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* Central Laboratory of the Netherlands Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands; and
Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands
Correspondence: Peter L. Hordijk, Ph.D., Central Laboratory of the Netherlands Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. E-mail: P_Hordijk{at}CLB.NL
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: human • cytokines • transmigration • IL-1ß, IL-6, IL-8
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Activation of endothelial cells by inflammatory stimuli promotes leukocyte infiltration through increased cellular adhesion-molecule expression, increased vascular permeability, and production of chemoattractants [1 , 6 7 8 9 10 11 12 ]. The molecular basis underlying transendothelial migration has been well described [9 , 10 , 13 , 14 ]. Granulocyte transmigration is triggered by various types of chemoattractants, such as chemokines, e.g., interleukin (IL)-8, lipid mediators, platelet-activating factor (PAF), bacteria-derived peptides, formyl-Met-Leu-Phe (fMLP) and complement fragments, and C5a. [15–17]. Migration of granulocytes is inhibited by antibodies that block ligand binding of granulocyte integrins [9 , 18 , 19 ], integrin-associated proteins such as CD47, or immunoglobulin (Ig)-like adhesion molecules such as CD31 [20 21 22 23 24 25 ].
Similar to the endothelium, the epithelium also plays an important role in leukocyte infiltration at sites of inflammation. However, whereas a large number of molecules are implicated in the control of transendothelial migration, only few molecules are known to be involved in the transmigration across epithelial monolayers. These include the leukocyte ß2-integrin CD11b/CD18 and the glycoprotein CD47 [15 , 26 , 27 ]. The epithelial ligand for the ß2-integrin has not yet been firmly established [27 ], although adhesion of eosinophils to human bronchial epithelium was recently described to depend on CD18/ICAM-1 interaction [28 ]. These results are in line with earlier reports of up-regulation of cellular adhesion molecules, such as ICAM-1, on activated epithelial cells [29 30 31 32 ]. In addition, activated epithelial cells release a variety of proinflammatory mediators, chemokines, and lipid mediators that may all modulate leukocyte infiltration [15 , 26 , 33 34 35 ]. For instance, bronchial epithelial cells of asthmatic patients have been shown to produce increased levels of IL-1ß, IL-6, IL-8, granulocyte/macrophage-colony stimulating factor, and IL-16 [35 ]. Finally, the orientation of the epithelial monolayer is essential to allow efficient in vitro transmigration. The physiologically relevant basolateral-to-apical migration of leukocytes is much more efficient than migration in the opposite direction, implicating the polarity of the epithelium as an additional regulatory factor of leukocyte transmigration [26 ].
Despite the large body of knowledge on migration across monolayers of endothelial or epithelial cells, possible interactions between these cell types and the resulting modulation of leukocyte transmigration have not been thoroughly studied. We have therefore developed a transmigration model in which we simultaneously culture monolayers of primary human lung epithelial cells and human umbilical cord vascular endothelial cells (HUVECs) on opposite sides of Transwell filters. We have characterized this model with respect to morphology and chemoattractant-induced transmigration of granulocytes and compared the results with those obtained with single endothelial or epithelial cell monolayers. The main results of this study reveal a paracrine interaction between the endothelial and epithelial monolayers resulting in increased release of cytokines and chemokines and enhanced transmigration of neutrophils.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Granulocyte isolation
Blood was obtained from healthy volunteers. Granulocytes were
isolated from a buffy coat of 500 ml of blood by density-gradient
centrifugation over isotonic Percoll (Pharmacia, Uppsala, Sweden)
[39
]. After lysis of the erythrocytes in the pellet
fraction with cold lysis buffer [155 mM NH4Cl, 10 mM
KHCO3, and 0.1 mM ethylenediaminetetraacetate (EDTA), pH
7.4], the granulocytes (>95% neutrophils) were washed in
phosphate-buffered saline (PBS) and resuspended in HEPES medium [132
mM NaCl, 6.0 mM KCl, 1.0 mM CaCl2, 1.0 mM
Mg2SO4, 1.2 mM KH2PO4,
20 mM HEPES, 5.5 mM glucose, and 0.5% (w/v) HSA, pH 7.4]. This
fraction is hereafter referred to as neutrophils.
Cell culture and experimental models
Endothelial cells.
