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of July 31, 2017.
Adhesion Molecule Mechanisms Mediating
Monocyte Migration Through Synovial Fibroblast
and Endothelium Barriers: Role for CD11/CD18,
Very Late Antigen-4 (CD49d/CD29), Very Late
Antigen-5 (CD49e/CD29), and Vascular Cell
Adhesion Molecule-1 (CD106)
Xiao-zhou Shang, Bianca J. Lang and Andrew C. Issekutz
J Immunol 1998; 160:467-474; ;
http://www.jimmunol.org/content/160/1/467
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Copyright © 1998 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Adhesion Molecule Mechanisms Mediating Monocyte
Migration Through Synovial Fibroblast and Endothelium
Barriers: Role for CD11/CD18, Very Late Antigen-4 (CD49d/
CD29), Very Late Antigen-5 (CD49e/CD29), and Vascular
Cell Adhesion Molecule-1 (CD106)1
Xiao-zhou Shang, Bianca J. Lang, and Andrew C. Issekutz2
T
he emigration of leukocytes, including monocytes from
blood into tissue, is a prominent feature of acute and
chronic inflammation in many diseases. For example,
rheumatoid arthritis is characterized by chronic inflammation with
infiltration of synovium and periarticular tissues by lymphocytes
and monocytes, which give rise to macrophages. These cells are
believed to play a role in joint destruction by secreting factors such
as cytokines, growth factors, and proteases (1). The mechanisms of
monocyte migration and accumulation in inflammatory joint tissues initially require adhesion to and migration through vascular
endothelium and then through synovial connective tissue. Monocyte transendothelial migration has been investigated by several
laboratories and is becoming relatively well defined. A number of
adhesion molecules, expressed on both the monocyte and the endothelium (e.g., selectins, b1 (CD29), and b2 (CD18) integrins,
Department of Pediatrics, Microbiology and Immunology, Dalhousie University,
Halifax, Nova Scotia, Canada
Received for publication May 15, 1997. Accepted for publication September
22, 1997.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Grant MT-7684 from the Medical Research Council of Canada to (A.C.I).
2
Address correspondence and reprint requests to Dr. Andrew C. Issekutz, Department of Pediatrics, IWK-Grace Health Center, 5850 University Avenue, Halifax, Nova Scotia, Canada B3J 3G9.
Copyright © 1998 by The American Association of Immunologists
vascular cell adhesion molecule-1 (VCAM-1),3 and intercellular
adhesion molecule-1 of the Ig supergene family) are involved (2–
8). In contrast, much less is known about how monocytes migrate
through connective tissues such as the synovium once the monocytes have traversed the vascular endothelium. It is likely that
monocyte chemotactic factors generated in the synovium and joint
fluid induce continued migration. As is the case in monocyte
transendothelial migration, this process may involve molecular interactions between monocytes and synovial cells and/or the extracellular matrix (ECM). To date, most of the studies regarding interaction between monocytes and synovial cells or ECM have
focused on the mechanism of adhesion (9, 10). However, in connective tissue the mechanisms for adhesion and migration may be
distinct, and excessive adhesion to connective tissue cells or matrix
proteins could potentially interfere with efficient migration.
It is well known that monocytes express b2 (CD11/CD18) integrins, which share a common b-chain (CD18) and have three
distinct a-chains noncovalently associated with CD18, i.e., CD11a
(lymphocyte function-associated-1 or LFA-1), CD11b (Mac-1),
and CD11c (p150,95) (11, 12). We and others have shown that
monocyte migration across unactivated HUVEC is largely CD18
dependent, but migration induced by chemotactic factors across
HUVEC activated by IL-1, TNF-a, or LPS is CD18 independent,
and can be mediated by very late Ag-4 (VLA-4) (a4b1, CD49d/
3
Abbreviations used in this paper: VCAM-1, vascular cell adhesion molecule-1;
VLA, very late Ag; ICAM-1, intercellular adhesion molecule-1; ECM, extracellular matrix; HSF, human synovial fibroblast; MCP-1, monocyte chemoattractant
protein-1; HSA, human serum albumin; PMNL, polymorphonuclear leukocyte;
CS-1, connecting segment-1; RGD, amino acid sequence Arg-Gly-Asp.
0022-1767/98/$02.00
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Monocytes migrate through vascular endothelium, and then in connective tissue. As a model of this process, we investigated
adhesion molecules involved in monocyte migration through HUVEC and a barrier of human synovial fibroblasts (HSF). Minimal
spontaneous monocyte migration (6 –7%) occurred through either cell barrier, but this increased markedly (27–35% of added
monocytes) when a C5a chemotactic gradient was present. Migration across unstimulated HUVEC was partially inhibited (40%)
by mAb to CD18 (b2 integrin) and completely blocked by anti-CD18 plus anti-a4 (CD49d; very late Ag-4 (VLA-4)) mAbs. In
contrast, migration across HSF induced by C5a or monocyte chemoattractant protein-1 was not inhibited by mAb to CD18 and
was only partially inhibited (33%) in combination with anti-a4 mAb. The CD18- and VLA-4-independent migration across HSF
was completely inhibited by mAb to a5 of VLA-5. The inhibitory effect of mAbs to VLA-4 and VLA-5 was on the monocyte and
required blockade of CD11/CD18 to be observed. In contrast to HSF, no role for VLA-5 in monocyte transendothelial migration
was detected. Both HSF and IL-1-stimulated HUVEC expressed vascular cell adhesion molecule-1 (VCAM-1). However, VLA4-mediated monocyte migration across HSF was only partially dependent on VCAM-1, in contrast to transendothelial migration,
which was completely blocked by anti-VCAM-1 mAbs. In conclusion, unlike transendothelial migration, for which VLA-4 is the
alternative mechanism to CD11/CD18 on monocytes, both VLA-4 and VLA-5 can mediate monocyte migration through fibroblast barriers. In addition to VCAM-1, other ligand(s) on HSF are also involved in the VLA-4-mediated migration. The Journal
of Immunology, 1998, 160: 467– 474.
