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IMMUNOBIOLOGY
From the Division of Cellular Immunology, National
Institute for Medical Research, The Ridgeway, Mill Hill, London,
United Kingdom.
The migration of lymphocytes from the bloodstream into lymph nodes
(LNs) via high endothelial venules (HEVs) is a prerequisite for the
detection of processed antigen on mature dendritic cells and the
initiation of immune responses. The capture and arrest of lymphocytes
from flowing blood is mediated by the multistep adhesion cascade, but
the mechanisms that lymphocytes use to penetrate the endothelial lining
and the basement membrane of HEVs are poorly understood. Matrix
metalloproteinases (MMPs) control the metastatic spread of tumor cells
by regulating the penetration blood vessel basement membranes. In this
study, synthetic and natural inhibitors were used to determine the role
of MMPs and MMP-related enzymes in regulating lymphocyte extravasation
in mice. Mice were treated systemically with the hydroxamate-based MMP
inhibitor Ro 31-9790 and plasma monitored for effective
levels of Ro 31-9790, which block shedding of L-selectin. The total
numbers of lymphocytes recruited into LNs were not altered, but
L-selectin levels were higher in mice treated with Ro
31-9790. A reduced number of lymphocytes completed diapedesis and
there was an increase in the number of lymphocytes in the endothelial
cell lining, rather than the lumen or the basement membrane of HEVs.
Lymphocyte migration and L-selectin expression in the spleen were not
altered by Ro 31-9790 treatment. Two MMP inhibitors,
TIMP1 and Ro 32-1541, did not block L-selectin shedding and had
no effect on lymphocyte migration across HEVs. These results suggest
that metalloproteinase activity is required for lymphocyte
transmigration across HEVs into LNs and provide evidence for the
concept that metalloproteinases are important players in some forms of
transendothelial migration.
(Blood. 2001;98:688-695) The constitutive trafficking of naïve
lymphocytes in and out of lymphoid organs is a prerequisite for the
detection of processed antigen on mature dendritic cells and the
initiation of immune responses.1 Lymphocytes enter lymph
nodes (LNs) from the bloodstream by migrating across the walls of high
endothelial venules (HEVs), which are postcapillary venules
structurally adapted to support lymphocyte trafficking.2
Although lymphocyte migration from HEVs does not depend on exogenous
inflammatory stimuli, it has features in common with the migration of
leukocytes to inflammatory sites. The capture and arrest of lymphocytes
from flowing blood onto the inner, endothelial surface is controlled by
the multistep adhesion cascade. In peripheral LNs of mice, L-selectin
mediates rolling and lymphocyte function-associated antigen 1 (LFA-1) integrin, the chemokine-dependent arrest of lymphocytes
in HEVs.3 The subsequent migration of lymphocytes across
the HEV wall into the LNs is thought to be directed by a
chemoattractant produced within the LN paracortex, which establishes a
gradient across the HEV wall. Secondary lymphoid tissue chemokine (SLC;
also known as 6Ckine, exodus2, or TCA-4) is produced by endothelial
cells lining HEVs and mediates chemokine-dependent arrest of rolling
lymphocytes in HEVs.4 Gene ablation studies have confirmed
that the expression of CCR7, a receptor for SLC, is required for normal
trafficking of lymphocytes into LNs5; however, the
chemokines that direct lymphocyte migration across the HEV wall to the
LN paracortex have not been identified.
