(Journal of Leukocyte Biology. 2000;68:131-136.)
© 2000
by Society for Leukocyte Biology
Proteasome-mediated regulation of interleukin-1ß turnover and export in human monocytes
Marlena A. Moors and
Steven B. Mizel
Department of Microbiology and Immunology, Wake Forest University Medical Center, Winston-Salem, North Carolina
Correspondence: Steven Mizel, Dept. of Microbiology and Immunology, Wake Forest University Medical Center, Winston-Salem, NC 27157. E-mail: smizel{at}wfubmc.edu
 |
ABSTRACT
|
|---|
Interleukin-1ß is a secreted protein that accumulates in the cytosol
as an inactive precursor (pIL-1ß) before processing and release of
biologically active protein. To understand the impact of this property
on IL-1ß production, we examined the intracellular stability of
pIL-1ß in lipopolysaccharide (LPS)-stimulated human monocytes.
Precursor IL-1ß was degraded with a relatively short half-life of
2.5 h in the promonocytic cell line, THP-1, and in primary
monocytes. MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) stabilized
pIL-1ß levels in THP-1 cells, suggesting that degradation was
proteasome-mediated, but this inhibitor was toxic for primary
monocytes, causing release of pIL-1ß as well as the cytoplasmic
enzyme, lactate dehydrogenase (LDH) into supernatants. In contrast,
clasto-lactacystin ß-lactone, a specific inhibitor of the
proteasome, caused a dose-dependent stabilization of intracellular
pIL-1ß, and this led to a corresponding increase in mIL-1ß and
pIL-1ß but not LDH release into culture supernatants. Therefore, by
regulating intracellular levels of precursor IL-1ß, the proteasome
plays an important and previously unrecognized role in controlling the
amount of biologically active IL-1ß that is exported by activated
monocytes.
Key Words: THP-1 MG132 lactacystin precursor interleukin-1ß degradation
 |
INTRODUCTION
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Interleukin-1ß (IL-1ß) is produced predominantly by monocytes
in response to inflammatory stimuli such as lipopolysaccharide (LPS)
and other bacterial products. It has a broad range of effects on immune
and nonimmune cells, but its major roles are in lymphocyte activation
and the induction of inflammatory responses [1
].
Injection of nanogram quantities of recombinant IL-1ß into humans
rapidly induces fever, hypotension, and the production of other
proinflammatory mediators [2
, 3
], effects
that are similar to those induced by LPS. In addition, IL-1ß has been
implicated in the etiology of certain inflammatory and autoimmune
diseases [reviewed in ref. 1
]. Given these biological effects, it is
important to understand the regulatory factors that control IL-1ß
production and release by inflammatory monocytes.
IL-1ß is synthesized as a 33-kDa precursor protein (pIL-1ß) that is
cleaved intracellularly by IL-1ß converting enzyme (ICE) to yield the
17-kDa mature form (mIL-1ß) [4
, 5
].
Monocytes have the capacity to secrete both pIL-1ß and mIL-1ß,
however, mIL-1ß is the biologically active form [6
,
7
]. Precursor but not mIL-1ß is found in cellular
lysates, therefore it is likely that processing and secretion are
intimately coordinated events [8
].
A feature of IL-1ß regulation that is of particular interest is its
capacity to be exported from the cell in the absence of a classic,
hydrophobic signal sequence. Export of IL-1ß proceeds via a novel
pathway that bypasses the endoplasmic reticulum and Golgi apparatus
[9
, 10
]. Although little is known about
this export mechanism, it has been demonstrated that, compared to
proteins secreted by the conventional, signal sequence-directed
pathway, release of IL-1ß is a relatively slow process. The precursor
protein accumulates in the cytosol before release [9
,
10
] and significant quantities of IL-1ß are not
detectable in monocyte supernatants until approximately 2 h after
synthesis [8
]. In contrast, proteins secreted by the
conventional pathway appear in supernatants within minutes after
synthesis [11
].
Proteins that reside in the cytosol for any length of time after
synthesis are potential targets for proteolysis. The proteasome, an
ATP-dependent multicatalytic protease complex, is responsible for
degrading the majority of normal and damaged cytosolic proteins, as
well as foreign proteins for immune recognition [12
].
