Subcellular
localization
Yaacov Matzner7 Alfred I. Tauber *Hematology Oncology, Laboratory,
Unit, Hadassah Boston
Israel Hadossah
of heparanase
Vlodavsky,t University
Matia
Hospital
University Hospital, City Hospital, Boston,
Mount
Em Karem, Mcssachasetts
Abstract The subcellular localization of a heparan sulfate degrading endoglycosidase, heparanase, was studied in human neutrophils. Unstimulated cells were disrupted by nitrogen cavitation and fractionated on a Percoll density gradient into three components, separating the plasma membranes, specific granules, and azurophilic granules. Heparanase activity was measured by gel uultration analysis of 35S-labeled degradation fragments released from subendothelial extracellular matrix (ECM) or produced during incubation with soluble, ECMderived, heparane sulfate proteoglycans. Heparanase activity was found mainly in fractions containing the specific granules; this activity was inhibited by heparin. Freezing and thawing was not needed for recovery of the enzyme from the subcellular fraction, confirming previous data about its ready release. The mechanism of the ready release of heparanase from the specific granules requires further investigation.J. Lcukoc. Biol. 51: 519-524;
in human
BarNer,t
Rivka
Scopus,
Jerusalem,
Israel;
Israel;
William
Jerusalem,
( mostly
lshaiMichaeli,t
and
heparan-
and
tDepartment B.
and
laminin, fibronectin, tion of freshly isolated labeled ECM resulted
degradation
neutrophils of Radiation
Costle
and
Hematology
Clinical
Research
dermatan-sulfate
proteoglycans),
and elastin [11, 12, 17, 28, 29]. Incubahuman neutrophils with metabolically in release of low molecular weight
products
shown
to
be
heparan
sulfate
frag-
ments. We defined the responsible degradative enzyme as heparanase and demonstrated that it was readily, and preferentially, released by simply incubating the cells at 4#{176}Cin the absence of added stimuli. Under these conditions, no release of other potentially harmful neutrophil constituents could be documented [21]. We thus suggested that hepara-
nase
activity
tion
and
may
be involved
diapedesis
in the early
of neutrophils
signal. The present study heparanase activity within tionation studies revealed the specific granules.
events
of extravasa-
in response
to a threshold
was undertaken to localize the neutrophil that by cell the enzyme primarily localized
the fracin
1992.
MATERIALS Key Words: neusrophil heparanase . diapedesis
.
subendothelial
extracellular
Materials
INTRODUCTION Neutrophil migration and diapedesis through the endothelial lining and subendothelial basement membrane are thought to be driven by a chemotactic factor(s) gradient, which at the inflamed area is high enough to induce discharge of granules and a subsequent #{176}2 production [31]. In contrast, while still
in circulation
the cells
exhibit
little
or no enzyme
release
and
02 production in response to low concentrations of the chemoattractant, and thus the vascular endothelial cell lining may be protected. It was therefore postulated that neutrophils may contain an additional distinct secretory compartment whose enzymes are excreted in response to a threshold signal, which may be mobilized in the early events of neutrophil chemotaxis without damaging the endothelial cells themselves. Such a role has been ascribed to a metalloproteinase (gelatinase) that is preferentially released from human neutrophils and is putatively stored in a previously undescribed
intracellular
AND METHODS
matrix
compartment
[7,
24].
Another
such activable intracellular compartment was identified in human neutrophils by measurements of latent alkaline phosphatase and tetranectin activities [5, 6]. We have described a heparan sulfate degrading endoglycosidase, heparanase, that is readily released from human neutrophils [21]. In those studies, we used the subendothelial extracellular matrix (ECM) deposited by cultured bovine endothelial cells, which closely resembles the vascular subendothelial basal lamina in its morphological appearance and molecular composition [11, 28]. This ECM preparation contains collagens (mostly types III and IV), proteoglycans
Polystyrene from Falcon
tissue Labware
CA).
fibroblast
Basic
culture dishes (35-mm) were Division, Becton Dickinson
growth
factor
was
obtained (Oxnard,
partially
purified
from bovine brain as described [10]. Dulbecco’s Eagles’s medium (DMEM), Roswell Park Memorial
tute
medium
(RPMI)
1640,
penicillin, streptomycin, obtained from Biological p-Nitrophenyl phosphate, N,N’-bis(2-ethanesulfonic bumin,
and
calf
serum,
fetal
calf
modified Insti-
serum,
and trypsin/EDTA solution were Industries (Beit-Haemek, Israel). 4-aminoantipyrene, piperazineacid) (PIPES), bovine serum al-
dextran
T-40
were
purchased
from
Sigma
Chemical Co. (St. Louis, MO). Na2[35S]04 and Biofluor scintillation fluid were from New England Nuclear (Boston, MA). Ficoll-Hypaque, dextran T-500, Sepharose 6B, Percoll, and colored density marker beads were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). All other chemicals were of reagent grade from Sigma Chemical Co.
Neutrophil
Isolation
and Fractionation
from normal human donors were isolated as predescribed [4]. Before cavitation, the cells were suspended in phosphate-buffered saline (PBS), pH 7.0, and incubated with 10 mM diisopropylfluorophosphate for 5 mm on ice. The serineNeutrophils
viously
Al)hreviations:
ECM.
extracellular
matrix:
PBS.
phosphate-buffered
saline. Received Reprint sity
Hospital.
Journal
August requests:
13,
1991;
Yaacov
Mount
of Leukocyte
Scopus,
accepted Matzner, Jerusalem.
Biology
November Hematology Israel,
Volume
18,
1991.
Unit,
Hadassah
Univer-
91240.
51, June
1992
519
protease inhibitor was washed out by the addition of PBS and centrifuged at 400 g for 5 mm [18]. The cells were then disrupted by nitrogen cavitation and fractionated on Percoll gradient as described in detail previously [3, 4]. Briefly, the tonicity the
and
final
pH
of Percoll
volume
(1 M KC1,
was
adjusted
of a 10-times
30 mM
NaCl,
by adding
concentrated
35 mM
MgC12,
buffer
10 mM
Na2ATP,
through a spinal needle. The nuclei and the few unbroken cells were pelleted by centrifugation at 500 g for 10 mm at 4#{176}C, and 8.5 ml of the supernatant (S1) were applied to the top of the gradient, which was centrifuged in a centrifuge (Beckman Instruments, Inc., Palo Alto, CA) using a SS34 at
4#{176}Cfor
20
mm
at 48,000g.