The HEVs comprise a layer of high endothelial cells (HECs) supported by
a pericyte-containing basement membrane.6,7 It has been
proposed that lymphocytes and other leukocytes need to degrade blood
vessel basement membranes to complete diapedesis, and proteinases are,
therefore, attractive candidates for regulating this step in leukocyte
migration. A family of zinc-dependent endopeptidases, the matrix
metalloproteinases (MMPs), plays a potentially important role in
basement membrane penetration by leukocytes. MMP2 (gelatinase A) and
MMP9 (gelatinase B) degrade type IV collagen, a major constituent of
basement membranes, and these enzymes regulate tumor cell invasion of
blood vessel basement membranes in vivo.8 T lymphocytes express MMP9,9,10 and both MMP2 and MMP9 expression can be up-regulated by cellular activation9 or integrin-dependent adhesion.11-14 MMP inhibitors block T-cell migration
across synthetic basement membranes in vitro9
suggesting that MMPs may regulate T-cell diapedesis in vivo. Until
recently, proteases had not been implicated in leukocyte-endothelial
interactions; however, MMP-related enzymes have been shown to regulate
L-selectin shedding15,16 and the rolling velocity of
neutrophils on L-selectin ligands.17,18
Direct demonstration of roles for MMPs in biologic systems has depended
on a combination of approaches including overexpression of activated
MMPs, gene ablation, and the use of selective inhibitors. Naturally
occurring MMP inhibitors are the tissue inhibitors of matrix
metalloproteinases (TIMPs). Of the 4 TIMPs known all are broad-spectrum
inhibitors of secreted MMPs and all inhibit the membrane-associated MMP
(MT-MMP),19 apart from TIMP1, which does not inhibit some
MT-MMPs.20,21 In addition some TIMPs, notably
TIMP3,22 inhibit members of a related family of
zinc-dependent endopeptidases, the metalloproteinase-disintegrins or
ADAMs.23 Manipulation of MMPs in the clinical setting has
been attempted using synthetic inhibitors. A number of
low-molecular-weight MMP inhibitors have been synthesized, which are
peptide analogues of collagen with a hydroxamic acid side chain to bind
the zinc ion in the active site.24 Structurally related
compounds differ in efficacy against individual MMPs and some inhibit
members of the ADAMs family, but they show little activity against
unrelated zinc-dependent endopeptidases.25 Hydroxamic
acid-based MMP inhibitors prevent or reduce the spread and growth of a
number of different malignant tumors in animal
models,26,27 and some of these inhibitors are in the
advanced stages of clinical testing for efficacy against solid
tumors.28 Anti-inflammatory effects of synthetic MMP
inhibitors as well as the TIMPs have been reported,29 but
their effects on leukocyte extravasation were not determined.
In this study, we have used hydroxamate-based MMP inhibitors and TIMPs
to determine the role of metalloproteinases in regulating lymphocyte
extravasation in mice. We show that L-selectin is down-regulated on
lymphocytes migrating into LNs, metalloproteinase inhibitors arrest
lymphocytes in the endothelial lining of HEVs, and the effect on
transendothelial migration correlates with inhibition of L-selectin
shedding. These results suggest that metalloproteinases regulate
transendothelial passage of lymphocytes and provide evidence for a
novel regulatory step in lymphocyte diapedesis.
Animals and inhibitors
L-selectin shedding assay
Angiotensin-converting enzyme assay Hydrolysis of the synthetic substrate furylacryloylphenylalanylglycylglycine (FAPGG) by angiotensin-converting enzyme (ACE) was measured as described previously.32 FAPGG (ACE reagent, Sigma, Poole, United Kingdom) and ACE from porcine kidney (ACE calibrator, Sigma) were reconstituted in water and used according to manufacturer's instructions. The synthetic ACE inhibitor pGlu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro (Sigma A0773) was dissolved at 12 mM in water. Ro 31-1541 and Ro 31-9790 were dissolved at 30 mM in DMSO and stored at 20°C in aliquots. ACE
reagent (1.0 mL) and ACE calibrator (0.1 mL) were preincubated with
inhibitors (final concentration of ACE inhibitor at 24 µM and
Ro 31-9790/Ro 32-1541 at 100 µM) or equivalent amounts of
vehicle (DMSO or water) for 15 minutes at 37°C before mixing.
Absorbance at 340 nm was measured immediately and following 30 minutes
incubation at room temperature using a Shimadzu UV mini-1240
spectrophotometer (Shimadzu Europa, Milton Keynes, United Kingdom). The decrease in absorbance was calculated and the
results are average change in absorbance calculated for triplicate
samples ± SD.
Migration of CFSE-labeled lymphocytes Lymphocytes were labeled with CFSE as described previously.33 For each experiment, 5 to 8 × 108 spleen cells were labeled at 5 × 107 cells/mL for 15 minutes at 37°C with 1 µM CFSE (Molecular Probes, Eugene, OR; 5 mM stock solution in DMSO stored at20°C) in PBS and excess CFSE removed by washing in PBS
containing 1% fetal calf serum (FCS). CFSE labeling did not affect
cell viability (determined by vital dye exclusion); expression of
L-selectin, CD44, LFA-1,  4 integrins, or ICAM-1; proliferation to
mitogens or anti-CD3; or lymphocyte adhesion to cultured endothelial
cells (data not shown and reference 33). CFSE-labeled cells
(40 × 106) in 0.1 mL PBS were injected intravenously
into mice (groups of 3 or 4) pretreated for 30 minutes with inhibitors
or vehicle alone and the number of CFSE-labeled lymphocytes in blood,
LNs, and spleen determined at different times by flow cytometric
analysis of cell suspensions. The localization of CFSE-labeled
lymphocytes in blood vessels of LNs and spleen was determined by
fluorescence microscopy of tissue sections.
