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*
Center For Blood Research and Department of Pathology, Harvard Medical School, Boston, MA 02115; and
Institut für Prophylaxe der Kreislaufkrankheiten, Klinikum Innenstadt, Ludwig-Maximilians-Universität, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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gradient on endothelium and increased transendothelial chemotaxis of
monocytes by a VLA-4-dependent mechanism. In contrast, endothelial
activation with IL-4 did not affect the time required for diapedesis of
monocytes itself. Hence, VCAM-1 may facilitate transendothelial
chemotaxis by supporting lateral migration of attached monocytes along
endothelium. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monocytes, eosinophils, and lymphocytes express the
ß1 integrin very late antigen
(VLA)-43
(4ß1, CD49d/CD29)
(14), which binds to domains 1 and 4 of VCAM-1, an Ig superfamily
member induced by cytokines on endothelium (15, 16, 17, 18). VLA-4 also binds
to an alternatively spliced domain of the extracellular matrix (ECM)
protein fibronectin, while VLA-5
(5ß1) binds to a
distinct domain containing the sequence RGD (19, 20). In vitro studies
suggest that ß2 integrins and ICAM-1 are
important in transendothelial migration of leukocytes; however,
interactions of VLA-4 and VCAM-1 additionally contribute to migration
across cytokine-stimulated endothelium (21, 22, 23, 24, 25). This is
consistent with in vivo studies showing a role of VLA-4 in migration of
monocytes and lymphocytes into inflammatory sites or cytokine-induced
lesions, following up-regulation of endothelial adhesion
molecules (26, 27, 28).
The cytoplasmic domain of 4 has been described
to exhibit specialized functional properties essential for
extravasation; as compared with the cytoplasmic tail of
5, it supported better migration and less
adhesion strengthening and spreading in transfectants expressing
chimeric subunits (29). We have recently shown that CC chemokines
sequentially regulate adhesiveness of VLA-4 and VLA-5 in monocytes (9).
Transient activation and deactivation of the adhesive strength of VLA-4
to VCAM-1 precedes activation of VLA-5 avidity for fibronectin.
Transient activation of VLA-4 may follow initial tethering and rolling
of leukocytes through selectins and 4
integrins and appears to facilitate transendothelial chemotaxis of
monocytes (9), whereas the late and prolonged activation of VLA-5 may
support interactions with the underlying basement membrane and with the
ECM. However, it remains to be elucidated whether the mechanism by
which VLA-4 facilitates transendothelial chemotaxis involves increases
in migration of attached leukocytes along endothelium or in diapedesis
through interendothelial cell junctions.
Here we demonstrate that the interaction of VLA-4 with VCAM-1, fibronectin, or a 40-kDa fragment of fibronectin (FN40) containing the CS-1 binding site for VLA-4 but not the binding of VLA-5 to a 120-kDa fragment of fibronectin (FN120) containing the RGD binding site support CC chemokine-induced lateral random migration (chemokinesis) of isolated blood monocytes. This appeared to be associated with the transient regulation of VLA-4 avidity by chemokines. The induction of VCAM-1 on endothelium facilitated lateral chemokinesis and transendothelial chemotaxis of monocytes by a VLA-4-dependent mechanism. The transient nature of VLA-4 avidity regulation by chemokines may promote transendothelial chemotaxis by facilitating lateral random migration along endothelium rather than diapedesis itself.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human recombinant chemokines and cytokines monocyte
chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1
(MIP-1), and IL-4 were from Genzyme (Cambridge, MA). Accudenz was
from Accurate Chemicals (Westbury, NY). Other reagents were from Sigma
(St. Louis, MO). The murine IgG myeloma X63 (isotype control), TS1/18
(CD18) (30), R6.5 (ICAM-1) (31), and CBR-IC2/2 (32) were purified with
protein A. Purified TS2/16 (ß1 activating) (33)
was a kind gift from Dr. M. E. Hemler (Dana-Farber Cancer
Institute, Boston, MA). Purified HP1/2 (CD49d) (34) was a kind gift
from Dr. R. Lobb (Biogen, Cambridge, MA), purified 1G11 (VCAM-1) was
from Camon (Wiesbaden, Germany), My4 (CD14) (35) was from Coulter
(Hialeah, FL), and CD32 mAb was from PharMingen (San Diego, CA).
