© 1995 American Heart Association, Inc.
Articles |
Suppressive Role of Endogenous Endothelial Monocyte Chemoattractant Protein–1 on Monocyte Transendothelial Migration In Vitro
From the Departments of Cardiology (M.T., U.I., K.S.) and Clinical Immunology (J.-I.M., S.K.); the Division of Hemopoiesis, Institute of Hematology (S.-I.K., M.S.); and the Department of Medical Biology and Parasitology (T.K.); Jichi Medical School, Tochigi, Japan.
Correspondence to Jun-Ichi Masuyama, MD, Department of Clinical Immunology, Jichi Medical School, Minamikawachi-machi, Tochigi 329-04, Japan.
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Abstract |
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Abstract Monocyte chemoattractant protein–1 (MCP-1, or monocyte chemotactic and activating factor) is thought to play an important role in monocyte infiltration into tissue, but little is known about its effect on monocyte-endothelium interaction. We examined the effect of MCP-1 produced by cytokine-activated endothelial cells (ECs) on monocyte-endothelium adhesion and subsequent transendothelial migration by using a double-chamber vessel model. Unstimulated ECs showed no MCP-1 expression, but exposure to interleukin-1ß (IL-1ß, 25 U/mL) induced marked MCP-1 mRNA expression and protein synthesis. When placed in the lower compartment, recombinant human (rh) MCP-1 (100 ng/mL) produced a 1.9-fold and a 2.7-fold increase in adhesion and migration, respectively, compared with a corresponding 51% and 59% decrease when placed in the upper compartment. Migration of monocytes was dependent on a gradient of rh–MCP-1 from the apical to basilar side of the EC layer. Furthermore, a forward gradient of MCP-1 induced adherent cells to increase their subsequent migration, whereas a reverse gradient induced the cells to detach and completely inhibited their subsequent migration. Pretreatment with IL-1ß for 4 and 24 hours produced a 20% and 63% increase in monocyte migration, respectively. In the presence of anti–MCP-1 antibody, the increase was further enhanced by 52% and 152%, respectively. These results suggest that endogenous endothelial MCP-1, when secreted by IL-1–stimulated ECs, suppresses monocyte migration in the presence of MCP-1 on the basilar side of the EC layer. This process may be useful for preventing excessive infiltration of monocytes from the circulating blood into atherogenic tissue during the early stages of atherogenesis.
Key Words: atherosclerosis • monocyte-endothelium interaction • chemokines
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Introduction |
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The transendothelial migration of leukocytes from circulating blood is an essential event in the development of atherosclerosis and inflammation.1 2 3 4 5 Recent evidence suggests that the entire process of leukocyte migration includes at least three separate steps. The first step is leukocyte rolling and loose binding to the endothelium via selectin molecules, as supported by some studies that have shown that neutrophils adhere to endothelial cells (ECs) via L-selectin under shear stress conditions in vitro.6 The second step is the "triggering" of functionally inactive integrins on leukocytes to become active and adhesive, and the third step is the induction by these activated integrins of tight leukocyte-endothelium adhesion and subsequent transendothelial migration.7 8 9 Although it is generally accepted that a transendothelial gradient of soluble chemoattractants is responsible for leukocyte migration, more recent studies have indicated alternative roles for these substances in the second step.10 11 12 In this alternative model, interleukin-8 (IL-8) and macrophage inflammatory protein–1ß (MIP-1ß), which are cytokines with chemotactic activity (ie, chemokines),13 14 are "captured" by the surface proteoglycans of the endothelium. These immobilized chemokines then activate the integrin molecules on rolling leukocytes to induce tight adhesion and the shedding of selectin. It is also speculated that subsequent transendothelial migration is caused by haptotaxis in response to immobilized chemokines on the endothelial surface.10
