UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)
UvA-DARE (Digital Academic Repository)
The border-crossing behavior of eosinophils and neutrophils in the lung
Zuurbier, A.E.M.
Publication date
2001
Link to publication
Citation for published version (APA):
Zuurbier, A. E. M. (2001). The border-crossing behavior of eosinophils and neutrophils in the
lung.
General rights
It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s)
and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open
content license (like Creative Commons).
Disclaimer/Complaints regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please
let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material
inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter
to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You
will be contacted as soon as possible.
CHAPTER V
Paracrine Interaction between Endothelial
Cells and Bronchial Epithelial Cells:
Effect on Adhesion Molecule Expression
( 'Ihiplcr I
Paracrine Interaction between Endothelial Cells and Bronchial Epithelial
Cells: Effect on Adhesion Molecule Expression
Astrid E.M. Zuurbier, Frederik P.J. Mul, Sandra van Wetering*, Pieter S. Hiemstra , Dirk Roos and Peter L. Hordijk
Central Laboratory oftlie Netherlands Blood Transfusion Service (CLB) and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
Dept. ofPulmonology, Leiden University Medical Center. Leiden. The Netherlands
To gain more insight in the process of granulocyte infiltration of the lung, we have studied the transmigration of' neutrophils and the expression of' adhesion molecules on primary endothelial cells after co-culture with primary bronchial epithelial cells. Co-culture enhanced the migration of neutrophils across endothelial monolayers, and the expression of adhesion molecules on the endothelial cells was affected as well. The expression of ICAM-1 on endothelial cells was greatly enhanced after co-culture with epithelial cells and increased to a lesser extent after incubation with IL-lß. Blocking anti-IL-1 ß mAb did not affect this co-culture-indiiced increase in ICAM-1 expression, indicating that another soluble mediator was responsible for the ICAM-1
upregulation. Furthermore, CD31 and ß,-integrin expression on endothelial cells were enhanced and ICAM-2 expression was reduced after co-culture with epithelial cells. Although these latter effects were sensitive to blocking IL-lß with a mAb, incubation with IL-lß alone had hardly any effect on the expression of these adhesion molecules, indicating that not only IL-l ß but aIL-lso other factors were invoIL-lved. In contrast, VCAM-1 expression on endotheIL-liaIL-l ceIL-lIL-ls was not affected by eo-culture or incubation with IL-lß. Taken together, these results suggest that enhanced neutrophil migration across endothelial cells co-cultered with epithelial cells is due to the production of cytokines and chemokines that activate and attract neutrophils, and that also modulate adhesion molecule expression on endothelial cells.
Introduction
In lung inflammation and asthmatic reactions, circulating leukocytes, primarily neutrophils and
eosinophils, extravasale and infiltrate the airways, where they release toxic agents and cause severe tissue damage ( 1:2). This infiltration is the result of a complex interplay between various cell types at the site of inflammation. In short, patrolling lymphocytes, such asTh2 lymphocytes, are actuated to release se\eral cytokines (3). e.g. interleukin (IU-4. IL-5, IL-10. IL-13 and granulocyte-macrophage colony-stimulating factor (GM-CSF). These cytokines in turn activate surrounding endothelial cells (4) and epithelial cells (5). and prime and actixatc subsets of peripheral and tissue leukocytes such as eosinophils and neutrophils. Primed eosinophils and neutrophils arc subsequently attracted to the site of inflammation by cytokines and chemokines (3:6:7) that are secreted by activated endothelial cells (4;8) and epithelial cells (5). For instance, endothelial as well as epithelial cells can be stimulated to release eotaxin (9; 10), IL-8 (11), PAF ( I I ) and RANTES (12:13). The migration of these actix ated leukocytes is further promoted through
'Jaraerine Interaction between Endothelial and Epithelial Cells
increased expression of cellular adhesion molecules required for leukocyte migration across the endothelium (14-17) and across the epithelium (2:5:18-22).
The process of transmigration initially involves selectin-mediatcd rolling of the granulocytes on the endothelial cell surface and is followed by firm adhesion to the endothelium. Adhesion involves binding of ß, and ß; integrins on the granulocytes to their endothelial counter-receptors, i.e. vascular cell
adhesion molecule (VCAM )-1 and intercellular adhesion molecule (ICAM )-1, respectively
( 1:2:14:15:23:24). The granulocytes subsequently crawl though the junctions between the endothelial cells and cross the basement membrane, a process that is mediated by homotypic and heterotypic interactions ofplatclet-endothclial cell adhesion molecule (PECAM)-I (CD3I ) (25:26). Finally, the granulocytes move through the interstitial matrix and cross the epithelial basement membrane before passing the epithelium (2;5;20-22;27). The transcpithclial migration is mediated by granulocyte ß:-integrins (28) and
the epithelial integrin-associated glycoprotein CD47 (20;29).