However, under certain circumstances other proteases, including the
calcium-activated calpains and lysosomal proteases, can also degrade
cytosolic proteins [13
, 14
]. Given the
unusual features of IL-1ß export, an important issue in the
regulation of IL-1ß production is the intracellular stability of the
precursor protein. We demonstrate here that cytosolic pIL-1ß is
degraded fairly rapidly by the proteasome and that blocking the
proteolytic activity of the proteasome enhances release of IL-1ß by
activated monocytes.
 |
MATERIALS AND METHODS
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Reagents
MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) was obtained
from Biomol Research Laboratories (Plymouth Meeting, PA). PD-150606 and
clasto-lactacystin ß-lactone were obtained from Calbiochem
(San Diego, CA). E-64d was obtained from Sigma Chemical (St. Louis,
MO). Inhibitors were prepared as stock solutions in dimethyl sulfoxide
(DMSO). The preparation and use of polyclonal anti-IL-1ß antibody has
been described previously [15
].
Cell culture
The human monocytic cell line, THP-1, obtained from the American
Type Culture Collection, was maintained at a density of 2.5 x
105/mL to 1 x 106/mL in RPMI 1640 medium
containing 10% fetal bovine serum (FBS) and 50 µg/mL gentamicin
sulfate (complete RPMI).
Isolation of human peripheral blood monocytes
Peripheral blood mononuclear cells from healthy donors were
purified by density gradient centrifugation and cultured as described
previously [16
]. Briefly, 5 x 106 to
1 x 107/well mononuclear cells per well were cultured
in 24-well dishes for 2 h in serum-free RPMI 1640, after which
nonadherent cells were removed by washing with phosphate-buffered
saline (PBS). Adherent cells were cultured for 1618 h in complete
RPMI before assay.
Pulse/chase analysis, THP-1 cells
For assays, 5 x 106 log-phase THP-1 cells per
sample were centrifuged and pellets were resuspended in 1.5 mL of
methionine- and cysteine-free RPMI 1640 (ICN Pharmaceuticals, Irvine,
CA) containing 5% dialyzed FBS (labeling medium). Cells were cultured
in six-well dishes for 1 h in the presence of 100 pg/mL
Escherichia coli LPS (Sigma Chemical) after which 250
µCi/mL Tran35S-Label (ICN) was added for an additional
hour. Labeled cells were centrifuged and cell pellets were immediately
lysed with 0.5 mL ice-cold immunoprecipitation buffer (150 mM NaCl,
0.4% Nonidet P-40, 50 mM Tris, pH 8.0, 10 mM EDTA) containing
Calbiochem Protease Inhibitor Cocktail Set 1, or were chased with RPMI
1640 containing 5% FBS and 5 mM methionine (chase medium). After the
indicated chase periods, cells were centrifuged at 4°C, supernatants
were collected and concentrated to approximately 0.5 mL using
Centricon-10 microconcentrators (Amicon, Danvers, MA), and cell pellets
were lysed as described above. Lysates were cleared of particulate
material by centrifugation at 4°C before immunoprecipitation.
Pulse/chase analysis, primary human monocytes
For assays, adherent cells were washed once with PBS and 0.25
mL/well labeling medium containing 100 pg/mL LPS was added. Cells were
cultured for 1 h and labeled for an additional hour as described
for THP-1 cells. After the labeling period, cells were washed once with
PBS and lysed with ice-cold immunoprecipitation buffer or chased with
0.25 mL/well chase medium. After the indicated chase periods,
supernatants were collected and gently cleared of detached cells by
centrifugation at 260 g, and monolayers were lysed and
cleared of particulate material as described above.
Immunoprecipitation
IL-1ß was immunoprecipitated from THP-1 or monocyte lysates
and supernatants using polyclonal anti-IL-1ß antibody as described
previously [17
]. Immunoprecipitates were resolved by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and the gels were processed for autoradiography as described previously
[17
]. In addition, dried gels were scanned and pIL-1ß
bands were quantitated using an Ambis radioanalytic imaging system.