In this context, if activated EC-derived chemokines such as IL-8 are released predominantly to the luminal side of the vessel, then the role of endothelial IL-8 in neutrophil-endothelium interaction may be more complex. Gimbrone et al15 and Luscinskas et al16 have reported that under static conditions, soluble IL-877 (a 77–amino acid variant of IL-8 with similar biological effects) and other chemoattractants not only inhibits attachment of neutrophils to cytokine-activated EC layers but also promotes detachment of tightly adherent neutrophils from the layers, despite the increased expression of neutrophil CD11b/CD18 involved in adhesion. Therefore, the endothelial IL-8 might diminish the transendothelial chemotactic gradient of IL-8, particularly for adherent neutrophils that are continuously exposed to the released IL-8, and result in inhibition of the subsequent migration. In contrast, Huber et al,17 using double-chamber culture dishes, have shown that neutrophil migration across IL-1–activated EC layers is markedly inhibited in the presence of anti–IL-8 antiserum, indicating that endogenous endothelial IL-8 upregulates neutrophil adhesion and migration. This finding also supports a recently proposed mechanism whereby the endothelial IL-8 that is trapped on the endothelial surface may increase neutrophil adhesion and migration.10 Thus, there still appear to be certain inconsistencies with regard to the possible role of endothelial IL-8.15 16 17
Monocyte chemoattractant protein–1 (MCP-1), a chemoattractant specific for monocytes, belongs to the chemokine family of molecules.13 14 18 MCP-1 is produced by a variety of cell types in vitro, including monocytes, vascular ECs, smooth muscle cells, cardiac myocytes, fibroblasts, and tumor cell lines.19 20 21 22 23 MCP-1 has been shown to be present in areas where macrophages infiltrate, such as atherosclerotic lesions,24 25 rheumatoid synovial fluid,26 and the lung in patients with idiopathic fibrosis.27 The MCP-1 produced in such lesions is assumed to attract monocytes from the circulating blood into the tissue. Because the endothelium is a major source of MCP-1, it is particularly important to determine the effect of endogenous endothelial MCP-1 on monocyte transendothelial migration. Shyy et al28 have recently reported that monocyte-derived colony stimulating factor stimulates MCP-1 gene transcripts and increases monocyte adhesion to ECs and that the increased adhesion is inhibited by anti–MCP-1 antibody (Ab). However, little is known about the influence of MCP-1 on monocyte-endothelium interaction in vitro. In this study, using a double-chamber vessel model, we examined the effect of a transendothelial gradient of soluble MCP-1 on monocyte-endothelium adhesion and subsequent migration. We then determined whether endogenous MCP-1 released from IL-1–activated EC layers inhibited or enhanced monocyte transendothelial migration.
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Methods |
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Reagents
Purified recombinant human (rh) MCP-1 and rh–IL-8 were prepared as described previously.29 rh–IL-1ß was obtained from Otsuka Pharmaceutical Co. Type I collagen solution extracted from porcine skin (Cellmatrix I-A) was purchased from Nitta Gelatin Co. Fetal calf serum and EC growth supplement (ECGS) were purchased from Cell Culture Laboratories and Collaborative Research, respectively. Bovine serum albumin (BSA), HEPES, gelatin, collagenase (type I-A), and quin 2-AM were obtained from Sigma Chemical Co. Porcine heparin was purchased from Nakarai Chemical Co. Medium 199 (M199), RPMI 1640, and Eagle minimum essential medium (EMEM) were obtained from GIBCO. Monoclonal antibodies (MAbs) directed against intercellular adhesion molecule–1 (ICAM-1), E-selectin, and vascular cell adhesion molecule–1 (VCAM-1) were purchased from British Biotechnology Ltd. MAbs against CD18, L-selectin (TQ-1), and very late antigen–4 (VLA-4) were gifts from Dr C. Morimoto (Dana Farber Cancer Institute, Boston, Mass). MAbs against CD11a and CD11b (Mac-1) were purchased from Serotec and Becton Dickinson, respectively. Anti–MCP-1 and anti–IL-8 Abs were developed and purified as described previously.30 FITC-conjugated goat anti-mouse IgG was purchased from Organon Teknika Co.
Human EC Cultures
Primary ECs were harvested from human umbilical cord veins
treated with 0.1% collagenase, as described elsewhere.31
The ECs were grown on 5% gelatin-precoated 60-mm culture dishes
(Nunclon) in M199 containing 20% heat-inactivated fetal calf serum,
1% penicillin/streptomycin solution, glutamine (2 mmol/L), HEPES (15
mmol/L), heparin (100 µg/mL), and ECGS (60 µg/mL) (EC medium).
