(Received for publication, November 14, 1995; and in revised form, February 6, 1996)
From the
Leucocyte (L)-selectin can be proteolytically cleaved in the membrane proximal extracellular region to yield a soluble fragment that contains the functional lectin and epidermal growth factor domains. A variety of stimuli are known to stimulate L-selectin shedding including chemoattractants, phorbol esters, and L-selectin cross-linking; however, the enzymes that regulate L-selectin expression are not characterized. In this study we have used phorbol ester to stimulate endoproteolytic release of L-selectin and identified a major role for a cell surface metalloproteinase (L-selectin sheddase) in this process.
The hydroxamic acid-based inhibitor of zinc-dependent matrix metalloproteinases Ro 31-9790 completely prevented shedding of cell surface L-selectin from leucocytes in mouse, rat, and man. L-selectin was susceptible to cleavage by known matrix metalloproteinases. Recombinant human fibroblast collagenase (MMP1) reduced the number of L-selectin-positive lymphocytes to a similar extent as phorbol ester activation, and stromelysin (MMP3) had a partial effect on L-selectin expression. Gelatinases A (MMP2) and B (MMP9) were without effect. Lymphocytes did not express fibroblast collagenase or stromelysin at the cell surface, and tissue inhibitor of metalloproteinases (TIMP) did not affect L-selectin levels. L-selectin sheddase was not detected in media harvested from phorbol ester-stimulated lymphocytes and was only able to cleave L-selectin in the cis but not the trans configuration.
These results suggest that endoproteolytic release of L-selectin from the leucocyte surface is mediated by a metalloproteinase (L-selectin sheddase), which is distinguishable from known matrix metalloproteinases. Understanding the regulation of L-selectin sheddase will be critical for controlling leucocyte migration from the blood.
Leucocyte (L)-selectin is a member of the selectin family of adhesion molecules, which are highly restricted in their distribution to leucocytes (L- and P-selectin) or vascular endothelial cells (E- and P-selectin)(1) . A specialized role for the selectins in mediating the binding of leucocytes from flowing blood has been demonstrated for all three selectins(2, 3, 4, 5, 6, 7) . L-selectin was first identified in 1983 as a peripheral lymph node homing receptor, which mediated the binding of lymphocytes to high endothelial venules and their subsequent migration into peripheral lymph nodes of mice(8) . L-selectin also mediates the binding of neutrophils to acutely inflamed postcapillary venules in the mesentery (7, 9) and subsequent migration into the peritoneal cavity (10) . The critical role of L-selectin in regulating the migration of leucocytes has been confirmed recently in studies of L-selectin ``knockout mice''(11) .
Of
the selectins, L-selectin shows the unique property of being
proteolytically cleaved in the membrane-proximal extracellular region
to yield a soluble fragment that contains the known functional lectin
and EGF ()domains. This provides a rapid mechanism for
regulating L-selectin levels on leucocytes and, therefore, interfering
with their ability to migrate into tissues. The stimuli that induce
endoproteolytic release of L-selectin leading to decreased cell surface
levels are varied. Rapid loss of L-selectin from the leucocyte surface
was first demonstrated following stimulation of neutrophils by
chemotactic factors(12) . Jung and Dailey (13) showed
that loss of L-selectin from the surface of lymphocytes within 30 min
of phorbol ester activation was due to proteolytic cleavage in the
extracellular domain of the molecule. This observation has been
elegantly confirmed in recent studies in which a cleavage site in human
L-selectin has been mapped to the extracellular membrane-proximal
15-amino acid region(14) . Proteolytic cleavage of L-selectin
from leucocytes is also initiated by reagents that cross-link
L-selectin (15) . L-selectin on T-lymphocytes is down-regulated
by cross-linking CD3 or following mitogenic stimulation; however, the
time couse is slower than that initiated by phorbol
esters(16) . The physiological role of rapid L-selectin loss is
not yet fully understood. It has been proposed that loss of L-selectin
allows the leucocyte to de-adhere from the luminal surface of the
vessel wall and start migrating between endothelial cells and across
the vessel wall into the underlying tissues(12) . The lack of
L-selectin on antigen-activated lymphocytes is thought to underlie the
altered migration of these lymphocytes away from lymph nodes and into
non-lymphoid tissues(17) .