The
supernatant
from
these density gradients (S2) and three visible bands, a and ‘y were separated by aspiration or, alternatively, the gradient was fractionated into 1.5-ml portions by aspiration from the bottom of the gradient. Percoll was removed from pooled fractions by centrifuging at 200,000g for 3 h in a SW41 rotor (Beckman Instruments, Inc.). Sedimentable biologic material is layered directly on a hard-packed pellet of Percoll, from which it was easily separated by aspiration and resuspended in PBS. ,
Preparation
of Sulfate-labeled
ECM-coated
Cultures of bovine cQrneal endothelial from steer eyes as described [8, 9]. DMEM (1 g glucose/l), 10% 5% dextran T-40, penicillin
Dishes
cells were Cells were
calfserum, (50 U/mi),
by
scintillation fluid. The by blue dextran and the
phenol
red.
[35S]04
[23,
material,
one-tenth
cavitation
12.5 mM EGTA, 100 mM PIPES, pH 6.8) [4]. A discontinuous gradient was obtained by layering 14 ml Percoll of density 1.120 g/cm3 under 14 ml Percoll of density 1.050 g/cm3
rotor,
Bio-fluor marked
in
5% fetal calf serum, and streptomycin
(50 sg/ml) at 37#{176}Cin a 10% CO2 humidified incubator. Basic fibroblast growth factor (100 ng/ml) was added every other day during the phase of active cell growth. Na2[35S]04 (540-590 mCi/mmol) was added 3 and 7 days after seeding
latter
was
High
representing
shown
relative
soluble
to comigrate
moiecuiar
with
free
weight-labeled
proteogiycans,
eiuted
from
the
column with Kay < 0.2 (Mr of 5 x 10 dalton) and was defined as peak I proteoglycan material. Fragments of heparan sulfate glycosaminoglycan chains were eluted with Kay between 0.5 and 0.75 (Mr of 5 x 10 1 x 10 dalton) and were defined as peak II heparan sulfate degradation production [21, 23, 27, 30]. Recoveries of labeled material applied on the columns ranged from 85 to 95% in different experiments. Each experiment was performed at least four times and differences in elution positions (Kay values) were 0
c
Degradation of 35S-labeled ECM by Subcellular Fractions of Human Neutrophils To
determine
the
DFP-treated tion The
subcellular
neutrophils
localization
were
disrupted
and fractionated on a discontinuous resulting a, 13, y, and cytosol (S2)
of by
Co Cs C CS
heparanase,
nitrogen
Percoll fractions
500 0. 0
cavitaI
gradient [3]. were assayed
for specific markers of azurophilic granules (myeloperoxidase), specific granules (vitamin B12-binding protein), and light (predominantly plasma) membranes (alkaline phosphatase), and the results shown in Table 1 were similar to our
by means
ofheparanase
activity
demonstrated ECM as a peak products (five side chains) Kay < 0.75).
after incubation of intact oflow relative molecular to six times smaller than eluted from Sepharose A similar elution pattern
the
containing
/3 band,
specific
[21]. This
activity
was
Fig.
have
previously
shown
that
neutrophil
heparin
(Fig.
2 B),
suggesting
that
little
incubated
Alkaline
heparanase
0.0039
-y
01987
f3 a
0.0227
(9.1)
0.0245
(9.8)
fractions is given
in
from parentheses.
B,2
(1.6)
gradients are
were representative
obtained of
of hu-
due
to
the
of the
primary
activity
potential
granules
in the
primary
inhibition
by
to the ECM. granules
The
was
myeloperoxidase
not
since
heparanase
activity
was
recovered
upon
freeze-
The
low
relative
released
from
tracellular ucts of
molecular
labeled
by
radioactive
human
neutrophils
supernatant were identified heparan sulfate side chains
Fractions
of Human
as degradation as indicated
Myeloperoxidase
(%)
fragments and
cx-
prodby their
Neutrophils
protein protein)
weight
ECM
(U/mg
protein)
Protein
(%)
(mg/mi)
0.11
(0.1)
0
(0)
2.45
260
(39)
0.21
(5.7)
4.03
(92.2)
0.52
(14.2)
7.75
2.93
(80.1)
10.25
257
results
fractions
Degradation of Soluble 35S-labeled Heparan Sulfate Proteoglycans by Subcellular Fractions of Human Neutrophils
bound/mg
6194
density The
of heparanase
B ,-binding
(795)
Percoll
to absorption
lack
in Subcellular
Vitamin (ng
due
A similar
of Markers
(%)
Cytosol
Subcellular
Distribution
by subcellular
thawing of the various fractions. Neither incubation of the bands at 37#{176}Cnor disruption ofthe granules by freezing and thawing emptied them of their heparanase activity as shown by a marked activity that remained in the post centrifugation pellet of the specific granules (results not shown).
or no hepar-
phosphatase
(U/mi)
activities
1.
ECM
myeloperoxidase-deficient cytoplasts [26] also lacked this activity (results not shown). The unfractionated cell cavitate ( S1) expressed heparanase activity that, in all experiments, was lower than the activity recovered from the specific granules. This may be due to the presence ofa heparanase inhibitor(s) that upon fractionation was distributed into separate (most likely soluble) compartments.
anase activity was present in the plasma membrane. The relative amount of heparanase in the specific granules was estimated by incubating the 35S-labeled ECM with various concentrations of the 3 fraction. As shown in Fig. 3, substantial activity was determined even at dilution of 1:100. Comparable activity was previously demonstrated with cxtracellular supernatant prepared from 1.25 x 106 neutro-
TABLE
of sulfate-labeled
phils incubated at 4#{176}C for 60 mm [21]. Obvious heparanase activity could not be detected in the plasma membrane fraction even at a 1:5 dilution (not shown). The cytosol and a band exhibited very little heparanase activity under the same conditions, which appeared to be
was
activity could be inhibited by heparin [21]. Addition of 10 g/ml heparin to the f3 fraction inhibited the enzyme, as reflected by a shift of the low relative molecular weight heparan sulfate degradation products to high relative molecular weight material (Kay < 0.3) (Fig. 2 A). No such obvious shift could be demonstrated when the ‘y band was pretreated
with
1. Degradation
man neutrophils. Subcdllular fractions from Percoll density gradient were obtained and the Percoll removed as described in Materials and Methods. The fractions were diluted 1:5 and adjusted to pH 6.2 (20 mM citrate phosphate buffer) and incubated (24 h, 37#{176}C)with “S-labeled ECM. Radioactive material released into the incubation medium was analyzed by gel filtration over Sepharose 6B. S1 (Ls), S2 (A), a (0), 3 (#{149}), y (0).