Results are expressed as the % IL-1ß remaining after the chase
period, relative to the time 0 (no chase) control.
Experiments were performed at least three times, and in each case, a
representative gel is shown.
Assay for IL-1ß release by primary human monocytes
Monocytes were prepared and cultured as described for
immunoprecipitations but were activated to synthesize IL-1ß for
3 h with 1 µg/mL LPS. This was followed by an additional
incubation in medium containing LPS with or without
clasto-lactacystin-ß lactone. Culture supernatants were
harvested after 5 h and gently cleared of detached cells by
centrifugation. The amount of pIL-1ß and mIL-1ß in the samples was
determined by enzyme-linked immunosorbent assay (ELISA; R & D Systems,
Minneapolis, MN). As determined by the manufacturer, the pIL-1ß ELISA
is specific for pIL-1ß and does not cross-react with mIL-1ß. The
mIL-1ß ELISA cross-reacts at a level of approximately 13% with
pIL-1ß. ELISAs were run in parallel on each sample, and values
reported for mIL-1ß have been corrected by subtracting 13% of the
total pIL-1ß value detected for each sample.
Assay for lactate dehydrogenase (LDH) activity
Supernatants were collected after the indicated chase periods
from monocyte cultures that were treated exactly as described for
pulse/chase analysis except the 35S-methionine label was
omitted. LDH activity released into supernatants was measured using the
Promega CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Madison, WI)
according to the manufacturers instructions. Percent cytotoxicity was
calculated as follows: [(experimental release - spontaneous
release)/(maximum release - spontaneous release)] x 100.
 |
RESULTS
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Half-life of pIL-1ß in THP-1 cells
To address the issue of protein half-life in the regulation of
IL-1 production, we examined the stability of pIL-1ß in the human
monocytic cell line, THP-1. To measure the rate of protein turnover due
to intracellular degradation, we stimulated the cells for 2 h with
100 pg/mL LPS. As shown by pulse/chase and immunoprecipitation
analysis, THP-1 cells stimulated with a low concentration of LPS
synthesize pIL-1ß but do not process or secrete the protein
(Fig. 1B
). As shown in Figure 1A and 1B
, the 33-kDa pIL-1ß protein is
degraded with a half-life of 2.53 h in THP-1 cells.

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Figure 1. Pulse/chase analysis of pIL-1ß in THP-1 cells. (A) IL-1ß was
immunoprecipitated from 35S-methionine-labeled THP-1 cell
lysates immediately after the labeling period (time 0) or
after chasing with cold methionine for 1, 2, 3, or 5 h. pIL-1ß
bands were quantitated using an Ambis radioanalytic imaging system and
results are expressed as the percent pIL-1ß remaining after the
chase, relative to the time 0 control (% control). (B)
IL-1ß was immunoprecipitated from 35S-methionine-labeled
THP-1 lysates and supernatants immediately after the labeling period
(time 0) or after chasing for 2.5 or 5 h. Percent
control values are indicated as in panel A. Molecular weight markers
are shown in the first lane. The arrow indicates the 33-kDa pIL-1ß
band.
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|
MG132 stabilizes pIL-1ß in THP-1 cells
Degradation of most cytosolic proteins is mediated by the
proteasome [12
]. To determine whether the proteasome
degrades pIL-1ß, THP-1 cells were pulse-labeled and chased for 2.5
and 5 h in the presence and absence of MG132, one of a group of
tripeptide aldehydes that block activity of the proteasome
[12
, 18
]. As shown in Figure 2
, treatment with 50 µM MG132 significantly stabilized pIL-1ß levels.
Approximately 90% of the total pIL-1ß synthesized was found in cell
lysates after a 2.5-h chase in the presence of inhibitor, compared with
60% in the absence of inhibitor. Similarly, after a 5-h chase, 79% of
the total pIL-1ß remained compared with 27% in the absence of
inhibitor. MG132 stabilized pIL-1ß levels almost completely at 1050
µM, the effective concentration range of tripeptide aldehydes for
inhibition of proteasome function in intact cells (data not shown)
[12
]. In no case was IL-1ß found in significant
amounts in THP-1 cell supernatants (Figs. 1
and 2)
. These results
suggest that degradation of pIL-1ß in THP-1 cells is
proteasome-mediated.

