Cell Surface Localization of Heparanase On Macrophages Regulates Degradation of Extracellular Matrix Heparan Sulfate

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RESEARCH ARTICLE | MARCH 15 2004
Cell Surface Localization of Heparanase on Macrophages Regulates
Degradation of Extracellular Matrix Heparan Sulfate1 
Norihiko Sasaki; ... et. al
J Immunol (2004) 172 (6): 3830–3835.
https://doi.org/10.4049/jimmunol.172.6.3830

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The Journal of Immunology

Cell Surface Localization of Heparanase on Macrophages


Regulates Degradation of Extracellular Matrix Heparan Sulfate1
Norihiko Sasaki,2* Nobuaki Higashi,* Tomohiro Taka,* Motowo Nakajima,† and
Tatsuro Irimura3*
Extravasation of peripheral blood monocytes through vascular basement membranes requires degradation of extracellular matrix
components including heparan sulfate proteoglycans (HSPGs). Heparanase, the heparan sulfate-specific endo-␤-glucuronidase,
has previously been shown to be a key enzyme in melanoma invasion, yet its involvement in monocyte extravasation has not been
elucidated. We examined a potential regulatory mechanism of heparanase in HSPG degradation and transmigration through
basement membranes in leukocyte trafficking using human promonocytic leukemia U937 and THP-1 cells. PMA-treated cells were
shown to degrade 35S-sulfated HSPG in endothelial extracellular matrix into fragments of an approximate molecular mass of 5

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kDa. This was not found with untreated cells. The gene expression levels of heparanase or the enzyme activity of the amount of
cell lysates were no different between untreated and treated cells. Immunocytochemical staining with anti-heparanase mAb
revealed pericellular distribution of heparanase in PMA-treated cells but not in untreated cells. Cell surface heparanase capped
into a restricted area on PMA-treated cells when they were allowed to adhere. Addition of a chemoattractant fMLP induced
polarization of the PMA-treated cells and heparanase redistribution at the leading edge of migration. Therefore a major regu-
latory process of heparanase activity in the cells seems to be surface expression and capping of the enzyme. Addition of the
anti-heparanase Ab significantly inhibited enzymatic activity and transmigration of the PMA-treated cells, suggesting that the cell
surface redistribution of heparanase is involved in monocyte extravasation through basement membranes. The Journal of Im-
munology, 2004, 172: 3830 –3835.