Blood and endothelial cell isolation
Blood was collected from healthy donors and citrate anticoagulated. Leukocyte-rich plasma was prepared by 0.6% dextran T500 sedimentation of erythrocytes for 45 min at room temperature. Monocytes were separated from lymphocytes by Accudenz 1.068 hyperosmotic gradient centrifugation of leukocyte-rich plasma (9, 24). Platelets were removed from monocytes by four washes at 300 g for 7 min. This protocol yielded a population of 85% monocytes as analyzed by CD14 staining and light scatter. Monocytes were not activated by this isolation procedure, inasmuch as expression of L-selectin, which is rapidly shed during monocyte activation, was identical with that on the monocyte fraction in minimally processed whole blood and L-selectin-mediated functions under flow were maintained (9). Isolation and culture of HUVEC was performed as described (9).
Time lapse videomicroscopy
VCAM-1 (a kind gift from Dr. R. Lobb), FN40, FN120, or
fibronectin (Life Technologies, Gaithersburg, MD) were coated onto
96-well plates at 4°C overnight at 1, 3, or 10 µg/ml, respectively,
and unspecific binding sites were blocked with 1% heat-treated human
serum albumin (HSA) for 2 h at 37°C. ICAM-1 (10) was
coimmobilized with VCAM-1 or coated alone at 10 µg/ml. HUVEC were
seeded into 96-well plates and were stimulated with or without IL-4
(500 U/ml) for 24 h. Isolated monocytes were kept on ice for no
longer than 3 h, and 104 cells were added to wells in
40 µl of assay medium (HBSS, 10 mM HEPES, 1 mM
Ca2+, 1 mM Mg2+, 0.5% HSA) with or without
TS2/16 (10 µg/ml) or Mn2+ (1 mM). HUVEC were also grown
on collagen-coated transparent 2.5-cm-diameter Transwell culture
inserts (Costar, 8 µm pore size) and treated with or without IL-4
(500 U/ml) for 24 h, and MCP-1 was added to 2 ml of assay medium
in six-well plates containing the inserts. Monocytes were resuspended
at 2 x 105 cells/ml in assay medium (1:1 RPMI
1640-medium 199, 0.5% HSA), and 500 µl were added to inserts. Cells
were allowed to settle for 10 min on the stage of a Nikon Diaphot
(Nikon, Instrument Group, Melville, NY) microscope in a
Plexiglas incubator box at 37°C and densities of 
10 cells/40x
high power field were obtained to avoid interactions between cells.
After addition of MCP-1 or MIP-1, cells were observed with phase
contrast optics, and images were recorded for 30 min at a speed 12-fold
slower than normal using a Panasonic AG-6730 time lapse video
recorder (Panasonic, Secaucus, NY. Sufficient experiments were
performed to obtain 30 cells for analysis. To trace individual monocyte
paths, images were captured as digitized movies, and cell edges were
enhanced, outlined, chronologically colorized, and projected over each
other, using National Institutes of Health image software (Bethesda,
MD). The image of a hemocytometer was used for size calibration. The
coordinates of the cell centroids were determined, and the lengths
of individual cell paths were quantitated as the sum of the linear
distances between the centroids after every 1-min interval,
representing a scalar parameter of cell migration. The locomotion
rate of every cell analyzed was determined by dividing the length of
its cell path by the time elapsed and was expressed as micrometers per
minute. The mean ± SD of all individual cells and the medians of
the sample distributions are given. As indicated, the data were
statistically analyzed by nonparametric signed rank test according to
Wilcoxon, or by analysis of variance by rank according to
Kruskal-Wallis, where appropriate. Locomotion rates were also presented
as the percentage of cells in velocity categories to reflect the
distribution of cells over the range occurring under different
conditions, as previously described for similar cell numbers (36).