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Figure 2. Stabilization of pIL-1ß in THP-1 cells by MG132. Pulse/chase analysis
was performed as described in Figure 1
, except that in some samples
MG132 was included during the chase period at a final concentration of
50 µM. IL-1ß was immunoprecipitated from THP-1 cell lysates and
supernatants at the indicated time points.
|
|
Degradation of pIL-1ß in primary human monocytes
Having obtained evidence that the proteasome degrades pIL-ß in
THP-1 cells, it was important to analyze the regulation of pIL-1ß
turnover in primary human monocytes. As for THP-1 cells, pIL-1ß was
degraded with a half-life of approximately 2.5 h (Fig. 3
), although some donor-to-donor variability was observed (mean
half-life = 2.3 ± 0.6 h; range 13 h;
n = 16). Again, little to no IL-1ß was detected in
monocyte supernatants, indicating that turnover of cytosolic pIL-1ß
was due to degradation and not to processing and secretion. In contrast
to THP-1 cells, MG132 failed to stabilize pIL-1ß in primary monocytes
(Fig. 4
). There was no change in the degradation of pIL-1ß in the presence
and absence of inhibitor as measured over 2.5- and 5-h chase periods.
However, significant quantities of pIL-1ß were found in supernatants
of cells treated with 50 µM MG132. To determine whether release of
pIL-1ß in the presence of MG132 was specific for IL-1ß or was a
result of general cytotoxicity, we measured levels of the cytoplasmic
enzyme lactate dehydrogenase (LDH) in parallel. LDH was detectable in
supernatants of cells treated with MG132 but not in culture
supernatants from control cells (Fig. 4)
. In a similar experiment, the
appearance of pIL-1ß and LDH in supernatants after a 2.5-h chase in
the presence of MG132 correlated with an increase in the percentage of
trypan blue-positive (TB+) cells (5 and 27.5%
TB+ cells in the absence and presence of MG132,
respectively). These results indicate that release of pIL-1ß in
MG132-treated cells was due to cytotoxicity. In addition, we were
unable to demonstrate stabilization of pIL-1ß at MG132 concentrations
that were not toxic for monocytes; treatment with 3.5-fold and 10-fold
lower concentrations of inhibitor did not stabilize pIL-1ß or induce
its release (data not shown).

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Figure 3. Pulse/chase analysis of pIL-1ß in primary human monocytes. IL-1ß
was immunoprecipitated from 35S-methionine-labeled monocyte
lysates (A) and supernatants (B) immediately after the labeling period
(time 0) or after chasing with cold methionine for 2, 3, or
5 h. pIL-1ß bands were quantitated, and results are expressed as
described in Figure 1
.
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Figure 4. MG132 induces release of pIL-1ß and LDH from primary human monocytes.
Pulse/chase analysis was performed as described in Figure 3
, except
that in some samples, the tripeptide aldehyde MG132 was included during
the chase period at a final concentration of 50 µM. pIL-1ß was
immunoprecipitated from monocyte lysates and supernatants at the
indicated time points. In parallel, LDH activity was measured in
supernatants using the Promega Cytotox96 kit, and results are expressed
as percent cytotoxity, calculated as described in Materials and
Methods.
|
|
Because MG132 stabilizes pIL-1ß in THP-1 cells but is toxic for
primary monocytes we evaluated the effects of three additional protease
inhibitors on pIL-1ß degradation in monocytes. Unlike the peptide
aldehyde class of inhibitors [12
]
clasto-lactacystin ß-lactone is a specific inhibitor of
the proteasome [19
, 20
]. PD-150606 is a
specific inhibitor of the calcium-activated µ- and m-calpains, which
are found in the cytosol [21
]. E-64d inhibits the
activity of cysteine proteases including cathepsins, which are present
in lysosomes, as well as the calpains [22
]. As shown in
Figure 5
, treatment of monocytes with clasto-lactacystin ß-lactone
stabilized pIL-1ß in a dose-dependent manner. In similar experiments,
50 µM PD-150606 and E-64d had no effect on degradation of pIL-1ß.