The transmigration of granulocytes across single endothelial cell and epithelial cell monolayers has been extensively studied in vitro. However, in vivo transmigration across a particular monolayer of endothelial cells is often followed by contact with a second cell type, e.g. epithelial cells in the lung. In an attempt to mimic this complex in vivo situation, we previously developed an experimental model to investigate granulocyte migration in the context of lung inflammatory disorders, using
monolayers of primary human umbilical vein endothelial cells (HUVECs) and primary lung epithelial cells cultured on opposite sides of the same Transwell filter (bi-layer system) (30). In the present paper, a paracrine interaction between the epithelial and the endothelial cells is demonstrated, which stimulates transmigration of neutrophilic granulocytes. To further investigate the roles of either cell type in the stimulated neutrophil migration across the bi-laycrs, we have studied the behavior of the endothelial and epithelial cells in a co-culture model (Fig. I ). We found that the expression of adhesion molecules on endothelial cells is affected by co-culture w ith epithelial cells. This effect was in part dependent on epithelial-derived IL-1 ß.
Endothelial monolayer
Epithelial monolayer
Figure 1. Schematic representation oj the co-culture model. Endothelial cells are cultured on top of a Transwell filler, whereas epithelial cells are cultured on the bottom of the lower compartment of the franswell system.
Cli api er I
Materials & Methods
Reagents
PAF, C5a, fMLP, bovine scrum albumin (BSA) and isoproterenol were from Sigma Chemical Co. (St. Louis, MO, USA). Recombinant human (rh) IL-8, IL-1JS and TNF-u were purchased from PeproTech (Rocky Hill, NJ, USA) and bFGF from Boehringer Mannheim (Mannheim, Germany). Anti-lCAM-2 and anti-VCAM-1 mAb were obtained from Immunotech (Marseille, France). MAb 8A2 directed against ß, integrins was a kind gift from Dr. J.M. Harlan (Harbour Mcdicaj Center, Seattle, WA, USA). CD31 monoclonal antibody (mAb) (IgGl; H EC 170) was isolated from the hybridoma supernatant by precipitation with 50% saturated ammonium sulfate and subsequent protein-A affinity chromatography. Anti-lL-lß mAb (IgGl; IC25-6101) was purchased from Instruchemie (Hilversum, The Netherlands). CD 14 mAb (IgGl: mAb SG3). anti-ICAM-1 mAb (IgGl: mAb 15.2). human scrum albumin (HSA)and fibronectin were obtained from our institute (CLB, Amsterdam, The Netherlands). PE-conjugated secondary antibodies were from Dako (Glostrup, Denmark). Vitrogen was obtained from Cohesion (Palo Alto, CA, USA). Calcein-AM was from Molecular Probes (Eugene, OR, USA), and RPMI 1640 was from Gibco (Gibco Life Technologies, Gaithersburg, MD, USA).
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) (31 ). After lysis of the erythrocytes in the pellet fraction with cold erythrocyte lysis buffer (155 mM NH4C1. 10 mM KHCO, and 0.1 mM EDTA, pH 7.4). the granulocytes (> 95% neutrophils) were washed in PBS and resuspended in HEPES medium ( 132 mM NaCI. 6.0 mM KCl. 1.0 mM C a C k 1.0 mM M g:S 04, 1.2 mM KH,P04, 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
All cell cultures were incubated in a cabinet with 5% C O: and maximal humidity at 37°C.
Freshly isolated primary HUVEC (32) were cultured in HUVEC medium (RPMI 1640 supplemented with 10% (v/v) heat-inactivated human serum. 100 U/'ml penicillin. 100 ug/ml streptomycin. 2 mM glutamine and I ng ml bFGF) in culture flasks coated w ith I mg/ml fibronectin. The 2"J-4lh passages of primary HUVEC were used for sub-culturing on fibronectin-coatcd polycarbonate Transwcll filters (3.0 urn pore size, 12 mm diameter; Costar, Cambridge, MA, USA).
The human king adenocarcinoma-derived epithelial cell line 11292 (American Type Culture Collection CRL I 848) (33) was grown in RPMI 1640 supplemented with 10% (v/v) heat-inactivated human scrum. 100 U/ml penicillin. 100 ug ml streptomycin and 2 mM glutamine. in uncoated culture flasks. The 4' -30' passages of H292 cells were used for sub-culturing on uncoated polycarbonate Transwcll filters
Subcultures of primary human bronchial epithelial cells were obtained from bronchial tissues w ith macroscopicallv normal appearance from patients undergoing lobectomy or pneumcctomv for lung cancer. The cells were cultured in serum-free keratinocyte medium (Keratinocyte-SFM, Gibco) with 1 mM isoproterenol (34) in culture Husks coated with 10 ug ml fibronectin, 30 ug ml vitrogen and 10 ug/ml BSA. After the monolayers had reached confluence, cell suspensions were obtained by mild trypsin/EDTA treatment (Gibco). The detached cells were washed once m PBS containing Soybean trypsin inhibitor type-II (Sigma) before seeding Ï he 3' -4' ' passages of the bronchial epithelial cells were used for sub-culturing
Paracrine Interaction between Endothelial and Epithelial Cells
in 50/50 medium consisting of 50% (v/v) keratinocytc medium and 50% (v/v) RPVII 1640 supplemented with 2.5 % (v/v) HSA and 2 mM glutamine (final CaCI; concentration 0.5 niM) on coated polycarbonate Transwell filters.