Flow cytometric analysis of lymphocyte suspensions Spleen and axillary, brachial, and inguinal LNs were collected separately from each recipient mouse into PBS (calcium and magnesium free) at 4°C. The spleen was cut in half and cell suspensions prepared from one piece of spleen and from one of each LN pair. Red blood cells were lysed with 3 to 5 mL lysis buffer (150 mM NH4Cl in 10 mM KHCO3/0.1 mM EDTA, pH 7.4) for 10 minutes at room temperature and the cells washed twice with PBS. Heparinized blood (300 µL) was also analyzed following lysis of red blood cells. CFSE-labeled lymphocytes were analyzed on a FACS Vantage (Becton Dickinson, Oxford, United Kingdom) using the fluorescein isothiocyanate filter. Acquisition was performed on 2 × 105 viable cells for spleen and blood and 5 to 10 × 105 viable cells for LNs and the data analyzed using WinMDI software (Joseph Trotter, Scripps Institute, La Jolla, CA). The percentage of CFSE-labeled cells recovered in the spleen, blood, and each group of LNs was determined and compared between control and treated animals. Expression of L-selectin on CFSE-labeled cells was determined following incubation with biotinylated MEL-14 and detection of bound antibody using phycoerythrin (PE)-conjugated streptavidin (Southern Biotechnology, Birmingham, AL). The percentage of CFSE-labeled cells expressing L-selectin and the mean fluorescence intensity of cells positive for L-selectin were determined.Fluorescence microscopy One inguinal LN, one brachial LN, and 5-mm slices of spleen from each mouse were fixed in 2% formaldehyde/5% sucrose in PBS for 2 hours, transferred to 20% sucrose in PBS for 2 hours, snap frozen in liquid nitrogen, and stored at70°C for up to 2 years. Cryostat
sections of 8 µm were cut and stored at 70°C for up to 1 year.
HEVs in LN sections were stained with MECA-79 (American Type Culture
Collection, Bethesda, MD) and Texas Red (TXRD)-conjugated goat
anti-rat immunoglobulins (Igs; Molecular Probes) to detect bound
antibody. The position of CFSE-labeled lymphocytes (green) in relation
to HEVs (red) was determined by fluorescence microscopy using a Biorad
MRC 600 confocal microscope (Biorad, Hemel Hempstead, United
Kingdom) and a × 20 objective. For each LN, 10 to 15 images containing complete cross-sections through 30 to 55 HEVs (average HEV
size 2500 µm2) were collected and analyzed using the
"NIH-Image" image analysis software for the Macintosh (public
domain software downloadable from the NIH Image FTP site,
http://rsbweb.nih.gov). The total cross-sectional area of HEVs and the
remaining area within each image were calculated. CFSE-labeled cells
"inside HEVs" were those attached to the luminal surface of the
vessel wall and within the HEV wall (Figure 3; arrows). The remaining
CFSE-labeled cells were scored as "outside HEVs" (Figure 3;
arrowheads). The numbers of CFSE-labeled cells inside and outside HEVs
were not significantly different in inguinal and brachial LNs of
individual mice or between animals in each experimental group. However,
there were differences between experimental groups, which reflect
variation in the absolute number of CFSE-labeled cells injected and the
exact time of LN collection. The results have been pooled from inguinal
and brachial LNs of mice within each experimental group and, where the
results were similar, between experimental groups and are expressed as means ± SEM CFSE-labeled cells/mm2 inside and outside
HEVs. Laminin in the HEV basement membrane was stained using rabbit
anti-mouse laminin34 (generously provided by C. Streuli,
Manchester, United Kingdom) and bound antibody detected with
Cy5-conjugated goat anti-rabbit Ig (Jackson Immunoresearch, West
Grove, PA) (Figure 4). LN sections were examined using an Olympus IX70
inverted microscope, and digital images of 40 to 50 HEVs were acquired
using a Photometrics CH35L liquid-cooled CCD camera and Deltavision
deconvolution software (Applied Precision, Issaquah, WA). The length of
laminin around each HEV was measured using the NIH-Image software. The
number of CFSE-labeled cells between the endothelial lining and laminin
and within the laminin layer were counted. Results are expressed as
number of CFSE-labeled cells/mm of laminin (mean ± SEM).