For PD-150606, 47% of the pIL-1ß synthesized was detected after a
2.5-h chase in the absence of inhibitor, versus 40% in the presence of
inhibitor. Similarly, 57% of pIL-1ß remained after a 2.5-h chase in
the absence of E64-d compared with 58% in the presence of E64-d. No
IL-1ß was detected in supernatants of cells treated with PD-150606 or
E-64d (data not shown). In three of four monocyte donors treated with
clasto-lactacystin ß-lactone, pIL-1ß was stabilized in a
dose-dependent manner and no IL-1ß was not detected in culture
supernatants by immunoprecipitation (data not shown). In the fourth
donor, shown in Figure 5
, despite stabilization of intracellular
pIL-1ß, some protein was detected in supernatants, and the amount
increased with increasing doses of inhibitor. This is in contrast to
the effect of MG132 on monocytes, in which stabilization of
intracellular pIL-1ß could not be demonstrated, and large quantities
of pIL-1ß were immunoprecipitated from supernatants.

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Figure 5. The proteasome degrades pIL-1ß in primary human monocytes.
Pulse/chase analysis was performed as described in Figure 3
, except
that in some samples, clasto-lactacystin ß-lactone was
included at the indicated concentrations during the 2.5-h chase period.
IL-1ß was immunoprecipitated from monocyte lysates and supernatants
at the indicated time points.
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|
Clasto-lactacystin B-lactone augments release of IL-1ß
from activated human monocytes
We next determined the effect of stabilizing intracellular
pIL-1ß on levels of IL-1ß released into supernatants. For these
experiments, we used an ELISA rather than radiolabeling and
immunoprecipitation to achieve greater sensitivity of detection. As
shown in Table 1
, both pIL-1ß and mIL-1ß levels in supernatants of LPS-activated
monocytes increased approximately twofold in the presence of 15 µM
lactacystin relative to controls without lactacystin. These results are
consistent with the results of pulse/chase experiments that
demonstrated an approximately twofold stabilization of intracellular
pIL-1ß levels in the presence of this concentration of lactacystin
(Fig. 5 , compare chase without lactacystin to chase with 15 µM
lactacystin). Unlike MG132 treatment, lactacystin treatment did not
induce release of significant quantities of LDH into culture
supernatants, indicating that toxicity did not account for the increase
in IL-1ß release. These results demonstrate that proteasome-mediated
degradation of intracellular pIL-1ß serves to restrict the amount of
IL-1ß released by activated monocytes.
 |
DISCUSSION
|
|---|
IL-1ß is an exported protein that accumulates in an inactive
precursor form before processing and release from activated monocytes.
This quality distinguishes IL-1ß from other proinflammatory cytokines
such as tumor necrosis factor
and IL-6 that have an amino-terminal
signal sequence, and are exported via the conventional secretory
pathway within minutes after synthesis [8
,
26
]. Using pulse/chase analysis and a series of
cell-permeable protease inhibitors, we have shown that one consequence
of this property is that the IL-1ß precursor protein is subject to
degradation by the proteasome, the major cytosolic protease, with an
intracellular half-life of approximately 2.5 h. That the
proteasome is responsible for the degradation of intracellular pIL-1ß
is consistent with immunostaining studies that have localized pIL-1ß
to the cytoplasm of activated monocytes [9
,
10
].