Co-culture model
The primary epithelial cells were cultured for 5-7 days in serum-free keratmocyte medium with I mM isoproterenol on the bottom of culture plates coated with 10 ug/ml fibronectin, 30 ug/ml vitrogen and 10 Ug/ml BSA. H292 cells were seeded on the bottom of uncoated culture plates and HUVECs were seeded on fibronectin-coated Transwell inserts, and both cell types were cultured separately in HUVEC medium for4 days. The different monolayers were subsequently washed, and the inserts with endothelial cells were placed in the wells with epithelial cells cultured on the bottom. The co-culture with H292 cells was performed in HUVEC medium, and the co-culture with primary epithelial cells was performed in 50/50 medium, both for 2 days.
Calcein-AM labeling and transmigration
Fresh medium was added to the Transwclls 4 hours before the start of the assay. The Transwells were washed twice with HEPES medium just before the start of the experiment. Neutrophils (10 /ml) were labeled with 4 ug/ml calcein-AM in HEPES medium for 45 min at 37°C prior to the start of the
transmigration assay (35). After labeling, the cells were washed twice and rcsuspended in HEPES medium (final cell concentration 106/ml). Calcein-labeled neutrophils (5x10"cells) were placed in the upper compartment, and chemoattractants were placed in the lower compartment. The chemoatrractant concentrations in the lower compartment were PAF, 100 nM; fMLP, lOnM; IL-8, 10 nM; orC5a, 10 nM. The Transwells were incubated for 40 min at 37°C with 5% C O: and maximal humidity. After the incubation, the upper and lower compartments «ere washed with HEPES medium, the fluids were collected and the membranes were excised. The cells in the collected fluids and in the excised membranes were lysed in lysis buffer (PBS supplemented with 0.1 % (v/v) Twcen-20, 0.2% (w/v) hexadecyl-trimethyl-ammoniumbromide (Sigma), 0.2 % (w/v) BSA and 20 mM EDTA). The extent of transmigration was quantified by means of fluorescence measurement, i.e. the levels of Calcein-AM in the upper
compartment, lower compartment and membrane were measured with a spectrofluonmctcr (Model RF-540, Shimadzu Coiporation, Kyoto, Japan; ^EX 485 nm; /., M 525 nm). The percentage of labeled cells that had transmigrated was calculated from the amount of fluorescence detected in the lower compartment in relation to the fluorescence of the originally added Calcein-AM-labeled granulocytes.
FACS analysis
The expression of surface antigens was measured by indirect itnmunofluoiescense with flow cytometry on granulocytes and on endothelial and epithelial cells detached by means of a 15-min incubation with PBS supplemented with 5 mM EDTA at 37°C. The cells were incubated with mAbs (5 ug/ml) as indicated in the text, for 30 min at 4°C. After the cells had been washed with a 30-fold excess of ice-cold PBS containing 1% (w/v) BSA and 14 ug ml a/ide. binding of mAb was detected by incubation with PE-conjugated goat anti-mouse-Ig for 30 min at 4°C. The fluorescence intensity of the cells was measured with a flow cvtometer (FACScan. Becton Dickinson, San Jose, CA, USA).
( 'Ihiph'r I '
Statistical ana h sis
Results were expressed as the mean + SEM of at least 3 independent experiments, performed with cells from different donors. Results were analyzed with the Student's /-test (indicated in the legend of the figures). Two-tailed P values were calculated, and P values exceeding 0.05 were considered not significant.
Results
The transmigration of neutrophils was studied in an in vitro co-culture model (Fig. I ). We have previously found that neutrophil migration across endothelial cell monolayers is increased when endothelial cells are co-cultured for two days with H292 lung epithelial cells (Fig. 2a)(30). Washing of endothelial monolayers co-cultured with primary bronchial epithelial cells just before the start of the transmigration assay prevented this increase in chemoattractant-driven migration (Fig. 2b). confirming the notion that co-cultured cells secrete mediators that stimulate neutrophil migration. The
spontaneous neutrophil transendothelial migration across these endothelial monolayers was slightly
but significantly enhanced as compared to migration across untreated, washed monolayers (Fig. 2b). This increase in spontaneous migration may be due to direct changes in the endothelial cells, induced bv mediators secreted during co-culture.
medium PAF fMLP IL-8 C5a medium PAF fMLP IL-8 C5a
Figure 2. Migration oj neutrophils across monolayers of primary III I 'EC co-cultured with epithelial cells. Transmigration was measured across untreated IIUVEC monolayers {open hurs) and across I (I JVLC monolayers dial were co-cultured with epithelial cells for 2 days Uil I cd bars). Neutrophils were allowed lo migrate towards medium alone or towards \anous chemotactic stimuli, i.e. 100 nM I'AF. 10 nM fMLP, 10 nM IL-8 and 10 nM ('5a. (a) Migration across I1UVHC monolayers co-cultured with 11202 lung epithelial cells Dala are mean ± SLM of 2-5 independent experiments with neutrophils from dliferent donors (b) Migration across washed HUVEC monolayers thai had previously heen co-cultured with primär) bronchial epithelial cells Data are mean ± SEM of 2-6 independent experiments with neutrophils from different donors. Student's paired (-test was performed to compare transmigration across untreated and co-cultured IIUVEC monolayers, -k : P<0.05.
