| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Pediatrics, Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
4
(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-4 mAb. The CD18- and VLA-4-independent
migration across HSF was completely inhibited by mAb to
5 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,
VLA-4-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. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
It is well known that monocytes express ß2 (CD11/CD18)
integrins, which share a common ß-chain (CD18) and have three
distinct -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-,
or LPS is CD18 independent, and can be mediated by very late Ag-4
(VLA-4) (4ß1, CD49d/CD29) integrins (2, 13). The ß1 integrins (also called VLA proteins) are
expressed by many different cell lineages including monocytes, and
comprise a common ß-chain (CD29) and at least nine distinct
-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 (5ß1)
integrin can mediate human monocyte adhesion to fibronectin, and
ß2 (CD11/CD18) integrin can mediate adhesion to laminin.
However, the role of ß1 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.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following adhesion function-blocking murine mAbs against
human Ags were used as purified IgG: 60.3
(anti-ß2 (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-4-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-5-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-6-chain of VLA-6, IgG1; gift from Dr.
S. J. Kennel, Oak Ridge National Laboratory, TN) (21), 3H11B9
(anti-pertussis 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-1 (sp. act. of 4 x
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 MEM (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
MEM-10% FBS, 50 µM 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-µm 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 x 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% of125I-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 µg/ml endothelial cell growth factor (Collaborative Research, Lexington, MA), and 22.5 µg/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 µg of human fibronectin (Collaborative Research) in 45 µl of water at 37°C for 2 h. The HUVEC (1.5 x 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 x g for 10 min at room temperature), the leukocyte pellet was resuspended in a Ca2+-, Mg2+-free Tyrode’s solution with 5% autologous platelet-poor plasma and labeled with 51Cr sodium chromate (25 µCi/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 Ca2+-, Mg2+-free Tyrode’s solution (360 mOsmol), 5% platelet-poor plasma, and resuspended in Ca2+-, Mg22+-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 x 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 x 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-1 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 x
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 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 µg/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, 51Cr-labeled 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-1 (0.5
ng/ml). The IL-1 was then removed by washing, and 100 µl of RPMI
1640/5% FBS/0.1% NaN3 containing mAb 4B9 to VCAM-1
(10 µg/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 µl 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
µl 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 µl 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We observed that monocyte migration across HSF or HUVEC barriers
in response to C5a was time dependent, reaching a maximum 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 ± 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.
|
Our previous studies and that of others have shown a role for
CD11/CD18 (ß2) and VLA-4
(4ß1) 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 4-chain of VLA-4, or with a
combination of mAb to CD18 plus mAb to 4 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 4, 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 4, 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 VLA-5 (5ß1) integrin, a
receptor for fibronectin, and VLA-6 (6ß1)
integrin, a receptor for laminin (14), are the two other
ß1 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 the
blocking mAb JBS-5 to the 5-chain of VLA-5 and mAb
450-30A1 to the 6-chain of VLA-6 to treat the monocytes.
As shown in Figure 3, treatment of
monocytes with the mAb to 5 alone, just as treatment
with the mAb to 4 alone, did not significantly inhibit
monocyte migration induced by C5a. The combination of mAb to
4 and 5 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
6 was added to the combination of mAbs to
4 and 5, there was also no significant
decrease in migration observed (Fig. 3).
|
4 (VLA-4), and 5 (VLA-5), with or
without mAb to 6 (VLA-6). As shown in Figure 4, treatment of monocytes with mAb to
CD18 plus mAb to 5 (VLA-5) slightly but significantly
inhibited monocyte migration across HSF barriers. Similarly, treatment
with mAb to CD18 plus mAb to 4 inhibited migration,
compared with monocytes treated with control mAb. However, treatment of
monocytes with a combination of mAb to CD18 plus mAbs to both
4 (VLA-4) and 5 (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 6 (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.
|
4 of VLA-4
and 5 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
4 and 5 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
4, 5, and CD18 were all present during
the assay. When the fibroblast barriers were pretreated with mAb to
4 and to 5 and washed before addition of
monocytes, which 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.
|
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 4 or 5
partially inhibited the migration. The migration was completely
inhibited by mAb to CD18 plus mAb to 4 and
5. We also observed that treatment of monocytes with mAb
to 4, 5, and 6 alone or in
combination did not inhibit migration (data not shown), as was also the
case for C5a (Fig. 3
).
|
Our previous results indicated that the mechanisms involved in
monocyte migration across IL-1-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-1 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-1-activated HSF, but addition of
mAb to 4 (VLA-4) or mAb to 5 (VLA-5)
to the mAb to CD18 partially blocked the migration. The combination of
mAb to CD18 plus mAb to 4 and 5
completely inhibited monocyte migration across IL-1-activated HSF,
as was the case with unactivated HSF.
|
VCAM-1 is a ligand for VLA-4 and can mediate VLA-4-dependent
monocyte migration across HUVEC activated with IL-1 (2, 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 = 0.680 ± 0.022, for HUVEC = 0.00.