468
Materials and Methods
Monoclonal Antibodies
The following adhesion function-blocking murine mAbs against human
Ags were used as purified IgG: 60.3 (anti-b2 (CD18) integrin, IgG2a, provided by Bristol-Myers Squibb, Seattle, WA) (16, 17); R15.7 (anti-CD18,
IgG1, provided by Dr. R. Rothlein, Boehringer Ingelheim, Ridgefield, CT)
(18); HP1⁄2 (anti-a4-chain of VLA-4 integrin); 4B9 (anti-domain 1 of
VCAM-1); and GH12 (anti-domain 4 of VCAM-1; all three mAbs are
IgG1 and are gifts from Dr. R. Lobb, Biogen Inc., Cambridge, MA) (19,
20); and JBS-5 (anti-a5-chain of VLA-5, IgG1; gifts from Dr. J. Wilkins,
University of Manitoba, Winnipeg, Canada) (15). The following mAbs
were used as ascites: 450-30A1 (anti-a6-chain of VLA-6, IgG1; gift from
Dr. S. J. Kennel, Oak Ridge National Laboratory, TN) (21), 3H11B9 (antipertussis toxin, IgG1; from Dr. S. Halperin, Halifax, Canada), mAb W6/32
(anti-HLA class I, IgG2a), mAb 543 (anti-CR1, IgG1), 3C10 (anti-CD14,
IgG1), and TS1/18 (anti-CD18, IgG1) were generated from the hybridomas, the latter four being purchased from the American Type Culture
Collection (Rockville, MD).
Reagents
Recombinant human IL-1a (sp. act. of 4 3 107 U/mg) was a gift from Dr.
D. Urdal (Immunex Corp., Seattle, WA). IL-1 was diluted immediately
before use in 0.1% LPS-free human serum albumin (HSA; Connaught Laboratories, Don Mills, Canada). Recombinant human C5a was a gift from
CIBA-Geigy Pharmaceuticals (Summit, NJ). Purified human recombinant
monocyte chemoattractant protein-1 (MCP-1) was produced at Genentech
Inc. (a gift from Dr. T. Schall, Genentech, Inc.).
Isolation and growth of HSF
HSF were aseptically isolated from synovium obtained at surgery or arthroscopy of knee or hip joints of patients with rheumatoid arthritis (provided by Dr. J. Hanly, Division of Rheumatology, Victoria General Hospital, Halifax, Canada), as reported previously (15). Briefly, the minced
tissue was digested with 2 mg/ml of collagenase type IV (512 U/mg) in
aMEM (both from Sigma Chemical Co., St. Louis, MO) containing 10%
heat-inactivated FBS (HyClone Laboratories, Logan, UT) by incubation in
a shaker (250 rpm) at 37°C for 4 h. Single cells were recovered by centrifugation, washed and cultured in aMEM-10% FBS, 50 mM 2-ME, and
penicillin G/streptomycin until cells grew confluent. The cultures became
homogeneous by the second passage and were used at the 3rd to the 12th
passage. Cells were harvested with 0.05% trypsin/0.02% EDTA (Sigma
Chemical Co.) and seeded onto Transwell polycarbonate filters bearing
5-mm pores in plate inserts (6.5 mm diameter, Transwell 3421; Costar,
Cambridge, MA), which were precoated overnight with 0.01% gelatin.
Seeding density was 3 3 104 in 0.1 ml of culture medium above the filter,
and 0.6 ml of the medium was added to the lower compartment beneath the
filter. After 6 to 7 days of culture, confluent monolayers had formed on the
filters, which allowed the diffusion of ,5% of 125I-HSA in 45 min compared with diffusion of 25 to 30% across bare filters.
Isolation and culture of endothelial cells
HUVEC were isolated and cultured as described by Jaffe et al. (22), and
HUVEC monolayers on filters were grown as described previously (23,
24). Briefly, endothelial cells isolated from umbilical cords by collagenase
treatment were grown in the complete medium: RPMI 1640 (Sigma Chemical Co.) containing 2 mM L-glutamine, 2-ME, sodium pyruvate, penicillin
G/streptomycin, 20% FBS (HyClone), 25 mg/ml endothelial cell growth
factor (Collaborative Research, Lexington, MA), and 22.5 mg/ml heparin
(Sigma Chemical Co.) in gelatin-coated flasks (Nunc, Naperville, IL; and
Life Technologies, Grand Island, NY). The HUVEC were detached with
0.025% trypsin/0.01% EDTA and cultured on the Transwell filters described above. The filters were prepared by coating with 0.01% gelatin
(37°C overnight) followed by application of 3 mg of human fibronectin
(Collaborative Research) in 45 ml of water at 37°C for 2 h. The HUVEC
(1.5 3 104 cells in 0.1 ml of complete medium) from first or second passage were added above the filter and 0.6 ml of medium was added to the
lower compartment beneath the filter. The cells became confluent and
formed a tight monolayer in 5 to 6 days, with permeability ,1.5% when
tested by 125I-HSA diffusion as described previously (23, 24).
