Tissue Inhibitor of Metalloproteinases-3 Inhibits Shedding of
L-selectin from Leukocytes*
Gillian
Borland,
Gillian
Murphy
, and
Ann
Ager§
From the Division of Cellular Immunology, National Institute for
Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, and the
School of Biological Sciences, University of East Anglia,
Norwich, Norfolk, NR4 7TJ, United Kingdom
 |
ABSTRACT |
Although the enzyme or enzymes mediating shedding
of L-selectin have not yet been identified, this activity can be
blocked by synthetic hydroxamic acid-based inhibitors of
metalloproteinases such as Ro 31-9790. However, the endogenous matrix
metalloproteinase inhibitor tissue inhibitor of metalloproteinases
(TIMP)-1 does not block L-selectin shedding. Here, we
report that TIMP-3, but not TIMP-2, inhibits L-selectin shedding from
mouse and human lymphocytes, Jurkat T cells, and human monocytes.
TIMP-3 has an IC50 of 0.3-0.4 µM on these
cell types compared with 0.7-4.8 µM for Ro 31-9790. A
metalloproteinase (tumor necrosis factor-
(TNF-
)-converting enzyme; ADAM17) has recently been identified which cleaves the pro-form
of TNF-
to produce soluble cytokine. We compared inhibition of
L-selectin shedding by TIMPs and Ro 31-9790 with inhibition of TNF-
shedding from human monocytes. TIMP-3 inhibited TNF-
shedding
(IC50 of 0.1 µM), as did Ro 31-9790 (IC50 of 0.4 µM). TIMP-2 had a partial
effect, and TIMP-1 did not inhibit. This study confirms that L-selectin
sheddase is a metalloproteinase, but not a matrix metalloproteinase,
and investigates the relationship between shedding of L-selectin and
TNF-
.
 |
INTRODUCTION |
L-selectin is one of three members of the selectin family and is
expressed on leukocytes (for review, see Ref. 1). Through binding to
mucin-like carbohydrate ligands on vascular endothelium, L-selectin mediates the tethering and rolling of
leukocytes, which precedes their migration into lymphoid organs and
sites of inflammation. L-selectin is rapidly down-regulated from the
leukocyte surface within minutes by a number of stimuli including
chemoattractants (2), phorbol ester (3, 4), and cross-linking of cell surface L-selectin (5), or more slowly by cross-linking of the T cell
CD3 complex (6). L-selectin is also down-regulated on leukocytes that
have migrated across cultured endothelial cells (7, 8) or into sites of
inflammation (9). The latter observation led to the suggestion that
L-selectin down-regulation from the leukocyte surface may be required
for transendothelial migration, although it is not necessary for
migration of neutrophils across human umbilical vein endothelial cells
(10).
Rapid down-regulation of L-selectin has recently been shown to be the
result of proteolytic cleavage in the membrane proximal extracellular
domain (11). The cleavage site has been mapped in human L-selectin to
Lys283-Ser284. The activity mediating this
proteolytic cleavage (L-selectin sheddase) is inhibited by the
hydroxamic acid-based inhibitors of zinc-dependent
metalloproteinases Ro 31-9790 (4), KD-IX-73-4 (12), and TAPI (13), but
not by inhibitors of other classes of proteases (4). L-selectin
sheddase is also not inhibited by tissue inhibitor of
metalloproteinases (TIMP)-11
(4), an endogenous inhibitor of matrix metalloproteinases (MMPs).
To characterize L-selectin sheddase further, we have tested the ability
of two additional members of the TIMP family, TIMP-2 and TIMP-3 (for
review, see Ref. 14), to inhibit L-selectin shedding from leukocytes.
These inhibitors are structurally homologous to TIMP-1, which we have
already shown does not inhibit shedding of L-selectin (4).
However, the expression patterns and inhibitory specificities of the
TIMPs differ (15, 16), suggesting that although all three proteins
inhibit the MMPs, they have some functional differences that may be
useful in the characterization of L-selectin sheddase.