M acrophages and related cells play essential roles in the HS. The HS moieties play essential roles in the interaction of
immune system, such as inflammation, defense against HSPG with a wide range of molecules including ECM components
microbial infection, immunity to foreign substances, (collagen, laminin, fibronectin, and others), cytokines (basic fibro-
wound healing, and angiogenesis. Monocytes circulate throughout blast growth factor, platelet-derived growth factor, hepatocyte
the body, extravasate through the endothelial lining of the blood growth factor, and others), and enzymes (lipoprotein lipase and
vessel wall, and enter the underlying tissue in response to local others) (1–3). Degradation of HS causes loss of mechanical integ-
inflammation. During the process, monocytes should pass through rity of basement membrane and release of soluble mediators.
the vascular basement membrane that supports the structure and The first indication that HS maintains the mechanical integrity
survival of endothelial cells and also prevents the vessels from of basement membrane came from a work in which heparitinase
mechanical destruction. The basement membrane mainly consists digestion of glomerular basement membranes resulted in a loss of
of type IV collagen, laminin, and heparan sulfate (HS)4 proteo- function (4). The ability of tumor cells to degrade basement mem-
glycans (HSPGs). Degradation of these basement membrane com-
brane was shown to be due to a HS-specific endo-␤-glucuronidase
ponents results in disintegration of the structure and it is conceiv-
(5, 6). cDNA cloning and expression of human heparanase have
able that such processes are a regulatory step for the extravasation.
recently been reported by four groups (7–10). The cDNA encodes
HSPGs are ubiquitous in extracellular matrices (ECMs) including
a unique protein of 543 amino acids that contains a potential signal
basement membranes, and consist of diverse core polypeptides and
peptide sequence and six putative N-linked glycosylation sites. It
was predicted that the 543-aa polypeptide formed a proenzyme
*Laboratory of Cancer Biology and Molecular Immunology, Graduate School of that was processed to be a mature 50 kDa active enzyme after
Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; and †Tsukuba Research removal of 157 N-terminal amino acids. The active enzyme has
Institute, Novartis Pharma, Tsukuba, Japan
been claimed to be a heterodimer that comprises the 50-kDa
Received for publication August 26, 2003. Accepted for publication January 7, 2004. polypeptide and a short fragment of 8-kDa peptide derived from
The costs of publication of this article were defrayed in part by the payment of page the N terminus of heparanase proenzyme (11). The active enzyme
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact. shows at least 100-fold HS degradation activity in comparison to
1
This work was supported by Grants-in-Aid from the Ministry of Education, Science, the proenzyme (8), therefore this processing could be one of the
Sports and Culture of Japan (11557180, 11672162, and 12307054), and from the critical regulatory steps of heparanase. Although it was assumed
Program for Promotion of Basic Research Activities for Innovative Biosciences.
that secreted or membrane-associated heparanase is responsible for
2
Current address: Division of Cell Biology, Institute of Life Science, Soka Univer- the degradation of ECM, the mechanisms involved in translocation
sity, Tokyo 192-8577, Japan.
3
of the enzyme have not been elucidated.
Address correspondence and reprint requests to Dr. Tatsuro Irimura, Graduate
School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Heparanase activity was reported in platelets, neutrophils,
Tokyo 113-0033, Japan. E-mail address: irimura@mol.f.u-tokyo.ac.jp monocytes, macrophages, Langerhans cells, astrocytes, activated
4
Abbreviations used in this paper: HS, heparan sulfate; HSPG, HS proteoglycan; (but not resting) rat T lymphocytes, and umbilical vein endothelial
ECM, extracellular matrix; FL-HS, fluoresceinamine-labeled HS; SVBCE, simian
virus bovine corneal endothelial cell; EDC, 1-ethyl-3-(3-dimethylaminopropyl)car- cells or smooth-muscle cells (12–19). It is likely that heparanase is
bodiimide hydrochloride; MMP, matrix metalloproteinase. required for extravasation of the cells in the immune system, and

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00


The Journal of Immunology 3831

the activity should be under tight regulation to avoid tissue dam- X-100, 1 mM PMSF, 0.2 mM AEBSF, 10 ␮g/ml leupeptin, 10 ␮g/ml
age. In the present study, we asked whether the heparanase is in- pepstatin A, 1 ␮g/ml aprotinin, pH 7.5) on ice for 30 min, followed by
volved in macrophage extravasation by use of macrophage-like centrifugation at 15,000 rpm for 10 min. Protein concentrations of the
supernatants were determined with bicinchoninic acid protein assay using
cell lines. Regulation of heparanase activity during macrophage BSA as a standard (Pierce). For preparation of fluoresceinated HS as the
differentiation and attachment to basement membranes was also heparanase substrate, HS (sodium salt, 1 mg), EDC (0.2 mg), and fluores-
investigated. A major regulatory process seems to be its unique ceinamine (FL; Fluka, Tokyo, Japan) (5 ␮g) were dissolved in water, and
spatial distribution. stirred for 1 h at room temperature, followed by dialysis overnight with
water. The solution was then concentrated with a Centricon 30 concentrator
(Amicon, Bedford, MA). The ratio of attached fluorescein to unmodified
Materials and Methods carboxyl group was determined using the carbazole-sulfuric acid method.
Chemicals An enzymatic reaction was conducted in a 100 ␮l mixture containing 25
mM sodium acetate buffer (pH 5.5), 5 ␮g of FL-HS, 20 mM D-saccharic
RPMI 1640 medium was purchased from Nissui Pharmaceuticals (Tokyo,
acid 1,4-lactone (Sigma-Aldrich), and cell lysates at 37°C for 24 h. The
Japan), FCS from BioWhittaker (Walkersville, MD), PMSF from Wako
reaction was stopped by the addition of 100 ␮g of heparin and subsequent
Biochemicals (Osaka, Japan), PMA (P8139), heparin (H3400), 4-(2-ami-
boiling for 5 min, followed by addition of solution (5 M NaCl, 250 mM
noethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), leupeptin, pep-
Tris-HCl, 0.5% Triton X-100, pH 7.4). The mixture was then centrifuged
statin A, aprotinin, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
at 1000 rpm for 5 min to precipitate the insoluble material. The products of
(EDC) from Sigma-Aldrich (St. Louis, MO), HS (sodium salt) obtained
FL-HS yielded by this reaction were analyzed by high-speed gel perme-
from bovine kidney from Seikagaku Kogyo (Tokyo, Japan), normal mouse
ation chromatography. Briefly, a 50 –100 ␮l aliquot of the supernatant was
IgG1 from Zymed Laboratories (South San Francisco, CA), and FITC-
injected into Superose 12 HR 10/30 column (Amersham Biosciences)
conjugated goat anti-mouse IgG(Fc) Ab F(ab⬘)2 from Sigma-Aldrich. Pu-

