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1 Department of Internal Medicine, Justus-Liebig University, Giessen 35392; and 2 Jerini AG, 10115 Berlin, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Monocyte chemoattractant
protein-1 (MCP-1) is an essential chemokine involved in
monocyte traffic across endo- and epithelial barriers both in vitro and
in vivo. However, the contribution of endothelial MCP-1 signaling via
its CCR2 receptor in monocyte adhesion to inflamed endothelium under
flow is incompletely understood. A sensitive flow chamber assay was
used to assess monocyte adhesion to TNF-
-activated primary human
pulmonary artery endothelial cells (HPAEC) during physiological shear
stress. Monocyte adhesion was markedly reduced (~45%) when
HPAEC-derived MCP-1 was either neutralized with anti-MCP-1 mAb or
inhibited by translational arrest of MCP-1 mRNA transcripts with MCP-1
antisense oligomers. Corresponding efficacy was observed for blockade
of monocyte CCR2 receptor function by anti-CCR2 mAb or MCP-1
antagonists (9-76 analog). The impact of endothelial MCP-1 on
monocyte-HPAEC adhesion occurred via 
2-integrin but not
via 1-integrin adhesion pathways. In this line,
pretreatment of monocytes with MCP-1 but not RANTES provoked a rapid
and transient neoepitope 24 expression on 2-integrin -chains, as analyzed by increased reporter mAb24 binding.
Collectively, our data show an important cross talk of endothelial
MCP-1 with monocyte CCR2 effecting monocyte firm adhesion to inflamed
HPAEC under physiological flow conditions.
monocytes/macrophages; adhesion molecules; chemokines; cell trafficking; monocyte chemoattractant protein-1
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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VARIOUS STUDIES in the past few years have demonstrated that monocyte chemoattractant protein (MCP)-1 is the major chemoattractant for monocyte recruitment across endothelial cells (EC) as well as epithelial cells both in vitro and in vivo (9, 17, 25). Intra-alveolar overexpression of MCP-1 in pulmonary type II alveolar epithelial cells of mice was found to provoke a massive accumulation of monocytes within the alveolar air spaces (9). More recently, studies from our laboratory showed that intra-alveolar deposition of recombinant murine JE/MCP-1 in mice elicited substantial intra-alveolar accumulation of peripheral blood-derived monocytes. This further suggests that locally liberated low-molecular-weight chemokines like MCP-1 increase chemoattraction of circulating monocytes across biological barriers including EC and epithelial cell layers (17, 19).
There are, however, only a few studies with inconsistent
results addressing the effects of MCP-1 on early monocyte recruitment events, such as adhesion to inflamed endothelium. MCP-1 was shown to be
a major contributor to the robust arrest of monocytes to inflamed
endothelium under shear stress conditions in vitro (5, 16). Furthermore, Palframan and co-workers (24)
recently demonstrated a specific role of exogenous MCP-1 mediating
monocyte arrest to high endothelial venule (HEV)-draining lymph nodes
of inflamed skin in vivo. In contrast, another report
(29) identified a specific contribution of growth-related
oncogene (GRO)- but not MCP-1 to the monocyte adhesion process to
inflamed human umbilical vein EC (HUVEC) in vitro. Also, a
recent report suggested a specific role of monokine induced by
interferon-
but not MCP-1 in the monocyte adhesion process to HEV
draining inflamed lymph nodes in vivo (12).
The present study was designed to analyze a possible role of
endothelium-derived MCP-1 on monocyte adhesion in the pulmonary vasculature. We specifically interrupted the cross-talk between MCP-1
and its receptor, CCR2, on the monocyte surface. MCP-1 antisense oligomers that result in translational arrest of endothelial MCP-1 synthesis, MCP-1 antagonists (9-76 analog) that block CCR2
downstream signaling, and neutralizing anti-MCP-1 or function-blocking
anti-CCR2 mAbs were employed in a highly sensitive flow chamber assay
of human pulmonary EC. The data show that MCP-1 liberated from inflamed human pulmonary artery endothelium is capable of effecting monocyte adhesion under physiological shear-stress conditions by promoting increased activation of 2-integrin adhesion pathways.