Proteolytic cleavage is a commonly used mechanism for down-regulation
of cell surface proteins (17). Like L-selectin, the shedding of a
number of these proteins is inhibited by the hydroxamic acid-based
inhibitors at concentrations much greater than those required to
inhibit the MMPs (18, 19). Recently, a cell surface metalloproteinase
has been identified which mediates cleavage of one of these cell
surface molecules, the pro-form of tumor necrosis factor
(TNF)-
(20, 21). This enzyme belongs to the ADAM (a
disintegrin and metalloproteinase) family of
zinc-dependent metalloproteinases. To determine the
relationship between these enzymes and L-selectin sheddase, we compared
the sensitivities of TNF-
shedding and L-selectin shedding from
human peripheral blood monocytes to inhibition by TIMPs and synthetic
hydroxamic acid-based inhibitors.
 |
MATERIALS AND METHODS |
Reagents and Antibodies--
Phorbol myristate acetate (PMA;
Sigma) was dissolved in dimethyl sulfoxide to a concentration of 1 mM and stored at
20 °C in aliquots. Ro 31-9790 (Roche
Products, Herts.) was dissolved in dimethyl sulfoxide to a
concentration of 30 mM and stored at
20 °C in
aliquots. Lipopolysaccharide (LPS; Sigma) was dissolved in RPMI 1640 to
a concentration of 1 mg/ml and stored at
20 °C in aliquots.
Recombinant TIMPs were produced as described in (22-24). Antibodies
used are described in Table I.
Cells--
Mouse lymphocytes were isolated from pooled axillary
and cervical and mesenteric lymph nodes of 6-8-week-old BALB/c mice
bred in the SPF unit at the NIMR. Tissues were collected into calcium- and magnesium-free phosphate-buffered saline (PBS-CMF) at 4 °C and
cell suspensions prepared. Human lymphocytes and monocytes were
prepared from buffy coat preparations of peripheral blood obtained from
the North London Blood Transfusion Service (Colindale, London).
Peripheral blood mononuclear cells (PBMC) were isolated by flotation
over Ficoll-Paque (Amersham Pharmacia Biotech). E6.1 Jurkat T cells
were grown in RPMI 1640 containing 10% fetal calf serum (FCS), 2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin at 37 °C in 5% CO2. For experiments, mouse
lymphocytes and Jurkat cells were resuspended at 5 × 107/ml in RPMI 1640 supplemented with 1% FCS and 10 mM Hepes. Human PBMC were resuspended at 2 × 106/ml in RPMI 1640 supplemented with 10% FCS.
L-selectin Shedding Assay--
Mouse lymphocytes or Jurkat T
cells (2.5 × 106 in 50 µl) were incubated with 100 nM PMA or an equivalent volume of dimethyl sulfoxide for 60 min. Cells were pretreated with inhibitor for 20 min and throughout the
assay. Inhibitors and PMA were removed by washing in PBS-CMF containing
0.2% bovine serum albumin, and cell surface L-selectin expression was
determined by flow cytometry. This assay was also carried out using
PBMC (106 in 0.5 ml) within 90 min of isolation from buffy coat.
TNF-
Shedding Assay--
PBMC (106 in 0.5 ml)
were incubated in the presence or absence of inhibitor for 20 min
followed by the addition of 1 µM LPS for 3 h.
Inhibitors and LPS were removed by washing in PBS-CMF containing 5%
FCS, and cell surface TNF-
expression was determined by flow
cytometry. The washing and staining procedures for flow cytometric
analysis were carried out on ice, using chilled buffer to prevent
proteolytic cleavage of cell surface TNF-
after washing out of inhibitor.
Flow Cytometry--
L-selectin was measured on mouse lymphocytes
and Jurkat T cells by direct immunofluorescence using fluorescein
isothiocyanate-conjugated MEL-14 and fluorescein
isothiocyanate-conjugated FMC46, respectively. L-selectin and cell
surface TNF-
on human PBMC were measured using two-color indirect
immunofluorescence. LAM1.3 was used to detect L-selectin and 101/4 to
detect TNF-
, followed by phycoerythrin-conjugated anti-mouse IgG1;
fluorescein isothiocyanate-labeled anti-human CD14 was used to identify
monocytes in PBMC. Cells were incubated with appropriate antibodies at
each stage of staining for 20 min at 4 °C; mouse lymphocytes and
Jurkat T cells were washed in PBS-CMF with 0.2% bovine serum albumin
(Sigma), and PBMC were washed in PBS-CMF with 5% FCS. Stained cells
were analyzed on a Becton Dickinson FACStar Plus or FACS Vantage. Data
for 10,000 events were collected for mouse lymphocytes or Jurkat T
cells and 30,000-50,000 events (depending on the proportion of
monocytes in the samples) for human mononuclear cells. Data were
analyzed using FACSplot software developed by John Green, Computing
Lab, NIMR, or using WinMDI (Joseph Trotter, Scripps Institute, CA).