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equilibrated with PBS and eluted at 0.5 ml/min. Eluates were monitored by
rified monoclonal anti-human heparanase mAb was prepared as described
fluorescence intensity at 520 nm (excitation at 492 nm). The activity was
elsewhere (Ref. 20 and T. Nobuhisa, M. Nakajima, J. Sagara, S. Taniguchi,
determined from HPLC chromatograms by measuring an area of the peak
T. Tanaka, and Y. Naomoto, manuscript in preparation).
of the intact FL-HS and degraded FL-HS.
Cell culture
Immunocytochemistry
U937 and THP-1 leukemia cells were cultured in RPMI 1640 medium
supplemented with 10% FCS at 37°C with 5% CO2. For macrophage- U937 cells (5 ⫻ 104 cells/well) cultured on a chamber slide (154534; Nalge
directed differentiation, U937 and THP-1 were plated at a density of 2.5 ⫻ Nunc, Naperville, IL) or those cultured in suspension and fixed on a cham-
106 cells in 10-cm culture dish (Falcon 3003; BD Labware, Franklin Lakes, ber slide by centrifugation (500 rpm, 5 min) were washed with Dulbecco’s
NJ) and incubated at 37°C with 5% CO2 for 24 h. Cells were collected and modified PBS (PBS containing 0.91 mM CaCl2 and 0.49 mM MgCl2), and
replated at a density of 3.0 ⫻ 106 cells in the same dish that was pretreated fixed with 4% paraformaldehyde supplemented with 8% sucrose. For per-
with siliconizing agents (Pierce, Rockford, IL) in the presence of 50 nM meabilization, the fixed cells were subsequently treated with 0.1% saponin.
PMA. The culture was maintained at 37°C with 5% CO2 for 24 or 48 h. After washing with Dulbecco’s modified PBS, the cells were blocked with
Simian virus bovine corneal endothelial cells (SVBCE) were maintained in 10 ␮g/ml normal goat IgG (Zymed Laboratories) in Dulbecco’s modified
1:1 (v/v) mixture of DMEM and Ham’s F-12 supplemented with 10% FCS. PBS for 30 min, treated with the first Abs (10 ␮g/ml) in PBS containing 3%
BSA and 2% normal goat serum overnight at 4°C, then treated with 5
Measurement of cell-mediated HSPG degradation ␮g/ml FITC-conjugated anti-mouse IgG(Fc) Abs F(ab⬘)2 for 15 min at
Cell-mediated HSPG degradation was measured as previously described 22°C. In permeabilized conditions, 0.1% saponin was added throughout the
elsewhere (21). Briefly, ECM prepared from SVBCE was metabolically whole procedure. Stained cells were examined on a confocal microscope
labeled with their sulfate with Na2[35S]O4. Subconfluent SVBCE was cul- (MRC-1024; Bio-Rad, Hemel Hempstead, U.K.) equipped with a krypton/
tured on a 6-well plate (MS-80060; Sumitomo Bakelite, Osaka, Japan) in argon laser.
serum-free RPMI 1640 medium containing Na2[35S]O4 (Muromachi For cell polarization assays, differentiated U937 cells (5 ⫻ 104 cells/
Chemical, Tokyo, Japan) for 48 h and depleted by 0.5% Nonidet P-40. well) were cultured for 1 h in a well of 4-well chamber slides in which solid
Cells were cultured on ECM at a density of 2.5 ⫻ 106 cells per well for agarose gel was prepared on one side of the well. (154526; Nalge Nunc).
24 h at pH 6.6. Cultured medium was collected, and subsequently applied A chemotactic peptide fMLP was added to the gel (10⫺5 M), and the
to a Sephacryl S-300 column (Amersham Biosciences, Piscataway, NJ). chamber slides were incubated for 6 h. After the removal of the gel, cells
Nondegraded HSPGs were eluted near to the V0, peak I, whereas fragments were fixed with 4% paraformaldehyde supplemented with 8% glucose and
of HS were eluted at 0.5⬍Kav⬍0.8, peak II. stained for heparanase by the same method previously described.