(37#{176}Cfor 24 h) with 35S-labeled ECM (Fig. 1). The ‘y band contained a marked proteolytic activity revealed by higher relative molecular weight degradation products (Kay < 0.3) [21]. However, significant radioactivity, though not appearing as a distinct peak, was eluted with Kay -.0.5-0.6, sug gesting that the plasma membrane may contain some heparanase activity as well.
We
20
Fraction
cells with 35S-labeled weight degradation intact heparan sulfate 6B column (0.5 < was obtained when
granules,
V0
0
previous report on the distribution of these subcellular fraction markers [4]. The fractions were further studied for heparanase activity by incubation with 35S-labeled ECM for 24 h. Gel filtration analysis was performed of labeled degradation products released into the incubation medium. As previously shown, unstimulated neutrophils degraded the heparan sulfate in
ECM
1000
0
(3.8)
as described five
in Materials
and
Methods.
The
percentage
distribution
of recovered
fractionations.
Matzner
ci al.
Neutrophil
heparanase
localization
521
15000
15000
A E 0. 0 -
10000
10000
>‘
>
0 Cs
a, Co
Cs C Cs Cs 0.
a) I
V
0
10
20
Vt40
30
0
v
30
20
40
50
0
Fraction Fig.
2.
Effect
fractions
of heparin
on
degradation
obtained
as
described
were
ECM
was
carried
out
in
the
of “S-labeled in
Materials
(#{149}) and
absence
ECM and
by
Fraction the
Methods,
presence
(0)
neutrophil-specific diluted
of
10 ag/ml
1:25, heparin.
resistance to chondroitinase ABC and papain digestion, sensitivity to deamination with nitrous acid, and precipitation with 0.05% cetylpyridinium chloride in 0.6 M NaCl [19, 21, 25]. We have previously demonstrated that a proteolytic activity is required for degradation of ECM-associated, but not soluble, heparan sulfate [21]. The previous experiments were therefore repeated using neutrophil supernatant and soluble “S-labeled heparan sulfate proteoglycans. As demonstrated in Fig. 4, soluble “S-labeled heparan sulfate eluted from Sepharose 6B at Kay 0.3 was used. Its incubation with supernatants of neutrophils, that were first incubated at room temperature or 4#{176}C for 1 h, resulted in the appearance of low relative molecular weight-labeled material (peak II, Kay 0.6-0.7) similar in its elution position to heparan sulfate degradation fragments released under the same
conditions
from
35S-labeled
ECM
[21].
granules and
adjusted
The
(A)
and
plasma
to pH
6.2
before
conditions
of the
incubation
membrane the
(B)
fractions.
experiment. and
gel
Percoll-free
Incubation filtration
with
are
described
pooled “S-labeled in
Fig.
1.
bation of the specific granules, /3 fractions, with 35S-labeled heparin sulfate proteoglycan revealed almost complete conversion to low relative molecular weight heparan sulfate degradation products (Kay 0.67). In contrast, the a and S2 fractions lacked heparan sulfate degradation activity, and the unfractionated cavitate, S1 expressed a low degradation activity. It should be noted that the amount of sulfate-labeled material eluted from the column after incubation of the soluble “S-labeled heparan sulfate with the a, S1, and S2 fractions was much lower than the radioactivity eluted after incubation with the ‘y and j3 fractions (Fig. 5). This is probably due to adherence of the intact labeled heparan sulfate to an undefined material present in these fractions, which was pelleted before gel filtration. The -y fraction degraded the soluble heparan sulfate proteoglycan to fragments of higher relative molecular weight (Kay 0.5) than those obtained
Incubation of the pooled subcellular fractions of human neutrophils with soluble “S-labeled heparan sulfate proteoglycans resulted in a degradation profile close to the one obtained with intact “S-labeled ECM (Fig. 5). Thus, incu-
2000
E 0. > Cs
1500
1100 1
1000 .
1000
Cs 0 0
900
0
E
800
Cs C Cs
Co
500
0. 0
0. 0
700
0 Cs
I
600
V0
0 Co
500
Cs C Cs Cs 0. 0
Fig. 4. Degradation human neutrophils.
100
I
Dilution 3.
Degradation
tion
of
the
total
cpm
sulfate eluted
522
specific in fractions
degradation from
Sepharose
Journal
20
30
V4#{176}
50
Fraction 10
Fig.
10
of sulfate-labeled granules 20-30
ECM
fraction. (peak
products)
as a function
Heparanase II,
low
relative
multiplied
by
of the
activity molecular the
Kay
concentra-
is expressed weight
of
the
peak
as
heparan fraction
6B.
of Leukocyte
Biology
Volume
51, June
1992
of soluble “S-labeled heparan sulfate Freshly isolated neutrophils suspended
proteoglycans in PBS (5
by x 106
cells/mi) were incubated at 4#{176}C for 60 mm. The cells were then centrifuged (300g, 10 mm) and the supernatant was adjusted to pH 6.2 and incubated (16 h, 37#{176}C)with “S-labeled peak I material isolated from ECM as described in Materials and Methods. After centrifugation, the radioactive material in the incubation mixture was analyzed by gel filtration over Sepharose 6B. Untreated “S-labeled heparin sulfate proteoglycans (0); “S-labeled heparan sulfate proteoglycans incubated with neutrophil supernatant (#{149}).
Consistent with our previous report [21], heparanase was readily released from the granules. Intact granules exhibited most of the activity. and there was only a small increase after freezing and thawing. In both intact and lysed preparations, significant heparan sulfate-degrading activity remained associated with the granules. The limitations of the heparanase assay and the possible presence of heparanase inhibitors in the cellular cavitate (S1) made accurate quantitation of the enzyme activity in the various compartments impossible.
E 0. 0
> 0 Cs
1000
a, Co
Cs C Cs
We were
500
Cs
a, I
20
V4#{176}
30
50
Fraction Fig.
5. Degradation
subcellular coll
fractions
density
described
in
sulfate
ned
of soluble
“S-labeled
of
neutrophils.
human
gradient
were
Materials
and
proteoglycans
and
out as described
in
teoglycans
(0);
gel
and
4.
the
of the
Untreated
heparan
with
incubated
was
Per-
removed
as
heparan
material
were
heparan with:
by
from
“S-labeled
radioactive
‘5S-labeled
sulfate
proteoglycans fractions
Percoli
Incubation
filtration
Fig.
sulfate
Subcellular
obtained Methods.