Measurement of Soluble L-selectin by ELISA--
These assays
were carried out as described in (4). 96-well ELISA plates (Nunc,
Maxisorb) were coated with MEL-14 (10 µg/ml) overnight at 4 °C,
blocked for 60 min with Dulbecco's PBS with 1% bovine serum albumin,
and washed three times with Dulbecco's PBS with 0.05% Tween 20. Doubling dilutions (from 1/4) of supernatants from mouse lymphocytes
(treated with PMA to induce L-selectin shedding in the presence or
absence of inhibitor) were added to the plate and incubated for 90 min
at room temperature. Captured L-selectin was detected using
biotinylated T28.45 (5 µg/ml) and streptavidin-horseradish peroxidase
conjugate (DAKO). The reaction was stopped with 2 M
H2SO4, and absorbance was read at 450 nm. A
standard curve was constructed using a mouse L-selectin-Ig fusion protein (LEC-Ig, generously provided by Dr. Susan Watson, Genentech) over the range of 0.75-100 ng/ml; absorbance values for an irrelevant fusion protein (mouse CTLA4-Ig; generously provided by Dr. Peter Lane,
Basel Institute for Immunology, Basel) were subtracted from the
standards and test samples.
Calculation of Percent Inhibition--
For mouse and human
lymphocytes and Jurkat T cells, 100% inhibition was set as the
percentage of cells positive for L-selectin in an untreated sample
(after flow cytometric analysis) or the concentration of soluble
L-selectin in the supernatant of an untreated cell sample (after
ELISA); 0% inhibition was set at the appropriate value for a
PMA-treated sample. For results from human monocytes, a different
calculation was needed to allow direct comparison of L-selectin and
TNF-
shedding. Therefore, in this case 100% inhibition was set as
the percentage of cells positive for L-selectin in the presence of PMA + 50 µM Ro 31-9790 or the percentage of cells positive
for cell surface TNF-
in the presence of LPS + 50 µM
Ro 31-9790 (doses of Ro 31-9790 which gave maximal inhibition); 0%
inhibition was set as the percentage of cells positive for L-selectin
or TNF-
in the presence of PMA or LPS, respectively. Intermediate
percent inhibition was calculated as follows:
|
(Eq. 1)
|
where x = value for sample under test,
y = value for sample set as 0%,
z = value for sample set at 100%.
Results are presented as percent inhibition ± S.E. for at least
three replicates for each point.
Calculation of IC50 Values--
The IC50
value for inhibitor in each system was defined as the concentration of
inhibitor which gave 50% inhibition, where 100% inhibition was set as
the percentage of cells positive for L-selectin or cell surface TNF-
in the presence of PMA + 50 µM Ro 31-9790 or LPS + 50 µM Ro 31-9790, respectively (doses of Ro 31-9790 which
gave maximal inhibition) and 0% inhibition was set as the percentage
of cells positive for L-selectin or TNF-
in the presence of PMA or
LPS alone, respectively.
 |
RESULTS |
Inhibition of Mouse Lymphocyte L-selectin Shedding by
TIMP-3--
We have shown previously that PMA-induced shedding of
L-selectin from mouse lymphocytes is inhibited by the synthetic
metalloproteinase inhibitor Ro 31-9790 but not by the endogenous MMP
inhibitor TIMP-1 (4). These studies have been extended by including
TIMP-2 and TIMP-3 in the same in vitro L-selectin shedding
assay. Lymphocytes isolated from mouse lymph nodes were treated with
PMA in the presence or absence of varying concentrations of each of the
inhibitors, and L-selectin remaining on the cell surface was detected
by flow cytometry. As reported previously, TIMP-1 had no effect on
PMA-induced shedding of cell surface L-selectin at concentrations up to
1.1 µM (30 µg/ml) (Fig.
