© 2000 American Heart Association, Inc.
Vascular Biology |
Proliferative Effect of Lipoprotein Lipase on Human Vascular Smooth Muscle Cells
From the CHUM Research Center, Notre-Dame Hospital, Department of Nutrition (J.-C.M., G.R.), and Laboratory of Molecular Cardiology (L.L.), University of Montreal, Quebec, Canada.
Correspondence to Dr Geneviève Renier, CHUM Research Center, Notre-Dame Hospital, J.-A. De Seve Pavilion, Door Y 3622, 1560 Sherbrooke St E, Montreal, Quebec, Canada H2L 4M1. E-mail renierg{at}ere.umontreal.ca
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Abstract |
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Abstract—Vascular smooth muscle cell (VSMC) proliferation is a key event in the development and progression of atherosclerotic lesions. Accumulating evidence suggests that lipoprotein lipase (LPL) produced in the vascular wall may exert proatherogenic effects. The aim of the present study was to examine the effect of LPL on VSMC proliferation. Incubation of growth-arrested human VSMCs with purified endotoxin-free bovine LPL for 48 and 72 hours, in the absence of any added exogenous lipoproteins, resulted in a dose-dependent increase in VSMC growth. Addition of VLDLs to the culture media did not further enhance the LPL effect. Treatment of growth-arrested VSMCs with purified human or murine LPL (1 µg/mL) led to a similar increase in cell proliferation. Neutralization of bovine LPL by the monoclonal 5D2 antibody, irreversible inhibition, or heat inactivation of the lipase suppressed the LPL stimulatory effect on VSMC growth. Moreover, preincubation of VSMCs with the specific protein kinase C inhibitors calphostin C and chelerythrine totally abolished LPL-induced VSMC proliferation. In LPL-treated VSMCs, a significant increase in protein kinase C activity was observed. Treatment of VSMCs with heparinase III (1 U/mL) totally inhibited LPL-induced human VSMC proliferation. Taken together, these data indicate that LPL stimulates VSMC proliferation. LPL enzymatic activity, protein kinase C activation, and LPL binding to heparan sulfate proteoglycans expressed on VSMC surfaces are required for this effect. The stimulatory effect of LPL on VSMC proliferation may represent an additional mechanism through which the enzyme contributes to the progression of atherosclerosis.
Key Words: lipoprotein lipase • vascular smooth muscle cell • protein kinase C • proteoglycans • atherosclerosis
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Introduction |
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Lipoprotein lipase (LPL) is the key enzyme for the hydrolysis and removal of chylomicrons and VLDLs from the circulation.1 LPL acts on triglyceride-rich lipoproteins at the vascular endothelium, where it is bound to heparan sulfate proteoglycans (HSPGs).2 LPL is synthesized by a variety of cells, including adipocytes,3 4 myocytes,5 and mammary epithelial cells.6 The enzyme is also produced by monocyte-derived macrophages and vascular smooth muscle cells (VSMCs), 2 prominent cellular components of the atherosclerotic lesion.7 8 9 Although plasma LPL activity tends to drive lipoprotein metabolism in a nonatherogenic direction,10 11 12 LPL produced in the vascular wall may act as a proatherogenic protein. Indeed, LPL has been shown to mediate uptake of lipoprotein particles by vascular cells,13 14 15 to promote lipoprotein retention to the extracellular matrix,16 17 to induce the expression of the proatherogenic cytokine tumor necrosis factor-
,18 19 and to enhance monocyte adhesion to
endothelial cells.20 21 Furthermore,
recent studies indicate that LPL enhances cellular proteoglycan
production22 and promotes foam cell formation and
atherosclerosis in vivo.23 VSMC migration and proliferation are typical features of intimal hyperplasia and atherogenesis.24 These biological processes are mediated by cytokines and growth factors released by infiltrating inflammatory cells and neighboring endothelial cells.25 The proliferative response of VSMCs to various stimuli involves protein kinase C (PKC) activation.26 27 28 In light of our previous study demonstrating that LPL activates PKC in human mononuclear cells,19 the present study was conducted to investigate whether LPL may exert a direct stimulatory effect on VSMC proliferation and to determine the mechanisms responsible for this effect.
