through TNF--stimulated epithelial cells (Fig. 7).
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). However, in contrast to A549
cells, CD11a, CD11c, and VLA-4 on monocytes, as well as ICAM-1 and
VCAM-1 on TI-HAEpC, were significantly involved in the spontaneous
transmigration process (Fig. 8). Stimulation of TI-HAEpC with TNF-
further increased the migration dependency on monocyte CD11a and VLA-4,
and the epithelial ligands ICAM-1 and VCAM-1 (Fig. 8). In line with the
findings in A549 cells, mAbs directed against PECAM-1 and L-selectin
had no effect on the monocyte migratory response (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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challenge
of the alveolar epithelial cells provoked strong polarized luminal
secretion of MCP-1 and RANTES, in companion with markedly enhanced
transepithelial monocyte migration in the basal-to-apical direction.
Assessment of epithelial adhesion molecule expression and employment of
various specific Abs suggested multiple adhesive interactions to
underlie the enhanced transepithelial monocyte traffic under these
conditions. These included the ß2 integrins
CD11a, CD11b, CD11c/CD18, the ß1 integrins
VLA-4, -5, and -6, and the integrin-associated Ag CD47 on the monocyte
side, as well as ICAM-1, VCAM-1, CD47, and presumably matrix components
on the epithelial side. Spontaneous monocyte migration in the absence
of inflammatory challenge, in contrast, appears to largely rely on
CD11b/CD18 and CD47. Marked spontaneous monocyte migration from both the basolateral to the apical as well from the apical to the basal compartment of the epithelium was noted in the absence of exogenous chemokine admixture. This observation is well in line with the fact that monocytes as the precursor cells of alveolar macrophages enter the alveoli under physiological conditions in vivo (47), but alveolar macrophages may also traverse the epithelium from the alveolar to the submucosal side when migrating into pulmonary lymph nodes (48). In the presence of MCP-1, this bidirectional monocyte migration was greatly enhanced, which supports the previously established role of this agent as a potent monocyte chemokine (6, 49, 50).
When mimicking inflammatory conditions by TNF- stimulation of
epithelial cells, marked enhancement of transepithelial monocyte
traffic was again noted, but then with predominant migration from the
basal to the apical compartment. This observation further supports the
view that the basolateral and the apical cell membranes of epithelial
cells have different functions (51). When analyzing this
directed migration in more detail, it was found to be largely ascribed
to polarized chemokine secretion with establishment of a chemotactic
gradient by the epithelial cells under TNF- challenge, as has been
described for rat alveolar epithelium (52). First, MCP-1
and RANTES were both noted to be preferentially released from the
epithelium into the apical compartment under these conditions. Second,
neutralizing Abs against MCP-1 and RANTES inhibited the TNF--induced
enhancement of the migratory response to the apical surface. And third,
the TI-HAEpC, but not the A549 cells, displayed some polarized MCP-1
secretion also under baseline conditions, concomitant with the finding
of some preferential basal-to-apical monocyte migration through the
nonstimulated TI-HAEpC but not the A549 barrier. Among the two agents
shown to be responsible for the currently noted chemotactic gradient,
MCP-1 may thus be even more relevant for the directed transepithelial
monocyte traffic than RANTES. It may also be more relevant than TGF-ß
and leukotriene B4, which displayed monocyte
chemotactic activity and were constitutively secreted by A549 cells
(12). The present findings are reminiscent of previous
observations in neutrophil movement across IL-1ß-stimulated airway
epithelial cells, in which preferential basal-to-apical migration also
occurred, dependent on the predominant luminal secretion of the
neutrophil chemokine IL-8 by the epithelial cells
(28).