Transendothelial chemotaxis assay
HUVEC were grown on collagen-coated 6.5-mm-diameter Transwell
culture inserts (Costar, 8 µm pore size) and treated with or without
IL-4 (500 U/ml) for 24 h. Transendothelial chemotaxis assays were
as reported (9). Monocytes were incubated with 5% human serum or CD32
mAb (10 µg/ml) for 30 min to block Fc receptors and to prevent
binding of monocytes via Fc receptors to mAbs added in inhibition
experiments. Cells were washed and resuspended at 5 x
106 cells/ml in assay medium (1:1 RPMI 1640-medium 199,
0.5% HSA). MCP-1 or MIP-1 was added to 24-well tissue culture
plates in 600 µl of assay medium. Transwells were inserted into
wells, cells were added to the top chamber in a final volume of 100
µl, and monocytes were allowed to transmigrate for 1 h.
Transmigrated and input monocytes were detached with 5 mM EDTA and
counted with a FACScan (Becton Dickinson, Mountain View, CA) using
forward and side scatter gates for monocytes. In each experiment, the
ability of HUVEC to prevent upward diffusion of low m.w. FITC-dextran
was assessed. For mAb inhibition studies, cells were preincubated with
mAb to ß2 (TS1/18, 20 µg/ml), VLA-4 (HP2/1,
10 µg/ml), or isotype control (X63, 20 µg/ml) for 20 min on ice.
Data were statistically analyzed using analysis of variance, where
indicated.
Flow cytometry
Monocytes were washed and reacted for 30 min with mAb on ice, stained with a 1:20 dilution of goat anti-mouse IgG FITC (Zymed, San Francisco, CA), and subjected to scatter-gated flow cytometry in a FACScan. Concentrations of mAb used for inhibition were saturating (data not shown). Surface expression of ICAM-1, ICAM-2, and VCAM-1 was analyzed by staining of HUVEC with mAb as described (37). After subtraction of unspecific staining, specific fluorescence intensity was expressed in channels.
Quantification of MCP-1 protein
The MCP-1 concentration in HUVEC supernatants was quantitated using a sandwich ELISA (R&D Systems, Minneapolis, MN). Supernatants were collected from resting and IL-4 (500 U/ml, 24 h)-stimulated HUVEC and sterile filtered. An ELISA was performed according to the manufacturer’s protocols using protein standards provided.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transient regulation of VLA-4 avidity by CC chemokines has been
suggested to promote chemotaxis of monocytes across VCAM-1-bearing
barriers, whereas prolonged activation of VLA-5 may mediate subsequent
localization within the extracellular matrix (9). To elucidate the
mechanism by which interactions of VLA-4 and not VLA-5 with their
ligands may support transendothelial chemotaxis, we studied lateral
random migration of monocytes on VCAM-1 and the fibronectin fragments
FN40 or FN120. On VCAM-1 (3 µg/ml), unstimulated monocytes showed
spreading and spontaneous random migration (Fig. 1
A and data not shown), and
the percentage of cells moving >30 µm within 30 min was 30% (Fig. 2A). CC chemokines induced a
marked cellular shape change with formation of leading filopodia and
lamellipodia (not shown). MCP-1 and MIP-1 induced lateral migration
in random directions (chemokinesis) (Fig. 1B and data not
shown), with 86% and 80% of cells crawling >30 µm within 30 min,
respectively (Fig. 2, B and C). The
activating ß1 mAb TS2/16 or Mn2+
strongly inhibited spontaneous and MCP-1-induced random migration,
restricting it to the area of cell attachment (Fig. 1, C and
D; Fig. 2, D–F; and data not
shown). This indicates that locking VLA-4 in a high avidity state
impaired chemokinesis.
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, respectively, moving for
>30 µm within 30 min (Fig. 2
, J–L). This
indicates that migration was not restricted to one VLA-4 ligand and
suggests that it is a function of the integrin. By comparison,
attachment to FN120 (10 µg/ml) which contains the binding site for
VLA-5, resulted in diffuse spreading but extension of few filopodia or
lamellipodia in unstimulated or MCP-1-induced cells (not shown).