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Methods |
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Reagents
DMEM and Coomassie brilliant blue were purchased from ICN Biomedicals Inc. FBS was obtained from Wisent Inc. Penicillin-streptomycin was purchased from Flow. Fatty acid–poor and endotoxin-free BSA fraction V, calphostin C, and chelerythrine were purchased from Calbiochem. Affinity-purified bovine LPL, lipopolysaccharide (LPS), heparin, heparinase III, human VLDLs, guanidine hydrochloride (GuHCl), and PMSF were obtained from Sigma Chemical Co. RPMI-1640 medium, PBS, polymyxin B sulfate, HBSS, and PKC assay kit were purchased from Gibco-BRL. Ethanol, methanol, and acetic acid were obtained from Fisher Scientific. Perchloric acid was purchased from BDH Inc. Mouse IgG1 isotype control (clone No. 11711.11), platelet-derived growth factor (PDGF)-BB, basic fibroblast growth factor (bFGF), transforming growth factor-ß (TGF-ß), and antibodies against these antigens were purchased from R&D Systems. The monoclonal anti-LPL antibody 5D2 (MAb 5D2) was a generous gift of Dr J.D. Brunzell, University of Washington, Seattle. [Methyl-3H]thymidine was obtained from NEN Life Science Products. The Silver Stain Plus kit was purchased from Bio-Rad. The chemiluminescence Western blotting kit was obtained from Amersham Pharmacia.
VSMC Isolation and Culture
Human VSMCs were isolated from saphenous veins obtained after
bypass surgery, with informed consent of the patients. Primary human
cells were grown in DMEM supplemented with 20% (vol/vol) FBS and 1%
(vol/vol) penicillin-streptomycin. Cell purity of human explant
preparations (passage 2 to 5) was determined by -actin
immunostaining. Briefly, VSMCs were cultured in chamber
slides, washed twice with PBS, and fixed for 10 minutes at 4°C in
cold methanol. After further washing, fixed cells were incubated for 30
minutes at room temperature with PBS containing 10% FBS. Chamber
slides were then incubated at 37°C for 2 hours with a monoclonal
anti–-smooth muscle actin antibody (Sigma) diluted in 1% FBS-PBS
(1/100), followed by incubation in the dark for 1 hour at 37°C with
the secondary goat anti-rabbit IgG antibody. After PBS washing, nuclei
were stained for 15 minutes with 0.01% Hoechst solution (Sigma). After
successive washes, slides were mounted with Fluorsave Reagent
(Calbiochem) and then observed with an epifluorescence
microscope.
Murine Macrophages
The J774 macrophage cell line was obtained from the
American Type Culture Collection. The cells were cultured in DMEM
containing 10% FCS and 100 µg/mL penicillin-streptomycin.
Purification of the LPL Preparations
The affinity-purified bovine LPL was dialyzed against saline
with 10 000 molecular weight cutoff Slide-A-Lyzer dialysis
cassettes from Pierce. The enzyme preparation was next filtered with an
endotoxin removal resin from Associates of Cape Cod.
The purity of the bovine LPL preparation was assessed by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE) using 4% to 12% gradient gels. Proteins were either
stained with Coomassie brilliant blue or silver or transferred to
nitrocellulose membranes for Western blot analysis. The
membranes were blocked for 3 hours at room temperature with PBS
containing 0.1% Tween 20 and 5% dry milk. After being washed, the
membranes were incubated overnight at 4°C with the appropriate
concentration of the primary antibody in PBS–0.1% Tween 20. At the
end of the incubation period, the membranes were washed and incubated
with a 1:1000 dilution of horseradish peroxidase–conjugated goat
anti-mouse IgG for 1 hour at room temperature. Blots were visualized by
enhanced chemiluminescence (ECL, Amersham Pharmacia). Silver and
Coomassie brilliant blue staining of the gels showed a single major
band in the molecular weight range expected of monomeric lipase
(Mr 55 kDa) (Figure 1A
and 1B, respectively). Because milk
contains some heparin-binding growth factors,29 the
purity of the bovine LPL preparation was further established by
immunoblotting with antibodies against growth factors
present in milk that might contaminate our LPL preparation. As
shown in Figure 1C, 1D, and 1E, PDGF, TGF-ß, and bFGF proteins
were not detected on immunoblots. Western blot
analysis using MAb 5D2 showed an immunoreactive band of 55 kDa
(Figure 1F).