The human papilloma virus-immortalized HUVEC cell line
[40
] or freshly isolated, primary HUVECs
[41
] were cultured in HUVEC medium [RPMI 1640
supplemented with 10% (v/v) heat-inactivated human serum, 100 U/ml
penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 1 ng/ml basic
fibroblast growth factor (bFGF)] in culture flasks coated with 1 mg/ml
fibronectin. The doubling time of the HUVECs was 
48 h. At
confluence, cell suspensions were obtained by trypsin/EDTA treatment.
The 2nd–4th passages of the primary HUVECs were used for subculturing
on fibronectin-coated polycarbonate Transwell filters (3.0 µm pore
size, 12 mm diameter; Costar, Cambridge, MA). HUVECs (150,000 in 0.5 ml
culture medium) were added to the upper compartment, and the Transwells
were cultured for another 4 days to obtain confluent HUVEC monolayers.
Epithelial cells.
The human lung adenocarcinoma-derived cell line H292 (American
Type Culture Collection, Rockville, MD, CRL 1848) [42
]
was grown in RPMI 1640 supplemented with 10% (v/v) heat-inactivated
human serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM
glutamine in uncoated culture flasks. The doubling time of the H292
cells was 24 h. At confluence, cell suspensions were obtained by
trypsin/EDTA treatment. The 4th–30th passages of H292 cells were used
for subculturing on the bottom side of Transwell filters, according to
Parkos et al. [43
], with minor modifications
[26
]. In brief, a sterile polyoxymethylene polyacetal
collar, with an inner diameter equal to the outer diameter of the
Transwell insert and with a height of 13 mm, was tightly fixed to the
bottom of the Transwell insert. Subsequently, 80,000 H292 cells (in a
volume of 0.5 ml culture medium) were allowed to attach for 18 h
(5% CO2, 37°C). Thereafter, the collars were removed,
and the Transwell inserts were placed upright in 12-well culture dishes
and incubated for 5 days.
Primary epithelial cells.
Subcultures of primary human bronchial epithelial cells were
obtained from bronchial tissues with macroscopically normal appearance
from patients undergoing lobectomy or pneumectomy for lung cancer. The
cells were cultured in serum-free keratinocyte medium
(Keratinocyte-SFM, Gibco) with 1 mM isoproterenol [44
]
in culture flasks coated with 10 µg/ml fibronectin, 30 µg/ml
vitrogen, and 10 µg/ml bovine serum albumin (BSA). After the
monolayers had reached confluence, cell suspensions were obtained by
mild trypsin/EDTA treatment (Gibco). The detached cells were washed
once in PBS containing soybean trypsin inhibitor type-II (Sigma) before
seeding. The 3rd–4th passages of the bronchial epithelial cells were
used for culturing at the bottom side of Transwell filters. The
inverted monolayers were created as described above for H292 cells,
except that 200,000 bronchial epithelial cells were added to coated
Transwell filters and cultured in 50% keratinocyte medium and 50%
RPMI 1640 supplemented with 2.5% HSA and 2 mM glutamine (final
CaCl2 concentration 0.5 mM; 50/50 medium).
Bilayer model.
The epithelial cells (75,000 cells/well) were allowed to adhere
to the bottom side of the Transwell filters as described above. After 1
day (H292) or 5–7 days (primary cells), the top side of the filters
was coated with 1 mg/ml fibronectin, and 150,000 HUVECs were seeded.
The bilayers composed of cell line cells were cultured in HUVEC medium,
and the bilayers composed of primary cells were cultured in 50/50
medium. The bilayers were cultured for another 4 days to allow
formation of confluent monolayers of epithelial and endothelial cells.
To confirm confluence, the cells at either side of the Transwell
filters were labeled by adding 4 µg/ml calcein-AM [45
]
in HEPES medium to the lower or upper compartment of the Transwell
system. The filters were washed after 15 min, mounted on glass slides,
and inspected by fluorescence microscopy (Dialux, Leitz, Germany).
Alternatively, cells on the filters were stained by
May-Grünwald/Giemsa. The different monolayers were consistently
found to reach confluence within the time frame of culture.
Coculture model.
The primary epithelial cells (75,000 cells/well) were cultured
for 5–7 days in serum-free keratinocyte medium with 1 mM isoproterenol
on the bottom of culture plates coated with 10 µg/ml fibronectin, 30
µg/ml vitrogen, and 10 µg/ml BSA. Primary HUVEC (150,000
cells/well) were seeded on fibronectin-coated Transwell inserts and
cultured in HUVEC medium for 4 days. The monolayers were subsequently
washed, and the inserts were placed in the wells with epithelial cells
cultured on the bottom. The coculture was cultured in 50/50 medium for
another day. The two cell types were subsequently separated, washed,
and cultured separately in 50/50 medium for another day.