1, A and B),
whereas the hydroxamic acid-based inhibitor Ro 31-9790 completely
inhibited PMA-induced shedding at a concentration of 10 µM (Fig. 1B). TIMP-2 was also unable to
inhibit L-selectin shedding (Fig. 1, A and B),
but TIMP-3 inhibited PMA-induced L-selectin shedding in a
dose-dependent manner with complete inhibition at 0.8 µM (20 µg/ml; Fig. 1B). To confirm that PMA
was inducing shedding of L-selectin, rather than internalization, soluble L-selectin was quantitated by ELISA in supernatants from untreated, PMA-treated, and PMA + inhibitor-treated mouse lymphocytes. Although there was a background level of soluble L-selectin present in
cell supernatants in the absence of PMA, an increase of approximately 100% was seen in response to phorbol ester (Fig. 1C); this
PMA-induced increase was inhibited completely by TIMP-3 and Ro 31-9790 but not by TIMP-1 or TIMP-2. This background level of L-selectin
shedding, detectable as soluble L-selectin in the supernatants of
untreated mouse lymphocytes, explains the >100% inhibition of
PMA-induced shedding by TIMP-3 and Ro 31-9790 (Fig. 1B);
these inhibitors prevent both the PMA-induced and the background
shedding, allowing cell surface L-selectin to increase to a level
greater than that seen on untreated lymphocytes. However, even in the
presence of TIMP-3 or Ro 31-9790 some residual soluble L-selectin can
be detected in cell supernatants by ELISA.

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Fig. 1.
TIMP-3 and Ro 31-9790 inhibit L-selectin
shedding from mouse lymphocytes. Panel A, flow
cytometry profiles showing inhibition of L-selectin down-regulation
from cells treated with 100 nM PMA in the presence and
absence of inhibitor. a, untreated lymphocytes stained with
control monoclonal antibody (open histogram) or MEL-14
(shaded histogram); b-f, lymphocytes stained
with MEL-14 after treatment with b, PMA alone; c,
PMA + 1.1 µM TIMP-1; d, PMA + 1.44 µM TIMP-2; e, PMA + 0.83 µM
TIMP-3. Vertical line indicates marker used to determine
percent positive; number in each box indicates
percent cells positive for cell surface L-selectin. Profiles are
representative of several independent experiments. Panel
B, cells were treated as in panel A, and cell surface
L-selectin was detected by flow cytometry. Results are presented as the
percent inhibition of shedding ± S.E. Panel C,
inhibition by TIMP-3 and Ro 31-9790 of soluble L-selectin appearance in
the supernatants of mouse lymphocytes treated as in panel A,
detected by ELISA. Background shedding of L-selectin (at approximately
350 ng/108 cells under these conditions) in the absence of
PMA can be seen. Results for inhibitor-treated samples are from a
representative experiment; the points showing the levels of soluble
L-selectin for untreated and PMA-treated are the mean ± S.E. for
at least three experiments and indicate the variable levels of soluble
L-selectin obtained from different samples. In panels B and
C, closed circles represent Ro 31-9790-treated
samples, open circles represent TIMP-1-treated samples,
closed squares represent TIMP-2-treated samples, and
open squares represent TIMP-3-treated samples. In
panel C alone, the closed triangle represents
untreated samples, and the open triangle represents
PMA-treated samples; these values are the mean ± S.E. for > six independent experiments.
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TIMP-3 Inhibits L-selectin Shedding from Human
Lymphocytes--
The TIMPs used in the experiments described above
were recombinant human TIMPs. It was possible that the absence of
inhibition of L-selectin shedding by TIMP-1 and TIMP-2 was caused by
the inability of the human TIMPs to function in a mouse cell system. Therefore, the ability of the TIMPs to inhibit shedding of L-selectin from human cells was investigated. Jurkat T cells were treated with PMA
in the presence and absence of inhibitors, and cell surface L-selectin
was detected using direct immunofluorescence. As found for
mouse lymphocytes, TIMP-3 and Ro 31-9790 inhibited PMA-induced shedding of L-selectin, whereas TIMP-1 and TIMP-2 were without effect
(Fig. 2A). Similarly, TIMP-3
and Ro 31-9790 (but not TIMP-1 or TIMP-2) inhibited L-selectin shedding
from human peripheral blood lymphocytes (Fig. 2B).