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Human LPL was purified from 100 mL of postheparin plasma by 2 successive affinity chromatography steps on heparin-Sepharose 4B columns as previously described.30 31 Briefly, plasma was mixed with 50 mmol/L NH4OH-HCl, pH 8.5, and applied to a preequilibrated heparin-Sepharose column. After washing, the LPL preparation was eluted with the same buffer containing 2.0 mol/L NaCl. LPL collected in the effluent was further purified on a second heparin-Sepharose column, and the enzyme was eluted with an NaCl gradient ranging from 0.3 to 2.0 mol/L. The fractions containing enzyme activity were pooled, dialyzed against 25 L of 0.005 mol/L ammonium bicarbonate, and lyophilized.
Murine LPL was isolated as previously described.32 Briefly, J774 macrophages were plated at a density of 5x106 cells per T75 flask and maintained for 2 days in DMEM supplemented with 10% FBS. Cells were then washed and incubated with 5 mL DMEM containing 0.5% BSA and 0.1 U/mL heparin. After 12 hours, supernatants were mixed with an equal volume of cold loading buffer (10 mmol/L sodium phosphate [pH 7.0], 0.15 mol/L NaCl, and 30% glycerol) and loaded into a heparin-Sepharose affinity column equilibrated with loading buffer. The column was washed overnight with loading buffer, and LPL was eluted with the same buffer containing 1.5 mol/L NaCl.
For inhibition of LPL activity, the enzyme was either boiled for 30 minutes or treated with GuHCl or PMSF. Guanidine-inactivated LPL was prepared by overnight dialysis at 4°C against 6 mol/L GuHCl, 10 mmol/L Tris-HCl, pH 8.5, followed by removal of guanidine by dialysis against PBS. PMSF-inactivated LPL was obtained by incubating the enzyme for 30 minutes at 4°C with PBS containing 1 mmol/L PMSF.
All LPL preparations were assayed for protein mass and enzymatic activities. Total protein content of the LPL preparations were measured according to the method of Bradford33 with a colorimetric assay (Bio-Rad) and BSA as standard. Enzymatic activities of the LPL preparations were determined with the Confluolip kit (Progen).34
Determination of Endotoxin Concentrations
Endotoxin content of all media and of all LPL preparations (1
µg/mL) was determined by the Limulus amoebocyte lysate
assay (Sigma Chemical Co) and was consistently found to be <10
pg/mL. Treatment of VSMCs from all donors with LPS 10 pg/mL did not
induce cell proliferation. Moreover, addition of an LPS
inhibitor, polymyxin B sulfate (100 µg/mL), did not
inhibit LPL-induced VSMC proliferation.
VSMC Proliferation Assay
Human VSMCs were trypsinized and cultured at a density of 7500
cells/cm2 in DMEM supplemented with 1% (vol/vol)
penicillin-streptomycin and 20% (vol/vol) FBS in 24-well plates
(Falcon, Becton Dickinson). After 24 hours, cultured cells were washed
with PBS and growth-arrested for 48 hours by serum
deprivation. VSMCs cultured in serum-free DMEM were then
treated with the appropriate experimental agent(s) for 24 hours and
incubated in the presence of sterile
[methyl-3H]thymidine (5 µCi/mL) for an
additional 48-hour period. Nonincorporated
[3H]thymidine was removed by washing with cold
PBS. Cells were next fixed with cold ethanol–acetic acid solution
(3:1) for 10 minutes at 4°C, washed twice, and incubated with
perchloric acid 0.5N for 15 minutes at 4°C. After further washing,
VSMCs were incubated with perchloric acid 0.5N for 30 minutes at 80°C
and allowed to detach. The level of
[3H]thymidine incorporation was determined by
scintillation counting (Packard).
Determination of VSMC proliferation was also assessed by hemocytometry. On the basis of the good correlation observed between changes in cell number and the level of [3H]thymidine uptake by VSMCs, the latter was used to evaluate the effect of LPL on VSMC proliferation in this study.
Measurement of PKC Activity
Adherent human VSMCs were recovered and homogenized
(Dounce; 15 strokes) in 500 µL of ice-cold buffer A (20 mmol/L
Tris [pH 7.5], 0.5 mmol/L EDTA, 0.5 mmol/L EGTA, 25 µg/mL
aprotinin, and 25 µg/mL leupeptin). The membrane and cytosolic
fractions were separated by ultracentrifugation
(100 000g for 30 minutes at 4°C). After recovery of
high-speed supernatants containing cytosolic PKC, the corresponding
membrane pellets were homogenized in 500 µL of buffer A
containing 0.5% Triton X-100. The enzyme from both fractions was
partially purified through DE52 chromatography columns.