Cells were used between passages 2 and 4.
Isolation of Monocytes
Mononuclear cells were prepared from heparinized venous blood
samples drawn from healthy adult donors by Ficoll-Conray density
gradient centrifugation. Monocytes were purified from the mononuclear
cells by centrifugal elutriation with a Hitachi SRR6Y elutriation rotor
(Hitachi Ltd,), as described previously.32 The monocyte
fractions contained 85% to 95% monocytes with 5% to 15%
lymphocytes, as determined by Giemsa staining of cytospin preparations.
Fractions were resuspended in M199 supplemented with 0.1% BSA, 1%
penicillin/streptomycin solution, and 2 mmol/L glutamine (assay
medium).
Northern Blot Analysis
Total RNA was prepared from ECs cultured in 60-mm dishes by the
guanidine isothiocyanate–CsCl method. Equal amounts of total RNA (10
to 15 µg) were size-fractionated by electrophoresis on denaturing
1.0% agarose/formaldehyde gels and transferred to nylon membranes
(Hybond N+, Amersham). Hybridization was performed
at 65°C for 24 hours with an excess of
[32P]dCTP-labeled human MCP-1 cDNA probe (specific
activity, >1x108 cpm/µg DNA) at 60°C for 24 hours.
The MCP-1 probe was a 0.4-kb EcoRI restriction fragment. At
the end of hybridization, the filters were washed twice in 0.2xSSC at
60°C (1xSSC contains 0.15 mol/L NaCl, 0.015 mol/L sodium citrate, pH
7.0) and then exposed to Kodak XAR-5 film overnight at -70°C with
one intensifying screen.
Radioimmunoassay for MCP-1
A competitive radioimmunoassay (RIA) for quantitation of MCP-1
was performed using 125I-labeled r–MCP-1 and polyclonal
rabbit anti-human MCP-1 Ab.30 Briefly, r–MCP-1 was
radioiodinated with Bolton Hunter reagent (Amersham). Fifty-microliter
aliquots of 125I–MCP-1 (2x104 cpm), sample,
and antiserum (diluted 1:3200) were mixed and incubated at 4°C
overnight. Then magnetic goat anti-rabbit IgG (Advanced Magnetics Inc)
was added; the mixture was incubated further for 2 hours at 4°C and
spun down, and the radioactivity in the pellet was counted. With these
procedures, 0.01 ng per tube (0.1 ng/mL) MCP-1 could be correctly
evaluated.
Adhesion and Migration Assay
For the monocyte transendothelial migration assay, we used inner
wells (cell culture insert, catalog No. 3095, Falcon) to divide each
well of a 24-multiwell plate (Falcon) into two compartments. To
evaluate by microscopy the chemoattractive effect of MCP-1 on monocyte
migration, a thin layer of collagen gel (100 µL per well) was placed
on the filters of the inner wells. The collagen gels were prepared as
described previously.33 34 The wells were then seeded with
ECs at a density of 6x104 cells per inner well in 300 µL
of EC medium and then placed in the wells of the multiwell plates, each
of which contained 500 µL of EC medium without ECGS. The EC layers
were grown to confluence and cultured further for at least 48 hours.
The EC layers were then treated for 4 hours with or without IL-1ß (25
U/mL) and washed prior to the assay. Monocyte suspensions containing
2x105 cells in 300 µL of assay medium were added to the
EC monolayers. After incubation at 37°C in a humidified incubator
under 5% CO2, unbound monocytes were gently removed
from the inner wells by washing with warmed assay medium. At this step,
monocytes adherent to the EC layers were counted. To determine the
number of monocytes that had migrated into the collagen gel across the
EC layers, the inner wells were incubated further for 60 minutes at
37°C with 0.4% EDTA in PBS. By phase-contrast microscopy, it was
confirmed that almost all adherent monocytes and ECs had been removed
from the surface of collagen gels by gentle pipetting. In some
experiments, we employed a convenient method that uses a 48-multiwell
plate (Falcon) without inner wells; ie, EC layers were grown on
collagen gels. The collagen gels that contained migrated cells were
fixed with 1% paraformaldehyde, and the numbers of adherent and
migrated cells were counted in at least eight fields under a
phase-contrast microscope (Nikon, Japan) at x100 magnification. The
experiments were done in duplicate or triplicate. The percentage of
monocyte adhesion and migration in the control wells ranged from
11.77% to 15.25% and 11.20% to 13.90%, respectively (experimental
n=6). The data in this study were expressed as index values relative to
controls (control=100% for the index value). Because monocyte adhesion
and migration with unstimulated and IL-1ß–stimulated EC layers
increased comparably during incubation periods of 3 and 5 hours, we
assessed the adhesion and migration at 1 hour of incubation in the
following experiments.