Paracrine Interaction between Endothelial and Epithelial Cell
\o investigate this, the cell-surface expression of adhesion molecules was assessed in
endothelial cells that had previously been co-cultured with primary bronchial epithelial cells for 2 days. We found that expression of ICAM-1 was greatly increased and that the expression of CD3 I and |i| integrin was augmented as well, whereas the expression of ICAM-2 was significantly down-regulated and the expression of VCAM-1 remained unchanged on co-cultured endothelial cells in comparison to untreated endothelial cells (Fig 3a). Co-culture w ith H292 lung epithelial cells yielded similar but less pronounced effects, i.e. enhanced expression of ICAM-1, CD3 1 and ß, integrin. reduced expression of ICAM-2. and hardly any expression of VCAM-1 on co-cultured endothelial cells (Fie 3b). 4000 3000 2000 • 1000 4000 3000 2000 1000 •
ICAM-1 CD31 |1, integrin ICAM-2 VCAM-1
Figure 3. Expression of adhesion
molecules on primary HUVEC co-cultured with epithelial cells. The surface
expression (mean fluorescence intensity (MFI)) of ICAM-1, CD31, ß,-integrin subunit, ICAM-2 and VCAM-1 was measured on untreated HUVEC (open
liars ) and on I It IVEC that had been
co-cultured with bronchial epithelial cells for 2 days (filial hars ).
(a) Expression on endothelial cells after co-culture with primary bronchial epithelial cells. Data are mean ± SEM of 2-4 independent experiments Student's unpaired Mest was performed to compare expression on untreated and co-cultured HUVEC. • : P<0.05.
iht Expression on endothelial cells after co-culture with 11292 lung epithelial cells. Data arc mean ± SEM of 2-6 independent experiments. Student's unpaired Mest was performed to compare expression on untreated and co-cultured HUVEC.
ICAM-1 CD31 (i, integrin ICAM-2 VCAM-1
Because we started this co-culture study with the aim to elucidate the mechanism underlying stimulated neutrophil migration across bi-lax ers (30), the expression of adhesion molecules on epithelial cells was also determined. We measured the expression of ICAM-1, ß, integrin. VCAM-1 and E-cadherin on epithelial cells with and without preceding co-culture with endothelial cells. However, we did not find any effect of the co-culture on the cell-surface expression of these molecules (data not shown).
( liaplcr I
T o determine w h i c h mediators initiated the observed modulation o f adhesion molecule expression in endothelial cells, we tried to m i m i c the co-culture by adding cytokines that have been described to activate endothelial cells and are k n o w n to be secreted by epithelial cells. For instance, epithelial cells produce I L - I ß (30), and this cytokine modulates adhesion molecule expression on endothelial cells (4). The expression o f adhesion molecules on endothelial cells was measured after stimulation for 2 days w i t h IL-1 ß. A g a i n , I C A M - 1 expression was increased, and no expression o f V C A M - 1 was f o u n d , but I L - I ß d i d not affect the expression o f C D 3 1 or ß, integrin subunit C D 2 9 (Fig. 4). The effect o f IL-1 ß on I C A M - 2 expression was not consistent, the expression was slightly up-regulated w i t h 2 1 % in one experiment and it was down-regulated w i t h 5 4 % in another. However, preliminary results revealed that a b l o c k i n g m A b to I L - l ß added to the co-cultures w i t h primary epithelial cells nearly completely prevented the down-regulation o f I C A M - 2 ( F i g . 5a). Furthermore, incubation w i t h this m A b d i d not affect the induced I C A M - 1 expression ( F i g . 5a & 5b) but partially prevented the up-regulation o f CD31 and ßi integrin expression.
2000
1500
1000
500
Figure 4. Expression of adhesion molecules on
primary III 'IEC'incubated with IL-lß. The
surface expression (MFI) of I C A M - 1 , CD31. ßi-integrin subunit, ICAM-2 and VCAM-1 was measured on untreated HUVEC (open bars) and on HUVEC that had been incubated with 5 ng ml IL-1 (! for 2 days {filled bars |. Data are mean ± SEM of 2-4 independent experiments.
ICAM-1 CD31 |1, integrin ICAM-2 VCAM-1
ICAM-1 CD31 ß, integrin ICAM-2
2500 2000 1500 1000 500 0
B
r-mvi
W/s
m//
ICAM-1 CD31 8A2Figure 5. Effect of anti-11.-I ßmAb on the expression of adhesion molecules on priinan III 'VEC co-i allured with
epithelial cells. The surface expression (MFI) of I C A M - 1 , ('D.M. ßi-inlegrin subunit and ICAM-2 was measured on
untreated HUVEC (open bars) and on HUVEC dial had been co-cultured with a) primär) epithelial cells and b) 11292 lung epithelial cells for 2 days in the absence (filled bars ) or presence of mAbs directed against II -Iß dum lied bars) Data are mean of 2 independent experiments.
Paracrine Interaction between Endothelial and Epithelial ( 'clIs
To gain more insight, the obtained results « e r e summarized and depicted as percentage of control expression instead of mean fluorescence intensity in figure 6. In summary, the expression of ICAM-I on endothelial cells was greatly enhanced after co-culture with epithelial cells and increased to a lesser extent after incubation with IL-I ß (Fig. 6a). Yet, blocking anti-IL-1 ß mAb did not have any effect on this increase in ICAM-1 expression (Fig. 5). indicating that in the co-culture model, not only IL-I ß but also other secreted mediators regulate the expression of ICAM-I in endothelial cells. Furthermore, CD31 and ß, integrin expression were significantly enhanced and ICAM-2 expression was significantly reduced after co-culture with epithelial cells (Fig. 6b). and these effects were sensitive to blocking the action of IL-I ß (Fig. 5). Yet. incubation with IL-Iß alone had hardly any effect on the expression of CD3 1. ß, integrin and ICAM-2 (Fig. 6b), indicating that other soluble mediators than IL-Iß were also involved in this process.