Furthermore, unstimulated monocytes showed little spontaneous random
migration on FN120 (Figs. 1E and 2G), and
chemokinesis was hardly stimulated by MCP-1 or MIP-1 (Figs. 1F and 2, H and I). MCP-1-induced chemokinesis on VCAM-1 and fibronectin is mediated by VLA-4 and optimal at intermediate substrate densities
To further define differential contributions of VLA-4 and VLA-5,
we studied lateral random migration on intact fibronectin and VCAM-1
coimmobilized with ICAM-1. On fibronectin (3 µg/ml), unstimulated
monocytes showed little random migration, with 14% of cells moving
>30 µm within 30 min. MCP-1 induced chemokinesis, with 47% of cells
moving >30 µm within 30 min (p < 0.05 vs
unstimulated monocytes, Fig. 3,
A and B). This increase in migration induced by
MCP-1 was completely inhibited by mAb HP1/2 to
4 (p < 0.05 vs
isotype control) so that motility resembled that on FN120 or that
without MCP-1, with 13% of cells crawling >30 µm within 30 min
(Fig. 3C). By contrast, a mAb to 5
increased locomotion rates (p < 0.05 vs
isotype control, Fig. 3C). On ICAM-1 (10 µg/ml), migration
of monocytes was induced by MCP-1 to an extent comparable to that on
fibronectin and was not impaired by 4 mAb
(Fig. 3, D–F). Since adhesion is a prerequisite
for migration, the ICAM-1 substrate represents an appropriate control
in which neither fibronectin nor VCAM-1 was included. Coimmobilizing
VCAM-1 with ICAM-1 did not affect migration of unstimulated monocytes
but markedly improved chemokinesis of MCP-1-stimulated monocytes as
compared with ICAM-1 alone (Fig. 3, G and H).
Similar to results seen on fibronectin, addition of mAb HP1/2 to
4 decreased chemokinesis on VCAM-1
coimmobilized with ICAM-1 to levels found on ICAM-1 alone (Fig. 3I). Thus, migration of monocytes on fibronectin or VCAM-1
was clearly mediated by interactions with VLA-4 and not VLA-5. We next
tested whether the rate of MCP-1-induced chemokinesis was dependent on
the concentration at which the substrate was adsorbed (Table I). Our results indicate that the
locomotion rates were optimal at intermediate substrate concentrations
of VCAM-1 and impaired at lower and higher substrate concentrations.
Similarly, however, less marked effects of the substrate concentration
were seen on fibronectin. In contrast, the chemokinetic locomotion rate
on FN120 was low and was little altered over a 10 -fold range of FN120.
Thus, our data suggest that intermediate site densities of VCAM-1 or
fibronectin appeared to optimally support VLA-4-dependent chemokinesis
of monocytes.
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The kinetics of VLA-4 regulation by MCP-1 showed transient
activation of adhesive strength to VCAM-1 with a peak at 10 min (9).
Hence, we analyzed the locomotion rate of MCP-1-stimulated monocytes at
different time points to correlate migration with the kinetics of
avidity regulation. Interestingly, the mean locomotion rate on VCAM-1
was markedly higher in the first 6 min (Fig. 4A) and between 13 and 25 min
(Fig. 4C) than between 6 and 13 min of stimulation (Fig. 4B). The percentage of cells crawling at >1 µm/min was
93% in the first 6 min, 64% between 7 and 12 min, and 93% between 13
and 25 min. Thus, the locomotion rates appeared to be inversely
correlated to the adhesive strength for VCAM-1. In particular,
deactivation of VLA-4 at later time points appeared to favor the
motility of monocytes on VCAM-1. These data implicate the transient
regulation of VLA-4 avidity in the induction of lateral migration of
monocytes by MCP-1.
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To study how interactions of VLA-4 with VCAM-1 may facilitate
migration across endothelium, we analyzed lateral random migration on
resting and IL-4-activated HUVEC monolayers. Resting HUVEC expressed
ICAM-1 and ICAM-2 but not VCAM-1 surface protein (Table II). Treatment of HUVEC with IL-4 for
24 h induced expression of VCAM-1 but did not affect that of
ICAM-1 or ICAM-2 (Table II) (38, 39). Only few unstimulated monocytes
crawled >30 µm in 30 min on resting or IL-4-activated HUVEC,
indicating that random migration in the absence of chemokine was not
altered by IL-4 activation (Fig. 5,
A and B). MCP-1 stimulated the migration of
monocytes on resting HUVEC (p < 0.001 vs
unstimulated monocytes) with 44% of cells moving >30 µm in 30 min
(Fig. 5C). On IL-4-activated HUVEC, MCP-1 more markedly
increased migration of monocytes (p < 0.0001
vs unstimulated monocytes) with 63% of cells crawling >30 µm in 30
min (Fig. 5D). VLA-4 mAb only slightly reduced the
locomotion rate of monocytes on resting HUVEC but markedly inhibited
chemokinesis of monocytes (p < 0.05 vs isotype
control) on IL-4-treated HUVEC (Fig. 5, E and F)
to levels comparable with those seen on resting HUVEC (compare Fig. 5, C and F). The chemokinesis of monocytes that
could still be observed on resting and IL-4-activated HUVEC after
treatment with 4 mAb was further blocked by a
mAb to ß2 (Fig. 5, G and
H). Induction of VCAM-1 expression by IL-4 may thus
facilitate migration along activated endothelium by a VLA-4-dependent
mechanism.