In a preliminary study, the capacity of confluent EC layers to maintain gradients of soluble factors was assessed by adding 125I–MCP-1 to the upper compartment of this system and then sampling the medium in both the upper and lower compartments and the collagen gels at 1 to 5 hours. Although 125I–MCP-1 diffusion in the subendothelial space increased with time, the amount of 125I–MCP-1 that diffused into the underlying gels was 11%, 13%, and 15% and in the lower compartment, 3%, 9%, and 20%, of the total added 125I-MCP-1 at 1, 3, and 5 hours of incubation, respectively. These data indicated that the EC layers and collagen gels were able to exclude soluble factors and therefore that the transendothelial gradient of MCP-1 concentration was essentially unchanged by the MCP-1 that diffused into the subendothelial space during the 1-hour assay period.
Detection of Adhesion Molecules on EC Layers and Monocytes
Cell surface expression of adhesion molecules on EC monolayers
was examined by a cellular enzyme-linked immunosorbent assay (ELISA)
method as described previously.35 Briefly, confluent EC
layers that had been preincubated with MCP-1 (100 ng/mL) for 4 hours in
96-multiwell plates (Falcon) were fixed with 1% paraformaldehyde and
then treated for 1 hour with 3% BSA in PBS to block nonspecific
binding. An appropriate amount of MAb against adhesion molecules
(ICAM-1, VCAM-1, and E-selectin) was added, followed by incubation for
2 hours at 37°C. After being washed, the ECs were treated for 1 hour
with the development Ab (horseradish-conjugated anti-mouse IgG). An
o-phenylenediamine mixture was reacted with the ECs, and the
optical density at 492 nm was measured by an ELISA reader (Titertek
Multiscan, Flow Laboratories).
Expression of adhesion molecules on monoytes was determined by flow cytometry. Monocytes (1x105 cells) treated with MCP-1 (100 ng/mL) for various times at 37°C were treated with saturating amounts of MAb for 20 minutes and then washed three times with PBS containing 0.1% BSA and 0.02% NaN3. After being washed, the monocytes were stained for 20 minutes with FITC-conjugated goat anti-mouse IgG and then washed three times with the same PBS. The cells (1x104 cells) were analyzed with a flow cytometer (FACScan, Becton-Dickinson). All procedures were carried out at 4°C.
Determination of Cytoplasmic Free Ca2+
Levels
Cytoplasmic free Ca2+ levels
([Ca2+]i) were measured by using the
fluorescent calcium indicator quin 2-AM as described
previously.36 Monocytes (1x107 cells/mL)
suspended in Hanks' balanced salt solution were equilibrated at 37°C
for 5 minutes. Quin 2-AM (15 µmol/L) was added, and the cells were
incubated at 37°C for 15 minutes in a shaking water bath. The cells
were diluted to 1x107 cells/mL with warm Hanks' balanced
salt solution and incubated for another 40 minutes at 37°C. After
loading, the cells were washed twice, suspended in HEPES buffer (145
mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L
Na2HPO4, 1 mmol/L
CaCl2, 5 mmol/L glucose, and 10 mmol/L HEPES, pH
7.4), and kept at room temperature until used. The fluorescence was
measured with a fluorescence spectrophotometer (F-4010, Hitachi Ltd)
equipped with a thermostatted cuvette holder. The cell suspension in
HEPES buffer was added to a 3-mL cuvette to obtain a final volume of 2
mL. Final cell concentration was 1x106 cells/mL.