Ctrl primary H292 cells IL-1 epithelial
cells
(i, integrin
CA.
ICAM-2
Figure 6. Modulation of expression o) adhesion molecules on primary III I 'EC. I he "percentage of control" is the expression of antigens (MFI) on treated HUVEC divided by the expression on untreated HUVEC limes 100%. The "percentage of control" is depicted for untreated IIUVEC (100%) (open bars) and for HUVEC alter co-culture with primär) bronchial epithelial cells [Jilted bins I or « ilh 11292 lung epithelial cells (hatched bars ) or aller incubation with II -I |1 (mottled bars), (a) "Percentage of control" of ICAM-1 expression Data are mean ± SEM of 2-6 independent experiments. Student's unpaired t-test ua.s performed to compare (he "percentage of control" to the control value 1100%). + . P O . 0 5 , • • : /'••() 01. (b) "Percentage of control" ofCD31, ßi-integrin and ICAM-2 expression. Data arc mean ± SEM of 2-6 independent experiments. Student's unpaired /-test was performed to compare the "percentages of control" to the control values ( 100%). + : P O . 0 5 , * * : P<0.0l.
( "liaplcr I
In contrast to the other adhesion molecules, the expression of VCAM-I was not affected by co-culture or IL-1 ß. This lack of effect was not due to the quality of the mAb we used to detect VCAM-1, because this mAb readily bound to the antigen when its expression was induced after a 24-h incubation with TNF-u (Fig. 7). It is interesting to note that TNF-u significantly reduced the expression of CD3I with 22%, next to the expected stimulation of ICAM-1 expression (Fig. 7).
10000
7500
5000
2500
Figure 7. Expression of adhesion molecules
on primary III 'I EC incubated it it h TNF-a.
The surface expression (MFI) of ICAM-1, CD31 ami VCAM-1 «as measured on untreated HUVEC (open bars) and on HUVEC thai had been incubated with 10 ng/ml TNF-a for 1 day (filled bars). Data are-mean + SEM of 2-3 independent experiments. Student's unpaired /-test was performed to compare the expression of untreated and '1 NF-a-treated HUVEC.
* : P O . 0 5 , • * : P<0.0\.
ICAM-1 CD31 VCAM-1
Discussion
We have previously found that when primary endothelial cells and primary bronchial epithelial cells are co-culturcd, bronchial epithelial cells are stimulated to produce IL-6 and that endothelial cells release more IL-8 in response to epithelial-derived IL-lß (30). Moreover, wc found that neutrophil migration is enhanced when these two cell types are cultured on the same Transwell membrane as a bi-layer, i.e. endothelial cells on top and epithelial hanging underneath the membrane (30). To substantiate the hypothesis that a paracrine interaction between endothelial cells and epithelial cells is involved in the enhanced neutrophil migration across endothelial-epithelial bi-layers (30), we have studied the behavior of these cells in a co-culture model (Fig. I ). The hypothesis is supported by the observation that neutrophil chemouttractant-driven transmigration is increased when endothelial cell and epithelial cell monolayers are co-cultured (30). Moreover, increased transendothelial migration was only observed when the co-cultured monolayers were not washed prior to the transmigration assay, presumably because secreted, soluble mediators were not removed under those conditions.
Besides the described secretion of mediators that activate or attract neutrophils, mediators that activate endothelial cells or epithelial cells during co-culture may also play a role. When the secreted mediators that accumulated in the culture supernatant were removed by washing just before the start of the transmigration assay, we found that the spontaneous neutrophil migration, i.e. in absence of chemoattractants. across co-cultured endothelial monolayers was enhanced. This could be due to modulated expression of adhesion molecules, because the cell-surface expression of ICAM-l. CD31 and ßr integrin was up-regulated and the ICAM-2 expression was down-regulated on co-cultured endothelial cells. The expression of ICAM-1, CD31, ß,-integrin protein and ICAM-2 seemed to be regulated by 1L-I ß in concert with other mediators present in the co-culture supernatant. The
Paracrine Inlenielioii between Endothelial und Epithelial Cells
regulation o f I C A M - 1 expression was found to be less dependent o f I f - 1 ß than the régulation o f C D 3 I. ß p i n t e g r i n and I C A M - 2 expression. I L - l ß is presumably derived from the epithelial cells because these cells produce much more IL-1 ß than cultured endothelial cells do (30).