After removal of unbound proteins by washing of the columns with buffer
B (20 mmol/L Tris [pH 7.5], 0.5 mmol/L EDTA, and 0.5
mmol/L EGTA), fractions containing PKC were eluted with buffer C
(20 mmol/L Tris [pH 7.5], 0.5 mmol/L EDTA, 0.5
mmol/L EGTA, 10 mmol/L ß-mercaptoethanol, and 0.2 mol/L NaCl).
Eluates were analyzed for PKC activity, following the optimum
conditions of the assay, by measuring the incorporation of
32P into the synthetic peptide Ac-myelin basic
protein (4–14). The specificity of the assay was determined by
subtracting the radioactivity obtained in the presence of the
pseudosubstrate inhibitor PKC19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 from total
radioactivity of the assay. Data were expressed as percentages
considering the control as 100% activity.
Determination of Cell Viability
Cell viability after treatment with experimental agents was
assessed by trypan blue exclusion. Viability was found to be
>90%.
Statistical Analyses
Data were analyzed by Student’s t test for
single comparisons and by 1-way ANOVA followed by the
Student-Newman-Keuls test for multiple comparisons. Results are
expressed as mean±SEM. A value of P<0.05 was considered
significant.
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Results |
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Direct Effect of LPL on Human VSMC Proliferation
Growth-arrested human VSMCs were treated for 24, 48, or 72 hours with exogenous bovine LPL in the absence of any added exogenous lipoproteins. Treatment of VSMCs with LPL for 24 hours did not induce human VSMC proliferation (data not shown). In contrast, exposure of VSMCs to LPL for 48 or 72 hours induced VSMC proliferation in a dose-dependent manner (Figure 2A
and 2B).
Compared with control, LPL significantly stimulated VSMC proliferation
in doses as low as 250 and 100 ng/mL after 48 and 72 hours of
induction, respectively. The extent of LPL effect on VSMC growth was
similar at both time points, with a maximal stimulation being observed
at a dose of 1 µg/mL LPL. Addition of VLDLs (20 µg protein/mL) to
the medium of VSMCs treated with LPL (1 µg/mL) did not further
enhance the stimulatory effect of bovine LPL on VSMC growth (VSMC
proliferation [% increase over control values]: LPL, 192±20,
P<0.001; LPL+VLDLs, 164±8, P<0.001). Treatment
of growth-arrested VSMCs with other sources of LPL, such as human and
murine LPL (1 µg/mL), induced VSMC proliferation to the same level of
stimulation as produced by bovine LPL (Figure 3A). To test whether LPL-induced VSMC
proliferation could be due to possible traces of growth factors in our
bovine LPL preparation, we next examined the effect of LPL on VSMC
proliferation in the presence or absence of antibodies against PDGF,
bFGF, and TGF-ß. Addition of these antibodies to the incubation
medium did not abrogate the LPL-induced VSMC proliferation
(Table). Antibodies against PDGF,
bFGF, and TGF-ß inhibited VSMC proliferation induced by these growth
factors (data not shown).
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To assess the specificity of the LPL effect on VSMC proliferation, the
enzyme was immunoneutralized with the anti-LPL antibody 5D2.
Immunoneutralization of LPL with MAb 5D2 totally suppressed LPL-induced
VSMC proliferation (Figure 3B
). In contrast, addition of an
irrelevant mouse IgG1 antibody did not affect the LPL effect (Figure 3B). As an additional control study, the effect of MAb 5D2 on
the murine LPL-induced VSMC proliferation was examined. The monoclonal
5D2 antibody, which does not recognize murine LPL, did not inhibit the
proliferative effect of this lipase (VSMC proliferation [% increase
over control values]: murine LPL, 180±18, P<0.05; murine
LPL+MAb 5D2, 214±27, P<0.05).