CFSE-labeled lymphocytes in the spleen were localized to the white pulp
or the surrounding marginal zone (MZ). Sections were incubated with rat
anti-mouse sialoadhesin 3D6 (generously provided by P. Crocker,
Dundee, United Kingdom), which is expressed by MZ
macrophages35 and bound antibody detected using
TXRD-conjugated goat anti-rat Ig. Images were collected using the
confocal microscope and a × 10 objective, the areas of MZ and white
pulp were calculated using NIH-Image software, and the numbers of
CFSE-labeled cells in these locations counted. The numbers of
CFSE-labeled cells in the MZ and white pulp were not significantly
different between animals and the results have been pooled from 6 mice.
Results are expressed as mean ± SEM CFSE cells/mm2 of
MZ or white pulp.
Statistical analysis Pooled data were computed as means ± SEM and were compared using Student t test.
Ro 31-9790 inhibits the major classes of secreted MMPs,
including interstitial collagenase (MMP1), the gelatinases (MMP2 and MMP9), stromelysin (MMP3), and neutrophil collagenase (MMP8), as well
as the membrane-associated ectoenzymes, MT1-MMP (MMP14) and MT4-MMP
(MMP17), with inhibitory concentration of 50% (IC50) values ranging from 2 to 200 nM.21,36,37 Ro
31-9790 also inhibits ADAM1722 and phorbol
ester-induced shedding of L-selectin from the surface of
lymphocytes.31 In contrast, Ro 32-1541, another hydroxamate MMP inhibitor,36 and the endogenous MMP
inhibitor TIMP1 did not inhibit L-selectin shedding (Figure
1A,B).16 Ro 31 9790 and Ro
32-1541 did not inhibit ACE (Table 1), a
zinc-dependent endopeptidase belonging to the thermolysin
family.23 We used the broad-spectrum MMP inhibitor,
Ro 31-9790, in the first instance, to determine the potential
role of metalloproteinases in regulating lymphocyte extravasation. Mice
were treated systemically with inhibitor and its effect was determined
locally by studying the migration of lymphocytes from the blood into
lymphoid organs. The effect of plasma from mice treated with Ro
31-9790 was compared with an optimal dose of Ro 31-9790 (30 µM31) on L-selectin shedding in vitro (Figure 1B,C).
Sufficient Ro 31-9790 to inhibit L-selectin shedding by more
than 90% was found in the bloodstream of mice 60 minutes following a
single intraperitoneal injection of at least 100 mg/kg, but Ro
31-9790 levels were suboptimal after 120 minutes (Figure 1C, solid
bars, 60 minutes; hatched bars, 120 minutes). Plasma from mice given a
similar dose of Ro 32-1541 or 33mg/kg TIMP1 had no effect on L-selectin
shedding (Figure 1C).
Lymphocytes migrate from the bloodstream into LNs and spleen during
normal trafficking to survey the body for invading
pathogens.1 Fluorescent (CFSE)-labeled lymphocytes were
injected 30 minutes following a single intraperitoneal injection of 100 mg/kg Ro 31-9790 and the localization of lymphocytes in LNs,
spleen, and blood determined 30, 60, and 120 minutes following
intravenous injection of cells. Plasma levels of Ro 31-9790 at each time point were measured using exogenous Ro 31-9790 in mouse plasma as a standard and confirmed by high-performance liquid
chromatography analysis (E. Worth, C.F., A.A., unpublished
observations, 1998). Circulating levels of Ro 31-9790 sufficient to completely inhibit L-selectin shedding (> 30 µM) were
found at 30 and 60 minutes, but levels were suboptimal (< 30 µM) at
120 minutes (Table 2). The recovery of
CFSE-labeled lymphocytes from LNs, spleen, or blood of Ro
31-9790-treated mice was not significantly different from control
mice (Figure 2A). The kinetics of
lymphocyte migration were similar to published studies using different
labeling protocols.38 The number of lymphocytes in the
blood stabilized after 30 minutes, whereas the number of lymphocytes
entering LNs and spleen increased between 30 and 60 minutes (Figure
2A); splenic localization was higher than LN localization at both time
points. Blood pressure and vascular permeability were not affected, as
reported previously using similar doses of Ro
31-979039 or other hydroxamate MMP
inhibitors.18 Ro 31-9790 was not cytotoxic or
proapoptotic to lymphocytes and histologic analysis of lymphoid organs
showed no adverse effects on tissue architecture (Figure 4B). The
numbers of lymphocytes positive for L-selectin recovered from LNs,
spleen, and blood were not affected by Ro 31-9790; however,
L-selectin expression was consistently higher in LNs of mice treated
with Ro 31-9790 (Figure 2B). This was not simply due to
increased recruitment of T lymphocytes, which express higher levels of
L-selectin,40 because the T/B cell ratio seen in LNs at
these early time points of about 4:138 was not affected by
Ro 31-9790 (data not shown). The critical role of L-selectin
in recruitment of lymphocytes by HEVs in peripheral LNs is confirmed by
the increased numbers of cells positive for L-selectin and the higher
level of expression on CFSE-labeled lymphocytes harvested from LNs in
comparison with blood or splenic lymphocytes. Ro 31-9790 did
not alter the expression of L-selectin on lymphocytes in the blood or
splenic compartments, indicating that the effect of Ro
31-9790 was restricted to lymphocytes migrating into LNs.