“S-labeled
heparan
sulfate (Lx),
5,
carpro-
52
(A),
We
also
reflecting in the
assayed
a much lower plasma membrane.
heparan
heparan
sulfate-
sulfate-degrading
activity
in
each fraction of the entire Percoll density gradient rather than in pooled fractions. As shown in Fig. 6, the fractionation resulted in efficient separation of the three compartments with very little contamination of plasma membranes by markers of specific granules. Heparan sulfate degradation activity could be recovered from one fraction in the area of maximal alkaline phosphatase activity and in four fractions
of the vitamin B12 binding protein peak. roborate the previous experiment indicating heparanase activity low activity detected to some contamination additional compartment
efficient ules
to
plasma
results cormost of the
i located in the specific granules. in the plasma membranes may of the /3 fraction or represents comprising little activity
translocation the
These that
of the
enzyme
from
the
able
to demonstrate
specific
trophils
with
resulted
in
Triton
X-100,
translocation
of
in-
while the
regular
alkaline
fractionation
phosphatase
to
the
The be due a true and/or
to
containing organelle” fuses with the plasma membrane, increasing both the amount of Mol expressed on the cell surface and the amount of gelatinase released into the extracellular medium. Evidence for translocation of granule constituents to the plasma membrane has been documented by
others
as well
[1, 3, 4, 15].
The
existence
of a distinct
addi-
tional intracellular compartment, however, is still controversial and at least one group has presented evidence that gelatinase is a component of the specific granule population and is not stored in a distinct secretory granule [14]. Using a double labeling immunogold technique, they found 80% collocalization of gelatinase and lactoferrin. They argued that the extensive release of gelatinase relative to other specific granule markers was due to an the gelatinase content remaining in the and cytosolic factors present during the
underestimation of cells since elastase assay of gelatinase
gran-
membrane.
DISCUSSION
demonstrated
activity
plasma membrane. We could not use detergent since it mactivates heparanase (unpublished results). Additional evidence for a “tertiary compartment” was published by Dewaid et al. [7]. and its importance was later emphasized by Petrequin et al. [24]. Each group showed that the metallo-proteinase, gelatinase, and the C3bi receptor,
Heparanase
By neutrophil compartments activity was was detected
heparanase
Mol, are localized in a “gelatinase containing organelle” Moreover, it was shown that upon exposure of neutrophils low concentrations of a chemotactic agent, the “gelatinase-
a (D), $ (#{149}), .y (U).
with 13 fraction, degrading activity
not
fraction corresponding to the novel intracellular compartment described by Borregaard et al. [5]. It should be emphasized, however, that these authors revealed the latent alkaline phosphatase activity peak only after permeabilizing the neu-
0.
++++
+
50
fractionation, we have identified the cellular that contain heparanase activity. Most of the found in the specific granules and small amount in the light membranes. We have previously that
a
proteolytic
activity
is
required
30
for
degradation of ECM-bound heparan sulfate by neutrophil heparanase. This finding was attributed to the conversion of the solid phase ECM proteoglycans into peak I-soluble proteoglycans, which provided for improved exposure of the heparan sulfate side chains to direct interaction with the neutrophil heparanase. In contrast, degradation of soluble peak I proteoglycans by the neutrophil heparanase was neither stimulated by added proteases nor inhibited by protease inhibitors [21]. The involvement of a proteolytic activity in the degradation of ECM heparan sulfate by specific granules was ruled out by experiments in which soluble peak I proteoglycans, rather than intact ECM, were substrate. The similar results obtained indicate that served degradation was primarily due to heparanase found in the specific granules.
40
used as the obactivity
20
10
10
20
Fraction Fig.
6. Distribution
tivity
in fractions
of enzyme from
supernatant
was
trifugation,
1.5-mi
the
gradient
quots
of
each
B12-binding (marked
layered
Matzner
and
fraction
heparan
were
assayed
After for
Neutrophil
Materials
degradation
gradient. gradient
by aspiration removal (U), and
heparanase
and
Post and
from
the
of the
myeioperoxidase
phosphatase in
sulfate
Percoll Percoll
collected Methods).
(#{149}), alkaline
#{247}), as described
et aL
and entire
a discontinuous were
Materials
protein as
on
fractions
(see
markers
a discontinuous
(0), heparanase
acnuclear
after
cen-
bottom
of
au-
Percoll, Vitamin activity
Methods.
localization
523
inhibit
gelatinase
activity.
The
finding
of a distinct
compart-
15. Higson,
ment, close to the plasma membrane and containing latent alkaline phosphatease, is difficult to establish since it can be demonstrated only after permeabilization with Triton X-100. As mentioned, the “latent alkaline phosphatase compartment” [5], as well as the gelatinase-containing organelle [24], were shown to fully translocate to the plasma membrane after stimulation with nanomolar concentrations of fMLP. It cannot be excluded that the readily released heparanase translocates from its intracellular compartment(s) to the plasma membrane during the neutrophil isolation. Thus, the question as to the source of heparanase released upon minimal stimulation requires further investigation.
F.K., Durbin, L., Pavlotsky, N., and Tauber, Al. of cytochrome B245 translocation in the PMA stimulation of the human neutrophil NADPH-oxidase. j ImmunoL 135, 519, 1985. 16. Kanofsky, JR., Wright, J., Miles-Richardson, G.E., and Tauber, Al. Biochemical requirements for singlet oxygen Studies
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2678,
18.
20.
N.,
tant regulated ment in human 6. Borregaard, N., and Clemmensen, set of human tetranectin and 408, 1990. 7.
Dewald,
B.,
9.
10.
11.
12.
13.
14.
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and
Springer,
TA.
Journal
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synthesis
of
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51, June
1992
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mobilization of a novel intracellular compartneutrophils. Science 237, 1204, 1987. Christensen, L., Bjerrum, O.W., Birgens, H.S., I. Identification of a highly mobilizable subneutrophil intracellular vesicles that contains latent alkaline phosphatase. j Clin. Invest. 85,
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and degradation proteoglycans
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1. Arnaut, MA. Structure and function ofthe leukocyte adhesion molecules CD11/CD18. Blood 75, 1037, 1990. 2. Bar-Ncr, M., Kramer, M.D., Schirmacher, V., Ishai-Michaeli, R., Fuks, Z., and Vlodavsky, I. Sequential degradation of heparan sulfate in the subendothelial extracellular matrix by highly metastatic lymphoma cells. In!. j Cancer 35, 483, 1985. 3. Borregaard, N., Heiple, J.M., Simons, ER., and Clark, R.A. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. j Cell BioL 97, 52, 1983. 4. Borregaard, N., and Tauber, A.!. Subcellular localization of the human neutrophil NADPH-oxidase: b cytochrome and associated flavoprotein. j BioL Chem. 259, 47, 1984.
by
74,
of hu-
vs. plastic. and
Fuks,
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matrix produced the thrombogenicity Thromb. Res. 28,
by 175,
1982.