Isolation of human monocytes
Monocyte isolation was performed as described previously (2). Briefly,
venous blood from healthy human volunteers was collected in EDTA plus
acid citrate dextrose anticoagulant. Dextran (Baxter Travenol, Dartmouth,
Canada) was added (final 1%) to induce RBC sedimentation at 1 g and the
leukocyte-rich plasma was harvested. After centrifugation (150 3 g for 10
min at room temperature), the leukocyte pellet was resuspended in a Ca21-,
Mg21-free Tyrode’s solution with 5% autologous platelet-poor plasma and
labeled with 51Cr sodium chromate (25 mCi/ml) (Amersham Corp.,
Oakville, Canada) by incubation for 30 min at 37°C. During this incubation, the osmolarity of the medium was gradually increased in three steps
from 290 to 360 mOsmol by addition of 9% NaCl. This improved the
monocyte purity and did not affect cell viability or function, as shown
previously (25, 26). The labeled leukocytes were washed once with Ca21-,
Mg21-free Tyrode’s solution (360 mOsmol), 5% platelet-poor plasma, and
resuspended in Ca21-, Mg221-free Tyrode’s solution (360 mOsmol) containing 0.2% EDTA, 10% platelet-poor plasma, and 56% Percoll (Pharmacia Fine Chemicals, Dorval, Canada) based on 100% being isotonic
Percoll. The leukocytes were separated on discontinuous Percoll gradient
of 73, 62, 56 (containing the labeled leukocytes), 50, 46, and 40% by
centrifugation (400 3 g, 25 min at room temperature). The purest monocyte fraction was recovered at the 46 to 40% Percoll interphase with .90%
purity, .95% viability by neutral red staining and trypan blue exclusion,
with minimal platelet contamination and no PMNL as described previously
(2). The monocytes were resuspended for migration studies at 7 3 105/ml
in RPMI 1640, 0.5% HSA (pyrogen free; Connaught Labs, Toronto, Canada) containing 10 mM HEPES (pH 7.4).
Monocyte migration across fibroblast and endothelium
barriers
The monocyte migration assay was performed as described previously (2).
Briefly, HSF or HUVEC monolayers on the filters and the lower compartment were washed with RPMI 1640 and incubated for 5 h in fresh RPMI
1640 with 10% FBS, or stimulated for 5 h by addition of cytokine IL-1a
to the medium. After incubation, the filters were washed on the upper and
lower surfaces with RPMI 1640 and transferred to a new, clean well (lower
compartment) of a 24-well plate. To this well, 0.6 ml of RPMI 1640-HSA
was added containing C5a or MCP-1 as a chemotactic stimulus. Before
immersion of the HSF or HUVEC-filter unit in the well, 0.1 ml of medium
containing 7 3 104 51Cr-labeled human monocytes were added above the
HSF or HUVEC-filter unit. After a 100-min incubation, the migration was
stopped by washing of the upper compartment twice with 0.1 ml RPMI
1640 to remove nonadherent monocytes. The undersurface of the filter was
then rinsed into the lower compartment and swabbed with a cotton swab
soaked in ice-cold PBS/0.2% EDTA solution. The upper compartment was
placed into 0.7 ml of 0.5 M NaOH to allow dissolution of adhered monocytes. The cells that migrated into the lower compartment were lysed by
addition of 0.5% Triton X-100, and this medium was combined with the
swab contents and analyzed for 51Cr to determine the total 51Cr monocytes
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CD29) integrins (2, 13). The b1 integrins (also called VLA proteins) are expressed by many different cell lineages including
monocytes, and comprise a common b-chain (CD29) and at least
nine distinct a-chains (14). There are at least six members of this
group, VLA-1 to VLA-6 (CD49a-f), which are expressed on
monocytes. Among them, VLA-6 is predominately expressed on
monocytes, followed by moderate amounts of VLA-5, VLA-4, and
VLA-2, and low amounts of VLA-1 and VLA-3 (14). These integrins serve as cellular receptors for ECM proteins including fibronectin, collagen, laminin, and vitronectin. Recently, Bauvois et
al. (10) reported that VLA-5 (a5b1) integrin can mediate human
monocyte adhesion to fibronectin, and b2 (CD11/CD18) integrin
can mediate adhesion to laminin. However, the role of b1 integrins
in monocyte migration in connective tissue such as in synovium is
not yet known. In a previous study, we examined polymorphonuclear leukocyte (PMNL) migration through a biologic barrier of
human synovial fibroblast (HSF) grown on a microporous filter to
model PMNL migration in connective tissue (15). In the current
study, we used this model to investigate the molecular mechanisms
involved in human monocyte migration through such tissue. Our
results indicate that chemotactic factors, such as C5a, induce migration of monocytes across unactivated and cytokine-activated
HSF barriers. This migration is mediated by the CD11/CD18,
VLA-4, and VLA-5 integrins on the monocyte. These mechanisms
are in part distinct from those required for migration through endothelium, where VLA-5 appears to have no significant role.
MONOCYTE MIGRATION ACROSS FIBROBLASTS
The Journal of Immunology
beneath the filter, referred to as migrated cells, The results are expressed as
the percentage of the total 51Cr monocytes added above the HSF or
HUVEC that migrated. All the treatment conditions were performed in
triplicate.