These results suggest that activation of a metalloproteinase
accompanies lymphocyte migration from the blood into LNs. It was
therefore possible that Ro 31-9790, although not affecting the number of lymphocytes that bind to blood vessels, could alter the
subsequent extravasation of lymphocytes. Lymphocytes migrate into LNs
from postcapillary venules called HEVs. CFSE-labeled lymphocytes in the
process of migrating across the endothelial lining of HEVs were
identified by counterstaining LN sections with mAb MECA 79, which
stains HECs in peripheral LNs,41 and recorded as inside
HEVs. The remaining CFSE-labeled cells were recorded as outside HEVs
(Figure 3). The number of lymphocytes inside HEVs decreased between 30 minutes and 120 minutes and
lymphocytes accumulated outside HEVs as cells completed diapedesis and
entered the LNs (Figure 3). Ro 31-9790 treatment of mice
increased the number of lymphocytes inside HEVs and decreased the
number of lymphocytes outside HEVs at 30 and 60 minutes. The effect of
Ro 31-9790 was greatest at 60 minutes with a 2-fold increase
in lymphocytes inside HEVs. This increase was seen in mice that had
been perfused intravenously prior to collection of LNs demonstrating
that it was not simply due to loosely bound lymphocytes in the HEV
lumen. However, the effect of Ro 31-9790 was transient and
completely reversible as shown by the normal numbers of lymphocytes
accumulating outside HEVs 120 minutes after injection. The
time-dependent effects of Ro 31-9790 on lymphocyte
extravasation correlated with the highest plasma levels at 60 minutes
and the lowest at 120 minutes (Table 2). The colocalization of CFSE
labeling and MECA 79 staining (Figure 3) did not allow the exact
position of lymphocytes in the HEV wall to be determined. LN sections
from mice that had been intravenously perfused with fixative to
maintain patency of HEVs were counterstained for laminin to determine
whether CFSE-labeled cells accumulated in the subendothelial basement
membrane. As reported above, the number of lymphocytes inside HEVs
increased (Figure 4A). There was a slight
increase in the number of lymphocytes either adjacent to or within the
HEV basement membrane of mice treated with Ro 31-9790;
however, it was not significant (Figure 4A), suggesting that the
increased number of lymphocytes inside HEVs was not simply due to
arrest at the basement membrane. Circulating Ro 31-9790 will
affect injected (CFSE-labeled) and host (unlabeled) lymphocytes
equally. To determine whether Ro 31-9790 affected lymphocyte
binding in the lumen of HEVs, we used ultrastructural analysis without
distinguishing between injected and host lymphocytes (Figure 4B). As
reported previously,42 about 90% of lymphocytes in HEVs
of control mice were within the endothelial lining, with very few bound
in the lumen (< 10%). Although the total number of lymphocytes
inside HEVs was visibly increased in Ro 31-9790-treated mice, the distribution of lymphocytes in the lumen and endothelial lining was not altered (Figure 4B). The ultrastructural appearance of
HEVs, basement membranes, and lymphocytes were unaltered in mice
treated with Ro 31-9790, and other types of leukocytes were not bound to HEVs. Ro 31-9790 did not inhibit lymphocyte
motility or chemotaxis per se because lymphocytes migrated normally
from the MZ to the white pulp of the spleen in Ro
31-9790-treated mice (Table 3).