30. Vlodavsky, I., Fuks, Z., Bar-Ncr, M., Ariav, Y., and Schirrmacher, V. Lymphoma cell mediated degradation of sulfated proteoglycans in the subendothelial extracellular matrix: relationship to tumor cell metastasis. Cancer I?es. 43, 2704, 1983. 31. Weissmann, G., Smolen, J.E., and Korchak, H.M. Release of inflammatory
j 32.
Med.
mediators
303,
Yahalom, of sulfated matrix
1984.
by
J.,
27, 1980. Eldor, A., proteoglycans
human
platelet
from
stimulated
Z., and
Fuks, in
the
heparitinase.
neutrophils.
Vlodavsky, subendothelial j
Clin.
N
EngI.
I. Degradation extracellular
Invest.
74,
1842,
localization
Yaacov Matzner7 Alfred I. Tauber *Hematology Oncology, Laboratory,
Unit, Hadassah Boston
Israel Hadossah
of heparanase
Vlodavsky,t University
Matia
Hospital
University Hospital, City Hospital, Boston,
Mount
Em Karem, Mcssachasetts
Abstract The subcellular localization of a heparan sulfate degrading endoglycosidase, heparanase, was studied in human neutrophils. Unstimulated cells were disrupted by nitrogen cavitation and fractionated on a Percoll density gradient into three components, separating the plasma membranes, specific granules, and azurophilic granules. Heparanase activity was measured by gel uultration analysis of 35S-labeled degradation fragments released from subendothelial extracellular matrix (ECM) or produced during incubation with soluble, ECMderived, heparane sulfate proteoglycans. Heparanase activity was found mainly in fractions containing the specific granules; this activity was inhibited by heparin. Freezing and thawing was not needed for recovery of the enzyme from the subcellular fraction, confirming previous data about its ready release. The mechanism of the ready release of heparanase from the specific granules requires further investigation.J. Lcukoc. Biol. 51: 519-524;
in human
BarNer,t
Rivka
Scopus,
Jerusalem,
Israel;
Israel;
William
Jerusalem,
( mostly
lshaiMichaeli,t
and
heparan-
and
tDepartment B.
and
laminin, fibronectin, tion of freshly isolated labeled ECM resulted
degradation
neutrophils of Radiation
Costle
and
Hematology
Clinical
Research
dermatan-sulfate
proteoglycans),
and elastin [11, 12, 17, 28, 29]. Incubahuman neutrophils with metabolically in release of low molecular weight
products
shown
to
be
heparan
sulfate
frag-
ments. We defined the responsible degradative enzyme as heparanase and demonstrated that it was readily, and preferentially, released by simply incubating the cells at 4#{176}Cin the absence of added stimuli. Under these conditions, no release of other potentially harmful neutrophil constituents could be documented [21]. We thus suggested that hepara-
nase
activity
tion
and
may
be involved
diapedesis
in the early
of neutrophils
signal. The present study heparanase activity within tionation studies revealed the specific granules.
events
of extravasa-
in response
to a threshold
was undertaken to localize the neutrophil that by cell the enzyme primarily localized
the fracin
1992.
MATERIALS Key Words: neusrophil heparanase . diapedesis
.
subendothelial
extracellular
Materials
INTRODUCTION Neutrophil migration and diapedesis through the endothelial lining and subendothelial basement membrane are thought to be driven by a chemotactic factor(s) gradient, which at the inflamed area is high enough to induce discharge of granules and a subsequent #{176}2 production [31]. In contrast, while still
in circulation
the cells
exhibit
little
or no enzyme
release
and
02 production in response to low concentrations of the chemoattractant, and thus the vascular endothelial cell lining may be protected. It was therefore postulated that neutrophils may contain an additional distinct secretory compartment whose enzymes are excreted in response to a threshold signal, which may be mobilized in the early events of neutrophil chemotaxis without damaging the endothelial cells themselves. Such a role has been ascribed to a metalloproteinase (gelatinase) that is preferentially released from human neutrophils and is putatively stored in a previously undescribed
intracellular
AND METHODS
matrix
compartment
[7,
24].
Another
such activable intracellular compartment was identified in human neutrophils by measurements of latent alkaline phosphatase and tetranectin activities [5, 6]. We have described a heparan sulfate degrading endoglycosidase, heparanase, that is readily released from human neutrophils [21]. In those studies, we used the subendothelial extracellular matrix (ECM) deposited by cultured bovine endothelial cells, which closely resembles the vascular subendothelial basal lamina in its morphological appearance and molecular composition [11, 28]. This ECM preparation contains collagens (mostly types III and IV), proteoglycans
Polystyrene from Falcon
tissue Labware
CA).
fibroblast
Basic
culture dishes (35-mm) were Division, Becton Dickinson
growth
factor
was
obtained (Oxnard,
partially
purified
from bovine brain as described [10]. Dulbecco’s Eagles’s medium (DMEM), Roswell Park Memorial
tute
medium
(RPMI)
1640,
penicillin, streptomycin, obtained from Biological p-Nitrophenyl phosphate, N,N’-bis(2-ethanesulfonic bumin,
and
calf
serum,
fetal
calf
modified Insti-
serum,
and trypsin/EDTA solution were Industries (Beit-Haemek, Israel). 4-aminoantipyrene, piperazineacid) (PIPES), bovine serum al-
dextran
T-40
were
purchased
from
Sigma
Chemical Co. (St. Louis, MO). Na2[35S]04 and Biofluor scintillation fluid were from New England Nuclear (Boston, MA). Ficoll-Hypaque, dextran T-500, Sepharose 6B, Percoll, and colored density marker beads were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). All other chemicals were of reagent grade from Sigma Chemical Co.