RNA isolation and competitive RT-PCR Flow cytometry


Total RNA was isolated with Ultraspec-RNA solution (Biotecx Laborato- U937 cells cultured in suspension were fixed in 4% paraformaldehyde
ries, Houston, TX) according to the manufacturer’s instructions. After re- supplemented with 8% sucrose. Normal goat IgG (Zymed Laboratories) at
verse transcription of 2 ␮g of total RNA by oligo(dT) priming, the resulting 10 ␮g/ml in Dulbecco’s modified PBS was applied to block nonspecific
single strand cDNA was amplified using TaqGold DNA polymerase bindings for 30 min. The cells were incubated with Abs (10 ␮g/ml) in PBS
(Roche, Indianapolis, IN) and an appropriate buffer solution. The PCR containing 3% BSA and 2% normal goat serum for 30 min on ice, then
primers used (8) were heparanase sense (5⬘-TTCGATCCCAAGAAG treated with 5 ␮g/ml FITC-conjugated anti-mouse IgG(Fc) Abs F(ab⬘)2 for
GAATCAAC-3⬘), heparanase antisense (5⬘-GTAGTGATGCCATGTA 15 min on ice. Five thousand cells were analyzed using an Epics Elite flow
ACTGAATC-3⬘), ␤-actin sense (5⬘-CCTTCATTGACCTCAACTAC-3⬘), cytometer (Coulter, Miami, FL).
and ␤-actin antisense (5⬘-ACCACAGTCCATGCCATCACT-3⬘). PCR
was done on 100 ng of single strand cDNA in the presence of 5 ␮M of each
oligonucleotide primer, 2 mM dNTPs, 10⫻ PCR buffer, and competitor Transmigration assay
cDNA, using TaqGold DNA polymerase. Competitor cDNA (5⬘-GTAGT Transwells (8-␮m pore size, ␾ ⫽ 6.4 mm; Asahi Techno Glass, Tokyo,
GATGCCATGTAACTGAATCGAGGCTGACCAACATCAGGAC-3⬘) Japan) were added with 50 ␮g of Matrigel (BD Labware, Bedford, MA)
was prepared according to the manufacturer’s instructions. Reaction mix- solubilized in 200 ␮l of PBS, and air-dried at room temperature. The
tures were incubated in a Perkin-Elmer DNA thermal cycler (Perkin- transwells were then assembled in a 24-well plate (MS-80240; Sumitomo
Elmer/Cetus, Norwalk CT; heparanase: 50 cycles, denaturation at 94°C for Bakelite). The lower chambers were filled with 600 ␮l of RPMI 1640
45 s, annealing at 60°C for 60 s, extension at 72°C for 60 s; ␤-actin: 25 medium supplemented with 10 ␮g/ml fibronectin and the upper chamber of
cycles, denaturation at 94°C for 30 s, annealing at 59°C for 30 s, extension each transwell was filled with 200 ␮l of U937 or differentiated U937 (2 ⫻
at 72°C for 60 s). Aliquots of 10 ␮l of the amplification products were 105 cells/ml). The plates were incubated under humidified conditions at
separated by 2.0% agarose gel electrophoresis, visualized by ethidium 37°C with 5% CO2 for 24 h. Nonmigrating cells on the upper surface were
bromide staining, and quantified by NIH image analysis. scraped gently and removed. The filters were fixed in ethanol and stained
Measurement of heparanase activity in the cell lysates with Giemsa solution. The number of cells per magnification at ⫻400
high-power field that migrated to the lower surface of the filter was counted
For preparation of cell lysates, cells (5 ⫻ 106) were collected and lysed under a microscope. A mean of cell counts in five independent high-power
with 250 ␮l of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton fields per filter was obtained and indicated.
3832 CELL SURFACE EXPRESSION OF HEPARANASE