Ab treatment
In some experiments, 51Cr-labeled monocytes were treated for 20 min at
room temperature (22°C) with a saturating concentration of mAb (30 –50
mg/ml) before being tested for migration. These concentrations were determined by flow cytometry. The Abs were present throughout the migration assay except when indicated otherwise. In some experiments, 51Crlabeled monocytes were treated with mAbs, washed with RPMI 1640/0.5%
HSA to remove free mAb, and tested for migration in the absence of mAb.
Alternatively where indicated, HSF barriers were pretreated with mAbs for
40 min at 37°C and washed three times with RPMI 1640 to remove free
mAbs, before monocytes were added to the HSF.
Measurement of VCAM-1 expression on HSF or HUVEC
The expression of VCAM-1 on the cell monolayers was determined by cell
ELISA as described previously (24). Briefly, HSF or HUVEC monolayers
in 96-well plates were incubated for 5 h in fresh RPMI 1640 with 10%
FBS, or stimulated for 5 h with IL-1a (0.5 ng/ml). The IL-1a was then
removed by washing, and 100 ml of RPMI 1640/5% FBS/0.1% NaN3 containing mAb 4B9 to VCAM-1 (10 mg/ml) or mAb 3H11B9 to pertussis
toxin as an isotype control mAb was added. After 60 min (37°C, 5% CO2),
the monolayers were washed four times and 100 ml of peroxidase-conjugated goat anti-mouse IgG (Bio-Can Scientific, Mississauga, Canada) (1:
10000 in RPMI 1640/5% FBS) was added for 60 min (37°C, 5% CO2). The
monolayers were washed four times and then 100 ml of substrate (o-phenylenediamine, 12.5 mg/ml; 0.1 M citrate-phosphate buffer, pH 5; 0.012%
H2O2) was added. The enzyme reaction was stopped by adding 100 ml of
4N H2SO4, and absorbance at 490 nm was measured. Expression of
VCAM-1 on HSF was also determined by immunofluorescence flow cytometry. The HSF were detached by brief treatment with 0.01% trypsin and
0.02% EDTA, and stained using a standard immunofluorescence
protocol (27).
Statistical analysis
Unpaired t test and ANOVA were used for statistical analysis of the data,
as indicated, with individual group means compared using post hoc Bonferroni analysis. p , 0.05 was considered to be significant.
Results
Migration of monocytes across synovial fibroblast barriers
induced by C5a
We observed that monocyte migration across HSF or HUVEC barriers in response to C5a was time dependent, reaching a maximum
FIGURE 2. The effect of mAb to CD18 and to VLA-4 on C5a-induced
monocyte migration across synovial fibroblast or HUVEC barriers. The
51
Cr monocytes were incubated with saturating concentration (30 mg/
ml) of mAb (60.3, TS1/18, or R15.7; results pooled) to CD18 and/or
mAb (HP1⁄2) to a4 integrin of VLA-4 for 20 min at room temperature,
and then added above the HUVEC or fibroblast barriers. The control
mAb (3C10) to CD14 or mAb (543) to CR1 were used and results were
pooled, since no effect on migration was observed. Migration was induced by an optimal concentration of C5a (0.5 nM) for both migration
systems, added to the lower compartment. Results are expressed as in
Figure 1. Values are means 6 SD of three to seven experiments performed in triplicate. *p , 0.05, **p , 0.001 compared with control
mAb-treated group analyzed by post hoc Bonferroni analysis.
after 90 to 100 min of incubation (2) (data not shown). Therefore,
migration was quantitated after 100 min. As shown in Figure 1,
some spontaneous migration of monocytes was observed (6.7 6
0.7% of added monocytes) across HSF barriers. This increased
significantly when C5a was added beneath the HSF-filter unit. The
migration response was C5a concentration dependent, with 0.5 nM
inducing the maximal monocyte migration. This concentration of
C5a was chosen for subsequent experiments.
The role of CD18 and VLA-4 on monocyte migration across
HSF barriers
Our previous studies and that of others have shown a role for
CD11/CD18 (b2) and VLA-4 (a4b1) integrins in monocyte migration through HUVEC (2, 13). To determine the role of CD11/
CD18 and VLA-4 in monocyte migration across HSF barriers in
comparison to HUVEC, labeled 51Cr monocytes were treated with
saturating concentration of adhesion-blocking mAbs (60.3, R15.7,
or TS1/18) to CD18, with mAb HP1⁄2 to the a4-chain of VLA-4, or
with a combination of mAb to CD18 plus mAb to a4 before being
added above the barriers. As shown in Figure 2, mAb to CD18
partially (40%) inhibited monocyte migration across HUVEC in
response to C5a, and in combination with mAb to a4, migration
across endothelium barriers was inhibited to the level of migration
in the absence of C5a. In marked contrast, monocyte migration
across HSF barriers, using the same monocyte preparations, was
not inhibited by mAb to CD18, and in combination with mAb to
a4, there was only partial (33%) inhibition (Fig. 2) when compared
with control mAb treatments (anti-CD14 or anti-CR1) ( p , 0.05).
These results suggest the presence of a CD18- and VLA-4-independent mechanism involved in monocyte transfibroblast migration, which is not operative in monocyte transendothelial
migration.