Electron microscopy
Transwell filters, with endothelial cells on the top of the
Transwell filter and epithelial cells on the bottom side, were fixed
with 2.5% glutaraldehyde (v/v) in 0.1 M cacodylate buffer (pH 7.2) for
1 h and post-fixed in 1% (w/v) osmium tetroxide in the same
buffer for 1 h. The filters were subsequently block-stained with
uranyl acetate, dehydrated, and embedded in LX-112. Thin sections were
stained with uranyl acetate and lead citrate and examined with a CM10
transmission electron microscope (Philips, Eindhoven, The Netherlands).
Calcein-AM labeling and transmigration
The endothelial and epithelial cell line monolayers and bilayers
were cultured in HUVEC medium, and the primary endothelial and
epithelial monolayers and bilayers, in 50/50 medium. Fresh medium was
added to the Transwells 4 h before the start of the assay. The
Transwells were washed twice with HEPES medium just before the start of
the experiment. Neutrophils (107/ml) were labeled with 4
µg/ml calcein-AM in HEPES medium for 45 min at 37°C prior to the
start of the transmigration assay [45
]. After labeling,
the cells were washed twice and resuspended in HEPES medium (final cell
concentration, 106/ml). Where indicated, neutrophils or
monolayers of endothelial or epithelial cells were pretreated for 15
min with 10 µg/ml antibody to ß2-integrins, CD31, or
CD14 as a control, followed by washing of the cells with HEPES medium.
Calcein-labeled neutrophils (0.5x106 cells) were placed in
the upper compartment, and chemoattractants were placed in the lower
compartment. The chemoattractant concentrations in the lower
compartment were PAF, 100 nM; fMLP, 10 nM; IL-8, 10 nM; and C5a, 10 nM.
The Transwells were incubated for 35 min at 37°C.
To quantify transmigration, cells in the upper and lower
compartments and cells attached to the filter were separately lysed in
lysis buffer [PBS supplemented with 0.1% (v/v) Tween-20, 0.2% (w/v)
hexadecyl-trimethyl-ammoniumbromide (Sigma), 0.2% (w/v) BSA, and 20 mM
EDTA]. The amount of fluorescence in each of these compartments was
measured in a spectrofluorimeter (Model RF-540, Shimadzu Corporation,
Kyoto, Japan; 
;iEX 485 nm; ;iEM 525 nm)
and related to the fluorescence of the total input (set at 100%).
IL-6 and IL-8 quantification
The concentration of IL-6 and IL-8 in the supernatants,
collected from the upper and lower compartments of the different
transmigration and coculture models, was determined by enzyme-linked
immunosorbent assay (ELISA; CLB), according to the manufacturer’s
instructions. The absorption was measured in a Multiscan Multisoft
microplate reader (Labsystems Oy, Helsinki, Finland) at 450 nm.
Statistical analysis
Results were expressed as the mean ± SEM of at
least three independent experiments, performed with cells from
different donors. Results were analyzed with the Student’s paired
t-test (indicated in the legend of the figures)
[46
]. Two-tailed P values were calculated,
and P values exceeding 0.05 were considered not significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemoattractant-induced transmigration
Having established the bilayer model, we tested whether the
characteristics of neutrophil migration across the bilayers were
different from those of migration across single monolayers.
Transmigration of neutrophils across single epithelial monolayers, as
induced by a series of chemoattractants, was lower when compared with
migration across single endothelial monolayers (Fig. 3A
). This may be because the relatively thick and compact epithelial
monolayer (Fig. 1B)
is a more difficult barrier to cross.
Interestingly, the percentage of neutrophils that migrated across the
bilayers equaled the percentage of cells that migrated across single
endothelial monolayers, despite the presence of the additional
epithelial monolayer. This was particularly evident for PAF, C5a, and
IL-8. fMLP already induced a relatively high migration of neutrophils
across epithelial monolayers, and the migration across the bilayer did
not differ significantly from the migration in the other models.
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The relative increase in neutrophil migration across the bilayer, as compared with the migration across single epithelial monolayers, was not simply because of increased adhesion. The percentage of neutrophils associated with endothelial monolayers cultured on filters with a pore size that does not allow passage of neutrophils (0.45 µm) was similar in the absence or presence of epithelial cells on the bottom of the filter (unpublished results). The interaction of neutrophils with the extracellular matrix deposited by endothelial cells onto the filter did not seem to play a role either, because the presence of a deposited HUVEC matrix or fibronectin coating onto the topside of the filter did not enhance subsequent transepithelial migration (unpublished results).