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A) (9). Treatment of HUVEC
with IL-4 for 24 h did not induce a significant up-regulation of
MCP-1 secretion (Table II) (40); hence, it did not appear likely to
interfere with the MCP-1 gradient. Activation of HUVEC with IL-4 for
24 h, which induced VCAM-1, increased transendothelial migration
of monocytes by up to 70% (p < 0.05, Fig. 6B). Inhibition with VLA-4 mAb revealed that the increase in
chemotaxis was mediated by VLA-4 (Fig. 6B). Pretreatment
with a combination of ß2 and
4 mAb almost completely blocked chemotaxis
across resting and activated HUVEC (data not shown). Visualizing the
transendothelial chemotaxis of monocytes in the Transwell system
revealed that MCP-1 in the lower chamber induced cell movement toward
the filter pores (Fig. 6, C and D). The color
code of the monocytes as they enter the filter pore corresponds to the
elapsed time. Monocytes migrating across IL-4-treated HUVEC expressing
VCAM-1 (Fig. 6D) reached the pores more rapidly than those
migrating across resting HUVEC (Fig. 6C). This was due to
higher locomotion rates on IL-4-activated HUVEC
(p < 0.001 vs resting HUVEC, Fig. 6, E and F). The locomotion rate for lateral
migration toward a gradient of MCP-1 (Fig. 6F) was higher
than for MCP-1-induced chemokinesis (Fig. 5D), probably due
to the more directed migration toward a chemotactic gradient. The
fraction of cells crawling at >2.5 µm/min before entering pores was
29% on resting vs 47% on IL-4-activated HUVEC. This complies with the
findings that interactions of VLA-4 and VCAM-1 are favorable for
monocyte chemokinesis. Monocytes that had transmigrated across
IL-4-activated HUVEC appeared to pull free of the pores more readily,
and particularly after early diapedesis continued to migrate on the
lower surface of the Transwell filters before they detached (Fig. 6, C and D). Interestingly, the time monocytes spent
between entering and exiting the pores was little changed by activation
of HUVEC with IL-4 (4.6 ± 1.9 min vs 5.2 ± 1.5 min in
resting HUVEC, mean ± SD), suggesting that diapedesis itself was
not affected. Similarly, MIP-1, a chemokine not produced by HUVEC,
induced increased monocyte transmigration across IL-4-activated HUVEC
that was VLA-4 dependent (Fig. 7,
A and B) and increased the rate of lateral
locomotion on IL-4-activated HUVEC (Fig. 7, C and
D). These data suggest that VCAM-1 facilitates
transendothelial chemotaxis by supporting lateral migration of attached
monocytes along the endothelium before diapedesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunits confer
not only differential activation of adhesiveness but also differences
in the potential to support lateral migration of monocytes. Our data suggest that the induction of chemokinesis of monocytes on VCAM-1 may be promoted by the transient regulation of VLA-4 by MCP-1 in a subset of cells with relatively low or intermediate adhesive strength. CC chemokines transiently increase but then reduce the adhesive strength of VLA-4 in monocytes, resulting in attachment and subsequent detachment on its ligands (9). Locking VLA-4 in a high avidity state with stimulating mAbs, such as TS2/16, or Mn2+ interferes with the avidity regulation by CC chemokines and transendothelial chemotaxis (9, 41), and as demonstrated here markedly impaired lateral migration. The locomotion rate of monocytes appeared to be inversely correlated with the adhesive strength of VLA-4; i.e., it was the lowest when adhesive strength was maximally activated between 6 and 12 min. These data may further support an association of lateral migration with the transient regulation of VLA-4 avidity. Controlled detachment assays on VCAM-1 reveal that of the CC chemokines, MCP-1 most rapidly increased and deactivated the adhesive strength of VLA-4 (9). As observed for this regulation, our data now show that chemokinesis induced by MCP-1 was also optimally regulated at intermediate substrate concentrations of VLA-4 ligands and impaired at lower and higher substrate concentrations. Previous results (42) have established that immobilization of VCAM-1/Fc or fibronectin at various concentrations results in different site densities. In accordance with our data, this report (42) has indicated that the efficiency of spontaneous lymphocyte migration was optimal at intermediate site densities of VLA-4 ligands and higher across membranes coated with VCAM-1/Fc than with fibronectin. Hence, it is reasonable to assume that the changes in the locomotion rates we observed for monocyte chemokinesis were due to differences in site density under otherwise identical conditions. These data are also consistent with previous predictions that intermediate adhesive strength leads to maximal motility (43) and with recent studies implicating adhesive strength, which can be influenced by receptor expression and affinity, substrate density, and organization of adhesive complexes, as an important regulator of cell migration (44).