The roles of VLA-5 and VLA-6 in monocyte migration across
HSF barriers
The VLA-5 (a5b1) integrin, a receptor for fibronectin, and VLA-6
(a6b1) integrin, a receptor for laminin (14), are the two other b1
integrins, besides VLA-4, which are relatively highly expressed on
monocytes. Therefore, we investigated the role of VLA-5 and
VLA-6 in monocyte migration across the HSF barriers by using
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FIGURE 1. The dose response of C5a-induced monocyte migration
through a synovial fibroblast barrier. Monocytes labeled with 51Cr
were added above the fibroblast barriers, and varying concentrations
of recombinant C5a were added beneath the fibroblast/filter unit to
stimulate migration as described in Materials and Methods. Results are
expressed as the percent of added monocytes that migrated through
the fibroblast/filter unit. Values are means 6 SD of two dose-response
experiments performed in triplicate.
469
470
the blocking mAb JBS-5 to the a5-chain of VLA-5 and mAb 45030A1 to the a6-chain of VLA-6 to treat the monocytes. As shown
in Figure 3, treatment of monocytes with the mAb to a5 alone, just
as treatment with the mAb to a4 alone, did not significantly inhibit
monocyte migration induced by C5a. The combination of mAb to
a4 and a5 also had no effect on monocyte migration, compared
with monocytes treated with control mAbs 543 to CR1 or 3C10 to
CD14 (Fig. 3). Furthermore, when mAb 450-30A1 to a6 was
added to the combination of mAbs to a4 and a5, there was also no
significant decrease in migration observed (Fig. 3).
Since blocking function of CD18 (b2), VLA-4, VLA-5, or
VLA-6 did not inhibit monocyte migration across HSF barriers, we
examined whether the b2 (CD18) and these b1 integrins might
function in concert to mediate optimal monocyte migration. Therefore, monocytes were treated with mAb to CD18 plus mAb to a4
(VLA-4), and a5 (VLA-5), with or without mAb to a6 (VLA-6).
As shown in Figure 4, treatment of monocytes with mAb to CD18
plus mAb to a5 (VLA-5) slightly but significantly inhibited monocyte migration across HSF barriers. Similarly, treatment with mAb
to CD18 plus mAb to a4 inhibited migration, compared with
monocytes treated with control mAb. However, treatment of
monocytes with a combination of mAb to CD18 plus mAbs to both
a4 (VLA-4) and a5 (VLA-5) completely inhibited monocyte migration across HSF barriers in response to C5a, i.e., to the level of
migration existing in the absence of C5a. Addition of mAb to a6
(VLA-6) to this mAb combination did not enhance the inhibition.
These results suggest that CD11/CD18, VLA-4, and VLA-5 can
each mediate monocyte migration across HSF barriers.
We investigated whether the effect of mAb to a4 of VLA-4 and
a5 of VLA-5 was on the monocytes or on the fibroblast, because
VLA-4 and VLA-5 are present on both cell types (14, 28, 29). The
monocytes were treated with mAb to CD18 plus mAb to a4 and a5
and then washed to remove free Abs. mAb to CD18 alone was
added back to ensure that any CD11/CD18 integrins mobilized
from intracellular stores during the assay would be blocked. As
shown in Figure 5, migration of these monocytes was inhibited as
much as when mAb to a4, a5, and CD18 were all present during
the assay. When the fibroblast barriers were pretreated with mAb
to a4 and to a5 and washed before addition of monocytes, which
FIGURE 4. The effect of mAb to CD18 plus mAbs to VLA-4 and
VLA-5 on C5a-induced monocyte migration across HSF barriers. The
51
Cr monocytes were treated as in Figure 2 with mAb (HP1⁄2) to the
a4-chain of VLA-4, mAb (JBS-5) to the a5-chain of VLA-5, mAb (45030A1) to the a6-chain of VLA-6, or mAb (60.3) to CD18 alone or in
combination as indicated. Control mAbs were as described in Figure 2.
Migration was in response to C5a (0.5 nM) added to the lower compartment. Results are expressed as in Figure 1. Values are means 6
SEM of two to eight experiments performed in triplicate. *p , 0.05,
**p , 0.001 compared with control mAb-treated group analyzed by
unpaired t test.
FIGURE 5. The effect of mAbs to VLA-4 and VLA-5 on monocyte
migration across synovial fibroblast barriers. The 51Cr monocytes were
pretreated with a saturating concentration (30 mg/ml) of mAb (HP1⁄2) to
the a4-chain of VLA-4, mAb (JBS-5) to the a5-chain of VLA-5, and mAb
(60.3) to CD18, or mAb to CD18 alone for 20 min at room temperature. Where indicated, monocytes were washed twice to remove unbound Abs. To the washed monocytes, mAb to CD18 alone was added
back before the migration assay. In other cases, HSF monolayers were
pretreated with the mAb to a4 and mAb to a5 for 40 min at 37°C and
then washed to remove mAb, before monocytes treated with mAb to
CD18 were added for migration. Control mAbs were as described in
Figure 2. Migration was in response to C5a (0.5 nM) added to the
lower compartment for 100 min. Results are expressed as in Figure 1.
Values are means 6 SEM of three to eight experiments performed in
triplicate. **p , 0.001 compared with control mAb treated group (unpaired t test).
were treated only with mAb to CD18, the migration was not inhibited. The results indicate that VLA-4 and VLA-5 on the monocytes, but not on the fibroblasts, mediate CD18-independent monocyte transfibroblast migration in response to C5a.