Neutrophil
Isolation
and Fractionation
from normal human donors were isolated as predescribed [4]. Before cavitation, the cells were suspended in phosphate-buffered saline (PBS), pH 7.0, and incubated with 10 mM diisopropylfluorophosphate for 5 mm on ice. The serineNeutrophils
viously
Al)hreviations:
ECM.
extracellular
matrix:
PBS.
phosphate-buffered
saline. Received Reprint sity
Hospital.
Journal
August requests:
13,
1991;
Yaacov
Mount
of Leukocyte
Scopus,
accepted Matzner, Jerusalem.
Biology
November Hematology Israel,
Volume
18,
1991.
Unit,
Hadassah
Univer-
91240.
51, June
1992
519
protease inhibitor was washed out by the addition of PBS and centrifuged at 400 g for 5 mm [18]. The cells were then disrupted by nitrogen cavitation and fractionated on Percoll gradient as described in detail previously [3, 4]. Briefly, the tonicity the
and
final
pH
of Percoll
volume
(1 M KC1,
was
adjusted
of a 10-times
30 mM
NaCl,
by adding
concentrated
35 mM
MgC12,
buffer
10 mM
Na2ATP,
through a spinal needle. The nuclei and the few unbroken cells were pelleted by centrifugation at 500 g for 10 mm at 4#{176}C, and 8.5 ml of the supernatant (S1) were applied to the top of the gradient, which was centrifuged in a centrifuge (Beckman Instruments, Inc., Palo Alto, CA) using a SS34 at
4#{176}Cfor
20
mm
at 48,000g.
The
supernatant
from
these density gradients (S2) and three visible bands, a and ‘y were separated by aspiration or, alternatively, the gradient was fractionated into 1.5-ml portions by aspiration from the bottom of the gradient. Percoll was removed from pooled fractions by centrifuging at 200,000g for 3 h in a SW41 rotor (Beckman Instruments, Inc.). Sedimentable biologic material is layered directly on a hard-packed pellet of Percoll, from which it was easily separated by aspiration and resuspended in PBS. ,
Preparation
of Sulfate-labeled
ECM-coated
Cultures of bovine cQrneal endothelial from steer eyes as described [8, 9]. DMEM (1 g glucose/l), 10% 5% dextran T-40, penicillin
Dishes
cells were Cells were
calfserum, (50 U/mi),
by
scintillation fluid. The by blue dextran and the
phenol
red.
[35S]04
[23,
material,
one-tenth
cavitation
12.5 mM EGTA, 100 mM PIPES, pH 6.8) [4]. A discontinuous gradient was obtained by layering 14 ml Percoll of density 1.120 g/cm3 under 14 ml Percoll of density 1.050 g/cm3
rotor,
Bio-fluor marked
in
5% fetal calf serum, and streptomycin
(50 sg/ml) at 37#{176}Cin a 10% CO2 humidified incubator. Basic fibroblast growth factor (100 ng/ml) was added every other day during the phase of active cell growth. Na2[35S]04 (540-590 mCi/mmol) was added 3 and 7 days after seeding
latter
was
High
representing
shown
relative
soluble
to comigrate
moiecuiar
with
free
weight-labeled
proteogiycans,
eiuted
from
the
column with Kay < 0.2 (Mr of 5 x 10 dalton) and was defined as peak I proteoglycan material. Fragments of heparan sulfate glycosaminoglycan chains were eluted with Kay between 0.5 and 0.75 (Mr of 5 x 10 1 x 10 dalton) and were defined as peak II heparan sulfate degradation production [21, 23, 27, 30]. Recoveries of labeled material applied on the columns ranged from 85 to 95% in different experiments. Each experiment was performed at least four times and differences in elution positions (Kay values) were 0
c
Degradation of 35S-labeled ECM by Subcellular Fractions of Human Neutrophils To
determine
the
DFP-treated tion The
subcellular
neutrophils
localization
were
disrupted
and fractionated on a discontinuous resulting a, 13, y, and cytosol (S2)
of by
Co Cs C CS
heparanase,
nitrogen
Percoll fractions
500 0. 0
cavitaI
gradient [3]. were assayed
for specific markers of azurophilic granules (myeloperoxidase), specific granules (vitamin B12-binding protein), and light (predominantly plasma) membranes (alkaline phosphatase), and the results shown in Table 1 were similar to our
by means
ofheparanase
activity
demonstrated ECM as a peak products (five side chains) Kay < 0.75).
after incubation of intact oflow relative molecular to six times smaller than eluted from Sepharose A similar elution pattern
the
containing
/3 band,
specific
[21]. This
activity
was
Fig.
have
previously
shown
that
neutrophil
heparin
(Fig.
2 B),
suggesting
that
little
incubated
Alkaline
heparanase
0.0039
-y
01987
f3 a
0.0227
(9.1)
0.0245
(9.8)
fractions is given
in
from parentheses.
B,2
(1.6)
gradients are
were representative
obtained of
of hu-
due
to
the
of the
primary
activity
potential
granules
in the
primary
inhibition
by
to the ECM. granules
The
was
myeloperoxidase
not
since
heparanase
activity
was
recovered
upon
freeze-
The
low
relative
released
from
tracellular ucts of
molecular
labeled
by
radioactive
human
neutrophils
supernatant were identified heparan sulfate side chains
Fractions
of Human
as degradation as indicated
Myeloperoxidase
(%)
fragments and
cx-
prodby their
Neutrophils
protein protein)
weight
ECM
(U/mg
protein)
Protein
(%)
(mg/mi)
0.11
(0.1)
0
(0)
2.45
260
(39)
0.21
(5.7)
4.03
(92.2)
0.52
(14.2)
7.75
2.93
(80.1)
10.25
257
results
fractions
Degradation of Soluble 35S-labeled Heparan Sulfate Proteoglycans by Subcellular Fractions of Human Neutrophils
bound/mg
6194
density The
of heparanase
B ,-binding
(795)
Percoll
to absorption
lack
in Subcellular
Vitamin (ng
due
A similar
of Markers
(%)
Cytosol
Subcellular
Distribution
by subcellular
thawing of the various fractions. Neither incubation of the bands at 37#{176}Cnor disruption ofthe granules by freezing and thawing emptied them of their heparanase activity as shown by a marked activity that remained in the post centrifugation pellet of the specific granules (results not shown).
or no hepar-
phosphatase
(U/mi)
activities
1.