These findings may be relevant for a further understanding of pulmonary
artery remodeling processes, as observed in different types of
pulmonary arterial hypertension and vasculitic processes involving the
lung (13).
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Monoclonal antibodies and reagents.
The following adhesion function-blocking murine mAbs specific for human
antigens were employed: clone 38 (anti-CD11a, R&D Systems;
Wiesbaden, Germany), clone 44 (anti-CD11b, R&D Systems), MEM
48 (anti-CD18, R&D Systems), HP2/1 [anti-4 chain of
VLA-4 (CD49d), Serotec; Oxford, UK], and SIE4A4 [anti-major
histocompatibility complex (MHC) class I, a generous gift from M. Hadam; Hannover, Germany]. The reporter mAb24 was a generous
gift from N. Hogg, Imperial Cancer Research Fund (London, UK).
Neutralizing murine mAbs against human MCP-1 and RANTES (regulated on
activation normal T cell expressed and secreted) were purchased from
R&D Systems, as was the function-blocking mAb specific for human CCR2
(23). The 9-76 antagonist of MCP-1 was synthesized
and purified at Jerini Biotools (Berlin, Germany), as previously
described (6, 7). The phosphorothioate-modified antisense
oligodeoxynucleotides (PS-ODN) (MCP-1 AS2) and corresponding scrambled
control oligodeoxynucleotides employed in the present study have been
characterized recently (18, 20, 21) and were synthesized
and purified at Biognostik (Göttingen, Germany).
Monocyte isolation and treatment. Human monocytes (from buffy coats of healthy blood donors) were isolated using Ficoll density gradient centrifugation (800 g, 30 min, 21°C), followed by counterflow centrifugal elutriation (Beckmann J2-21 M/E centrifuge with JE-B6 elutriator rotor, standard elutriation chamber, Beckmann Instruments; Palo Alto, CA). The monocyte fraction always consisted of 93-97% monocytes, 3-7% lymphocytes, and 0-1% granulocytes, and viability of monocytes was always >95%, as determined by trypan blue dye exclusion.
In some experiments, monocytes were pretreated, for 30 min at room temperature, with saturating amounts of anti-CCR2 mAbs (5-10 µg/ml), 9-76 analog (5-10 µg/ml), or various function-blocking mAbs directed against1- and
2-integrins (5 µg/ml), as indicated. Subsequently,
monocytes were washed twice in RPMI medium supplemented with 10%
heat-inactivated FCS before perfusion over appropriately pretreated EC.
FcIgG receptors were blocked by preincubation with human Ig
(10 µg/ml, Octagam, Octapharma; Langenfeld, Germany).
Culture and treatment of EC. Human pulmonary artery EC (HPAEC; Cascade Biologics; Portland, OR) were maintained at 37°C, 5% CO2 in a humidified atmosphere, as previously described (18, 20). HUVEC were isolated (11) and maintained in MCDB 131 medium supplemented with 10% heat-inactivated FCS, 2 mM glutamine, penicillin-streptomcyin, microvascular growth supplement, and Na-pyruvate. HPAEC and HUVEC were all grown to confluence on gelatin-coated Thermanox membranes (Nunc; Roskilde, Denmark) before use in flow assays. The supplemented media and reagents were routinely analyzed for endotoxin contaminations by a limulus amoebocyte lysate assay (COATEST, Chromogenix; Mölndal, Sweden) and contained <10 pg/ml endotoxin, the lower detection limit of the assay.