To gain further insight into the role of L-selectin shedding from the surface of leucocytes in regulating their migration pathways we have used phorbol esters to induce proteolytic cleavage of L-selectin. L-selectin is susceptible to chymotrypsin cleavage(18) , and we have compared the mechanism of phorbol ester-stimulated endoproteolytic release of L-selectin with that of chymotrypsin. We report here that a metalloproteinase (L-selectin sheddase) regulates cell surface levels of L-selectin on leucocytes. We also demonstrate that fibroblast collagenase and, to a lesser extent, stromelysin can cleave L-selectin from the leucocyte surface. However, the endogenous enzyme is distinguishable from fibroblast collagenase and other known matrix metalloproteinases. The regulation of this enzyme at the cell surface may be critical in the control of leucocyte migration from the blood.
Figure 1: Chemical structure of Ro 31-9790, a hydroxamic acid-based inhibitor of zinc-dependent metalloproteinases.
Maximal loss of L-selectin from the surface of mouse
lymphocytes was induced with 50 nM PDBu. Loss was detectable
within 5 min and was maximal after 30 min (data not shown), the number
of L-selectin positive cells being reduced to between 10 and 40% after
60 min at 37 °C ( Table 1and Fig. 2). The staining
profiles were similar using mAbs MEL-14 or T28.45, which recognize
epitopes in the lectin and EGF domains, respectively(25) . The
effect of PDBu was completely inhibited by inclusion of staurosporine
or the specific protein kinase C inhibitor, Ro 31-8220 (24) , in the assay ( Table 1and data not shown). PDBu
did not affect the expression of other cell surface adhesion molecules
such as 4 integrins, CD44, or LFA-1 (Table 1) or of CD45,
MHC antigens, CD4, and CD3 (data not shown). Enzymatic removal of
L-selectin was maximal after 30 min using 40 µg/ml chymotrypsin and
was more effective than PDBu (Fig. 2); similar staining profiles
were obtained using mAbs MEL-14 and T28-45 demonstrating loss of
both the lectin and the EGF domains. PDBu and chymotrypsin stimulated
loss of human L-selectin from the mouse 300.19 pre-B cell
transfectants, and, as found using lymphocytes, the effect of
chymotrypsin was greater than that of PDBu (data not shown). The
residual L-selectin staining following PDBu treatment represented cells
expressing low levels of L-selectin (Fig. 2).
Figure 2:
Phorbol dibutyrate-stimulated proteolysis
of L-selectin is blocked by a zinc-dependent metalloproteinase
inhibitor, Ro 31-9790. Lymphocytes were incubated with 50 nM PDBu for 60 min at 37 °C (b and d) or 40
µg/ml chymotrypsin for 20 min at 37 °C (c and e) in the absence (b and c) or presence of
30 µM Ro 31-9790 (d and e), and
the expression of L-selectin compared with lymphocytes incubated in
control buffer (a). The percentage of cells positive for
L-selectin is given. The y axis gives cell number(0-400)
and the x axis gives fluorescence intensity on a log scale
(10-10
channels).
Figure 3:
Inhibition of L-selectin shedding by the
hydroxamic acid-based metalloproteinase inhibitor Ro 31-9790.
Lymphocytes were pretreated with Ro 31-9790 for 20 min prior to
addition of phorbol dibutyrate and incubated for a further 60 min. The
number of L-selectin-positive lymphocytes was determined by flow
cytometric analysis. Dashed lines represent expression levels
in the absence (-PDBu) and presence (+PDBu) of phorbol
ester. The IC is the amount of Ro 31-9790 that
inhibits shedding by 50%.
