1 Department of Nutrition and Physiology, Nihon University College of Bioresource Sciences, Tokyo 154-8516, Japan; 2 Departments of Human Genetics and Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618; and 3 Department of Pharmacology, Merck Research Laboratories, Merck & Company, Rahway, New Jersey 07065
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ABSTRACT |
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Type 1 plasminogen activator inhibitor (PAI-1),
a major physiological inhibitor of plasminogen activation, is an
important component of the hepatic acute phase response. We studied the acute phase regulation of murine hepatic PAI-1 in response to systemic
toxicity and local tissue injury in both wild-type mice and in mice in
which the interleukin (IL)-1 gene had been inactivated by gene
targeting. Endotoxin induced plasma PAI-1 antigen levels and PAI-1 mRNA
accumulation in liver to the same extent in both wild-type and
IL-1
-deficient mice. In contrast, turpentine increased plasma PAI-1
and hepatic PAI-1 mRNA accumulation in wild-type mice but not in
IL-1
-deficient mice. Intraperitoneal injection of murine IL-1
rapidly increased plasma PAI-1 and hepatic PAI-1 mRNA in both wild-type
and IL-1
-deficient mice. These results suggest that IL-1
is a
critical inducer of hepatic PAI-1 gene expression during the acute
phase response to local tissue injury. In situ hybridization studies
revealed that hepatocytes are the cells primarily responsible for the
hepatic expression of the PAI-1 gene induced by lipopolysaccharide and turpentine.
gene inactivation; knockout; mouse; acute phase response; turpentine; in situ hybridization
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INTRODUCTION |
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the acute phase (AP) response is the coordinated
systemic reaction of an organism to tissue injury and is characterized
by dramatic changes in the synthesis of diverse sets of proteins, known
as AP reactants, synthesized and secreted by the liver. The
physiological role of AP reactants is to reduce the systemic effects of
tissue damage by enhancing clot formation, reducing proteolysis, and
facilitating the clearance of toxic metabolites. The mediators of the
AP response are primarily cytokines, including interleukin (IL)-1,
IL-6, and tumor necrosis factor- (TNF-
), which are released by a
variety of cells, particularly inflammatory cells (3, 33).
Type 1 plasminogen activator inhibitor (PAI-1), a 50-kDa glycoprotein, is the major physiological inhibitor of both tissue-type and urokinase-type plasminogen activators and plays an important role in determining net fibrinolytic activity in vivo (4, 20, 36). The level of plasma PAI-1 is increased in patients with cardiovascular and thromboembolic disease, sepsis, and as part of the AP response to trauma, infection, and surgery (17, 18, 21); increased plasma PAI-1 may be the cause of the fibrinolytic shutdown and increased risk of thrombosis after surgery (18). We have demonstrated by in situ hybridization that human hepatocytes express PAI-1 and that PAI-1 transcripts are more abundant in liver specimens from subjects who died of sepsis, in which plasma PAI-1 levels are known to be elevated, than in normal transplant donor liver (35). We have also reported that IL-1 is a potent inducer of PAI-1 gene transcription in Hep G2 human hepatoma cells (14) and AML12 mouse hepatocytes in tissue culture (31). However, the mechanism of regulation of liver PAI-1 expression during the AP response has not yet been characterized.
The high degree of similarity in the qualitative and quantitative
patterns of AP reactants across species suggests that hepatic AP
reactants have been evolutionarily conserved. Nevertheless, there are
important interspecies differences in the AP response. For example, in
humans, C-reactive protein and serum amyloid A (SAA) are major AP
reactants, whereas, in the rat,
2-macroglobulin and
1-acid glycoprotein are the
major AP reactants and SAA is not expressed at all (3, 33). PAI-1 is an
AP reactant in humans and mice (17, 18, 21, 30, 35) but not in rats (25, 28, 34). Thus the mouse, but not the rat, provides an appropriate
model system in which to study AP regulation of PAI-1.
The present study characterizes the AP regulation of hepatic PAI-1 gene
expression in the mouse following systemic toxicity and local tissue
injury caused by lipopolysaccharide (LPS) and turpentine, respectively.
To demonstrate the role of IL-1, a potent inducer of PAI-1 gene
transcription in hepatocytes in vitro, we have compared PAI-1
regulation in wild-type mice and in mice in which the IL-1 gene has
been genetically inactivated by homologous recombination (IL-1
/
mice) (38). We report here that IL-1
is a critical
inducer of hepatic PAI-1 gene expression during the AP response to
local tissue injury and that the liver may be an important source of
plasma PAI-1 during the AP response to local tissue injury.
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EXPERIMENTAL PROCEDURES |
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Animals and treatments.
All animal experiments were performed in accordance with the guidelines
established by the Merck Laboratory Animal Care and Use Committee. Mice
were 10-12 wk of age when used for experiments and had access to
food and water ad libitum. Treatment groups were equally mixed male and
female, as closely as possible. For the first set of experiments,
homozygous IL-1
/
mice in a mixed C57BL/6J and 129SvEv
background (obtained from H. Zheng, Merck, Rahway, NJ) were used (38).