Treatment of EC with antisense or scrambled control PS-ODN as well as sequences of employed PS-ODN have recently been described in detail (18, 20, 21). Pretreatment of HPAEC with antisense MCP-1 oligomers (500 nM final concentration) specifically and effectively reduced TNF--induced MCP-1 synthesis by >85%, without affecting
IL-8 secretion or VCAM-1 and ICAM-1 cell surface expression and
endothelial transcription factor pools, including Sp1 and NF-
B or
activator protein-1 as well as their subunit composition (18, 20,
21).
Neutralization of endothelium-derived MCP-1 in response to TNF-
activation was performed by preincubating EC with saturating amounts of
neutralizing anti-MCP-1 mAb (5 µg/ml, determined in pilot
experiments) for 30 min at 37°C, 5% CO2, as
indicated. HPAEC monolayers were mounted in the flow chamber
and washed to remove the neutralizing anti-MCP-1 mAb before being
perfused with the appropriately pretreated monocytes.
Flow chamber assay.
Monocyte adhesion to confluent monolayers of TNF--activated (2.5 ng/ml, 6 or 12 h) HPAEC was investigated using a parallel plate
flow chamber. Briefly, monocytes (1 × 106 cells/ml)
resuspended in RPMI-1% FCS were allowed to flow over HPAEC under
physiological shear-stress conditions using a perfusion pump adjusted
to a constant shear rate of 1.5 dyn/cm2 at 37°C (model
WPI-SP100I, World Precision Instruments; Berlin, Germany). Monocyte
interaction with the endothelial monolayer was visualized by
videomicroscopy [model TK-C1381, JVC digital charge-coupled device
camera connected to an inverted microscope (Zeiss Axiovert S-100;
Wetzlar, Germany)] using ×100 magnification. After a 5-min
observation period, monocytes firmly attached to the HPAEC monolayer
were counted per high-power field (HPF) (mean ± SD of 5 randomly
chosen HPF evaluated). Each experiment was repeated at least three to
six times.
Flow cytometry of reporter mAb24 expression on monocytes in
response to MCP-1, RANTES, or PMA and on lymphocytes in response to
RANTES.
Induction of 24 neoepitope expression was used to evaluate the
different effects of MCP-1 and RANTES on the activation state of
2-integrin -chains. Monocytes were elutriated under
Ca2+/Mg2+-free conditions [PBS without
Ca2+/Mg2+ containing 5 mM EDTA (Seromed;
Berlin, Germany)], washed, and resuspended at 5 × 106 cells/ml in Ca2+/Mg2+-free HBSS
supplemented with 1 mM MgCl2 (Sigma; Deisenhofen, Germany) and 0.5% human serum albumin (HSA; Baxter; Unterschleissheim, Germany)
for 30 min at 37°C, 5% CO2. Subsequently,
incubation of cells with mAb24 (1:10 dilution of cell culture
supernatant) or control IgG in microtiter plates (Becton-Dickinson;
Heidelberg, Germany) was performed in the absence or presence of MCP-1,
RANTES, or PMA for various time periods, as indicated. The microtiter plates were then immediately placed onto ice, and the samples were
washed once in ice-cold HBSS-1 mM MgCl2-0.5% HSA,
supplemented with 0.01% NaN3, followed by incubation of
the samples with phycoerythrin-conjugated rat anti-mouse reagent (Dako;
Hamburg, Germany). In selected experiments, lymphocytes
isolated under the same experimental conditions as described for
monocytes were stimulated with recombinant RANTES for different time
points. Analysis of monocyte or lymphocyte mAb24 expression was
performed using a FACScan station and CellQuest software
(Becton-Dickinson; San Jose, CA).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of MCP-1 antisense oligomers or anti-MCP-1 monoclonal
antibodies on endothelial adhesion of monocytes under physiological
flow.