ECM
myeloperoxidase-deficient cytoplasts [26] also lacked this activity (results not shown). The unfractionated cell cavitate ( S1) expressed heparanase activity that, in all experiments, was lower than the activity recovered from the specific granules. This may be due to the presence ofa heparanase inhibitor(s) that upon fractionation was distributed into separate (most likely soluble) compartments.
anase activity was present in the plasma membrane. The relative amount of heparanase in the specific granules was estimated by incubating the 35S-labeled ECM with various concentrations of the 3 fraction. As shown in Fig. 3, substantial activity was determined even at dilution of 1:100. Comparable activity was previously demonstrated with cxtracellular supernatant prepared from 1.25 x 106 neutro-
TABLE
of sulfate-labeled
phils incubated at 4#{176}C for 60 mm [21]. Obvious heparanase activity could not be detected in the plasma membrane fraction even at a 1:5 dilution (not shown). The cytosol and a band exhibited very little heparanase activity under the same conditions, which appeared to be
was
activity could be inhibited by heparin [21]. Addition of 10 g/ml heparin to the f3 fraction inhibited the enzyme, as reflected by a shift of the low relative molecular weight heparan sulfate degradation products to high relative molecular weight material (Kay < 0.3) (Fig. 2 A). No such obvious shift could be demonstrated when the ‘y band was pretreated
with
1. Degradation
man neutrophils. Subcdllular fractions from Percoll density gradient were obtained and the Percoll removed as described in Materials and Methods. The fractions were diluted 1:5 and adjusted to pH 6.2 (20 mM citrate phosphate buffer) and incubated (24 h, 37#{176}C)with “S-labeled ECM. Radioactive material released into the incubation medium was analyzed by gel filtration over Sepharose 6B. S1 (Ls), S2 (A), a (0), 3 (#{149}), y (0).
(37#{176}Cfor 24 h) with 35S-labeled ECM (Fig. 1). The ‘y band contained a marked proteolytic activity revealed by higher relative molecular weight degradation products (Kay < 0.3) [21]. However, significant radioactivity, though not appearing as a distinct peak, was eluted with Kay -.0.5-0.6, sug gesting that the plasma membrane may contain some heparanase activity as well.
We
20
Fraction
cells with 35S-labeled weight degradation intact heparan sulfate 6B column (0.5 < was obtained when
granules,
V0
0
previous report on the distribution of these subcellular fraction markers [4]. The fractions were further studied for heparanase activity by incubation with 35S-labeled ECM for 24 h. Gel filtration analysis was performed of labeled degradation products released into the incubation medium. As previously shown, unstimulated neutrophils degraded the heparan sulfate in
ECM
1000
0
(3.8)
as described five
in Materials
and
Methods.
The
percentage
distribution
of recovered
fractionations.
Matzner
ci al.
Neutrophil
heparanase
localization
521
15000
15000
A E 0. 0 -
10000
10000
>‘
>
0 Cs
a, Co
Cs C Cs Cs 0.
a) I
V
0
10
20
Vt40
30
0
v
30
20
40
50
0
Fraction Fig.
2.
Effect
fractions
of heparin
on
degradation
obtained
as
described
were
ECM
was
carried
out
in
the
of “S-labeled in
Materials
(#{149}) and
absence
ECM and
by
Fraction the
Methods,
presence
(0)
neutrophil-specific diluted
of
10 ag/ml
1:25, heparin.
resistance to chondroitinase ABC and papain digestion, sensitivity to deamination with nitrous acid, and precipitation with 0.05% cetylpyridinium chloride in 0.6 M NaCl [19, 21, 25]. We have previously demonstrated that a proteolytic activity is required for degradation of ECM-associated, but not soluble, heparan sulfate [21]. The previous experiments were therefore repeated using neutrophil supernatant and soluble “S-labeled heparan sulfate proteoglycans. As demonstrated in Fig. 4, soluble “S-labeled heparan sulfate eluted from Sepharose 6B at Kay 0.3 was used. Its incubation with supernatants of neutrophils, that were first incubated at room temperature or 4#{176}C for 1 h, resulted in the appearance of low relative molecular weight-labeled material (peak II, Kay 0.6-0.7) similar in its elution position to heparan sulfate degradation fragments released under the same
conditions
from
35S-labeled
ECM
[21].
granules and
adjusted
The
(A)
and
plasma
to pH
6.2
before
conditions
of the
incubation
membrane the
(B)
fractions.
experiment. and
gel
Percoll-free
Incubation filtration
with
are
described
pooled “S-labeled in
Fig.
1.
bation of the specific granules, /3 fractions, with 35S-labeled heparin sulfate proteoglycan revealed almost complete conversion to low relative molecular weight heparan sulfate degradation products (Kay 0.67). In contrast, the a and S2 fractions lacked heparan sulfate degradation activity, and the unfractionated cavitate, S1 expressed a low degradation activity. It should be noted that the amount of sulfate-labeled material eluted from the column after incubation of the soluble “S-labeled heparan sulfate with the a, S1, and S2 fractions was much lower than the radioactivity eluted after incubation with the ‘y and j3 fractions (Fig. 5). This is probably due to adherence of the intact labeled heparan sulfate to an undefined material present in these fractions, which was pelleted before gel filtration. The -y fraction degraded the soluble heparan sulfate proteoglycan to fragments of higher relative molecular weight (Kay 0.5) than those obtained
Incubation of the pooled subcellular fractions of human neutrophils with soluble “S-labeled heparan sulfate proteoglycans resulted in a degradation profile close to the one obtained with intact “S-labeled ECM (Fig. 5). Thus, incu-
2000
E 0. > Cs
1500
1100 1
1000 .
1000
Cs 0 0
900
0
E
800
Cs C Cs
Co
500
0. 0
0. 0
700
0 Cs
I
600
V0
0 Co
500
Cs C Cs Cs 0. 0
Fig. 4. Degradation human neutrophils.
100
I
Dilution 3.
Degradation
tion
of
the
total
cpm
sulfate eluted
522
specific in fractions
degradation from
Sepharose
Journal
20
30
V4#{176}
50
Fraction 10
Fig.
10
of sulfate-labeled granules 20-30
ECM
fraction. (peak
products)
as a function
Heparanase II,
low
relative
multiplied
by
of the
activity molecular the
Kay
concentra-
is expressed weight
of
the
peak
as
heparan fraction
6B.
of Leukocyte
Biology
Volume
51, June
1992
of soluble “S-labeled heparan sulfate Freshly isolated neutrophils suspended
proteoglycans in PBS (5
by x 106
cells/mi) were incubated at 4#{176}C for 60 mm. The cells were then centrifuged (300g, 10 mm) and the supernatant was adjusted to pH 6.2 and incubated (16 h, 37#{176}C)with “S-labeled peak I material isolated from ECM as described in Materials and Methods. After centrifugation, the radioactive material in the incubation mixture was analyzed by gel filtration over Sepharose 6B. Untreated “S-labeled heparin sulfate proteoglycans (0); “S-labeled heparan sulfate proteoglycans incubated with neutrophil supernatant (#{149}).