In addition of being driven by the chemotactic gradient, the
transepithelial migration of monocytes evidently engaged different
adhesive interactions, summarized as a hypothetical cartoon in Fig. 9. In the absence of TNF-, the
monocyte interaction with both A549 and TI-HAEpC displayed strict
dependence on CD11b/CD18, and optimal transepithelial migration
additionally demanded the contribution of the
ß1 integrins VLA-4, -5, and -6. However, for
A549, blocking the constitutively expressed epithelial
ß2 and ß1 integrin
ligands ICAM-1 and VCAM-1 with mAbs had no effect. Such finding might
be explained by a strictly apical expression of ICAM-1 by the
epithelium, as had been suggested for the directed luminal migration of
granulocytes through intestinal epithelial cells, in which inhibition
was achieved by anti-CD11b/CD18 but not anti-ICAM-1 mAbs
(53). Although polarized expression of epithelial adhesion
molecules was not directly addressed in the present study, such strict
apical expression of ICAM-1 or VCAM-1 on the A549 cells is unlikely, as
a role of these adhesion molecules for basolaterally offered monocytes
became obvious under conditions of enhanced MCP levels (
Figs. 5–8).
Alternatively, predominant interaction of CD11b/CD18 and VLA4, -5, and
-6 with extracellular matrix proteins secreted by the epithelium
(30, 31) rather than with A549-expressed ICAM-1 and VCAM-1
may underlie the lack of efficacy of the anti-CAM Abs in
unstimulated A549 cells. With respect to native TI-HAEpC, some
inhibitory capacity of anti-ICAM-1 and anti-VCAM-1 was noted,
suggesting that in addition to ß1 and
ß2 integrin-matrix interactions, binding to
these CAMs was also relevant for transepithelial monocyte migration
under baseline conditions. Activation of leukocyte adhesion molecules
has been reported to be a prerequisite for the interaction of
lymphocytes and granulocytes with airway epithelium (17, 18). In addition, up-regulation of monocyte
ß1 and ß2 integrin
expression and function by MCP-1 has been described (54, 55). So one might speculate that exogenous MCP-1 added to A549
cells, or MCP-1 constitutively secreted by TI-HAEpC, initiated the
interaction of ß1 and
ß2 integrins with epithelial ICAM-1 and
VCAM-1.
|
was
additionally observed, but the assessment required the employment of
PE- instead of fluorescein-labeled mAbs for flow cytometric phenotyping
because of the high autofluorescence of cultured HAEpC. VCAM-1
expression has been previously demonstrated on renal and bronchial
epithelium (17, 27, 28), but was questioned for alveolar
epithelium (19, 20). In the present study, VCAM-1 was
found to be constitutively expressed by both A549 and TI-HAEpC but not
by TII-HAEpC, and VCAM-1 responded with up-regulation of gene and
protein expression upon TNF- stimulation. In summary, TNF- thus
increased luminal secretion of monocyte-specific chemokines and
expression of epithelial adhesion molecules, both contributing to the
enhancement of monocyte transmigration across the epithelial barrier.
As indicated by the inhibitory effects of the respective Abs, the
"high flux" transepithelial monocyte migration under conditions of
TNF- stimulation obviously demanded the parallel engagement of
several adhesive interactions, including the ß1
integrins VLA-4, -5, and -6, the ß2 integrins
CD 11a, CD11b, and CD11c/CD18, the epithelial counterparts ICAM-1 and
VCAM-1, and, most probably, as discussed for the experiments in the
absence of TNF-, matrix components. This finding contrasts to the
transendothelial migration of monocytes, for which CD11/CD18 and VLA-4
were noted to be alternative mechanisms, with lack of efficacy if only
one system was blocked (9, 43). The present data are also
at variance with a previous study in rats, in which anti-CD18 mAbs
did not significantly inhibit accumulation of radiolabeled monocytes in
the bronchoalveolar compartment after intratracheal application of
endotoxin (11). Blocking solely VLA-4 had also no effect
in that study, whereas the combination of anti-CD18 and
anti-VLA-4 decreased alveolar monocyte recruitment by 80%. One
possible explanation for the difference in the present study may be the
fact that currently freshly isolated monocytes faced the alveolar
epithelium, whereas in the in vivo situation monocytes had already
undergone the transendothelial passage, which is assumed to cause some
activation of these cells, which in turn may affect their preconditions
for the subsequent transepithelial passage (56, 57).