The cell suspension was maintained by means of a magnetic flea and
stirrer. The excitation and emission wavelengths were set at 339 and
492 nm, respectively.
Statistical Analysis
All values are given as mean±SE. For comparisons between two
groups, probability values were calculated by Student's t
test. In experiments that involved comparisons of multiple groups, the
probability that differences existed between the group means was
determined by ANOVA and the least significant difference for multiple
comparisons. Values of P<.05 were considered statistically
significant.
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Results |
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Expression of MCP-1 mRNA and Protein in ECs
We first examined the expression of MCP-1 mRNA and protein in ECs by stimulation with IL-1ß. Northern blot analysis revealed that MCP-1 mRNA expression was not detectable in unstimulated ECs, but its transcripts were clearly detected after exposure to IL-1ß (25 U/mL) for 4 hours, and the levels were sustained for at least 24 hours (Fig 1
). Furthermore, coculture of resting ECs
(2x106 cells) with human monocytes
(1x107 cells) also induced MCP-1 mRNA expression after 4
hours, but the levels declined during the subsequent 24 hours.
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Because IL-1ß–stimulated ECs were expected to produce substantial
amounts of MCP-1, we determined MCP-1 protein levels in the upper and
lower compartments of the double-chamber assay system. After culture of
the EC layers with or without IL-1ß (25 U/mL) for 4 or 24 hours, both
compartments were washed and the ECs were incubated further for 1 hour
with assay medium. MCP-1 levels in the conditioned media of both
compartments were measured serially by RIA. ECs pretreated with IL-1ß
for 4 hours secreted a total of 0.71 ng MCP-1 into the medium, 89.7%
(0.64±0.03 ng) of the total into the upper compartment and 10.3%
(0.07±0.04 ng) of the total into the lower (Fig 2).
Similar results were obtained for ECs pretreated with IL-1ß for 24
hours.
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) and lower (
) compartments was measured by
radioimmunoassay. Values represent mean±SE for three
independent experiments.
Effect of MCP-1 on Monocyte Adhesion to Unstimulated EC Layers
and Subsequent Migration
We then examined the effect of rh–MCP-1 (exogenous MCP-1) on
monocyte adhesion to and migration across EC layers in our assay
system. We added rh–MCP-1 at various concentrations to the upper
and/or lower compartment(s) and examined monocyte adhesion to the EC
layers and subsequent migration. As shown in Fig 3,
MCP-1 at 10 and 100 ng/mL in the lower compartment increased adhesion
by 1.4-fold and 1.9-fold, respectively, and increased migration by
1.7-fold and 2.7-fold, respectively, in comparison with control
conditions (13.5% and 12.8%, respectively, of the total cells added;
n=6) during which no MCP-1 was added to either compartment. However,
the enhanced adhesion and migration observed at 100 ng/mL MCP-1
decreased as the concentration of MCP-1 in the upper compartment was
increased from 0 to 10 ng/mL, and no enhancement of adhesion and
migration was observed when the concentration of MCP-1 in both
compartments was 10 and 100 ng/mL. In contrast, when MCP-1 (100 ng/mL)
was added only to the upper compartment, adhesion and migration were
inhibited by 51% and 59%, respectively. On the other hand, rh–IL-8
(25 ng/mL), which is also secreted from IL-1ß–stimulated ECs but
does not act on monocytes, did not affect the adhesion and migration of
monocytes. It is unlikely that this MCP-1–induced modulation of
adhesion and migration was caused by changes in the expression of cell
adhesion molecules, because MCP-1 showed no effect on monocytes with
regard to the expression of L-selectin, CD11a, CD11b, CD18, and VLA-4
and on ECs with regard to the expression of ICAM-1, VCAM-1, and
E-selectin, as revealed by flow cytometric analysis and cellular
ELISA (data not shown).