Consistent with our previous report [21], heparanase was readily released from the granules. Intact granules exhibited most of the activity. and there was only a small increase after freezing and thawing. In both intact and lysed preparations, significant heparan sulfate-degrading activity remained associated with the granules. The limitations of the heparanase assay and the possible presence of heparanase inhibitors in the cellular cavitate (S1) made accurate quantitation of the enzyme activity in the various compartments impossible.
E 0. 0
> 0 Cs
1000
a, Co
Cs C Cs
We were
500
Cs
a, I
20
V4#{176}
30
50
Fraction Fig.
5. Degradation
subcellular coll
fractions
density
described
in
sulfate
ned
of soluble
“S-labeled
of
neutrophils.
human
gradient
were
Materials
and
proteoglycans
and
out as described
in
teoglycans
(0);
gel
and
4.
the
of the
Untreated
heparan
with
incubated
was
Per-
removed
as
heparan
material
were
heparan with:
by
from
“S-labeled
radioactive
‘5S-labeled
sulfate
proteoglycans fractions
Percoli
Incubation
filtration
Fig.
sulfate
Subcellular
obtained Methods.
“S-labeled
heparan
sulfate (Lx),
5,
carpro-
52
(A),
We
also
reflecting in the
assayed
a much lower plasma membrane.
heparan
heparan
sulfate-
sulfate-degrading
activity
in
each fraction of the entire Percoll density gradient rather than in pooled fractions. As shown in Fig. 6, the fractionation resulted in efficient separation of the three compartments with very little contamination of plasma membranes by markers of specific granules. Heparan sulfate degradation activity could be recovered from one fraction in the area of maximal alkaline phosphatase activity and in four fractions
of the vitamin B12 binding protein peak. roborate the previous experiment indicating heparanase activity low activity detected to some contamination additional compartment
efficient ules
to
plasma
results cormost of the
i located in the specific granules. in the plasma membranes may of the /3 fraction or represents comprising little activity
translocation the
These that
of the
enzyme
from
the
able
to demonstrate
specific
trophils
with
resulted
in
Triton
X-100,
translocation
of
in-
while the
regular
alkaline
fractionation
phosphatase
to
the
The be due a true and/or
to
containing organelle” fuses with the plasma membrane, increasing both the amount of Mol expressed on the cell surface and the amount of gelatinase released into the extracellular medium. Evidence for translocation of granule constituents to the plasma membrane has been documented by
others
as well
[1, 3, 4, 15].
The
existence
of a distinct
addi-
tional intracellular compartment, however, is still controversial and at least one group has presented evidence that gelatinase is a component of the specific granule population and is not stored in a distinct secretory granule [14]. Using a double labeling immunogold technique, they found 80% collocalization of gelatinase and lactoferrin. They argued that the extensive release of gelatinase relative to other specific granule markers was due to an the gelatinase content remaining in the and cytosolic factors present during the
underestimation of cells since elastase assay of gelatinase
gran-
membrane.
DISCUSSION
demonstrated
activity
plasma membrane. We could not use detergent since it mactivates heparanase (unpublished results). Additional evidence for a “tertiary compartment” was published by Dewaid et al. [7]. and its importance was later emphasized by Petrequin et al. [24]. Each group showed that the metallo-proteinase, gelatinase, and the C3bi receptor,
Heparanase
By neutrophil compartments activity was was detected
heparanase
Mol, are localized in a “gelatinase containing organelle” Moreover, it was shown that upon exposure of neutrophils low concentrations of a chemotactic agent, the “gelatinase-
a (D), $ (#{149}), .y (U).
with 13 fraction, degrading activity
not
fraction corresponding to the novel intracellular compartment described by Borregaard et al. [5]. It should be emphasized, however, that these authors revealed the latent alkaline phosphatase activity peak only after permeabilizing the neu-
0.
++++
+
50
fractionation, we have identified the cellular that contain heparanase activity. Most of the found in the specific granules and small amount in the light membranes. We have previously that
a
proteolytic
activity
is
required
30
for
degradation of ECM-bound heparan sulfate by neutrophil heparanase. This finding was attributed to the conversion of the solid phase ECM proteoglycans into peak I-soluble proteoglycans, which provided for improved exposure of the heparan sulfate side chains to direct interaction with the neutrophil heparanase. In contrast, degradation of soluble peak I proteoglycans by the neutrophil heparanase was neither stimulated by added proteases nor inhibited by protease inhibitors [21]. The involvement of a proteolytic activity in the degradation of ECM heparan sulfate by specific granules was ruled out by experiments in which soluble peak I proteoglycans, rather than intact ECM, were substrate. The similar results obtained indicate that served degradation was primarily due to heparanase found in the specific granules.
40
used as the obactivity
20
10
10
20
Fraction Fig.
6. Distribution
tivity
in fractions
of enzyme from
supernatant
was
trifugation,
1.5-mi
the
gradient
quots
of
each
B12-binding (marked
layered
Matzner
and
fraction
heparan
were
assayed
After for
Neutrophil
Materials
degradation
gradient. gradient
by aspiration removal (U), and
heparanase
and
Post and
from
the
of the
myeioperoxidase
phosphatase in
sulfate
Percoll Percoll
collected Methods).
(#{149}), alkaline
#{247}), as described
et aL
and entire
a discontinuous were
Materials
protein as
on
fractions
(see
markers
a discontinuous
(0), heparanase
acnuclear
after
cen-
bottom
of
au-
Percoll, Vitamin activity
Methods.
localization
523
inhibit
gelatinase
activity.
The
finding
of a distinct
compart-
15. Higson,
ment, close to the plasma membrane and containing latent alkaline phosphatease, is difficult to establish since it can be demonstrated only after permeabilization with Triton X-100. As mentioned, the “latent alkaline phosphatase compartment” [5], as well as the gelatinase-containing organelle [24], were shown to fully translocate to the plasma membrane after stimulation with nanomolar concentrations of fMLP. It cannot be excluded that the readily released heparanase translocates from its intracellular compartment(s) to the plasma membrane during the neutrophil isolation. Thus, the question as to the source of heparanase released upon minimal stimulation requires further investigation.
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