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FIGURE 3. The effect of mAb to VLA-4, VLA-5, or VLA-6 on C5ainduced monocyte migration across HSF barriers. The 51Cr monocytes
were treated as in Figure 2 with mAb (HP1⁄2) to the a4-chain of VLA-4,
mAb (JBS-5) to the a5-chain of VLA-5, or mAb (450-30A1) to the a6chain of VLA-6 alone or in combination as indicated. The control
mAbs were as described in Figure 2. Migration was in response to C5a
(0.5 nM). Results are expressed as in Figure 1. Values are means 6
SEM of three to eight experiments performed in triplicate.
MONOCYTE MIGRATION ACROSS FIBROBLASTS
The Journal of Immunology
Monocyte migration induced by MCP-1
We further investigated monocyte migration across the HSF barrier in response to MCP-1, since this chemotactic factor is selective
for monocytes and is also present in inflamed synovial connective
tissue. The optimal concentration of MCP-1 for monocyte transfibroblast migration was 1.7 to 2 nM (data not shown). As shown
in Figure 6, treatment of monocytes with mAb to CD18 alone did
not inhibit the migration induced by MCP-1, and mAb to CD18
plus either mAb to a4 or a5 partially inhibited the migration. The
migration was completely inhibited by mAb to CD18 plus mAb to
a4 and a5. We also observed that treatment of monocytes with
mAb to a4, a5, and a6 alone or in combination did not inhibit
migration (data not shown), as was also the case for C5a (Fig. 3).
Monocyte migration across IL-1-activated HSF barriers
Our previous results indicated that the mechanisms involved in
monocyte migration across IL-1a-activated endothelium were
quantitatively different from migration across unactivated endothelium, in that the VLA-4-/VCAM-1-mediated migration pathway was enhanced by activation of endothelium (2). Therefore, we
investigated whether stimulation of HSF monolayers with IL-1a
influenced the mechanisms mediating monocyte transfibroblast
migration. As shown in Figure 7, mAb to CD18 did not inhibit
monocyte migration induced by C5a through IL-1a-activated HSF,
but addition of mAb to a4 (VLA-4) or mAb to a5 (VLA-5) to the
mAb to CD18 partially blocked the migration. The combination of
mAb to CD18 plus mAb to a4 and a5 completely inhibited monocyte migration across IL-1a-activated HSF, as was the case with
unactivated HSF.
Expression of VCAM-1 on HSF monolayers and its
contribution to VLA-4-mediated monocyte migration
VCAM-1 is a ligand for VLA-4 and can mediate VLA-4-dependent monocyte migration across HUVEC activated with IL-1 (2,
FIGURE 7. The effect of mAb to CD18, VLA-4, and VLA-5 on C5ainduced monocyte migration across unactivated or IL-1a-activated synovial fibroblast barriers. Synovial fibroblast monolayers were incubated with medium alone or IL-1a (0.5 ng/ml) for 5 h, after which
medium was exchanged and 51Cr monocytes were added above the
HSF barriers. The 51Cr monocytes were treated as in Figures 2 and 3
with mAb (HP1⁄2) to the a4-chain of VLA-4, mAb (JBS-5) to the a5-chain
of VLA-5, or mAb (60.3) to CD18 alone, or in combination as indicated. Migration was in response to C5a (0.5 nM) added to the lower
compartment. Results are expressed as in Figure 1. Values are
means 6 SEM of two to eight experiments performed in triplicate. *p ,
0.05; **p , 0.001 compared with no mAb-treated group; 1p , 0.05,
compared with unactivated HSF analyzed by unpaired t test.
13). In contrast to unstimulated HUVEC, which does not express
VCAM-1, unstimulated HSF expressed considerable levels of
VCAM-1, in fact to a greater degree than even IL-1-stimulated
HUVEC, when measured by whole cell ELISA on viable cells. By
this assay, absorbance values for unstimulated HSF 5 0.680 6
0.022, for HUVEC 5 0.00. After IL-1a (0.5 ng/ml for 5 h) stimulation of HUVEC, the absorbance increased to 0.219 6 0.005.
Stimulation of HSF with IL-1a under the same conditions increased VCAM-1 expression from the baseline of 0.680 up to
0.930 6 0.038 OD units. Flow cytometry analysis revealed that
41% of unstimulated HSF expressed VCAM-1 (mean fluorescence
U 5 9.6; nonbinding isotype control mAb 3H11B9 (anti-pertussis
toxin) 5 3.4), and this increased to 75% upon IL-1 stimulation
(mean fluorescence U 5 21.4; control mAb 5 3.3). The histograms showed a unimodal distribution in the case of the unstimulated and the IL-1-stimulated HSF cell populations. Following
IL-1 stimulation of HUVEC, 55% of cells became strongly
VCAM-1 positive with a bimodal distribution, as reported
previously (5).
Having determined that VCAM-1 was constitutively expressed
on HSF, we investigated its contribution to monocyte migration
through the HSF barriers. Although multiple integrins, i.e., CD11/
CD18, VLA-4, and VLA-5, contribute to monocyte migration, as
shown above, VCAM-1 is believed to function as a counterligand
only for VLA-4 or a4b7. Therefore, the role of VCAM-1 was
studied using monocytes that were treated with mAbs to CD18 and
VLA-5, so that all C5a-stimulated migration was mediated by the
VLA-4 pathway. In comparison, VLA-4-dependent monocyte migration across HUVEC was assessed using monocytes treated with
mAb to CD18 only since, as shown above (Fig. 2), VLA-5 does
not contribute to transendothelial migration. To assess the contribution of VCAM-1 on HSF and HUVEC to monocyte transmigration, two blocking anti-VCAM-1 mAbs were used. One was directed at domain 1 on VCAM-1 (mAb 4B9), and one was reactive
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FIGURE 6. The effect of mAbs to CD18, VLA-4, and VLA-5 on