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Mechanisms of Diminished Monocyte Adhesion by Addition of MCP-1 to
the Upper Compartment
We have demonstrated that exogenous MCP-1 in the upper compartment
diminishes monocyte adhesion and migration. There are at least three
explanations for this finding. First, homologous desensitization of
monocytes to MCP-1 could be considered. It has been suggested that
chemotaxis of monocytes is reduced by this desensitization at high
concentrations (>100 ng/mL) of MCP-1 in the Boyden chamber
assay.37 To test for this desensitization, we examined
[Ca2+]i in monocytes. As shown in Fig 4, desensitization was observed with multiple challenges
at an MCP-1 concentration of 100 ng/mL. However,
[Ca2+]i levels were obviously
increased by 100 ng/mL MCP-1 following the first challenge of 10 ng/mL
MCP-1. These results suggest that desensitization to MCP-1 is not
involved at a concentration as low as 10 ng/mL MCP-1, which induced a
significant reduction in monocyte adhesion and migration (Fig 3
).
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The other two possibilities are that MCP-1 may abolish the ability of
monocytes to adhere to ECs before the two cells interact and that MCP-1
may detach adherent monocytes and decrease subsequent migration. To
investigate the former possibility, monocytes were added to
unstimulated EC layers after pretreatment with rh–MCP-1 (100 ng/mL)
for 1 hour and then washed. Because adhesion and migration were not
altered by this pretreatment (data not shown), the first aforementioned
possibility seemed unlikely. To address the latter possibility, we
investigated the effect of adhesion and migration of monocytes that had
already adhered to EC layers. After nonadherent and loosely adherent
monocytes were removed from the endothelial surface by gentle washing
after the first 20 minutes of incubation (control), an additional
incubation was performed with or without rh–MCP-1 in the upper or
lower compartment for 1 hour. As shown in Fig 5,
although there were no significant differences in adhesion with regard
to the presence or absence of rh–MCP-1 (100 ng/mL) in the lower
compartment, addition of rh–MCP-1 to the upper compartment induced
significant detachment of adherent monocytes from the EC layers.
Transendothelial migration increased approximately twofold during the
additional incubation without MCP-1 in both compartments in comparison
with the control. Migration was further augmented by addition of
rh–MCP-1 (100 ng/mL) to the lower compartment, whereas migration was
inhibited almost completely when rh–MCP-1 was added to the upper
compartment. On the other hand, adhesion and migration were not
affected by addition of rh–IL-8 (25 ng/mL) to the lower compartment.
Thus, these results strongly suggest that MCP-1 on the apical side of
EC layers not only detaches adherent monocytes but also inhibits their
subsequent migration.
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Effect of Anti–MCP-1 Ab on Monocyte Adhesion and Migration
Our results support the premise that endogenous MCP-1 secreted
from activated ECs into the apical side suppresses adhesion and
migration of monocytes. To test this hypothesis, we assessed monocyte
migration across IL-1–stimulated EC layers in the presence of
anti–MCP-1 Ab. To preclude any influence of MCP-1 diffusion into the
lower compartment, the inner wells were transferred to new outer wells
just before the transendothelial migration assay. As shown in Fig 6, in comparison with unstimulated EC layers,
pretreatment with IL-1ß for 4 and 24 hours produced a 20% and 63%
increase of migration, respectively. In the presence of anti–MCP-1 Ab
(12.5 µg/mL) in the upper compartment, migration was further enhanced
by 52% and 152%, respectively, although migration across unstimulated
EC layers was not affected. In contrast, no enhancement of migration
was observed when Ab directed against IL-8 (12.5 µg/mL) was added to
the upper compartment. Thus, these results suggest that endogenous
endothelial MCP-1 has a suppressive effect on monocyte migration across
IL-1ß–stimulated EC layers.
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Discussion |
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Several investigators have demonstrated MCP-1 secretion from activated ECs and arterial smooth muscle cells,19 20 28 38 39 indicating a potentially important function for MCP-1 in atherogenesis. Our present study strongly suggests that the role of endogenous endothelial MCP-1 is complex and could have inhibitory effects on monocyte adhesion to the endothelium and subsequent transendothelial migration in vivo. This assumption is based on several experimental findings. Monocyte adhesion and migration were influenced by the location at which MCP-1 was added exogenously, ie, the apical or basilar side of the EC layers. When MCP-1 in the lower compartment formed a transendothelial gradient, monocyte migration was increased. This result is consistent with the generally accepted notion that soluble chemotactic gradients are responsible for transmigration. Despite the lack of enhancement of adhesion molecule (CD11a, CD11b, CD18, VLA-4, L-selectin, ICAM-1, VCAM-1, and E-selectin) expression on ECs or monocytes by MCP-1 (data not shown), increased adhesion of monocytes to ECs was observed. This increase may be caused by a change in the conformation of adhesion molecules, as proposed recently as an alternative chemoattractant mechanism for chemokines.10
On the other hand, when MCP-1 was added to the upper compartment so
that the gradient of MCP-1 between the upper and lower compartments
decreased, monocyte adhesion and migration were inhibited in a
dose-dependent manner. MCP-1 in the upper compartment only at
concentrations of 10 and 100 ng/mL decreased adhesion and migration to
levels lower than those of controls. There are several possible
mechanisms that might account for the inhibitory effect of MCP-1.