129SvEv mice (Taconic, Germantown, NY) were used as wild-type controls.
Because these IL-1
null mice subsequently became unavailable,
homozygous IL-1
/
mice that had been backcrossed with
B10.RIII mice for three generations (obtained from J. Mudgett, Merck)
were used in the second set of experiments. Their homozygous IL-1
+/+ littermates served as wild-type controls.
Immunologic assays.
ELISAs were developed for murine IL-1 and IL-6 using antibodies as
described by Molineaux et al. (24). Monoclonal antibody to murine
IL-1
was kindly provided by Dr. D. Chapman (Washington University,
St. Louis, MO). The ELISA for TNF-
was devised using a monoclonal
antibody to murine TNF-
(Celltech PLC, Slough, UK) and a polyclonal
antibody to murine TNF-
(Genzyme). The lower range of sensitivity
for the above ELISAs was 0.05 ng/ml plasma. Plasma PAI-1 antigen was
measured by ELISA using monoclonal antibodies to murine PAI-1 (6, 7),
kindly provided by Dr. P. J. Declerck (Katholieke Universiteit, Leuven, Belgium).
RNA isolation and analysis. RNA was isolated from murine liver and lung samples by a modification (27) of the method of Chomczynski and Sacchi (5) or by using TRIzol (Life Technologies, Gaithersburg, MD) according to the manufacturer's specifications. Northern blots were performed as previously described (15, 31). RNase protection assays were carried out essentially as described (2). Briefly, total cellular RNA (40 µg) was incubated with the appropriate radiolabeled riboprobes for 16-20 h at 50°C. After RNase A plus RNase T1 digestion, the samples were subjected to electrophoresis through 6% polyacrylamide-8 M urea gels. Radioactivity associated with a specific RNA fragment was determined using a model 400 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The amount of PAI-1 (or SAA) mRNA was normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and is presented as the ratio of experimental to control.
In situ hybridization. Histologic sections (5 µm) from paraformaldehyde-fixed, paraffin-embedded liver samples were prepared on 3-aminopropyltriethoxysilane (Nacalai Tesque, Kyoto, Japan)-coated slides. The sections were immersed sequentially in xylene (10 min, 3 times), graded ethanol solutions, 0.1 M phosphate buffer (pH 7.4) (15 s × 3), 1 µg/ml proteinase K (15 min × 1), 4% paraformaldehyde (15 min × 1), 0.2 M HCl (10 min × 1), and phosphate buffer (1 min × 1). The sections were then treated with 0.1 M triethanolamine in 0.25% acetic anhydride and dehydrated through graded ethanol solutions. Digoxigenin (DIG)-labeled riboprobe (1 µl) was applied to the tissue sections in 200 µl of hybridization buffer [50% deionized formamide, 10 mM Tris · HCl (pH 7.6), 200 µg/ml salmon sperm DNA, 1× Denhardt's solution, 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, and 1 mM EDTA]. Hybridization was conducted at 50°C in a humidified chamber for 16 h, after which the samples were washed sequentially in 2× sodium chloride-sodium citrate (SSC) with 50% formamide for 30 min at 50°C and in TNE [10 mM Tris · HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA] for 10 min at 37°C. The samples were then incubated with RNase A (10 µg/ml) for 30 min at 37°C and washed sequentially in TNE for 10 min at 37°C and then twice with both 2× SSC and 0.2× SSC (20 min at 50°C). The specifically hybridized DIG-labeled riboprobes were detected with anti-DIG Fab fragment labeled with peroxidase (Boehringer Mannheim, Indianapolis, IN) and a commercially available tyramide signal amplification kit (TSA-Indirect ISH kit, DuPont NEN, Boston, MA). After the hybridization, the sections were counterstained with hematoxylin (Sigma, St. Louis, MO).
Probes.
Mouse PAI-1 cDNA was obtained from Dr. D. Ginsburg (University of
Michigan, Ann Arbor, MI) (26). The mouse PAI-1 probe used in the first
set of experiments was constructed by inserting the 515-bp
Pst
I-Xho I mouse PAI-1 fragment into the
Pst I and
Xho I sites of pBluescript
KS (Stratagene, La Jolla,
CA). Sca I digestion generates a
template for synthesis of a 380-nt mouse PAI-1 riboprobe. Because the
protected fragment migrated close to a nonspecific band on RNase
protection assays, additional PAI-1 riboprobes were generated. The
mouse PAI-1 probe used in the second set of experiments was constructed by inserting the 650-bp EcoR
I-Msc I mouse PAI-1 fragment into the
EcoR I and
EcoR V sites of pBluescript
KS
.