Less than 5% of untreated monocytes adhered to resting HPAEC or HUVEC
(2 ± 0.5 cells/HPF, mean ± SD, n = 7) under
physiological shear-stress conditions. Inflammatory activation of HPAEC
or HUVEC with TNF- strongly increased firm adhesion of monocytes
under these conditions (HPAEC: 97.7 ± 3 cells/HPF and HUVEC:
95.5 ± 2.5 cells/HPF, means ± SD, n = 7;
Fig. 1, A and B).
Depletion of endothelium-derived MCP-1 by pretreatment of HPAEC with
MCP-1 AS2, a PS-ODN that translationally arrests MCP-1 mRNA
transcripts, strongly reduced monocyte adhesion under flow by ~45%
(P < 0.05; Fig. 1A). In contrast, monocyte
adhesion to HUVEC under the same experimental conditions was only
slightly suppressed by MCP-1 AS2 (Fig. 1B). Treatment of
both HPAEC and HUVEC with a scrambled PS-ODN did not affect monocyte
adhesion. The strongly reduced monocyte adhesion to MCP-1
AS2-pretreated, TNF--activated HPAEC was fully restored by
preincubating monocytes with exogenous recombinant MCP-1
(500-1,000 pg recombinant MCP-1/106 cells; Fig. 1,
A and B) but not heat-inactivated MCP-1 (data not
shown). This was also true for the slightly reduced adhesion to HUVEC.
Furthermore, pretreatment of TNF--stimulated EC with neutralizing
anti-MCP-1 mAb strongly reduced the firm arrest of monocytes to HPAEC
(P < 0.05; Fig. 1, A and B),
whereas monocyte adhesion to HUVEC was only weakly suppressed (Fig. 1,
A and B). Treatment of TNF--activated EC with
neutralizing anti-RANTES mAb exerted only a moderate, statistically
nonsignificant effect (Fig. 1).
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Effect of anti-CCR2 monoclonal antibodies or MCP-1 antagonists on
monocyte adhesion to HPAEC or HUVEC under physiological flow
conditions.
Preincubation of monocytes with a function-blocking anti-CCR2 mAb
significantly reduced adhesion of monocytes to TNF--activated HPAEC
by ~45% (Fig. 2A), with
only a minor impact on HUVEC (Fig. 2B). Pretreatment of
monocytes with either an isotypic control IgG or anti-MHC class I mAbs
was ineffective, demonstrating that antibody binding to the cell
surface per se did not interfere with monocyte-endothelium interaction
under flow conditions. In addition, interruption of MCP-1 cross-talk
with CCR2 by preincubation of monocytes with a 9-76 MCP-1
antagonist similarly reduced monocyte adhesion to inflamed HPAEC
(~40%; Fig. 2A) with moderate effects on HUVEC (Fig.
2B). However, pretreatment of monocytes with
heat-inactivated 9-76 analog was ineffective (Fig. 2, A
and B).
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Effect of endothelium-derived MCP-1 depletion on 2-
and 1-integrin-dependent adhesion of monocytes to HPAEC
under flow.
To evaluate whether MCP-1 depletion differentially affected
1- versus 2-integrin-dependent pathways
of monocyte adhesion to HPAEC, endothelium-derived MCP-1 depletion
procedures were performed subsequent to blockade of one of these
integrins. In agreement with a previous report (14),
monocyte adhesion to TNF--activated HPAEC under physiological shear
stress was dependent on 2-integrins LFA-1 and Mac-1 and
on 1-integrin VLA-4. Inhibition of either integrin
-chain by specific adhesion function-blocking mAbs reduced monocyte
adhesion by ~40%, whereas isotype-matched control antibody
was without effect (Fig. 3A).