First, because monocytes are desensitized by multiple challenges with
MCP-1 at a high concentration (ie, 1000 ng/mL [100
nmol/L],40 41 inhibition might result from MCP-1–induced
homologous desensitization. However, we have shown that adhesion and
migration were significantly inhibited at a concentration as low as 10
ng/mL MCP-1 and that the desensitization was minimal at this
concentration in calcium influx experiments (Figs 3 and 4).
Furthermore, it should be noted that MCP-1 in the upper compartment
only reduced adhesion and migration to levels lower than those of
controls, in which about 13.5% and 12.8% of total added monocytes
adhered and migrated, respectively, in the absence of MCP-1 (Fig 3).
This finding cannot be explained by a desensitization effect. Thus, it
is unlikely that desensitization exerts the inhibitory effect of MCP-1
on adhesion and migration.
Second, it is possible that MCP-1 changes the adhesive capacity of monocytes before they interact with the EC layers. However, our unpublished data have shown that monocyte pretreatment for 1 hour with MCP-1 (100 ng/mL) before the interaction does not affect adhesion and migration. This result seems to conflict with evidence that chemotactic stimulation not only activates integrins on neutrophils and monocytes but also increases their adhesion to EC layers under static conditions.6 10 17 42 43 Because T-cell–EC adhesion induced by such stimuli as phorbol myristate acetate and CD3144 45 has been shown to be temporary, adhesion of T cells to VCAM-1–coated wells increases as early as 15 minutes after the triggering of MIP-1ß and declines to near control levels 1 hour later (Y. Tanaka, personal communication), indicating that the adhesive effect of chemokines is also temporary. Similar to MIP-1ß, the effect of MCP-1 on the adhesion capacity of monocytes might be transient and disappear during the 1-hour pretreatment.
Finally, it could be assumed that MCP-1 detaches adherent monocytes and
decreases subsequent migration. This possibility is more likely,
because addition of MCP-1 to the upper compartment caused the
detachment of monocytes that had already adhered to the EC layers (Fig 4). This detachment may be induced by perturbation of the mechanisms
that mediate cell-to-cell adhesive interaction, including cell
locomotion. It has been suggested that cell locomotion involves the
polymerization and depolymerization of actin, the chief cytoskeletal
protein.46 Motile cells show polarization by exhibiting a
leading edge and a tail known as a uropod.47 The leading
edge is rich in actin-containing microfilaments and
receptors.48 A recent study has shown that IL-8 causes
rapid alterations in the molecular conformation and redistribution of
actin microfilaments in neutrophils.16 49 Because integrin
molecules, which mediate cell adhesion, are associated with
cytoskeletal proteins, such redistribution of actin microfilaments
would affect the adhesive function of the integrins. Although the
mechanism involved is unclear, IL-8 may deplete focal adhesion sites at
the leading edge, leading to subsequent detachment of neutrophils.
Similarly, MCP-1 could be speculated to induce rearrangement of, and
conformational changes in, the actin microfilaments of monocytes.
In contrast to our findings, Shyy et al28 have reported that ECs pretreated with monocyte-derived colony stimulating factor increase the gene expression of MCP-1 and the binding of monocytes and that the increased binding is blocked by anti–MCP-1 Ab. There is a similar controversy about the role of IL-8 on the adhesive interaction between neutrophils and ECs.15 16 17 The reverse results may be caused largely by the differences in the methods employed. For example, Shyy et al used EC layers cultured on plastic chambers, whereas we used a culture system that more closely mimics blood vessels in vivo. Because localization of chemokines (ie, the apical or basilar side of the endothelium) is assumed to profoundly influence the chemotactic movement of leukocytes, including their adherence to ECs, the assay system used for adhesion and migration might be an important factor in causing contradictory observations in experiments with chemokines.