After IL-1 (0.5 ng/ml for 5 h) stimulation of HUVEC, the
absorbance increased to 0.219 ± 0.005. Stimulation of HSF with
IL-1 under the same conditions increased VCAM-1 expression from the
baseline of 0.680 up to 0.930 ± 0.038 OD units. Flow cytometry
analysis revealed that 41% of unstimulated HSF expressed VCAM-1 (mean
fluorescence U = 9.6; nonbinding isotype control mAb 3H11B9
(anti-pertussis toxin) = 3.4), and this increased to 75% upon IL-1
stimulation (mean fluorescence U = 21.4; control mAb = 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
4ß7. 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
with domain 4, i.e., mAb GH12, because previous studies (5) indicated
that both domains of VCAM-1 could contribute to VLA-4-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(ab')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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) 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, 32, 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.
|
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
anti-VLA-5, with or without anti-VLA-4, treatments were
combined (data not shown). The reason for this difference is not
apparent but may be related to the form or distribution of fibronectin
synthesized by endothelium and HSF, since fibronectin is the likely
ligand for VLA-5 (14, 35). VLA-6 on monocytes does not appear to be involved in mediating migration. When mAb to VLA-6 was added to the other mAb treatments, it failed to inhibit migration. Interestingly, this is distinct from the situation for PMNL migration through HSF or dermal fibroblasts, a process in which PMNLs do utilize VLA-6 as well as VLA-5, as shown recently (15, 36). This would indicate that HSF express ligands for VLA-6, but monocyte VLA-6 may not be activated to engage this mechanism. Aside from this discrepancy between PMNLs and monocytes, PMNLs also have additional mechanisms to migrate through fibroblast barriers, since blocking CD11/CD18, VLA-4, VLA-5, and VLA-6 in combination could not inhibit PMNL migration to the unstimulated level (15, 37).
It is well established that treatment of endothelium with IL-1 and
TNF- up-regulate the expression of adhesion molecules such as
E-selectin, ICAM-1, and VCAM-1. This also enhances VLA-4-dependent
monocyte transendothelial migration (see Table I) (2, 5, 11, 12, 38, 39). Since these cytokines may be generated in inflamed connective
tissue, we investigated whether the mechanisms of monocyte migration
across cytokine-activated HSF barriers may be modified by such
treatment. Under the activation condition utilized here (5 h), IL-1 did
not increase the migration induced by C5a, although the migration
across IL-1-activated HSF barriers in the absence of C5a was
significantly increased (Fig. 7). The adhesion molecule mechanisms
mediating C5a induced migration across IL-1-activated HSF barriers were
comparable to the ones mediating migration across unactivated HSF
barriers, i.e., VLA-4, VLA-5, and CD11/CD18 (Figs. 4 and 7, and Table I
summary). Thus, unlike the mechanisms in monocyte transendothelial
migration that are markedly altered by IL-1 stimulation, likely due to
increased VCAM-1 expression on the HUVEC (2, 4, 5, 13, and Table I
summary), IL-1 has minimal qualitative or quantitative effects on this
process with HSF. This is likely due to the high level of VCAM-1
expressed constitutively on these cells.
The ligands for VLA-4 include VCAM-1 (40) and the non-RGD sequence in
the connecting segment-1 (CS-1) of fibronectin (12, 39). We and others
have observed that VCAM-1 on IL-1-activated HUVEC is a major ligand for
VLA-4-mediated monocyte transendothelial migration, as summarized in
Table I (2, 4, 5, 13). As shown in Figure 8, blocking both VLA-4
binding domains on VCAM-1 inhibited monocyte migration across activated
HUVEC to the baseline level, in contrast to migration across HSF, where
most of the VLA-4-mediated migration was not inhibitable by
anti-VCAM-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 |
|---|
|
Footnotes |
|---|
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.
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.