These results suggest that Ro 31-9790 does not alter lymphocyte binding to the luminal surface of HEVs or the initiation of
migration across the endothelial lining but prevents lymphocytes from
successfully completing transendothelial migration or increases their
transit time, thus resulting in accumulation within the endothelial
lining.
To explore the potential role of metalloproteinases further, 2 MMP
inhibitors that do not block L-selectin shedding were tested. The
recovery of CFSE-labeled lymphocytes from LNs 60 minutes after intraperitoneal injection of a similar dose of Ro 32-1541 (100 mg/kg)
was similar to that in control mice (data not shown) and the number of
lymphocytes inside and outside HEVs was not altered (Table
4 and data not shown). Intraperitoneal
administration of 33 mg/kg recombinant human (h)TIMP1 yielded
circulating levels of hTIMP1 ranging from 310 to 400 µg/mL (11-14 µM); however, lymphocyte transendothelial migration across HEVs was
not altered in TIMP1-treated mice (Table 4) and CFSE-labeled
lymphocytes accumulated in normal numbers in the LN paracortex
(127 ± 7 cells/mm2 outside HEVs in control mice and
126 ± 8 cells/mm2 in TIMP1-treated mice).
Immunocytochemical staining showed that hTIMP1 was evenly distributed
throughout the HEV wall and the LN paracortex (data not shown).
Using the broad-spectrum MMP inhibitor Ro 31-9790 we have shown that L-selectin is down-regulated on lymphocytes migrating into LNs from the bloodstream via HEVs. In addition, although the number of lymphocytes that enter LNs is not altered, lymphocytes accumulate in the endothelial lining of HEVs in inhibitor-treated mice. Together these results suggest that metalloproteinases regulate transendothelial migration of lymphocytes across HEVs and L-selectin shedding during migration from the blood into LNs. The effects of Ro 31-9790 were dose dependent and the effective systemic doses and plasma levels achieved were similar to those reported by others to block L-selectin shedding from neutrophils18,39 and tumor metastasis26,27 in rodents. A structurally related hydroxamate-based MMP inhibitor, Ro 32-1541, did not block L-selectin shedding and had no effect on lymphocyte transmigration across HEVs suggesting that the effects of Ro 31-9790 were specific to this compound. Although Ro 31-9790 inhibits both MMPs and ADAMs, marimastat, which is structurally similar to Ro 31-9790, does not inhibit the MMP-related astacin/tolloid metalloproteinase family43 suggesting that Ro 31-9790 could exhibit selectivity within the metzincin superfamily of metalloproteinases.23 Ro 31-9790 did not simply function as a nonselective inhibitor of metalloproteinases due to its zinc chelating properties because it was ineffective against ACE, a zinc-dependent endopeptidase belonging to the thermolysin family, which is distinct from MMPs.23 To explore the potential role of MMPs further we tested a naturally occurring MMP inhibitor, TIMP1, which is structurally unrelated to the hydroxamate inhibitors. We had previously shown recombinant hTIMP1 does not inhibit phorbol ester-induced L-selectin shedding from mouse lymphocytes.16 Interestingly, TIMP1 had no affect on lymphocyte transendothelial migration across HEVs in mice. Human TIMP1 inhibits murine MMP944 and the invasion of amnion membranes by B16-F10 murine melanoma cells in vitro.45 In addition administration of hTIMP1 to mice reduces the colonization of lungs by B16-F10 melanoma cells46 indicating cross-species inhibition, although the full spectrum of murine MMPs that are inhibited by human TIMP1 is not known. The lack of effect of hTIMP1 is unlikely to be due to insufficient inhibitor because the circulating plasma level achieved in this study (~15 µM/350 µg/mL) was 50- to 150-fold higher than that shown to completely inhibit murine MMP9 and matrix degradation by mouse blastocysts.44 The dose of hTIMP1 used was similar to that used to block experimental metastasis in mice and melanoma cell invasion of basement membranes in vitro.45,46 The requirement for metalloproteinase activity during transendothelial migration across HEVs has not been shown before and metalloproteinase-dependent down-regulation of L-selectin expression during migration into LNs provides independent evidence for metalloproteinase activation, but further experiments will be required to identify the enzyme activities responsible. It is possible that L-selectin shedding and transmigration across HEVs are regulated by different metalloproteinases that are both inhibited by Ro 31-9790; identification of all murine metalloproteinases that are inhibitable by