Simultaneous blockade of both 2-integrin -chains
CD11a and CD11b reduced monocyte adhesion by ~60%. The strongest
inhibition of monocyte adhesion was observed with simultaneous blockade
of both 2-integrins LFA-1 and Mac-1 plus blockade of
1-integrin VLA-4 (~75%). These data demonstrate that
monocyte firm adhesion to TNF--stimulated HPAEC under flow
conditions is dependent on both 2- and
1-integrins (Fig. 3A), which only partially
act as alternative adhesion pathways. Simultaneous blockade of both
2- and 1-integrin adhesion pathways plus
additional depletion of HPAEC-derived MCP-1 did not further decrease
monocyte firm adhesion observed with combined anti-CD11a plus
anti-CD11b plus anti-VLA-4 blockade (Fig. 3B). This finding demonstrates that residual monocyte adhesion pathways independent from
LFA-1, Mac-1, or VLA-4 mediating firm arrest on inflamed endothelium
are not sensitive to endothelium-derived MCP-1. Similarly, 1-integrin (VLA-4)-dependent adhesion of anti-CD11a-
plus anti-CD11b-pretreated monocytes was only marginally affected by
depletion of endothelium-derived MCP-1, suggesting a minor role for
MCP-1 in 1-integrin-mediated adhesion. In contrast,
2-integrin (CD11a and CD11b)-dependent adhesion of
anti-VLA-4-treated monocytes was further markedly reduced by depletion
of endothelium-derived MCP-1, which clearly suggests a primary effect
of MCP-1 on 2-integrin (CD11a and CD11b) adhesion
involved in monocyte arrest to inflamed endothelium. Moreover,
depletion of endothelium-derived MCP-1 enhanced the inhibitory effect
of anti-CD11a alone or anti-CD11b alone treatment of monocytes,
demonstrating that MCP-1 exerts its effect on both 2-integrin -chains LFA-1 and Mac-1.
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2-integrin-dependent adhesion pathways (Fig.
3C). In contrast, when monocytes were treated simultaneously
with anti-CD11a and anti-CD11b followed by incubation with recombinant
MCP-1 before perfusion over MCP-1-depleted HPAEC, no significant
increase in monocyte adhesion was observed, suggesting that depletion
of MCP-1 primarily affects 2-integrin rather than
1-integrin monocyte adhesion pathways. Furthermore, stimulation of anti-CD11a plus anti-CD11b plus anti-VLA-4 treated monocytes with recombinant MCP-1 before perfusion over MCP-1-depleted HPAEC did not increase the numbers of arresting cells. This suggests that adhesion pathways other than 1- and
2-integrins mediating the observed residual adhesion of
monocytes to MCP-1-depleted HPAEC (Fig. 3B) were not MCP-1 responsive.
Effect of MCP-1, RANTES, and PMA on activation-dependent 24 neoepitope expression on 2-integrin -chains.
To further investigate the effect of MCP-1 on monocyte
2-integrin -chain activity, we analyzed the
"reporter" expression of the 24 neoepitope on
2-integrin -chains. Stimulation of monocytes with
MCP-1 (Fig. 4, A-E)
induced a rapid and transient expression of the 24 neoepitope on
2-integrin -chains peaking between 30 and 150 s
posttreatment, whereas stimulation of monocytes with RANTES was
ineffective (Fig. 4, F-H). Stimulation of monocytes with PMA (Fig. 4, I-N), which is known to activate
integrins independently of G protein-linked receptors, induced a
sustained 24 neoepitope expression that was still detectable at 20 min
posttreatment. Contrary to the results observed with monocytes,
stimulation of lymphocytes with recombinant RANTES induced a rapid
expression of the 24 neoepitope peaking at 150 s posttreatment
with a decline thereafter, demonstrating that the recombinant RANTES
preparations used were biologically active (Fig. 4,
O-R).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Interrupting the MCP-1-based cross-talk between EC (MCP-1
synthesis) and monocytes (expression of the corresponding CCR2
receptor) profoundly reduced monocyte adhesion to inflamed HPAEC under
physiological shear-stress conditions. Depletion of HPAEC-derived MCP-1
was found to primarily affect 2-integrin (LFA-1 and
Mac-1)-mediated monocyte adhesion pathways. Importantly, pretreatment
of monocytes with exogenously administered recombinant MCP-1 fully
restored firm adhesion of monocytes to MCP-1-depleted HPAEC under
physiological flow conditions. Moreover, MCP-1 rapidly and transiently
induced the 24 neoepitope on monocytes, reflecting increased
chemokine-induced 2-integrin -chain activity. These
data support the hypothesis that endothelium-derived MCP-1 primes
monocytes for increased adhesion to inflamed endothelium under
physiological shear-stress conditions, primarily by enhancing the
activity of monocyte 2-integrin adhesion molecules.