Role of ß2-integrins and CD31 in neutrophil
transmigration
Neutrophil transmigration in the three different models was almost
completely CD18-dependent, because CD18-blocking antibodies inhibited
>95% of the transmigration across endothelial and epithelial
monolayers and across bilayers (Fig. 4
). Pretreatment of the endothelial cells with a blocking antibody
to CD31 inhibited transmigration of neutrophils across the bilayers and
across single HUVEC monolayers for 64% and 47%, respectively. The
CD31 antibody did not block migration across single epithelial
monolayers, which is explained by the absence of CD31 on epithelial
cells. These data show that the formation of the bilayer does not
significantly alter the role of prototypic adhesion molecules in
neutrophil adhesion and transmigration. When neutrophils were
pretreated with the CD31 antibody, transmigration was inhibited and
adhesion to the endothelial monolayers was increased (unpublished
results), possibly because of CD31-mediated activation of
ß1- and/or ß2-integrins on the surface of
neutrophils [22
, 49
].
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In an initial effort to address the notion that these agents are involved in the efficient transmigration of neutrophils across the bilayers, we measured the concentration of IL-1ß, IL-6, and IL-8 in the culture supernatant by means of ELISA. IL-1ß was found in the supernatant of primary epithelial cells, whereas in the supernatant of primary endothelial cells, hardly any IL-1ß was detected (Fig 5A ). Measurement of the IL-6 concentration revealed that the supernatant of monolayers of primary endothelial cells, H292 epithelial cells, and primary epithelial cells contained hardly any IL-6. Yet, the IL-6 level in the supernatant of the primary bilayers was significantly higher (Fig. 6 ). Moreover, a substantial amount of IL-6 was detected in the supernatant of primary epithelial cells that had previously been cocultured with primary endothelial cells, whereas the IL-6 level in the endothelial supernatant was low with or without previous coculture with epithelial cells (Fig. 5B) . In contrast, the supernatant of H292 epithelial cells did not contain more IL-6 after coculture with primary endothelial cells (unpublished results). Thus, the augmented IL-6 level in the supernatant of primary bilayers appears to be a result of increased IL-6 production by epithelial cells in response to soluble factors secreted by endothelial cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The characterization of this bilayer model by electron microscopy and immunocytochemistry showed that cellular morphology, distribution of the junctional proteins CD31 and E-cadherin, and the integrity of the endothelial and epithelial monolayers were unaltered in the bilayer model. In addition, the bilayer barrier was found to be less permeable to 3 kD FITC-conjugated Dextran than the single endothelial and epithelial monolayers (unpublished results).
Epithelial cells were found to grow into pores (size 3 µm) of the filters. This phenomenon has also been described for endothelial cells by Mackarel et al. [51 ] and is likely to be a general phenomenon. We found no indications for specialized structures or basement membranes at the zones of contact between endothelial and epithelial cells. Moreover, the molecular basis of neutrophil migration in the bilayer models appeared to be unaltered; i.e., the migration was largely mediated by ß2-integrins and CD31. Yet, the transmigration of neutrophils in the bilayer model was more efficient than in the single epithelial monolayer model and equaled the migration in the single endothelial monolayer models. Several mechanisms may be implicated in this phenomenon.
We tested whether the endothelial monolayer in the context of the bilayer model would represent a more adhesive surface for neutrophils, as compared with a single endothelial monolayer. However, neutrophil adhesion to the endothelium was not different in the bilayer model, indicating that increased adhesion does not occur. Currently, we cannot exclude that qualitative changes in neutrophil adhesion, i.e., the use of additional types of adhesion molecules other than ß2-integrins or CD31, play an important role in the migration in the bilayer model. Future research will therefore include the analysis of the adhesion molecule repertoire on the endothelial cells in the absence or presence of epithelial cells.
Transendothelial migration may enhance leukocyte motility, thus facilitating subsequent passage across an epithelial monolayer. Such effects may, for example, result from the interaction with endothelial CD31, because CD31-mediated interactions have recently been shown to stimulate the rate of integrin-supported neutrophil migration [52 ]. The interaction with the extracellular matrix at the basal side of the endothelial cells could enhance subsequent neutrophil transepithelial migration as well. However, the presence of a matrix deposited by endothelial cells did not alter the migration of neutrophils across the epithelial monolayer.