Therefore, similar results were obtained with each of the TIMPs using
both mouse and human cells.

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Fig. 2.
TIMP-3 and Ro 31-9790 inhibit PMA-induced
shedding of L-selectin from human lymphocytes. Jurkat T cells
(panel A) or human peripheral blood lymphocytes (panel
B) were treated with 100 nM PMA in the presence and
absence of inhibitors, and cell surface L-selectin was detected by flow
cytometry. In panel B, results are shown only for single
concentrations of TIMP-1 and TIMP-2 (1.1 µM and 1.44 µM respectively; 30 µg/ml); these were the maximal
concentrations used in other experiments and had no effect on
PMA-induced L-selectin shedding from any cell type tested. In both
panels A and B, closed circles
represent Ro 31-9790-treated samples, open circles represent
TIMP-1-treated samples, closed squares represent
TIMP-2-treated samples, and open squares represent
TIMP-3-treated samples.
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|
TIMP-3 Inhibits Both L-selectin Shedding and TNF-
Shedding on
Human Monocytes--
The cytokine TNF-
is a well characterized
example of a cell surface molecule that is shed by proteolytic
cleavage. This molecule is produced as a 26-kDa cell surface protein
and is processed by zinc-dependent metalloproteinases to
generate the 17-kDa soluble cytokine. We compared the abilities of
TIMP-1, TIMP-2, TIMP-3, and Ro 31-9790 to inhibit shedding of
L-selectin and TNF-
from human monocytes in order to investigate the
relationship between these enzyme activities on the same cell. Human
peripheral blood monocytes express L-selectin, therefore PMA was used
to induce shedding, and cell surface L-selectin was measured as
described for lymphocytes. Cell surface TNF-
is not normally
detectable on monocytes. To measure the effect of metalloproteinase
inhibitors on TNF-
shedding, monocytes were stimulated with LPS, to
up-regulate TNF-
synthesis, and the level of TNF-
which
accumulated at the cell surface, and which was released into the cell
supernatant, in the presence and absence of inhibitors was determined.
As found for lymphocytes, TIMP-3 and Ro 31-9790 (0.83 and 30 µM, respectively) blocked PMA-induced L-selectin shedding
from human monocytes (Fig. 3 and Table
II). TIMP-3 and Ro 31-9790 also mediated
accumulation of TNF-
on the surface of human monocytes (Fig. 3 and
Table II), indicating a block in the TNF-
shedding pathway. This
accumulation of TNF-
on the cell surface was mirrored by decreased
soluble TNF-
in supernatants of cells treated with LPS and TIMP-3
(data not shown). In contrast, TIMP-1 and TIMP-2 had no effect on
shedding of L-selectin from monocytes (Table II). TIMP-1 was also
without effect on cell surface TNF-
on monocytes, but inclusion of
TIMP-2 at 1.44 µM resulted in an increase in the
percentage of cells positive for TNF-
, from 39.3% to 55.7% (31%
inhibition), although this was less than that obtained with TIMP-3
(Table II).

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Fig. 3.
TIMP-3 and Ro 31-9790 inhibit shedding of
L-selectin and TNF- from human monocytes.
Human peripheral blood monocytes were either treated with 100 nM PMA in the presence and absence of inhibitor and cell
surface L-selectin was detected by flow cytometry or were treated with
1 µg/ml LPS in the presence and absence of inhibitor, and cell
surface TNF- was detected. The effects of TIMP-3 (panel
A) and Ro 31-9790 (panel B) are shown on the shedding
of both L-selectin and TNF- . In both panels A and
B, closed circles represent the percent cells
positive for cell surface L-selectin, and open circles
represent the percent cells positive for cell surface TNF- .