Chymotrypsin has been used to cleave L-selectin from the surface of leucocytes for functional studies of L-selectin(18) ; however, direct proteolysis has not been demonstrated. Since some zinc-dependent metalloproteinases require extracellular proteolytic processing for activation we determined whether chymotrypsin mediates cleavage of L-selectin via activation of L-selectin sheddase. Ro 31-9790 had no effect on chymotrypsin-mediated cleavage of L-selectin from either mouse lymphocytes or 300.19 transfectants ( Fig. 2and data not shown), demonstrating that chymotrypsin-mediated proteolysis is independent of metalloproteinase activation. To determine whether proteases other than chymotrypsin might be required for activation of the sheddase a range of inhibitors were tested including PMSF (25 µM), TPCK (15 µM), TLCK (30 µM), leupeptin (30 µM), chymostatin (15 µM), antipain (5 µg/ml), bestatin (30 µM), diprotin A (30 µM), ebalactone (30 µM), E64 (30 µM), and pepstatin (30 µM) and found to have no effect on L-selectin shedding stimulated by PDBu ( Fig. 4and data not shown). PMSF and TPCK, but not TLCK, inhibited chymotrypsin-mediated proteolysis.
Figure 4: Phorbol dibutyrate-stimulated proteolysis of L-selectin is not blocked by serine protease inhibitors. Lymphocytes were incubated with 50 nM PDBu for 60 min at 37 °C (b, d, and f) or 40 µg/ml chymotrypsin for 20 min at 37 °C (c, e, and g) in the presence of either 25 µM PMSF (d and e) or 15 µM TPCK (f and g), and the expression of L-selectin compared with lymphocytes incubated in control buffer (a). Results are presented as described in the legend to Fig. 2.
Figure 5:
Matrix metalloproteinase-mediated
proteolysis of L-selectin on mouse lymphoid cells. Lymphocytes (a-f) or 300.19 cells transfected with human L-selectin (g-i) were incubated with control buffer for collagenase
and stromelysin (a and g), 4 µM stromelysin (c), 2 µM human fibroblast
collagenase in the absence (e and h) or presence (i) of 30 µM Ro 31-9790, control buffer for
gelatinases (b), 4 µM gelatinase A (d),
or 4 µM gelatinase B (f) for 20 min at 37 °C,
and the expression of L-selectin was determined by flow cytometric
analysis. Solid lines represent staining for L-selectin and dashed lines for a non-reactive control antibody. The
percentage of cells positive for L-selectin is given. The y axis gives cell number(0-400) and the x axis gives
fluorescence intensity on a log scale (10-10
channels).
Figure 6: Localization of L-selectin sheddase activity. Lymphocytes were incubated for 60 min at 37 °C in the absence (a) and presence (b) of 100 nM PDBu. The effect of PDBu on L-selectin shedding was completely inhibited using 300 nM staurosporine (c). Staurosporine was added to PDBu-activated lymphocytes. L-selectin levels on fresh lymphocytes were determined following 20 min of incubation with cell-free supernatants from unactivated (d) and PDBu-activated (e) lymphocytes. L-selectin levels on a population of lymphocytes labeled with CFSE were determined following 20 min of incubation with either unactivated (f) or PDBu-activated (g) lymphocytes. Results are presented as described in the legend to Fig. 5. Similar results were obtained using 10 nM Ro 31-8220 to inhibit excess PDBu.
Although proteolytic cleavage of L-selectin from the surface of leucocytes has been known for a number of years, the enzyme(s) that mediate L-selectin shedding are unknown. In this study we have used a peptide derivative of hydroxamic acid, Ro 31-9790, to identify a role for a metalloproteinase in this process. Ro 31-9790 inhibited the proteolytic cleavage of L-selectin from leucocytes in mouse, rat, and man as measured by maintained cell surface expression of L-selectin as well as reduced shedding of L-selectin. L-selectin was cleaved by exogenous fibroblast collagenase (MMP1) and stromelysin (MMP3) but not other matrix metalloproteinases such as gelatinase A (MMP2) or gelatinase B (MMP9). However, the endogenous metalloproteinase (L-selectin sheddase) differs sufficiently from fibroblast collagenase and other known matrix metalloproteinases to suggest that it may represent a novel enzyme. Matrix metalloproteinases are normally secreted in zymogen form, are rarely associated with the cell surface, and expression in response to phorbol esters is generally regulated at the transcriptional level over a prolonged time course of 6-48 h(27) , which contrasts sharply with the rapid activation of L-selectin sheddase. Lymphocytes did not express cell surface collagenase or stromelysin, and L-selectin shedding was not inhibited by TIMP-1, a natural inhibitor of all known matrix metalloproteinases(23) . The close association of L-selectin sheddase with the cell membrane and its ability to be regulated by intracellular signaling events suggests that L-selectin sheddase may be related to a recently described transmembrane matrix metalloproteinase (28) or an entirely new class of metalloproteinase.