Preparation of peripheral blood monocytes


Peripheral blood monocytes were isolated from venous blood drawn from
normal healthy volunteers at the Tokyo Metropolitan Red Cross Blood
Center (Tokyo, Japan) by using an anti-CD14 mAb-coated microbeads and
magnetic cell separation system (MACS; Miltenyi Biotec, Bergisch Glad-
bach, Germany) as described elsewhere (22, 23). Purity of monocytes was
91.3 ⫾ 1.7% (n ⫽ 3, mean ⫾ SD), judged as the percentage of CD14⫹
cells by flow cytometric measurements.

Results
PMA-treated U937 and THP-1 cells degrade ECM HS
Promonocytic leukemia cell lines U937 and THP-1 before or after
PMA treatment were used in this study. Size distribution of re-
leased 35S-sulfated materials from endothelial ECM was deter-
mined after incubation with these cells. The released radioactivity
from ECM after incubation with untreated U937 or THP-1 cells
eluted at the void volume fraction (Fig. 1, A and B). The size

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distribution of 35S-sulfated materials released by the presence of
PMA-treated U937 or THP-1 cells indicated that the degraded
ECM components had a molecular mass of ⬃5 kDa (Fig. 1, C and
D). Degradation of the 35S-sulfated component was not observed
when incubation was performed in the presence of heparin, an FIGURE 2. Competitive RT-PCR of the heparanase gene. A, Compet-
inhibitor for heparanase (Fig. 1, E). itive RT-PCR was performed with total RNA obtained from U937 and
THP-1 cells cultured with PMA for 0, 24, and 48 h. Primers for the hepara-
nase gene provided 564 bp of amplified fragment. B, The results from A
Expression level of heparanase gene did not alter after PMA were quantified and are indicated. The values were normalized by ␤-actin.
treatment The experiments are repeated three times and similar results were obtained.
The degradation of HS by these PMA-treated cells should be de-
pendent on heparanase because only one heparanase gene was
identified in humans (24). Using an internal sequence of the Heparanase activity levels in cell lysates were not altered by
heparanase gene as a competitor, semiquantitative RT-PCR of PMA treatment
heparanase mRNA expression was performed. The products were
found to migrate to a similar position as human heparanase mRNA A possibility that processing of heparanase proenzyme (7) modi-
from human breast carcinoma cell lines (8) (Fig. 2A). The amount fied heparanase activity after PMA treatment was assessed. Cell
of PCR-amplified products of U937 and THP-1 cells before and lysates from untreated and PMA-treated cells were compared for
after the PMA treatment did not differ (Fig. 2B), although degra- heparanase activity. FL-HS was used as a substrate (25).
dation of ECM HS by these cells was observed only after treatment Percentages of degraded HS were estimated and used as indi-
with PMA. cators of heparanase activity in the lysates. The degradation
seemed to be due to heparanase because pretreatment of the U937
cell lysate with anti-heparanase mAb (10 ␮g/ml) inhibited as much
as 80% of the degradation (data not shown). PMA treatment did
not influence the relative quantity of the enzymatic activity in
U937 and THP-1 cell lysates (Fig. 3). Therefore, we concluded
that untreated and PMA-treated cells contained similar amounts of
active heparanase.