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Table II
Inhibition of L-selectin and TNF- shedding from human monocytes by
TIMP-3 and Ro 31-9790
Cells were either treated with 100 nM PMA in the presence
and absence of inhibitor and cell surface L-selectin was detected, or
cells were treated with 1 µg/ml LPS in the presence and absence of
inhibitor and cell surface TNF- was detected by flow cytometry.
Results shown are the percentage of cells positive for L-selectin or
TNF- under each treatment and are representative of at least three
experiments.
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IC50 Values--
The IC50 values obtained
for TIMP-3 and Ro 31-9790 on each of the cell types tested
are shown in Table III. TIMP-3 gave very similar IC50 values for inhibition of L-selectin shedding
on all cell types tested (0.30-0.40 µM). However, this
inhibitor prevented TNF-
shedding with a lower IC50 of
0.1 µM, indicating possible differences between TNF-
and L-selectin shedding activities. IC50 values obtained
for Ro 31-9790 on L-selectin shedding showed more variation between
cell types, but, like TIMP-3 this inhibitor was more potent in
inhibiting TNF-
shedding on monocytes than inhibiting L-selectin
shedding on any cell type tested. IC50 values obtained for
both TIMP-3 and Ro 31-9790 by measurement of soluble L-selectin
released from mouse lymphocytes were similar to the values obtained by
analysis of flow cytometry data (data not shown). The IC50
for inhibition by TIMP-3 of soluble TNF-
release from LPS-stimulated
human monocytes (0.17 ± 0.02 µM) was also similar to that obtained by measurement of cell surface TNF-
under identical conditions.
 |
DISCUSSION |
The enzyme or enzymes mediating L-selectin shedding from
leukocytes have yet to be identified. However, using a new category of
MMP inhibitors based on hydroxamic acid, L-selectin shedding has been
shown to be blocked in a variety of cell types in response to both
physiological and nonphysiological stimuli (4, 10, 12, 13), suggesting
that L-selectin shedding in these different circumstances is mediated
by similar metalloproteinase dependent mechanisms. In this
report, we extend our previous work and demonstrate that the endogenous
metalloproteinase inhibitor TIMP-3 inhibits L-selectin shedding from
mouse and human lymphocytes and human monocytes. We have shown
previously that TIMP-1 did not inhibit L-selectin shedding (4), and we
demonstrate here that the closely related inhibitor TIMP-2 is also ineffective.
Some MMPs, and collagenase in particular, mediate limited L-selectin
proteolysis (4), suggesting that the L-selectin sheddase activity may
be an MMP or an MMP-related enzyme. However, the work reported here and
a previous study (4) demonstrate that phorbol ester-induced L-selectin
shedding is not inhibited by two members of the TIMP family, TIMP-1 and
TIMP-2. Between them, these two proteins inhibit the catalytic
activity of all known MMPs, and the inability of TIMP-1 and
TIMP-2 to prevent shedding of L-selectin therefore suggests that
L-selectin sheddase is not a known MMP. This is supported by the
evidence that L-selectin sheddase is a cell-associated, rather than a
soluble, activity (4, 25), as the MMPs are secreted enzymes. Recently,
a subgroup of transmembrane MMPs (membrane-type MMPs or MT-MMPs) has
been identified (26). Like L-selectin sheddase, MT1-MMP is not
inhibited by TIMP-1 (27). However, this cell surface enzyme can be
excluded as a potential candidate for the L-selectin sheddase because
it is inhibited by both TIMP-2 and TIMP-3 (27), and the data presented here show that L-selectin shedding is not inhibited by TIMP-2.