What determines different functions of VLA-4 and VLA-5? Directional
cell migration involves formation of adhesive complexes and interaction
with force generating components of the cytoskeleton to provide
traction and detachment at the rear, allowing the cell to advance over
its substrate (13, 45). Intermediate adhesive strength is a major
determinant of cell speed (44) and may be different for VLA-4 and
VLA-5. Extracellular ligand binding can induce an attachment of
ß1 integrins to the retrograde-moving
cytoskeleton critical for organized receptor movement and migration of
adherent cells (46). In MCP-1-stimulated monocytes on VCAM-1,
immunostaining revealed actin polymerization and asymmetrical VLA-4
distribution into uropod-like structures (C. Weber, unpublished data).
Hence, migration may be associated with spatial redistribution of
VLA-4. By contrast, VLA-5 has been implicated in the strengthening of
integrin-cytoskeleton linkages stimulated by the ECM rigidity (47).
Studies in Chinese hamster ovary transfectants indicated that integrin
mutations increasing cytoskeletal organization reduce locomotion rates,
whereas mutations impairing cytoskeletal association and focal adhesion
complex formation decrease haptotaxis but increase random migration
(48). Whereas VLA-5 may thus favor a more rigid reinforcement of
cytoskeleton linkages with high adhesive strength, spreading, and lower
locomotion rates, the cytoskeletal associations of VLA-4 and its
transient avidity regulation by chemokines may allow faster retrograde
diffusion and better cell migration. Studies in K562 and Chinese
hamster ovary transfectants showed that the 4
cytoplasmic tail compared with that of 5
promotes migration but reduces localization into focal adhesions and
spreading and that 4 cytoplasmic tail deletion
decreases clustering and lateral diffusion of VLA-4 (29, 49). Thus,
subunit cytoplasmic domains may confer differences crucial for the
ability of integrins to support locomotion of monocytes, a cell type
without focal adhesions.
We assessed the contribution of VCAM-1 to the lateral random migration
of MCP-1-stimulated monocytes on endothelium. Induction of VCAM-1
expression by activation of HUVEC with IL-4 resulted in a
VLA-4-dependent increase in chemokinesis. Further analysis revealed
that the chemokinesis on resting and IL-4-activated HUVEC after
blocking VLA-4 was mediated by interactions of
ß2 integrins with their ligands, as seen on
ICAM-1 substrates. This is consistent with previous findings on the
involvement of ß2 integrins in lateral and
transendothelial migration of leukocytes and suggests that VLA-4-VCAM-1
interactions represent a major but not an exclusive pathway for lateral
migration before transmigration (31, 50). To study the relevance of our
findings in a chemotactic context, we analyzed chemotaxis across
IL-4-activated HUVEC. Monocytes use either ß2
integrins or VLA-4 during spontaneous transendothelial migration or
chemotaxis (23, 24). Activation of endothelium has been shown to
support VLA-4-mediated monocyte transmigration (23), consistent with an
essential role of VLA-4 in monocyte emigration to acute inflammatory
sites in vivo (27, 28). Similar to findings with TNF-stimulated HUVEC
(9), we found that mAb to ß2 or
4 independently inhibited transendothelial
chemotaxis. However, while the relative importance of
ß2 integrins was reduced, that of VLA-4 was
increased across IL-4-activated HUVEC expressing VCAM-1. This suggests
that VLA-4 and ß2 integrins may synergize at
distinct steps to achieve optimal transmigration across activated
endothelium.