PMA-treated cells express heparanase molecule on the cell


surface
Because the difference in HS degradation between untreated and
PMA-treated U937 cells was observed only when intact cells were
used, cell surface distribution or secretion of heparanase was
thought to be responsible for the difference. We therefore per-
formed immunocytochemical detection of this enzyme using
anti-heparanase mAb.
When the cells were permeabilized, heparanase associated with
unidentified granule-like structures in the cytoplasm irrespective of
FIGURE 1. Cell-mediated degradation of HSPG in endothelial cell-de- PMA treatment (Fig. 4, A and D). When the cells were not per-
rived ECM. U937 cells (A), THP-1 cells (B), U937 treated with PMA (C) meabilized, heparanase was distributed discretely on the surfaces
for 24 h, and THP-1 treated with PMA (D) for 24 h were added in a well
of PMA-treated cells (Fig. 4E). Flow cytometric analysis con-
precoated with ECM that had been labeled with Na2[35S]O4 and cocultured
for 24 h in the absence (F) or presence (E) of 100 ␮g/ml heparin. The
firmed the surface expression of heparanase (Fig. 4F). Cell surface
resultant supernatants were applied onto Sephacryl S-300 gel permeation localization of heparanase was not observed in untreated cells (Fig.
chromatography, and radioactivity of each fraction is shown. Arrows in- 4, B and C). The clear difference suggested that the difference in
dicate Vo and Vt. The experiments are repeated three times and similar cell-mediated HSPG degradation by untreated and PMA-treated
results were obtained. cells may be explained by the distribution of this enzyme.
The Journal of Immunology 3833

FIGURE 4. Intracellular and cell surface distribution of heparanase.


Intracellular (A and D) and cell surface (B and E) heparanases were im-
munostained and observed using confocal laser scanning microscopy.
Simultaneously, the fixed U937 cells in suspension were also stained with
anti-heparanase mAb (bold lines) or mouse IgG1 (broken lines) and ap-

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plied to flow cytometer (C and F). U937 (A–C) and U937 treated with
PMA (D–F) for 24 h are shown. Horizontal bar indicates 10 ␮m. The
experiments are repeated three times and similar results were obtained.