The hydroxamic acid-based inhibitors were originally designed as
inhibitors of MMPs, and Ro 31-9790 inhibits recombinant human fibroblast collagenase (MMP1) with an IC50 of 4.9 nM. As described here and elsewhere, hydroxamic acid-based
inhibitors prevent phorbol ester-induced shedding of cell surface
L-selectin with IC50 values in the low micromolar range (4,
12, 13) i.e. they are about 1,000-fold less potent in
inhibiting L-selectin shedding in cell-based assays than in inhibiting
recombinant MMPs in cell-free assays. When considered together with the
inability of TIMP-1 and TIMP-2 to inhibit L-selectin shedding from all
of the cell types tested, it is unlikely that the L-selectin-shedding
enzyme(s) is an MMP. Proteolytic cleavage of a number of other cell
surface molecules, such as angiotensin-converting enzyme (ACE) (28),
amyloid precursor protein (29), transforming growth factor-
(18),
and the pro-form of TNF-
((20, 21) and this study), is also
inhibited by synthetic hydroxamate inhibitors at micromolar
concentrations, suggesting that the activities mediating shedding of
these molecules and L-selectin shedding may be related.
An enzyme has recently been cloned, called TNF-
converting enzyme
(TACE; ADAM17) which correctly cleaves both a pro-TNF-
cleavage site
peptide and full-length pro-TNF-
(20, 21). In addition, leukocytes
from TACE-deficient mice have a much reduced ability to secrete soluble
TNF-
, suggesting an important role for TACE in mediating TNF-
shedding in vivo. TACE is a member of a recently
characterized family of zinc-dependent metalloproteinases known as the ADAM family (for review, see Ref. 30). An association between TACE and L-selectin shedding has recently been identified: thymocytes from TACE-deficient mice do not shed L-selectin in response
to PMA stimulation.2 In
addition, high concentrations of recombinant catalytic domain of TACE
have been shown to cleave a peptide representing the cleavage site of
L-selectin.2 This suggests that TACE may be a physiological
L-selectin sheddase. A second member of the ADAM family (ADAM10) also
cleaves a TNF-
cleavage site peptide but has yet to be shown to
cleave full-length pro-TNF-
correctly (31, 32). We therefore
investigated the possible relationship between enzymes mediating
shedding of L-selectin and TNF-
by comparing the inhibitory profiles
of the TIMPs and Ro 31-9790 on L-selectin and TNF-
shedding from human monocytes. We show here that TIMP-3 mediates
accumulation of TNF-
on the surface of human monocytes in
vitro. TIMP-1 has no effect, and TIMP-2 is less effective than
TIMP-3. Solomon et al. (33) have shown that although the
level of cell surface TNF-
on human peripheral blood monocytes may
not quantitatively reflect alterations in TNF-
shedding (because of
degradation of unprocessed pro-TNF-
), this measurement can be used
as a marker of inhibition of shedding at the time point utilized in our
TNF-
-shedding assays (3 h). Measurement of soluble TNF-
in
supernatants from monocytes stimulated with LPS in the presence or
absence of TIMP-3 gave an IC50 very similar to that
obtained from cell surface expression. This indicates that both types
of measurement can be used in our assay. Therefore differences can be
found in the inhibitory profiles of the TIMPs and Ro 31-9790 on
shedding of L-selectin and TNF-
from human monocytes and of Ro
31-9790 on shedding of L-selectin from different cell types, suggesting
the potential involvement of more than one enzyme in these proteolytic
events. However, it is also possible that the differences obtained
result from differential accessibility of enzymes(s) to the inhibitors,
particularly since shedding of the two proteins could not be
investigated under identical experimental conditions.
Interestingly, TIMP-3 has recently been reported to inhibit
processing of a peptide encoding the cleavage site of pro-TNF-
by
recombinant catalytic domain of TACE (34). As in our cell-based assays,
TIMP-1 had no effect on peptide proteolysis by TACE, and TIMP-2 was
less effective than TIMP-3. Shedding of cell surface interleukin 6 receptor, which is sensitive to the hydroxamic acid-based inhibitors,
has also recently been found to be blocked by TIMP-3 (35). The effect
of TIMPs on the activity of other non-MMP zinc-dependent metalloproteinases is not known. Although the TIMPs are structurally homologous and can all inhibit the MMPs (except MT1-MMP) equally well,
they exhibit different expression patterns, with TIMP-3 alone being
restricted to the extracellular matrix (16). The ability of TIMP-3 to
inhibit L-selectin shedding and TNF-
processing suggests either that
TIMP-3 is functionally less specific than TIMP-1 and TIMP-2 or that it
can more easily gain access to the L-selectin and TNF-
shedding
enzymes and so inhibit their activity. TIMP-3 is the only TIMP that
binds heparan sulfate
proteoglycans,3 which are
expressed at the cell surface, and binding to these glycosaminoglycans
may localize TIMP-3 and enable interaction with cell surface
metalloproteinases. There may be additional motifs within TIMP-3 which
contribute to the ability of this protein, but not TIMP-1 or
TIMP-2, to inhibit L-selectin shedding.