Induction of VCAM-1 by IL-4 increased both monocyte chemokinesis on HUVEC and transendothelial chemotaxis by a VLA-4-dependent mechanism. Notably, the time monocytes spent within Transwell pores was unchanged by activation of HUVEC with IL-4, suggesting that the process of diapedesis was not affected. Hence, VCAM-1 expression may facilitate the transendothelial chemotaxis by supporting lateral migration along endothelium. Diapedesis of monocytes across endothelium is promoted by a soluble gradient of MCP-1 rather than by haptotactic stimulation through immobilized MCP-1 (51). This may be due to a higher efficiency of migration with a directed gradient of soluble MCP-1. Indeed, locomotion rates for lateral migration toward a directed gradient were higher than for chemokinesis on IL-4-activated HUVEC. The extent of lateral migration on VCAM-1 may support transendothelial chemotaxis by improving the probability of encountering interendothelial cell junctions.
Alternatively, activation of endothelium with IL-4 may facilitate monocyte migration via distinct mechanisms, i.e., by inducing antiadhesive molecules or structures on endothelium. The VCAM-1-dependent adhesion of T lymphocytes to endothelium has been described to induce a 72-kDa gelatinase (52). Similarly, induction or activation of proteolytic enzymes by interaction of VLA-4 with VCAM-1 or by IL-4 activation of endothelial cells may contribute to the detachment of monocytes required for completing transmigration. This would comply with our findings that monocytes that had transmigrated across IL-4-activated HUVEC appeared to pull free of the pores more readily after diapedesis. Hence, it may be possible that migration on integrin ligands on the luminal side of IL-4-activated endothelium may transduce positive signals not only for completing monocyte diapedesis but also for the subsequent ß1 integrin-mediated migration on ECM proteins, likely to be present on the lower face of the Transwell filters, possibly generating a cross-talk to regulate sequential integrin functions (53). On the other hand, the crawling of monocytes on the lower surface of the Transwell filters after transgression of the filter pores may involve VLA-4-dependent lateral migration on components secreted into the subendothelial matrix, as shown on fibronectin substrates.
A version of the multistep model defined a role for
4 integrins in bridging between selectins and
ß2 integrin-mediated events (54). Distinct
functions have been found for integrins in the interaction of monocytes
with activated endothelium in flow, where VLA-4 participates in
attachment, while ß2 integrins mediate
transmigration (55). The VLA-4-dependent lateral migration induced by
CC chemokines may follow initial rolling and attachment of leukocytes
supported by selectins and 4 integrins (11, 12). This may mediate the migration of arrested leukocytes along
endothelium to interendothelial cell junctions where diapedesis can
occur. In contrast, the ß2 integrin LFA-1 may
be more important in transendothelial diapedesis itself, as also
suggested by the crucial role of changes in its avidity for
transendothelial chemotaxis (56). Migration and spreading on
fibronectin associated with prolonged VLA-5 activation (9) may support
interactions of leukocytes with the underlying basement membrane or
ECM. Fig. 8 illustrates the putative
sequence of monocyte extravasation with a potential role of
VLA-4-mediated lateral migration following monocyte arrest on
endothelial cells and preceding diapedesis and subendothelial
localization. Thus, the functional specialization among integrins that
we have demonstrated may be important to accomplish the complex process
of leukocyte extravasation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. C. Weber, Institut für Prophylaxe der Kreislaufkrankheiten, Pettenkoferstrasse 9, D-80336 München, Germany.
3 Abbreviations used in this paper: ECM, extracellular matrix; MIP, monocyte inflammatory protein; MCP, monocyte chemoattractant protein; VLA, very late antigen; FN40, 40-kDa fragment of fibronectin; FN120, 120-kDa fragment of fibronectin; HSA, human serum albumin.
Received for publication April 21, 1998. Accepted for publication August 18, 1998.
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References |
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