Matrix metalloproteinases are normally secreted as zymogens, and a range of reagents have been shown to activate latent metalloproteinases including serine proteases, alkylating agents, and organomercurials (29, 30) . We demonstrate that chymotrypsin-mediated proteolysis of L-selectin is not dependent on activation of the putative sheddase. The lack of effect of a range of protease inhibitors including serine proteases, cysteine proteases, and other inhibitors of zinc-dependent endopeptidases, such as phosphoramidon(31) , is in broad agreement with results of other studies(15, 32) . 1,10-Phenanthroline is widely used to inhibit zinc-dependent metalloproteinases; however, it was toxic to lymphocytes, although it has been used on human neutrophils(33) . Iodoacetamide and N-ethylmaleimide have both been used to induce p80 TNFR shedding from the myeloid cell line U937(34) , but effective doses were toxic to lymphocytes. Effective doses of the organomercurial, 4-aminophenylmercuric acetate, which is used to activate matrix metalloproteinases in vitro(23) , were also toxic.
Peptide derivatives of
hydroxamic acid that have a high affinity for zinc, such as Ro
31-9790, have been developed as non-toxic inhibitors of matrix
metalloproteinases to study the biological roles of these
enzymes(26) . The use of these compounds has identified roles
for metalloproteinases in the processing of pro-tumor necrosis
factor- leucocytes(35, 36, 37) .
Subsequent studies demonstrated metalloproteinase-dependent cleavage of
cell surface p80 TNFR(38) , Fas ligand(39) , IL-6R, and
p60 TNFR(40) . However, this is the first report of a
metalloproteinase regulating cell surface levels of L-selectin. It is
notable that, in our studies, the IC
of Ro 31-9790
for L-selectin sheddase was 2 µM, which is at least
400-fold higher than that for soluble fibroblast collagenase. It is
therefore possible that the metalloproteinase activities identified
using hydroxamic acid-based inhibitors in this and other
studies(35, 36, 37, 38, 39, 40) may
represent several enzymes.
The regulation and distribution of
L-selectin sheddase are clearly important areas for study. The basal
release of L-selectin from leucocytes seen here and in other studies (13, 41) suggests that L-selectin sheddase is normally
active. Phorbol esters may increase the activity of the enzyme or alter
the conformation and/or the distribution of L-selectin, increasing its
susceptibility to enzymatic cleavage. Our studies demonstrate that
L-selectin sheddase is tightly associated with the membrane rather than
a secreted enzyme. Mutational analysis of the extracellular
membrane-proximal 15-amino acid region containing the putative primary
cleavage site of human L-selectin (Lys-Ser
)
has demonstrated that the distance of this site from the plasma
membrane regulates cleavage(42, 43) . The inability of
L-selectin sheddase to operate in the trans configuration and
cleave L-selectin from the surface of adjacent cells may be dictated by
its precise topographical location with respect to L-selectin.
Alternatively, the ectodomain of L-selectin may be processed in an
intracellular compartment. Future studies will address the localization
of the sheddase using cell-free assay systems.
The physiological role of rapid L-selectin cleavage from the leucocyte surface has not been determined because of the lack of reagents to inhibit this process. L-selectin is shed from unactivated leucocytes in vitro(13, 41) , although the rate of shedding is significantly increased following activation. Accelerated shedding of L-selectin from neutrophils is also detectable following binding to IL-1-activated cultured endothelial cells(44) . Soluble L-selectin is detectable in the circulation at levels that could interfere with its function on leucocytes(45) . L-selectin down-regulation may be required to break ligand interactions for leucocyte rolling per se or to facilitate integrin-dependent binding and transendothelial migration, and these possibilities should be tested. The loss of cell surface L-selectin from leucocytes has profound effects on their migration pathways in vivo. An inhibitor of L-selectin shedding will be useful for determining its role in regulating leucocyte traffic.