Human fibronectin was used as a chemoattractant in these exper-


iments. The number of PMA-treated U937 cells that migrated
FIGURE 3. Measurement of heparanase activity in cell lysates. A, Cell through the membrane was approximately double that of untreated
lysates were obtained from U937 and THP-1 cells cultured with PMA for cells. The number of cells in the lower chamber was significantly
0, 24, and 48 h. The mixture of lysates (200 ␮g) and FL-HS were incubated smaller when heparin or anti-heparanase mAb was added to the
for 24 h at pH 5.5 and applied onto Superose 12 gel permeation chroma-
upper chamber ( p ⬍ 0.05) (Fig. 7). These results suggest that cell
tography. B, Cell lysates obtained from U937 were diluted and incubated
surface heparanase is involved in the transmigration of PMA-
with FL-HS for 24 h immediately after the preparation of the cell lysates
obtained in three independent experiments. The mixture was analyzed as in treated U937 cells through basement membranes.
A. U937 (F), U937 treated with PMA (f) for 24 h, and U937 treated with
Capping of heparanase on the cell surface of peripheral blood
PMA (E) for 48 h indicate mean values of the three independent results.
Error bars indicate the SD. monocytes
A remaining question to be clarified is whether the cell surface
Transport of heparanase to the cell surfaces was inhibited by expression and capping of heparanase occurs in nontransformed
pretreatment with nocodazole before the PMA treatment under macrophages and related cells. CD14⫹ peripheral blood mono-
conditions in which microtubule structures were completely dis- cytes were stained with anti-heparanase mAb under nonpermeabi-
rupted (Fig. 5C). Expression of heparanase and HSPG degradation lized condition. A large portion of monocytes (68.7 ⫾ 7.1%, n ⫽
was completely blocked by the treatment (Fig. 5, D and E). 4, mean ⫾ SD) was stained with anti-heparanase mAb. Of the
heparanase-positive monocytes, 34.3 ⫾ 10.3% (n ⫽ 4, mean ⫾
Cell adhesion-dependent capping of heparanase SD) of the cells showed capping formation (Fig. 6, G and H).
When PMA-treated U937 cells were detached from the surface,
placed on the surface of a glass plate by centrifugation, and cell
surface localization of heparanase was examined, a discrete peri-
cellular staining around the rim of the cells was observed as de-
scribed earlier. However, when the cells were placed on a glass
plate and incubated, cell surface heparanase (Fig. 6D) relocalized
and became concentrated on one of the pericellular rims of the
cells. This so-called capping was observed as early as 5 min after
incubation at 37°C. Approximately 50% of the cells showed this
profile within 1 h (Fig. 6E). When the cells were placed on plates
coated with fibronectin, laminin, Matrigel, or BSA, we observed
similar capping distribution. When agarose gel containing a che-
motactic peptide fMLP was placed on the glass plate, the local-
ization of heparanase on the cellular rim was directed toward the FIGURE 5. Inhibition of transport of cell surface heparanase abrogates
concentration gradient of fMLP (Fig. 6F). This apparently indi- cell-mediated HSPG degradation. Before the PMA treatment for 24 h,
cated that heparanase accumulated on the leading edge of the mi- U937 cells were pretreated with 10 ␮M nocodazole for 5 h (C and D) or
vehicles (A and B). Intracellular (A and C) and cell surface (B and D)
grating cells. Such unidirectional capping was not observed when
heparanases were immunostained. Simultaneously, the treated cells were
a chemotactic stimulus was not provided (Fig. 6E).
added in a well precoated with labeled ECM and cocultured for 24 h. The
resultant supernatants of nocodazole-treated (solid line) or vehicle-treated
Heparanase is involved in transmigration through ECM
(broken line) cells were applied onto Sephacryl S-300 gel permeation chro-
Two chamber culture systems separated by membranes coated matography. Horizontal bar indicates 10 ␮m. The experiments are repeated
with Matrigel were used to examine transmigration of U937 cells. three times and similar results were obtained.
3834 CELL SURFACE EXPRESSION OF HEPARANASE