Increased I C A M - 1 expression undoubtedly facilitates neutrophil transcndothelial migration, because increased neutrophil migration across activated endothelial cells is known to be I C A M 1 -dependent (36). As to the increased CD? 1 expression, to our know ledge, this is the first report o f inducible CD31 expression on endothelial cells. Up-regulation has not y d been reported but down-regulation o f CD3 1 has been described by Rival and co-workers (36). i.e. they found that the combination o f IFN-y and T N F - a causes CD31 to disappear from the endothelial intercellular junctions by means o f internalization and reduction in CD31 synthesis. T N F - a alone has been shown to induce CD31 redistribution from the cell junctions to the apical surface (37) and to reduce the expression o f CD31 by 9 % (36), whereas we measured a reduction o f 22%. This apparent difference in reduction o f I C A M - 1 expression may be due to the sensitivity o f endothelial cells to T N F - « (36). The response o f endothelial cells to T N F - « may depend on several factors, such as the concentration o f T N F - « , the incubation time, the number o f passages o f the endothelial cell cultures and the culture conditions (36;38). The observed CD3 I downregulation on I F N -y/TNF-cc-treated endothelial cells is accompanied by reduced neutrophil transcndothelial migration (36). In our co-culture model, w c found an increase in CD31 expression, but w hethcr or not increased CD31 expression also results in stimulated neutrophil passage remains to be elucidated.
I C A M - 2 is an adhesion molecule expressed at the j u n c t i o n s and on the apical surface o f endothelial cells (39) that binds to the ß: integrins L F A - I and M a c - I and is thought to be involved in leukocyte transcndothelial migration (40). As to the migration o f neutrophils. I C A M - 2 seems not to be absolutely essential for neutrophil extravasation since antibody to I C A M - 2 does not inhibit neutrophil recruitment into the peritoneum in an animal model o f inflammation (38). Nevertheless, this molecule has been shown to play a role in in-vitro neutrophil transcndothelial migration (41 ). T N F - « and I L - l ß down-regulate I C A M - 2 expression, particularly at the intercellular junctions (38). L i k e w i s e , in our co-culture model. I C A M - 2 expression is presumably down-regulated by epithelial-cell-derived I L - l ß in combination w i t h another stimulatory mediator secreted in the co-culture supernatant. The
consequence o f the reduction in I C A M - 2 expression for transcndothelial migration has not yet been established.
We also found stimulated expression o f the ß p i n t e g r i n component on primary H U V E C . ß, integrins are concentrated in focal adhesions, which mediate adhesion o f the endothelial cells to the extracellular matrix. Increased expression o f ß, integrins after activation by cytokines is probably accompanied by an increased number o f interactions o f ßri n t e g r i n s w i t h their ligands in the extracellular matrix, and this leads to strengthening o f the adhesion plaques. This cytokine-induced strengthening presumably serves to prevent disruption o f the cell layer when leukocytes pass. Thus, the augmented ß, integrin expression seems to be involved in protection o f the invaded tissue and not in the actual transmigration process o f leukocytes.
In summary, enhanced neutrophil migration across bi-layers may be due to the production o f cytokines that attract neutrophils, e.g. IL-8 (30). and cytokines that modulate adhesion molecule expression in endothelial or epithelial cells, e.g. I L - l ß . However, other factors may still play a role. For instance, the effect o f co-culture on the organization and r i g i d i t y o f the cytoskeleton and the intercellular j u n c t i o n s may be crucial for neutrophil transmigration. Confocal microscopy analysis indeed revealed that incubation o f endothelial cells w i t h cytokines k n o w n to be present in the
co-( 'haplcr I
culture supernatant, e.g. IL-1 ß, IL-6 and IL-S. induce formation of filamentous actin (F-actin). The actin filaments are organized in bundles, which run along the longtudinal axis of the cells (data not shown). These changes are similar to those reported in several other studies (38;42).
The precise mechanism underlying stimulated neutrophil migration across bi-layers of endothelial cells and bronchial epithelial cells is not clear yet. Nevertheless, the current findings underscore the notion that the actuation state of endothelial cells and epithelial cells is partly dependent on environmental stimuli, such as locally present cytokines (43;44). The effects of these stimuli arc not implicated in currently used in-vitro transmigration models. However, our data indicate that the role of these stimuli in inflammation should not be underestimated. For instance, they could be implicated in vivo in the neutrophil recruitment in the lungs during inflammation or allergic asthmatic reactions, but this may also hold for (allergic) inflammation in other organs, such as the skin, the kidnev and the uut.
References
Fiikacs.N.W., Stricter,K.M.. and Kunkel,S.I.. 1995. Leukocyte infiltration in allergic airway inflammation. Am.J.Respir.Cell Mol.Biol. 13:1.
Knol.F.F. and Roos.I). 1996. Mechanisms regulating eosinophil extravasation in asthma. Ew.RespirJ. 9:136s.
3. Bellanti.J.A. 1998. Cytokines and allergic diseases: Clinical aspects. Allergy Asthma Proc. 19:337. 4. Mantovani.A.. Bussolino.F.. and Introna.M. 1997 Cytokine regulation of endothelial cell function: From
molecular level to the bedside Immunol. Today 5:231.
Van der Velden,Y.H.J.. Savelkoul.H.F.J., and Versnel,M.A. 1998. Bronchial epithelium: Morphology, function, and pathophysiology in asthma. Eur.Cytokine Nehv. 4:585.
Lampinen.M., Rak.S., and Venge.P. 1999. The role of interleukin-5, interleukin-8 ami RAN 1 IS in the chemotactic attraction of'eosinophils to the allergic lung. Clin.Exp.Allergy 29:314.
7. Lezcano-Meza,D. and Teran.F.M. 1999, Occupational asthma and interleukin-8. Clin.Exp.Allergy 29:1 301 8. Kitayama.J., Maekay.C.R., Ponath.P.D., and Springer.'I'.A. 1998. I he C-C chemokine receptor CCR3
participates in stimulation of eosinophil arrest on inflammatory endothelium in shear flow ../.( 'lin.Invest 9:2017.