MCP-1 induced monocyte migration across HSF barriers. The 51Cr
monocytes were treated as in Figure 2 with mAb (HP1⁄2) to the a4-chain
of VLA-4, mAb (JBS-5) to the a5-chain of VLA-5, mAb (450-30A1) to
the a6-chain of VLA-6, or mAb (60.3) to CD18 alone or in combination
as indicated. Migration was in response to MCP-1 (1.7 nM) added to
the lower compartment. Results are expressed as in Figure 1. Values
are means 6 SD of two experiments performed in triplicate. *p , 0.05,
**p , 0.001 compared with no mAb-treated group analyzed by unpaired t test.
471
472
with domain 4, i.e., mAb GH12, because previous studies (5) indicated that both domains of VCAM-1 could contribute to VLA4-dependent monocyte transendothelial migration (5). Figure 8
shows that migration of monocytes across IL-1-stimulated
HUVEC or unstimulated HSF using monocytes pretreated as
above was completely inhibited to background levels by adding
mAb to VLA-4. In the case of HUVEC, pretreating the HUVEC
with a combination of mAbs to domain 1 plus domain 4 of
VCAM-1 inhibited migration to the same degree as anti-VLA-4
mAb. This contrasts with monocyte migration across HSF on
which blocking both domain 1 and domain 4 of VCAM-1 only
partially inhibited migration despite using saturating mAb treatments. These results were observed even when F(ab9)2 forms of
anti-VCAM-1 mAbs were used with the HSF as utilized by us
previously with HUVEC (5). The current experiments with HSF
were run in parallel with additional HUVEC experiments for comparison and to further verify mAb efficacy. Furthermore, pretreating the HUVEC or HSF with control mAbs reactive with HLA
class I (W6/32) did not affect monocyte transmigration. The results
suggest that monocyte VLA-4 utilize predominantly VCAM-1 as a
ligand on HUVEC. However, on connective tissue cells such as
HSF, additional ligand(s) may serve to mediate VLA-4-dependent
migration. Although not shown in Figure 8, stimulation of HSF
with IL-1 to further increase VCAM-1 expression before monocyte migration had no effect on the inhibitory effects of anti-VLA-4
or the VCAM-1 mAbs, indicating that the mechanisms of VLA-4
ligand dependence were not altered due to IL-1 effects on HSF.
Discussion
Monocyte interaction with and migration in connective tissue during inflammation are important events in many inflammatory dis-
eases, including arthritis. To model this in synovial connective
tissue, monocyte migration across a synovial fibroblast barrier was
examined. Fibroblasts grown on polycarbonate filters form a semipermeable barrier and produce ECM, which supports attachment
and growth of endothelial cells (30). The barrier is much less restrictive to macromolecules than to endothelium, as is the case in
connective tissue. For example, permeability to albumin is four- to
fivefold greater for the HSF than the HUVEC barriers (see Materials and Methods). The fibroblast/filter unit allows quantitation of
migrated leukocytes and control of assay conditions by allowing
ready exchange of medium and removal of secreted soluble fibroblast factors, which may influence monocyte migration. Fibroblasts grown in an ECM three-dimensional gel also could serve as
a useful model. However, quantitation of monocyte migration into
the gel and control of assay conditions due to trapping of fibroblast-derived factors in the gel during the prolonged culture may
be problematic in such a system. The results demonstrate that
monocyte migration across the HSF barrier is greatly enhanced by
C5a or MCP-1, which are relevant chemotactic factors present in
inflammatory synovial fluid. The migration response was strictly
concentration dependent, with an optimal response at a concentration of 0.5 nM C5a (Fig. 1) or 1.7 to 2 nM MCP-1 (data not
shown). The bell-shaped concentration dependence shown in Figure 1 is characteristic of the chemotactic response in a Boyden
chamber filter chemotaxis system (31–33). These results indicate
that addition of a biologic barrier to this system does not alter this
concentration-dependent relationship.
A parallel comparison here of monocyte migration mechanisms
utilized in transendothelial and transfibroblast migration revealed
common and distinct mechanisms. These are summarized in Table
I based on results from this study and previous studies as referenced in the table. The CD11/CD18 and VLA-4 on monocytes
mediate all the migration across HUVEC, as has more recently
been shown in vivo (3, 8). In marked contrast, using the same
preparation of monocytes, migration across the HSF barrier was
much less dependent on CD18 and VLA-4 function, as shown in
Figure 2 and summarized in Table I.
It is known that b1 integrins on leukocytes can in part mediate
leukocyte adhesion to ECM proteins (14, 34), which are produced
by fibroblasts. Of the b1 integrin family, VLA-4, VLA-5, and
VLA-6 are expressed on monocytes (14). Our results indicate that
of the b1 integrins, monocytes utilize VLA-5 as well as VLA-4
during the migration across HSF. This is not specific for C5a, since
this was also the case when MCP-1 was the stimulus (Figs. 4 and
6). This role for VLA-5 and VLA-4 was only apparent when
CD11/CD18 mechanisms were blocked, indicating that CD11/
CD18 alone could mediate optimal transfibroblast migration (Fig.