Recent reports have shown that overexpression of TIMP-3 in some cell
types results in increased apoptosis of the cells (36, 37). These
studies used non-hemopoietic cells and time courses of several days,
with little apoptosis occurring before 12-24 h of cell culture,
whereas the assays reported here used much shorter incubations with
TIMP-3 (1-3 h). However, to exclude the possibility that leukocytes
are more sensitive to TIMP-3-induced apoptosis, we used an annexin V
binding apoptosis assay to compare levels of apoptosis in
TIMP-3-treated and control cell samples. No differences were found
using either Jurkat T cells or human peripheral blood
monocytes.4 Thus, TIMP-3 does
not appear to induce apoptosis of these cells within the times used in
our assays.
Shedding of many different cell surface proteins, in addition to
L-selectin and TNF-
, is inhibited by hydroxamic acid-based inhibitors (18, 19). The close relationship between the shedding mechanisms involved in proteolytic cleavage of these molecules has been
revealed through the use of a mutant Chinese hamster ovary cell line,
defective in the shedding of transfected L-selectin, transforming
growth factor-
, interleukin-6 receptor,
-amyloid precursor
protein, and a number of unidentified endogenous proteins present in
the supernatants of parental, but not mutant cells (18). There is
evidence that this cell line differs from the parental line by
alteration in only a single gene, suggesting that a common mechanism
underlies shedding of many cell surface proteins, possibly a
"pan-sheddase." In support of this theory, TACE-deficient cells are
also defective in the shedding of a number of other cell surface
molecules, including L-selectin.2 Further studies
identifying the sheddases for other metalloproteinase-cleaved cell
surface molecules, including L-selectin sheddase, should clarify the
relationship between the sheddases. The results presented here address
this relationship and show differences in the susceptibility of
L-selectin and TNF-
shedding to inhibition by the TIMPs and also in
the IC50 values obtained with TIMP-3 and Ro 31-9790. This raises the possibility that shedding of L-selectin and TNF-
from human monocytes may be mediated by different enzymes, although other
explanations exist.
 |
ACKNOWLEDGEMENTS |
We thank Chris Atkins for help with flow
cytometry and Frank Johnson for preparing the figures. We also thank
Dr. Andrew Nesbitt and Dr. Andrew Docherty of Celltech for gifts of
antibody and useful discussions.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.:
44-181-959-3666 (ext. 2465); Fax: 44-181-913-8529; E-mail:
a-ager{at}nimr.mrc.ac.uk.
The abbreviations used are:
TIMP, tissue
inhibitor of metalloproteinases; MMP, matrix metalloproteinase; TNF-
, tumor necrosis factor-
; ADAM, a
disintegrin and metalloproteinase; PMA, phorbol myristate acetate; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; CMF, calcium- and magnesium-free; PBMC, peripheral blood mononuclear cells; FCS, fetal calf serum; FACS, fluorescence-activated cell sorter; ELISA, enzyme-linked immunosorbent
assay; MT-MMP, membrane-type matrix metalloproteinase; ACE, angiotensin-converting enzyme; TACE, TNF-
-converting enzyme.
2
Peschon, J. J., Slack, J. L., Reddy, P.,
Stocking, K. L., Sunnarborg, S. W., Lee, D. C., Russell, W. E.,
Castner, B. J., Johnson, R. S., Fitzner, J. N., Boyce, R. W., Nelson,
N., Kozlosky, C., Wolfson, M. F., Rauch, C. T., Cerretti, D. P.,
Paxton, R. J., March, C. J., and Black, R. A. (1998) Science
282, 1281-1284.
3
G. S. Butler and G. Murphy, unpublished observations.
4
G. Borland, G. Murphy, and A. Ager, unpublished observations.
 |
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