Received for publication May 15, 1997. Accepted for publication September 22, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, RANTES, and PAF but inhibits migration to MCP-1: a regulatory role for endothelium-derived MCP-1. J. Leukocyte Biol. 58:71.[Abstract]
4 epitopes. J. Biol. Chem. 266:10241.6ß4. Hybridoma 9:243.[Medline]
induced polymorphonuclear leukocyte-endothelial cell adhesion and transendothelial migration in vitro: the effect of apical versus basal monolayer stimulation. Immunol. Lett. 25:331.[Medline]
. Immunology 79:600.[Medline]
4ß1 and 5ß1. J. Immunol. 154:3128.[Abstract]
4ß1. J. Biol. Chem. 269:4005.This article has been cited by other articles:
|
|
 |
|
  H. P. Kiener, D. M. Lee, S. K. Agarwal, and M. B. Brenner Cadherin-11 Induces Rheumatoid Arthritis Fibroblast-Like Synoviocytes to Form Lining Layers in Vitro Am. J. Pathol., May 1, 2006; 168(5): 1486 - 1499. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Yoshimi, S. Yamaji, A. Suzuki, W. Mishima, M. Okamura, T. Obana, C. Matsuda, Y. Miwa, S. Ohno, and Y. Ishigatsubo The {gamma}-Parvin-Integrin-Linked Kinase Complex Is Critically Involved in Leukocyte-Substrate Interaction J. Immunol., March 15, 2006; 176(6): 3611 - 3624. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Valencia, J. M.G. Higgins, H. P. Kiener, D. M. Lee, T. A. Podrebarac, C. C. Dascher, G. F.M. Watts, E. Mizoguchi, B. Simmons, D. D. Patel, et al. Cadherin-11 Provides Specific Cellular Adhesion between Fibroblast-like Synoviocytes J. Exp. Med., December 20, 2004; 200(12): 1673 - 1679. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Mayer, S. Meyer, M. Reinholz-Muhly, U. Maus, M. Merfels, J. Lohmeyer, F. Grimminger, and W. Seeger Short-Time Infusion of Fish Oil-Based Lipid Emulsions, Approved for Parenteral Nutrition, Reduces Monocyte Proinflammatory Cytokine Generation and Adhesive Interaction with Endothelium in Humans J. Immunol., November 1, 2003; 171(9): 4837 - 4843. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Schram, N. Chaturvedi, C. Schalkwijk, F. Giorgino, P. Ebeling, J. H. Fuller, and C. D. Stehouwer Vascular Risk Factors and Markers of Endothelial Function as Determinants of Inflammatory Markers in Type 1 Diabetes: The EURODIAB Prospective Complications Study Diabetes Care, July 1, 2003; 26(7): 2165 - 2173. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. J. Bellingan, P. Xu, H. Cooksley, H. Cauldwell, A. Shock, S. Bottoms, C. Haslett, S. E. Mutsaers, and G. J. Laurent Adhesion Molecule-dependent Mechanisms Regulate the Rate of Macrophage Clearance During the Resolution of Peritoneal Inflammation J. Exp. Med., December 2, 2002; 196(11): 1515 - 1521. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Luppi, C. Haluszczak, D. Betters, C. A. H. Richard, M. Trucco, and J. A. DeLoia Monocytes are progressively activated in the circulation of pregnant women J. Leukoc. Biol., November 1, 2002; 72(5): 874 - 884. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Mayer, M. Merfels, M. Muhly-Reinholz, S. Gokorsch, S. Rosseau, J. Lohmeyer, N. Schwarzer, M. Krull, N. Suttorp, F. Grimminger, et al. omega -3 Fatty acids suppress monocyte adhesion to human endothelial cells: role of endothelial PAF generation Am J Physiol Heart Circ Physiol, August 1, 2002; 283(2): H811 - H818. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. E. de Vries, J. J. A. Hendriks, H. Honing, C. R. de Lavalette, S. M. A. van der Pol, E. Hooijberg, C. D. Dijkstra, and T. K. van den Berg Signal-Regulatory Protein {alpha}-CD47 Interactions Are Required for the Transmigration of Monocytes Across Cerebral Endothelium J. Immunol., June 1, 2002; 168(11): 5832 - 5839. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. H. Jacobson, P. Thylen, and J. Lundahl Three monocyte-related determinants of atherosclerosis in haemodialysis Nephrol. Dial. Transplant., September 1, 2000; 15(9): 1414 - 1419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Rosseau, J. Selhorst, K. Wiechmann, K. Leissner, U. Maus, K. Mayer, F. Grimminger, W. Seeger, and J. Lohmeyer Monocyte Migration Through the Alveolar Epithelial Barrier: Adhesion Molecule Mechanisms and Impact of Chemokines J. Immunol., January 1, 2000; 164(1): 427 - 435. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