3 and Table I). On the other hand, either VLA-4 or VLA-5 alone
could mediate a good migration response (Fig. 4) and, in concert
(e.g., when only CD11/CD18 was blocked), an optimal migration
via VLA-4 and VLA-5 mechanisms could occur. The inhibitory
effect observed with mAb to VLA-4 and VLA-5 is unlikely to be
nonspecific, since treatment of monocytes with control mAb (to
CR1 or CD14) did not inhibit migration (Fig. 4). Although VLA-4
and VLA-5 are present on fibroblasts as well as on monocytes, the
results in Figure 5 demonstrate that mAb blockade of the VLA-4
and VLA-5 only on the monocyte was required to inhibit migration. Thus, VLA-4 and VLA-5 are alternative mechanisms to
CD11/CD18 in mediating monocyte migration induced by C5a
across unactivated HSF barriers. This is in part distinct from
monocyte transendothelium migration, where VLA-4 appears to be
the only alternative mechanism to CD11/CD18 (Figs. 2 and 8,
Table I summary) (2, 13). No role for VLA-5 in monocyte migration across HUVEC was detectable, even when anti-CD18 plus
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FIGURE 8. Effect of mAb to domain 1 or 4 of VCAM-1 on monocyte
migration across unactivated HSF or IL-1a-activated HUVEC barriers.
The HSF or HUVEC barriers were incubated with medium alone or
IL-1a (0.5 ng/ml) for 5 h before treatment with saturating amounts of
mAb to domain 1 (4B9) or mAb to domain 4 (GH12) of VCAM-1, as
indicated, for 40 min at 37°C. Then 51Cr-labeled monocytes treated
with control mAbs as in Figure 2 or anti-CD18 mAb (60.3) alone or in
combination with mAb (JBS-5) to a5, with or without mAb (HP1⁄2) to
a4, were added above the monolayer as indicated. Migration was in
response to C5a (0.5 nM) added to the lower compartment. Results are
expressed as in Figure 1. Values are means 6 SEM of four experiments
performed in triplicate. *p , 0.05; **p , 0.001 compared with control
mAb-treated group; 1p , 0.05; 11p , 0.001; #p , 0.05 analyzed by
post hoc Bonferroni analysis.
MONOCYTE MIGRATION ACROSS FIBROBLASTS
The Journal of Immunology
473
Table I. Involvement of adhesion molecules in monocyte migration through HUVEC or HSF a
Resting HUVEC
Adhesion
Molecules
IL-1-Stimulated HUVEC
Resting or IL-1-Stimulated
HSF
Monocyte
HUVEC
Monocyte
HUVEC
Monocyte
HSF
NA
11
(2, 4, 13)
11
(2, 4, 13)
0
0
NA
NA
11
NA
ND
1
0
VLA-5
VLA-6
VCAM-1
111
(2, 4, 13)
1
(2, 4, 13)
0
0
NA
1
0
NA
0
ND
1
CS-1 FN
NA
ND
ND
111
(2, 4, 5)
1/2
(4, 5)
NA
1?
CD11/CD18
VLA-4
ND
ND
ND
1
(2, 4, 5)
1/2
(4, 5)
NA
a
The results are a summary of this study and previous studies referenced in parentheses. The degree of adhesion molecule involvement in the migration is expressed
as a 0 to 41 scale. NA, not applicable.
to the baseline level, in contrast to migration across HSF, where
most of the VLA-4-mediated migration was not inhibitable by antiVCAM-1 mAbs. The alternative ligand on HSF may be CS-1 fibronectin, as suggested by Meerschaert and Furie (4), based on
their observations that the CS-1 peptide (EILDVPST) could partially block monocyte migration through HUVEC grown on amnion. However, one must be cautious in this conclusion because
CS-1 peptide can interfere with VLA-4 ligand recognition in general and not only in the binding to CS-1 fibronectin (41). Other
approaches such as the use of function-blocking mAbs to CS-1
fibronectin, which are not yet readily available, may be required to
dissect the contribution of each ligand to VLA-4-mediated transfibroblast migration. Fibronectin is also a major ligand for VLA-5,
the recognition sequence being Arg-Gly-Asp (RGD) (14), which is
expressed in fibronectin on connective tissue cells. Recently L-1,
an Ig supergene member surface molecule on neural cells, has also
been shown to contain an RGD binding sequence for VLA-5 in the
mouse (42). Whether this interaction occurs on human cells and
whether L-1 is expressed on HSF, serving as a ligand for monocyte
migration, will require further investigation.
In conclusion, to our knowledge, this is the first study to show
that monocytes migrate across a connective tissue cell barrier, and
that there are at least three integrin mechanisms involved in this
process. Some of these (i.e., the CD11/CD18 and VLA-4 pathway)
are also involved in transendothelial migration. However, in connective tissue, the VLA-5 integrin may also be important as a
mediator of migration. Although it remains to be seen whether the
same adhesion proteins have the same relative importance when
monocytes are migrating through a structure more closely resembling “authentic connective tissue,” with a three-dimensional array
of ECM proteins and interspersed fibroblasts, the results here provide some insight into monocyte interactions in connective tissue
in vivo. The multiple integrins on monocytes, which may function
in monocyte migration in a connective tissue setting, indicate that
regulation of this process may require some novel approaches.
Acknowledgments
We are grateful to numerous colleagues, listed under Materials and Methods, who supplied valuable Abs and reagents for these studies. We also are
indebted to all those who donated blood for these studies. The outstanding
technical assistance of D. Rowter and C. Jordan and the expert secretarial
help of M. Hopkins are gratefully acknowledged.
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