Ro 31-9790, but not by Ro 32-1541 or TIMP1, will be useful in this regard. MMPs have been implicated in T-lymphocyte transmigration across the blood-brain barrier of mice from studies using the encephalitogenic mouse CD4+ T-cell clone C19. MMP2 is induced in C19 cells following binding to VCAM-112 and overexpression of MMP2 by C19 cells facilitates their migration across the blood-brain barrier in vivo.47 Transmigration across VCAM-1-expressing microvascular endothelial cells grown on collagen-coated filters is inhibitable by TIMP212 and the synthetic hydroxamate MMP inhibitor GM6001 prevented C19 cells from penetrating the collagen-coated Transwells,48 supporting a role for MMPs in basement membrane penetration. However, the metalloproteinase-dependent transendothelial migration of lymphocytes across HEVs that we report here is distinguishable in that it is a rapid event (half-life 30 minutes),49 whereas VCAM-1-induced MMP expression is maximal after 5 hours.11,12 In addition, MMP2 and MMP9 do not cleave cell surface L-selectin31 and binding to VCAM-1 does not stimulate L-selectin shedding within the time frame of transmigration studied here (A.A. and G.P., unpublished obervations, 2000). Studies so far have shown clearly that L-selectin sheddase cleaves L-selectin in the same but not an adjacent membrane suggesting that the metalloproteinase responsible for L-selectin shedding is expressed by lymphocytes. However, we cannot rule out the possibility that the metalloproteinase responsible is expressed on the HEV membrane and cleaves L-selectin from the lymphocyte surface at sites of close membrane apposition (4 nM), which occur during transmigration.50 Although we cannot draw any conclusions about the role of L-selectin shedding in lymphocyte transmigration across HEVs from this study, the correlation between inhibition of shedding and transendothelial migration is interesting and warrants some discussion. Studies of human neutrophils rolling on L-selectin ligands (MECA 79 antigen) in vitro17 and of mouse neutrophils rolling in vivo in exteriorized postcapillary venules on unidentified L-selectin ligands18 have shown that the rolling velocity is reduced in the presence of the hydroxamate-based MMP inhibitor KD-IX-73-4. Although L-selectin expression on rolling neutrophils was not measured, these authors concluded that rolling was regulated by limited proteolysis of L-selectin induced by ligand engagement. An increase in the number of neutrophils that bound from flow was also noted, but the effect of KD-IX-73-4 on the diapedesis of neutrophils in vivo was not determined. Whether L-selectin shedding regulates lymphocyte rolling in HEVs and the relationship between rolling velocity and the subsequent extravasation of leukocytes remain to be determined. We found that systemic treatment with Ro 31-9790 did not increase the binding of lymphocytes or other leukocytes in the lumen of HEVs, where L-selectin mediates adhesion3 and shedding is thought to occur, nor did it increase the overall recruitment of lymphocytes by HEVs; however, it did inhibit L-selectin down-regulation on lymphocytes entering LNs. Because lymphocytes were able to initiate transendothelial migration, we conclude that the effect of Ro 31-9790 is downstream of events in the lumen of HEVs and, therefore, may be completely independent of L-selectin shedding. It is unlikely that the metalloproteinases responsible regulate lymphocyte chemotaxis because Ro 31-9790 did not affect chemokine-dependent migration of lymphocytes from the MZ into the splenic white pulp, which, like LN entry, is dependent on CCR7.5 However, the generation and maintenance of a gradient of chemokine across the endothelial layer lining HEVs could be dependent on metalloproteinases, as shown for dorsoventral patterning in the embryo, which is regulated by another MMP-related family of metalloproteinases, the tolloid/astacins.51 Metalloproteinase activation could be just one step in a cascade of
events required for lymphocytes to find and penetrate interendothelial
cell adherens junctions and the underlying basement membrane to
complete diapedesis and enter the LN parenchyma. Recent studies of
monocyte transmigration across human umbilical vein endothelial cells
have shown that staining for the