Interaction of monocytes with endothelial cells might modify the
monocyte integrin function, and tight adhesion to the basolateral
membrane of endothelium or epithelium might prevent the monocytes from
subsequent further transepithelial migration into the alveolar space
(58). Such effects might also explain the difference
between the presently observed high percentage of monocytes crossing
the alveolar epithelial barrier under conditions of TNF- stimulation
in vitro and the low percentage of radiolabeled monocytes entering the
alveolar space after intratracheal endotoxin challenge in vivo
(11). Finally, the use of different inflammatory stimuli
(LPS vs TNF-) might contribute to these differences.
VLA-6, the integrin-ligand for the extracellular matrix component
laminin, has been previously described to play a prominent role for
granulocyte migration through fibroblast barriers (59, 60). In contrast, although expressed on monocytes, VLA-6
mechanisms did apparently not contribute to monocyte migration across
these cells (60). The present study indicates a
significant involvement of VLA-6 in transepithelial monocyte migration
already under baseline conditions, further enhanced after TNF-
stimulation of the epithelium. Alveolar epithelial cells determine the
molecular composition of the extracellular matrix (30, 31), and inflammatory stimuli probably modulate this composition
via impact on the epithelium. Such an effect may explain why
anti-VLA-6 Abs inhibited a higher percentage (75%) of the
migratory response through the TNF--stimulated epithelium as
compared with the epithelium under baseline conditions (50%).
A striking observation of the present study was the strong inhibitory
capacity of anti-CD47 Abs on transepithelial monocyte migration: a
90% reduction of the migratory response was achieved under all
experimental conditions in both epithelial cell types, whether
targeting epithelial or monocyte CD47. Previous studies noted a major
contribution of this integrin-related protein to neutrophil migration
through intestinal epithelial barriers (25), but its
impact on transepithelial monocyte migration has, to the best of our
knowledge, never been described. Further studies will be necessary to
clarify the function of CD47 in the interaction of alveolar epithelial
cells and monocytes in a more detailed fashion.
In conclusion, the present investigation demonstrated a prominent role
of polarized alveolar epithelial chemokine liberation for
basal-to-apical transepithelial monocyte migration, which is markedly
enhanced in response to epithelial challenge with TNF- as prototype
inflammatory stimulus. Adhesive interactions during the migratory
process are based in particular on CD11b/CD18, possibly interfering
with matrix components, and the integrin-related Ag CD47 under baseline
conditions. Upon up-regulation of epithelial ICAM-1 and VCAM-1 in
response to TNF-, the additional contribution of these cellular
adhesion molecules and the monocyte integrins VLA-4 and CD11a to the
enhanced transepithelial monocyte movement becomes more prominent.
These findings further support the concept of a prominent role of the
alveolar epithelium in directing monocyte traffic into the alveolar
compartment both under physiological and inflammatory conditions.
|
Acknowledgments |
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Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Simone Rosseau, Department of Internal Medicine, Justus-Liebig-University, Klinikstrasse 36, 35385 Giessen, Germany. E-mail address:
3 Abbreviations used in this paper: VLA, very late Ag; PECAM, platelet-endothelial cell adhesion molecule; MCP, monocyte chemoattractant protein; MIP, macrophage- inflammatory protein; HAEpC, human alveolar epithelial cell; TI-HAEpC, type I HAEpC; TII-HAEpC, type II HAEpC; RPMI-FCS, RPMI 1640 containing 10% heat-inactivated FCS.
Received for publication July 20, 1999. Accepted for publication October 13, 1999.
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) or the apical-to-basal direction (
) for 120 min in absence or
presence of 1, 10, or 100 ng/ml MCP-1 beneath the epithelium.
Right, Time-dependent influence of TNF-
,
p < 0.005 compared with native A549 cells in the
presence of MCP-1 in Fig. 6