9 Ying.S.. Robinson.D.S.. Meng.Q., Rottman.J., Kennedy.R.. Ringler.D.J.. Maekay.C.R.,
Daugherty.B.L., Springer.M.S.. Durham.S.R. et al. 1997. Enhanced expression of eotaxin and CCR3 niRNA and protein in atopic asthma. Association vv itli airway hyperresponsiv encss and predominant co-localization of eotaxin mRNA to bronchial epithelial and endothelial cells. EurJ.Immunol. 27:3507. 10 Shinkai.A., Yoshisiie.H.. Koike,M., Shoji.F... Nakagavva.S.. Saito.A.. Takeda.T.. Imabeppu.S., Kato.Y..
Hanai.V. Anazawa.H., Kuga.T.. and \ishi T. 1999. A novel human CC chemokine. eotaxin-3, which is expressed in IL-4- stimulated vascular endothelial cells, exhibits potent activity toward eosinophils.
J.Immunol. 163:1602.
11. Liu.L., Mul,F.P.J., Kuijpers,T.W., Lutter.R., Roos.D.. and Knol,F.F. 1996. Neutrophil transmigration across monolayers of endothelial cells and airway epithelial cells is regulated by different mechanisms.
Ann.NYAcad.Sci. 796:21.
12 Marfaing-Koka A„ Devergne.O.. Gorgone.G., Portier.A., Schall.T.J.. Galanaud.P., and Fmilie.D. 1995 Regulation of the production of the RANTES chemokine by endothelial cells. Synergistic induction by IFN-y plus TNF-a and inhibition bIFN-y IL-4 and IL-13. J.Immunol. 154:1870.
13 Stellato.C. Beck.L.A., Gorgone.G.A.. Proud.D.. Schall.T.J.. Ono.S.J.. l.ichtcnstein.L.M.. and Schleimer.R.P. 1995. Expression of the chemokine RANTES by a human bronchial epithelial cell line: Modulation by cytokines and glucocorticoids. J.Immunol. 155:.
14. Moser.R.. Fchr.J., OlgiatiX. and Bruijnzeel.P.L.B. 1992. Migration of primed human eosinophils across cytokine-activated endothelial cell monolayers. Blood 792937.
15. Springer.T.A. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 76:301.
16. lademarco.M.F., Barks.J.F.. and Dean,I).C. 1995 Regulation of vascular cell adhesion molecule-] expression by IL-4 and TNF-a in cultured endothelial cells. J.Clin.Invest 95:264.
Paracrine Interaction between Endothelial and Epithelial ( 'cils
17. Bochner.B.S., klunk.D.A.. Sterbinsky.S.A., Coffman.R.L., and Schlcimcr.R.P. 1995 [1.-13 selective!) induces vascular cell adhesion molecule- 1 expression in human endothelial cells. J.lmmimol. 154:799. IS. Cunningham.A.C. and Kirby.J.A. 1995 Regulation and function of adhesion molecule expression h\
human alveolar epithelial cells. Immunology 86:279.
19 Atsuta.J., Sterbinsky.S.A.. Plitt.J.. SchwiebcrtX.M., Bochner.B.S.. and SchleimerJR.P. 1997 Phenotyping and cytokine regulation of the BEAS-2B human bronchial epithelial cell: Demonstration of inducible expression of the adhesion molecules VC'A VI-1 and ICAM-1. Am J Respir.Cell Mol.Bio! 17:571. 20. Parkos.C.A. 1997. Molecular events in neutrophil transepithelial migration. BioEssays 19:865.
21 Si mon.R. H. and Paine I U.R. 1995. Participation of pulmonary alveolar epithelial cells in lung inflammation. J.Lab Clin.Med. 126:108.
22 Bloemen,P.G.M., Van den Tvveel.M.l'., Henricks.P.A.J.. Engels.F., Van de Velde.M.J.V.,
Blomjous.F.J., and Nijkamp.F.P. 1996. Stimulation of both human bronchial epithelium and neutrophils is needed for maximal interactive adhesion. AmJ. Physiol Lung Cell Mol.Physiol 270:L80.
23. Luscinskas.F.W., Cubulsky.M.I., Kiely,J.-M., Peckins.C.S., Davis.V.M.. and Gimbrone,M.A.,Jr. 1991 Cytokine-activated human endothelial monolayers support enhanced neutrophil transmigration via a mechanism involving both endolhelial-leukocyte adhesion 1 and intracellular adhesion molecule-I. J. Immunol. 146:1617
24 Hakkert.B.C. Kuijpcrs.T.W., LeeuwenbergJ.F.M., Van Mourik.J.A.. and Roos.D. 1991 Neutrophil and monocyte adherence to and migration across monolayers of cytokine-activated endothelial cells: 1 he contribution ofCD18, ELAM-1, and VI.A-4. Blood 78:2721.
25. Mnller.W.A., Weigl.S.A., Deng.X.. and Phillips.D.M. 1993. PECAM-1 is required for transendothelial migration of leukocytes J.Exp.Med. 1 78:449.