Effect of LPL Inactivation on LPL-Induced VSMC
Proliferation
To determine whether enzymatic activity was required for the LPL
effect on VSMC proliferation, we initially tested the ability of
GuHCl–inactivated LPL to induce VSMC growth. Treatment of
LPL with GuHCl did not significantly decrease the stimulatory effect of
LPL on VSMC growth (VSMC proliferation [% increase over control
values]: LPL, 205±33%; GuHCl-treated LPL, 142±82), whereas heat
inactivation of LPL by boiling of the enzyme for 30 minutes and
inactivation of the hydrolytic activity of the enzyme by the
serine-specific suicide substrate PMSF (1 mmol/L) totally
abolished the effect of LPL on VSMC proliferation (Figure 4).
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Effect of PKC Inhibitors on LPL-Stimulated VSMC
Proliferation
To investigate whether PKC activation is involved in LPL-induced
VSMC proliferation, the effect of the specific PKC
inhibitors calphostin C (1 µmol/L) and chelerythrine
(0.5 µmol/L) on LPL-induced VSMC proliferation was next
evaluated. Preincubation of VSMCs with these 2 PKC
inhibitors suppressed the LPL-stimulated VSMC growth
(Figure 5A and 5B).
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Effect of LPL on Human VSMC PKC Activity
Because the LPL-induced VSMC proliferation appeared to involve PKC
activation, we also measured the direct effect of LPL on PKC activity
in VSMCs. A maximal increase in PKC activity in the membrane fraction
of human VSMCs (2.6-fold increase over control values,
P<0.05) was observed after a 30-minute exposure of VSMCs to
LPL (1 µg/mL) (Figure 6). Both PMSF-
and heat-inactivated LPL failed to induce PKC translocation
in VSMCs. In addition, heparinase treatment of VSMCs before LPL
exposure significantly decreased LPL-induced PKC activity (Figure 6).
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Effect of Heparinase on LPL-Induced VSMC Proliferation
To establish the role of HSPGs expressed on the VSMC surface in
the LPL-induced VSMC proliferation, VSMCs were pretreated with
heparinase (1 U/mL) for 1 hour at 37°C before being incubated with
LPL. As shown in Figure 7, heparinase
treatment of VSMCs totally abolished the LPL-induced VSMC
proliferation.
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Discussion |
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The present study demonstrates for the first time that LPL exerts a direct stimulatory effect on human VSMC proliferation. Our finding that immunoneutralization of bovine LPL with MAb 5D2 suppresses the LPL-induced VSMC proliferation clearly demonstrates the specificity of the LPL effect. Our data showing that MAb 5D2, which does not recognize murine LPL,35 did not affect the proliferative effect of this lipase provide an additional evidence of the specificity of the LPL effect on VSMC proliferation. Evidence that LPL enzymatic activity is required for its effect on VSMC growth is given by our results demonstrating that heat inactivation or irreversible inhibition of LPL with PMSF totally abolished the lipase effect. The lack of inhibitory effect of GuHCl-inactivated LPL on VSMC growth is not surprising, considering the mild dissociation of LPL induced by GuHCl and the residual enzymatic activity in the guanidine-treated LPL preparations.36 Given the key role of fatty acids in the regulation of VSMC function,37 38 39 LPL could theoretically promote VSMC proliferation through fatty acids derived from its lipolytic action on triglyceride-rich lipoproteins. Our data showing that LPL induces VSMC proliferation in the absence of exogenous lipoproteins and that addition of exogenous lipoproteins to the incubation medium does not further enhance its stimulatory effect do not support this hypothesis. However, one cannot rule out that LPL exerts its effect on cell proliferation through fatty acids generated by the hydrolysis of membrane phospholipids. This possibility is supported by the recent finding that purified milk LPL indeed hydrolyzes phospholipids.40 Because LPL-induced phospholipid hydrolysis is less dependent on apoprotein C-II than triglyceride hydrolysis,40 the absence of apoprotein CII under our experimental conditions does not rule out the involvement of this process in the stimulatory effect of LPL on VSMC growth. Whether LPL binding to the VSMC cell surface actually causes the generation of fatty acids, lysophosphatidylcholine, or even diacylglycerol from cell membranes or plasma membrane vesicles will be determined in future studies.