The role of MCP-1 as a major chemoattractant for monocytes has been clearly established both in vitro and in vivo. Various mouse models have revealed a causal relationship between increased local release of MCP-1 and monocyte accumulation at extravascular sites (4, 8, 9). We recently used, in mice, a newly established technique for identifying monocytes recruited to the lung alveoli to demonstrate that local (intra-alveolar) deposition of recombinant JE/MCP-1 in mice provoked substantial and dose-dependent recruitment of circulating monocytes into the alveolar spaces (17, 19). This observation suggests that low-molecular-weight chemokines such as MCP-1 are capable of passing biological cell barriers, such as epithelial cells and EC, to establish a chemokine gradient capable of attracting monocytes to extravascular sites (8, 17). In fact, interruption of the MCP-1 cross-talk with its receptor, CCR2, has been previously shown to significantly affect leukocyte adhesion to cremaster muscle microvascular endothelium in knockout mice lacking the MCP-1 receptor CCR2, thereby decreasing the number of transmigrating monocytes (15).
To study the impact of endothelial MCP-1 on firm monocyte-endothelium
adhesion in more detail, we used both immunologic and translation
regulatory interventions to interrupt MCP-1/CCR2-based cross-talk in a
flow chamber assay of monocyte arrest on TNF--activated HPAEC under
physiological flow conditions. We used a recently developed
antisense-based approach that specifically and effectively (>85%)
blocks TNF--induced MCP-1 synthesis in HPAEC by translational arrest of MCP-1 mRNA transcripts. This blockage does not affect endothelial ICAM-1 and VCAM-1 cell surface expression (18,
20). This molecular strategy markedly reduced monocyte arrest to
TNF--activated HPAEC, which was reproduced by employment of
anti-MCP-1 but not anti-RANTES monoclonal antibodies. Notably, the
antisense-induced inhibition of monocyte firm adhesion was fully
restored when monocytes were treated with low concentrations of
recombinant MCP-1 immediately before perfusion through the flow
chamber. Interestingly, a sensitive cell surface ELISA did not detect
either TNF--induced endothelial MCP-1 or exogenously added
recombinant MCP-1 immobilized on the HPAEC cell surface (data not
shown). This suggests that immobilization on the EC surface is not a
precondition for MCP-1 to promote monocyte adhesion to endothelium
under flow.
In another study (10) investigating the adhesion of
monocytes to atherosclerotic lesions of carotid arteries from
ApoE
/ mice ex vivo, a significant contribution of
KC/GRO- but not JE/MCP-1 to monocyte arrest was observed.
In contrast, we found that blockade of the CCR2 receptor on the
monocyte cell surface with either anti-CCR2 mAbs (23) or a
specific 9-76 MCP-1 antagonist (6, 7) significantly
reduced monocyte adhesion to inflamed HPAEC to a similar extent as
observed for anti-MCP-1 mAb or MCP-1 antisense pretreatment of HPAEC.