VE-cadherin/ An interesting observation was that none of the MMP inhibitors tested blocked lymphocyte transmigration across the basement membranes of HEVs in mice. Hydroxamate inhibitors and TIMP1 have been used repeatedly to block tumor cell and lymphocyte migration across basement membranes in vitro and these observations formed the basis for the in vivo study reported here. However, the role of MMPs in regulating the extravasation of tumor cells has been called into question recently.53 Studies of TIMP1-expressing B16 melanoma cells have shown reduced colonization of mouse lungs54 and chick embryos55 following intravenous injection in comparison with wild-type B16 cells. However, intravital microscopy of blood vessels in the chicken chorioallantoic membrane showed that the expression of TIMP1 have no effect on the extravasation of injected B16 melanoma cells but reduced the growth and number of tumors after extravasation.56 Systemic treatment of mice with batimastat, another hydroxamate-based MMP inhibitor, reduced B16 melanoma cell growth in the liver, but intravital microscopy showed that extravasation of tumor cells was normal in inhibitor-treated mice.57 These studies do not eliminate a role for MMPs in tumor cell extravasation because, as discussed above, TIMP1 does not inhibit all MMPs and the efficacy of these inhibitors against murine MMPs is not worked out. However, they do highlight the difficulties in reproducing the complex interactions that regulate diapedesis in in vitro assays. It would be interesting to test Ro 31-9790 in models of B16 melanoma cell extravasation because we have shown that, in contrast to TIMP1, it is an effective inhibitor of transendothelial migration. In conclusion, the use of MMP inhibitors with differing selectivites for MMP-related enzymes has identified a novel role for metalloproteinases in controlling lymphocyte transendothelial migration. Identification of the metalloproteinase, the development of selective and long-lived synthetic inhibitors, and the effective delivery of inhibitors to the blood vessel wall will all be required for further analysis of this step in extravasation. It will be interesting to determine whether transendothelial migration of leukocytes at inflammatory sites or of metastasizing tumor cells is also metalloproteinase dependent. Identification of the metalloproteinases that affect lymphocyte transendothelial migration is therefore a major goal for the future.
Thanks go to Liz Hirst for help with confocal microscopy, Stamatis Pagakis for image analysis, and Joe Brock for the figures. We gratefully acknowledge Bill Luscinskas, Dylan Edwards, Gillian Murphy, and Ian Frayling for helpful comments on the manuscript and John Nixon and Kevin Bottomley for advice about inhibitors.
Submitted July 17, 2000; accepted March 21, 2001.
Supported by the Medical Research Council (United Kingdom), European Union grant CT-1999-01036, and a Wellcome Trust Travelling Fellowship (to C.F.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Ann Ager, Division of Cellular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, United Kingdom; e-mail: ann.ager{at}nimr.mrc.ac.uk.
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Top
Abstract
Introduction
apparent Ki) are means pooled from
published data using peptide or protein substrates in cell-free
assays
) or without (
) PMA. (C) Plasma from mice
pretreated with Ro 31-9790 inhibits L-selectin shedding.
Maximal inhibition was seen 60 minutes after a single injection of more
than 3 mg/mouse but inhibitor levels had fallen by 120 minutes. Plasma
from mice treated with Ro 32-1541 or TIMP1 did not inhibit L-selectin
shedding. Results are mean number of L-selectin-positive lymphocytes
of duplicate observations following incubation with PMA and plasma from
mice pretreated with inhibitor for 60 minutes (
). *1, 2, 3 and
6 mg/mouse equivalent to 33, 66, 100, and 200 mg/kg, respectively.
) or
vehicle alone (
) and 40 × 106 CFSE-labeled
lymphocytes injected. The number of CFSE-labeled cells in LNs, spleen,
and blood was measured 30 minutes and 60 minutes after injection. Bars
are mean percent CFSE-labeled cells ± SEM and the number of mice
analyzed is given below each bar. (B) CFSE-labeled lymphocytes
harvested from LNs, spleen, and blood were analyzed for L-selectin
expression. The total number of L-selectin-positive lymphocytes in
each tissue was not affected by Ro 31-9790. However, the
level of L-selectin was higher on lymphocytes harvested from LNs, but
not the spleen or blood, of mice treated with Ro 31-9790. Bars are mean fluorescence intensities of L-selectin-positive
lymphocytes (± SEM, n = 3) in mice treated with Ro 31-9790 (
-catenin/plakoglobin complex is lost from
adherens junctions at sites of monocyte penetration of the endothelial
layer and rapidly regained after transmigration,
topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases.
Protein Sci.
1995;4:823-840