Finally, the endothelial and epithelial cells may influence each other such that neutrophil transmigration across both monolayers is enhanced, i.e., the epithelial cells may secrete soluble factors that promote neutrophil transendothelial migration, and, vice versa, endothelial cells may secrete factors that promote migration across epithelial monolayers. Our results show that indeed transendothelial migration is increased when epithelial cells are cocultured on the bottom of the Transwell culture plate for two days and that transepithelial migration is increased when endothelial cells are cocultured. Thus, physical contact between these cell types is not required for the increase in transmigration. Instead a paracrine interaction between the epithelial and the endothelial cells seems to be implicated in the increase of neutrophil transmigration.
Our present results further support this idea; i.e., coculture of endothelial and epithelial cells dramatically increases the release of particular cytokines, an as yet undescribed phenomenon. For instance, the concentration of IL-6 was significantly and synergistically increased when primary endothelial and epithelial cells were cocultured because of stimulated epithelial IL-6 production. IL-6 enhances survival of neutrophils in vitro [53 ] and has been shown to decrease cell-cell associations of carcinoma cells [54 ]. In addition, IL-8, a potent neutrophil chemoattractant that has been described to be secreted by epithelial and stimulated endothelial cells [35 ], was found to be significantly and synergistically increased in the bilayer model as a result of spontaneous epithelial IL-8 production and stimulated endothelial IL-8 production. In general, we found that the primary lung epithelial cells produce more IL-6 and IL-8 than did the H292 cell line cells, an effect that may also contribute to the relatively efficient migration across primary vs. H292 epithelial monolayers.
Previously, IL-8 release induced by IL-1 has been shown to promote migration of neutrophils across monolayers of endothelial and lung epithelial cells [55 , 56 ]. In parallel, our data suggest that in the bilayer model, epithelial-derived IL-1ß induces IL-8 production by endothelial cells. Coculture with epithelial cells increases the endothelial IL-8 production, whereas the spontaneous IL-8 production of epithelial cells is unaffected by coculture with endothelial cells. In addition, epithelial cells spontaneously produce high levels of IL-1ß, in contrast to endothelial cells, which hardly produced any IL-1ß. Moreover, antibodies to IL-1ß prevented almost completely the production of IL-8 in epithelial monolayer and bilayer cultures (unpublished results). These data show that IL-1ß regulates epithelial IL-8 secretion via an autocrine loop. Whether epithelial cell-derived IL-1ß is indeed the initiating factor in the stimulated neutrophil migration across the bilayers remains to be established.
The enhanced release of IL-6 and IL-8 may contribute to the increase in neutrophil migration across the bilayer. This may occur as a result of enhanced chemotaxis but may also involve cytokine-mediated changes in the endothelial or epithelial monolayers. Regardless of the mechanism involved, a strong chemotactic stimulus was still required for neutrophil transmigration, because the spontaneous migration across the bilayers remained as low as across the single monolayers.
Recently, a similar transmigration model for neutrophils was described, combining HUVECs with the A549 lung epithelial cell line [57 ]. Although the experimental set-up of this work is similar to ours, these studies did not address the relative role for adhesion molecules in the migration or reveal any paracrine communication involving cytokines or chemokines between epithelial and endothelial cells. Moreover, in this model, the migration across the bilayers was not increased when compared with migration across the individual monolayers. The differences with our findings may be related to the alveolar, rather than the bronchial, origin of the A549 tumor line, and these cells may behave differently with respect to production and release of soluble mediators.
In conclusion, our current results with the bilayer model provide new insights in the molecular basis of neutrophil transmigration. This model adds new aspects to the research on leukocyte migration in lung inflammatory disorders by providing an extra level of complexity, i.e., the "cross-talk" between monolayers of different cell types and the concomitant effects on leukocyte passage. These interactions between endothelial and epithelial cells are likely to be relevant for inflammatory disorders in the lung and may also play a role in other tissues where endothelial and epithelial cell linings are in close proximity.
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ACKNOWLEDGEMENTS |
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Received October 25, 1999; revised February 29, 2000; accepted March 1, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, P < 0.05. (B)
Transmigration across primary endothelial monolayers, primary lung
epithelial monolayers, and bilayers cultured in 50/50 medium was
determined as in A. Data are mean ± SEM of three to
six independent experiments. Student’s paired t-test was
performed to compare transmigration across the bilayers with migration
across the epithelial monolayers. 