A previous study designed to address the kinetics of IL-1 release
reported a similar turnover rate for pIL-1ß in monocytes
[8
]. However, in that study, monocytes were activated
with a 100-fold higher concentration of LPS than was used in this study
and the intracellular half-life of pIL-1ß was a function of
processing and release as well as degradation. To focus on the impact
of protein stability on the regulation of IL-1ß production, we have
used a low concentration of LPS to promote synthesis of pIL-1ß, but
not processing and secretion [27
]. This approach allowed
us to accurately determine the rate as well as the mechanism of
degradation of precursor protein restricted to the cytosol. Increasing
the activating dose of LPS from 100 pg/mL to 1 µg/mL did not change
the intracellular half-life of pIL-1ß as measured by pulse/chase
analysis over 2.5 h (data not shown), but it did induce processing
of pIL-1ß and release of both forms of IL-1ß into supernatants
(Table 1)
[27
]. Clasto-lactacystin
ß-lactone, a specific inhibitor of the proteasome, stabilized
intracellular pIL-1ß levels approximately twofold and enhanced
IL-1ß release approximately twofold without a concomitant increase in
LDH release. Therefore, enhancement of IL-1ß release by lactacystin
is, unlike MG132, not related to a cytotoxic effect. These data
indicate that susceptibility of pIL-1ß to proteasome-mediated
degradation is functionally relevant.
The susceptibility of a given cytosolic protein to degradation is a
function of multiple factors, including primary sequence,
posttranslational modification and folded structure of the protein, and
subcellular location [28
]. Reported half-lives of normal
cytoplasmic proteins range from less than 30 min, as exemplified by
ornithine decarboxylase, to greater than 200 h, as exemplified by
phosphoglycerate kinase [29
, 30
]. In this
context, the 2.5-h intracellular half-life of pIL-1ß is relatively
short. The relative instability of cytosolic pIL-1ß has important
implications for IL-1ß regulation, especially in light of a study
which demonstrated that significant quantities of IL-1ß are not
released from LPS-activated monocytes until approximately 2 h
after synthesis of the precursor protein [8
]. Our
results expand on that observation by showing that the relatively slow
IL-1ß release mechanism exposes pIL-1ß to proteasome-mediated
digestion in the cytosol. The rate of pIL-1ß degradation is therefore
a critical factor that dictates availability of pIL-1ß for processing
and release by activated monocytes. Because the half-life of pIL-1ß
and the delay in release are similar, the data collectively suggest
that a delicate balance of two pathways, degradation versus processing
and release, controls the amount of IL-1ß released by activated
monocytes.
Proteasome inhibitors have been reported to exert cytotoxic effects on
various cell types [23
24
25
]. We have found that for
monocytes, the severity of this effect is donor-dependent as well as
inhibitor-dependent. MG132 induced release of IL-1ß as well as LDH by
primary monocytes, whereas lactacystin induced release of IL-1ß but
not LDH. Unlike primary monocytes, we did not observe MG132-induced
cytotoxicity with THP-1 cells, at least by the criteria of pIL-1ß and
LDH release. This variable toxicity might be explained simply by
concentration effects and/or may be due to different levels of in
vivo priming among donors; that is, more than one signal may be
required to sensitize monocytes to proteasome inhibitor-mediated
toxicity. Variability among monocyte donors was clearly reflected in
the small but significant differences observed for pIL-1ß half-life
(see Results). Because we were able to demonstrate effects of MG132 in
THP-1 cells and lactacystin in primary monocytes in the absence of
cytotoxicity, these observations were not central to our conclusions,
and we did not investigate them further.
IL-1ß is a potentially toxic cytokine, and as such its production and
activity are regulated at many levels, including transcription, message
stability, translation, and the production of a receptor antagonist
[reviewed in 1, 31]. In particular, transcription of the IL-1ß gene
in response to LPS is rapid and vigorous, but begins to decline within
several hours of stimulation [31
]. The opportunity to
synthesize high levels of IL-1ß is therefore relatively transient.
The results of this study indicate another limiting step in IL-1ß
production, imposed by proteasome-mediated degradation of the inactive
IL-1ß precursor. This represents an additional and previously
unrecognized regulatory mechanism that may serve two purposes: first to
restrict intracellular levels of pIL-1ß until the monocyte receives
an appropriate activation signal, and second, to limit the amount of
biologically active IL-1ß secreted upon monocyte activation.
 |
ACKNOWLEDGEMENTS
|
|---|
This work was supported by a grant from Ciblex Corporation and by
the Signal Transduction Mechanisms and Cell Function training program,
Grant CA-09422, from the National Institutes of Health.
Received October 22, 1999;
revised February 28, 2000;
accepted February 29, 2000.
 |
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