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FIGURE 7. Transmigration assay of U937 cells. A, U937 cells were
FIGURE 6. Capping of the heparanase molecule in response to adhe- treated with PMA for 0 or 24 h and plated on the upper chamber coated
sion and chemotactic stimulus. Light images (A–C) and cell surface hepara- with Matrigel in the absence (䡺) or presence (f) of 100 ␮g/ml heparin and
nase distribution (D–F) were observed in the U937 cells treated with PMA cultured for 24 h. The number of migrated cells below the chamber was
(A and D) for 24 h, the PMA-treated U937 cells (B and E) that adhered to counted in triplicate. B, A transmigration assay was performed as in A in
the glass slides for 1 h, and the PMA-treated U937 cells under the gradient the absence (䡺) or presence (f) of 50 ␮g/ml mouse IgG1 or anti-
of fMLP, for which concentrated fMLP solution was added on top (C and heparanase mAb (^). Data are expressed as the mean ⫾ SE of counts
F). Arrowheads indicate capping sites of the heparanase molecule. The measured in triplicate. ⴱ, p ⬍ 0.05.
experiments are repeated three times and similar results were obtained.
Horizontal bar indicates 10 ␮m. G and H, Monocytes were allowed to
adhere on a chamber slide for 1 h. Cell surface expression of heparanase in blood monocytes (Fig. 6, G and H), the cellular distribution of hepara-
peripheral blood monocytes was visualized by immunostaining followed nase we demonstrate in this study seem to represent a unique feature
by alkaline phosphatase colorization (red). Arrowheads indicate capping of a certain subpopulation of monocytes.
sites of the heparanase molecule. Anti-heparanase mAb (G) and mouse Heparanase was previously reported to be expressed in cells in
IgG1 (H) are shown. The experiments are repeated four times and similar the immune system, and these cells are capable of transmigrating
results were obtained. Horizontal bar indicates 10 ␮m. through vascular basement membranes in response to chemoat-
tractants and inflammatory stimuli. For example, expressions in
neutrophils, monocytes, macrophages, Langerhans cells, and acti-
Discussion vated (but not resting) T lymphocytes have been previously re-
Heparanase is an endo-␤-glucuronidase specific for HS. Hepara- ported (12, 14 –18). This enzyme is potentially involved in the
nase in metastatic melanoma cells is thought to be constitutively degradation of HSPGs in ECM by all of these cells. Alternatively,
active outside cell surfaces and capable of destroying surrounding heparanase might function as an adhesion molecule to ECM that
tissue, whereas the enzyme should be strictly regulated in cells of facilitates leukocyte adhesion as predicted previously (26). We
the immune system. We used U937 and THP-1 cells as models to demonstrated that migration of PMA-treated U937 cells through
study the spatial regulation of heparanase in macrophages. PMA- the reconstituted basement membrane (Matrigel) was significantly
treated cells exhibited degradation of HSPGs that were contained inhibited by addition of the anti-heparanase mAb (Fig. 7). There-
in endothelium-derived ECM (Fig. 1). This was not found with fore, heparanase is proven to be involved in the cell transmigration
untreated cells. Heparanase mRNA expression and the enzyme ac- through the reconstituted basement membrane. This was not due to
tivity in cell lysates before and after the PMA treatment was very the cytotoxic effect of the Ab because previous studies indicated
similar (Figs. 2 and 3). The supernatant of PMA-treated cells did that the transmigration was dependent at least in part on matrix
not show any detectable heparanase activity (data not shown), metalloproteinases (MMP) and other matrix degrading enzymes
therefore degradation of HSPGs was not due to released enzymes. (27). Our immunocytochemical study showed that one of the mem-
A most remarkable difference between the two populations was brane-associated MMP, MT1-MMP, colocalized with heparanase
cellular distribution of heparanase, i.e., cell surface expression in in PMA-treated U937 cells (data not shown). Cellular distribution
PMA-treated cells (Fig. 4). The requirement of microtubule-de- of MT1-MMP in macrophages has not been extensively studied. In
pendent molecular migration for the cell surface expression of the tumor cells, MT1-MMP has been shown to be an enzyme that
enzyme is achieved by the use of an inhibitor nocodazole (Fig. 5). localizes preferentially in caveolin-rich membrane (28), accumu-
After adhesion of the PMA-treated cells, capping of heparanase lates at the invadopodia (29), degrades and activates MMP-2, and
was induced (Fig. 6, A–F). Heparanase seems to be required for mac- thus facilitates ECM degradation (30). Other proteolytic enzymes
rophage transmigration through Matrigel that contains HS because such as seprase and DPP IV distribute at the invasive edge of
invasion was significantly inhibited by the addition of anti-heparanase tumor cells and fibroblasts (31, 32). Colocalization of a number of
mAb (Fig. 7). Taken together, we postulate that cell surface transport matrix degradation enzymes at the leading edge of macrophages
and capping of the heparanase are a series of important regulatory would be preferable to achieve effective transmigration through
mechanisms for heparanase activity in macrophages. As the hepara- basement membrane. Untreated U937 cells also showed some in-
nase is expressed on the cell surface in a large portion of peripheral vasive capacity that was not inhibited by either anti-heparanase Ab
The Journal of Immunology 3835

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A 300-kDa mannose phosphate receptor on activated T lympho-

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