The present study has shown that MCP-1 above an EC layer inhibits monocyte-EC adhesion events and subsequent migration. This indicates that the MCP-1 released from activated ECs might have effects similar to MCP-1 added exogenously. It has been shown that IL-1 stimulates monocyte adhesion and migration via the expression of endothelial adhesion molecules, such as ICAM-1 and VCAM-1.33 However, IL-1–stimulated ECs continued to secrete a significant amount of MCP-1 even after removal of IL-1; therefore we speculate that monocyte migration across IL-1–stimulated EC layers could be suppressed by endogenous MCP-1 released continuously from ECs. Augmentation of monocyte migration across IL-1–stimulated EC layers in the presence of an anti–MCP-1 Ab supports this notion. Despite a marked increase of adhesion molecules on IL-1–stimulated ECs,34 the increase in monocyte migration was only 20% after a 4-hour stimulation with IL-1 and 63% after a 24-hour stimulation. Other investigators have reported that treatment of ECs with IL-1 did not increase transendothelial migration.50 The reason for the small degree or lack of increase in monocyte migration upon stimulation of ECs with IL-1 may be attributable to the secretion of endogenous endothelial MCP-1.
MCP-1 as well as other cytokines and chemokines would be produced in abundance by infiltrating leukocytes, ECs, and vascular smooth muscle cells in atherosclerotic tissue in vivo. Nelken et al25 and Herttuala et al26 have shown that expression of the MCP-1 gene and protein was detectable in subendothelial tissues by in situ hybridization and immunohistochemistry in progressing atheromatous plaques. On the other hand, in the early stages of atherogenesis, the production of MCP-1 could occur in ECs; stimulation of ECs with various substances such as IL-1, lipopolysaccharide, or minimally modified LDL has been shown to induce MCP-1 expression in ECs in vivo.19 51 We also revealed that the interaction between monocytes and ECs induced MCP-1 mRNA expression. However, in view of the pathological implications, is it possible that endogenous MCP-1 remains on the upper side of ECs in vivo? Under conditions of blood flow, soluble endothelial MCP-1 might be rapidly washed away from the atherogenic focus. Recently, however, Tanaka et al11 12 have shown that MIP-1ß, which belongs to the C-C chemokine family, is captured by the surface proteoglycans of ECs. Because MCP-1 is similar to MIP-1ß in structure, it is therefore possible that MCP-1 secreted by ECs binds to the endothelial surface. Furthermore, monocytes that adhere to ECs could be directly exposed to MCP-1 secreted from activated ECs. Taken together, it appears that substantial amounts of MCP-1 could exist on the upper side of ECs in the early stages of atherogenesis in vivo, which could affect a gradient of MCP-1 across ECs.
In summary, the present findings suggest that endogenous endothelial MCP-1 could play a suppressive role in monocyte transendothelial migration when it is present on the apical side of EC layers. This process may be useful for preventing excessive infiltration of monocytes from the blood into atherogenic tissue during the early stages of atherogenesis. Thus, we conclude that monocyte adhesion to the endothelium and subsequent migration are regulated by the relationship between the local concentrations of endothelial MCP-1 and the soluble or immobilizable gradient of MCP-1 produced in subendothelial tissue: the former suppresses extravasation of circulating monocytes, whereas the latter promotes it.
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Acknowledgments |
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This work was supported in part by grants from the Ministry of Education, Culture and Science (No. 5670632) and the Study Group of Molecular Cardiology (Japan).We thank Dr Yoshiya Tanaka for helpful discussions and Drs Chikao Morimoto, Naofumi Mukaida, and Koji Matsushima for gifts of MAbs. We also thank Mamiko Semba, Atsuko Okamoto, and Toshiko Kambe for their technical assistance.
Received June 21, 1994; accepted March 1, 1995.
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