26 l.iao.F.. Huynh.H.K.. Firoa.A.. Greene.T., Polizzi.E., and Muller.W.A. 1995. Migration of monocytes across endothelium and passage through extracellular matrix involve separate molecular domains of PECAM-1. J.Exp.Med. 182:1337.
27. Huber.l).. Balda.M.S.. and Matter.K. 1998. Transepithelial migration of neutrophils. Invasion Metastasis 18:70.
28. I.in,I,.. Mul,F.P.J.. l.utter.R.. Roos.D.. and Knol.F.F. 1996. Transmigration of human neutrophils across lung epithelial cell monolayers is preferentially in the physiological basolateral-lo-apieal direction.
AmJ.Respir.Celi Mol.Biol. 15:771.
29 Parkos.C.A.. Colgan.S.P., Liang. TVS '., Nusrat.A.. Bacarra.A.E.. Carnes.D.K.. and Madara.J.L. 1996 CD47 mediates post-adhesive events required for neutrophil migration across polarized intestinal epithel ia.
J.Cell Biol. 132:437.
30. Mul.F.P.J.. Ziiiirbier.A.E.M., Janssen,H., Calafat.J.. Van Wetering.S.. Hiemstra.P.S.. Roos.D.. and Hordijk.P.L. 2000. Sequential migration of neutrophils across monolayers of endothelial and epithelial cells. J.Leukoc.Biol. 68:529.
31. Roos.D.and De Boer.M. 1986. Purification and cryopreservation of phagocytes from human blood. In:
Methods Eniymol. Volume 132 Immunochemical Techniques Part .1 Phagocytosis and Cell-Mediated
Cytotoxicity (G Di Sabato and .1 Everse, eds.), Academic Press. New York 225.
32. Brinkman.H.-J.. Mertens.K., Iliillhnis.J.. Zwart-HuininkX-A., Grijm.K., and Van Mourik.J.A. 1994. '1 he actuation of human blood coagulation factor X on the surface of endothelial cells: A comparison with various vascular cells, platelets and monocytes. Br.J.Haematol. 2:332.
33 Van Schillgaarde.M.. Van Alphen.L.. Fijk.P.. Everts.Y.. and Dankert.J. 1995 Paracytosis of
Haemophilus influenzae through cell layers of NC1-H292 lung epithelial cells. Infect.Immun. 63:4729.
34. Bloemen.P.G.M.. Nan den Tweel.M.G. Henricks.P.A.J.. Fngels.F.. Wagenaar.S.S., Rutten.A.A.J.J.I... and Nijkamp.F.P. 1993. Expression and modulation of adhesion molecules on human bronchial epithelial cells. AmJ.Respir.Celi Mol.Biol 9:586.
35. Akeson.A.L. and Woods.C'.W. 1993. A fluorimetric assay for the quantification of cell adherence to endothelial cells. J.Immunol.Methods 2:181.
36. Rival,Y., Del Mascliio.A.. Rabiet,M.-J., Dejana.E., and Duperray.A. 1996. Inhibition of platelet endothelial cell adhesion molecule-1 synthesis and leukocyte transmigration in endothelial cells by the combined action of TNF-a and IFN-y. J.Immunol. 157:1233.
37 Romer.I.H.. McLean.N.V.. Yan.H.-C Daise.M.. Sun.J., and DeLisser.H.M. 1995 IFN-y and TNF-a induce redistribution of PECAM-1 (CD3I ) on human endothelial cells. J.Immunol. 154:6582.
38. McLaughlin.F.. Hayes.B.P., Horgan.C.M.T., Beesley.J.E.. Campbell.C.J., and Randi.A.M. 1998 Tumor necrosis factor (TNF)-a and mterleukin (IL)-l [Î down-regulate intercellular adhesion molecule (ICAM)-2 expression on the endothelium. Cell Adhes.Commun. 6:381.
( 'Ihiplcr I
39. Bradley,.!.R., Thiru.S.. and Pober.J.S. 1995. Hydrogen peroxide-induced endothelial retraction is accompanied by a loss of the normal spatial organization of endothelial cell adhesion molecules
Am J.Pathol. 147:627.
4Ü. De Fougerolles.A.R., Stacker.S.A.. Schwarting,R., and Springer.T.A. 1991 Characterization of ICAM-2 and evidence for a third counter-receptor for LFA-1. J.Exp.Med. I 74:253.
41. Issekutz.A.C, Rowter.D.. and Springer.T.A. 1999 Role of ICAM-1 and ICAM-2 and alternate CD1 L C D 18 ligands in neutrophil transendothelial migration. J.Leukoc.Biol. 65:1 17.
42. Pober.J.S. and Cotran.R.S. 1990. Cytokines and endothelial cell biology. Physiol Rev. 70:427. 43 Rood.P.M.L., Gerritsen.W.R., Kramer.l).. R a n z i j n . C , Von dem Borne.A.E.G.Kr., and Van der
Schoot,CE. 1999. Adhesion of hematopoietic progenitor cells to human bone marrow or umbilical vein derived endothelial cell lines: A comparison. Exp.Hemalol. 27:1306.
44. Zhu.I).. Cheng,C--F., and Pauli.B.C. 1991. Mediation o f lung metastasis of murine melanomas by a lung-specific endothelial cell adhesion molecule. Proc.Natl.Acad.Sei.USA 88:9568.