LPL binds with high affinity to several receptors expressed on the VSMC
surface. These receptors include the LDL receptor–related
protein/2-macroglobulin receptor, the
VLDL receptor, and HSPGs.41 42 43 Our finding that treatment
of VSMCs with heparinase suppresses LPL-induced VSMC proliferation
suggests that LPL binding to HSPGs expressed on the VSMC surface is
essential for its effect on cell proliferation. However, because the
formation of a proteoglycan-rich pericellular matrix may be involved in
VSMC proliferation,44 inhibition of VSMC growth by
heparinase treatment of the cells could also be due to alteration of
the extracellular matrix integrity. Recently, Silver et
al45 showed that locally delivered heparinase reduced
medial VSMC proliferation induced by balloon catheter injury in rat
carotid arteries. Furthermore, these authors found that heparinase
inhibited both PDGF-BB– and bFGF-mediated increase in rat VSMC
proliferation,45 thus confirming that HSPGs also act as
coreceptors for growth factors involved in the stimulation of
arterial cell proliferation.46 47 Because
ligand clustering of the syndecan family of HSPGs is associated with
signaling events,48 49 one may also hypothesize that LPL
could induce intracellular signaling pathway(s) in VSMCs by binding to
members of the syndecan family of HSPGs expressed on the VSMC cell
surface.
The PKC signal transduction pathway plays a central role in many VSMC
functions, including cell proliferation.26 27 28 50 It was
recently demonstrated that bacterial phospholipase C, an enzyme
competing with LPL for binding to VSMCs, activates PKC in
VSMCs.51 Our data showing that LPL induces PKC activation
in VSMCs and that heat inactivation and PMSF inactivation of the enzyme
or heparinase treatment of VSMCs inhibit LPL-induced PKC activation
clearly suggest that the enzyme may promote the growth of these cells
by stimulating this signaling pathway. The involvement of PKC
activation in the direct stimulation of VSMC proliferation by LPL is
strengthened by our observations that pharmacological inhibition of PKC
by specific PKC inhibitors, including calphostin C and
chelerythrin, completely suppresses LPL-induced VSMC proliferation. The
PKC isoforms , ß1,
ß2, 
, and 
have been identified in human
VSMCs.52 It has been shown that the PKC isoforms and
ß1 are located primarily in the soluble
fraction of the cell and translocate to the particulate fraction on
12-o-tetradecanoylphorbol 13-acetate
stimulation, whereas the isoforms ß2, , and
are found primarily in the particulate
fraction.52 Our results showing that LPL induces the
translocation of PKC from the cytosol to the particulate fraction in
human VSMCs suggest that LPL-induced VSMC proliferation may involve the
translocation of the [Ca2+]-sensitive PKC
isoforms and ß1. This possibility is
further supported by our results demonstrating that calphostin C, which
interacts with the calcium-dependent regulatory domain of PKC,
suppresses LPL-induced VSMC proliferation. The contribution of
individual PKC isoforms in LPL-induced human VSMC proliferation will be
investigated in further studies.
Although the implications of our in vitro observations to atherosclerosis remain unclear, several lines of evidence suggest that LPL may regulate VSMC growth in the vascular wall. First, it has been demonstrated that monocyte-derived macrophages and VSMCs secrete LPL within atherosclerotic lesions.53 54 Second, association of the enzyme with the VSMC cell surface has been documented.55 Third, LPL has been shown to interact with the extracellular matrix, where it enhances the retention of atherogenic lipoproteins.56 The lipase capable of stimulating VSMC proliferation may therefore be derived from either newly secreted LPL or LPL bound to extracellular matrix. Although LPL from monocyte-derived macrophages may exert a paracrine effect by inducing the proliferation of neighboring VSMCs, LPL secreted by VSMCs may induce VSMC proliferation in an autocrine manner. Whether sufficient LPL is present in the vicinity of the artery wall for local regulation of VSMC function, however, remains to be determined.
In summary, our study demonstrates that LPL exerts a direct stimulatory effect on human VSMC proliferation and that this effect requires PKC activation and LPL binding to HSPG expressed on the VSMC surface. These results and the previous observation that human LPL induces the proliferation of natural killer cells57 support a new role for LPL, that of acting as a growth factor.
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Acknowledgments |
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This study was supported by grants from the Medical Research Council of Canada and from the Heart and Stroke Foundation of Canada. The authors thank Dr J.D. Brunzell (University of Washington, Seattle) for providing the MAb 5D2 antibody.
Received May 23, 2000; accepted July 11, 2000.
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