However, both pretreatment of monocytes with the 9-76 MCP-1
antagonist or anti-CCR2 mAb as well as MCP-1 antisense or anti-MCP-1
mAb pretreatment of HUVEC was much less effective in reducing
endothelial monocyte adhesion compared with HPAEC. Thus, from data
currently available, it appears that both EC from different organs and
their inflammatory activation (acute versus chronic inflammation) may
considerably affect their inducible chemokine/cytokine profiles and
related monocyte adhesion events. Against the background that HUVEC are
often considered as a "standard" EC type, it is particularily
relevant that there are actually many differences in phenotype between
EC from different vascular beds (22, 26). Future studies
with macro- and microvascular EC isolated from different organs should
lead to a better understanding of endothelium-derived
chemokine-dependent leukocyte arrest under flow conditions.
Characterization of molecular pathways of monocyte adhesion to
TNF--activated HPAEC revealed that both 2-integrins
LFA-1 and Mac-1 as well as the 1-integrin VLA-4
contributed equally to firm adhesion under physiological flow
conditions, in agreement with a previously published report
(14). Interestingly, blockade of LFA-1, Mac-1, or VLA-4
alone and subsequent perfusion of monocytes over MCP-1-depleted HPAEC
further significantly reduced monocyte firm adhesion. In particular,
anti-1-integrin (VLA-4)-pretreated monocytes showed a
significantly reduced adhesion to MCP-1-depleted HPAEC compared with
control EC, and this was fully restored by exogenous administration of
recombinant MCP-1. In contrast, anti-2-integrin -chain (CD11a and CD11b)-pretreated monocytes showed a comparable degree of firm adhesion to MCP-1-depleted HPAEC as to
non-MCP-1-depleted HPAEC. Furthermore, such pretreated monocytes did
not respond with increased firm adhesion to exogenous MCP-1
stimulation. These findings indicate that endothelium-derived MCP-1
predominantly affects 2-integrin-based monocyte
adhesive interactions with HPAEC, whereas the contribution of VLA-4 to
monocyte endothelium adhesion is largely MCP-1 independent.
Chemokine modulation of leukocyte integrin activity has been reported
for various leukocyte subpopulations (1, 27, 28). Therefore, we tested whether MCP-1 was capable of increasing
2-integrin -chain activity, using the 24 neoepitope
expression as a "reporter" to monitor this -chain activity
(2, 3). Indeed, we found that stimulation of monocytes
with MCP-1 but not RANTES induced a very rapid and transient activation
of 2-integrin -chains, occurring as early as ~30 s
after induction. This MCP-1-induced 24 neoepitope expression on the
monocyte cell surface corresponds well to the time frame necessary to
restore reduced monocyte adhesion to MCP-1-depleted HPAEC by
pretreatment of the monocytes with exogenous MCP-1 (~30 s).
With the use of several various translation regulatory and immunologic
interventions to interrupt MCP-1 cross-talk with its receptor on the
monocyte cell surface, we demonstrated that endothelial-derived MCP-1
primes monocytes for increased firm adhesion to TNF--activated HPAEC
under physiological flow conditions. Inhibition of HPAEC-derived MCP-1
synthesis only weakly affected 1-integrin (VLA-4) but
strongly diminished 2-integrin (LFA-1 and Mac-1)
adhesion-dependent pathways, which was fully rescued by exogenous
recombinant MCP-1. Together with the analysis of monocyte
2-integrin activity changes in the presence of MCP-1,
these data suggest that endothelial MCP-1 exerts its effect on firm
monocyte adhesion to inflamed endothelium via modulation of
2-integrin -chain activity.
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ACKNOWLEDGEMENTS |
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The authors thank R. Maus and G. Mansouri for expert technical assistance.
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FOOTNOTES |
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This study was supported by Deutsche Forschungsgemeinschaft Grant SFB 547 "Cardiopulmonary Vascular System."
Address for reprint requests and other correspondence: U. A. Maus, Dept. of Internal Medicine, Klinikstrasse 36, Justus-Liebig Univ., Giessen 35392, Germany (E-mail: Ulrich.A.Maus{at}med.uni-giessen.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 29, 2002;10.1152/ajpheart.00349.2002
Received 18 April 2002; accepted in final form 17 August 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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