Sca I digestion generates a template
for synthesis of a 182-nt mouse PAI-1 riboprobe. An additional mouse
PAI-1 probe was optimized for use in the in situ hybridization
experiments. This riboprobe was constructed by inserting the
EcoR
I-Pst I mouse PAI-1 fragment into the
EcoR I and
Pst I sites of pBluescript
KS
.
EcoR I digestion generates a template
for synthesis of a 390-nt antisense riboprobe, and
Xba I digestion generates a template for synthesis of a 410-nt sense riboprobe. DIG-labeled riboprobes were
synthesized using the DIG RNA labeling kit (Boehringer Mannheim) according to the manufacturer's specifications. Mouse
SAA1 cDNA was
obtained from Dr. J. Sipe (Boston University, Boston, MA) (37). The
construction of the template for synthesis of the rat GAPDH riboprobe
has been described (16).
32P-labeled riboprobes were
synthesized by in vitro transcription using T3 or T7 RNA polymerase and
[32P]UTP (Amersham,
Arlington Heights, IL) as described (2). For Northern blot analysis,
32P-labeled DNA was prepared by
the random primer method (12) using the large fragment of Klenow DNA
polymerase I and
[
-32P]dCTP (Amersham).
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RESULTS |
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To establish the role of IL-1 in the AP regulation of hepatic PAI-1,
we investigated its regulation in both wild-type mice and in mice in
which the IL-1
gene had been genetically inactivated by homologous
recombination (38). In our initial set of experiments (Figs.
1-3), wild-type (129SvEv) and IL-1
/
(mixed 129SvEv and C57BL/6J background) mice were used.
To simulate systemic toxicity, mice were injected intraperitoneally
with a sublethal concentration (200 µg) of bacterial endotoxin (LPS).
To create a sterile abscess to simulate local tissue injury, mice
received 100 µl of turpentine injected subcutaneously into one
hindlimb. Mice were euthanized at various times postinjection, and
blood and tissue samples were obtained. As previously reported (38),
assay of plasma cytokine levels by ELISA indicated a striking increase
in levels of IL-6 and TNF-
after LPS treatment in both wild-type and
IL-1
/
mice (Fig. 1,
B and
C). The level of these two cytokines
was significantly elevated at 3 h (the time at which the first blood
sample was taken) and then rapidly declined. IL-1
was modestly
increased by LPS in wild-type mice and, as expected, was undetectable
in IL-1
/
mice (Fig.
1A). In contrast, IL-1
and
TNF-
antigen were undetectable in the plasma of either wild-type or
IL-1
/
mice treated with turpentine (data not shown).
Only a small increase in IL-6 antigen was observed (10% of level
observed in LPS-treated mice) in wild-type mice injected with
turpentine but not in the IL-1
/
mice (data not
shown). These results are consistent with previously published reports
(8, 13, 38).
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PAI-1 antigen in plasma was measured by ELISA and found to be ~1
ng/ml in untreated mice. Plasma PAI-1 was dramatically induced by LPS
in both wild-type and IL-1
/
mice (Fig.
2A).
Turpentine induced a more modest increase in plasma PAI-1 antigen in
wild-type mice; no induction of plasma PAI-1 by turpentine was observed in IL-1
/
mice (Fig.
2A).
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RNase protection analysis was used to assay PAI-1 mRNA accumulation in
liver and lung. In untreated animals, PAI-1 mRNA was barely detectable
in liver, whereas weak expression was detected in the lung. LPS
dramatically induced PAI-1 mRNA accumulation in the lungs of both
wild-type and IL-1
/
mice (Fig.
2C) and induced hepatic PAI-1 mRNA
~15-fold in both wild-type and IL-1
/
mice (Fig.
2B). In contrast, turpentine induced
hepatic PAI-1 mRNA ~10-fold in wild-type mice but not at all in
IL-1
/
mice (Fig.
2B). Turpentine failed to induce
expression of PAI-1 mRNA in the lungs of either wild-type or IL-1
/
mice (Fig. 2C).
SAA is a major AP reactant in humans and mice. Therefore, we examined
induction of hepatic SAA mRNA accumulation by LPS and turpentine in
both wild-type and IL-1
/
mice. SAA mRNA accumulation was induced by LPS in both wild-type and IL-1
/
mice
(Fig. 3A), as
assayed by Northern blot analysis. Turpentine caused a 200-fold
increase in the expression of SAA mRNA in wild-type mice, but the
induction was reduced by >90% in IL-1
/
mice (Fig. 3B). Together, these results suggest
that the AP response to local tissue injury is dependent on IL-1
and
that IL-1
is a critical inducer of hepatic PAI-1 gene expression
during the AP response.
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Because the IL-1 null mice used in the experiments shown in Figs. 2
and 3 were no longer available, we had the opportunity to examine the
effect of a different genetic background on PAI-1 induction in response
to systemic toxicity and to local tissue injury in wild-type and
IL-1
/
mice. In this set of experiments (Figs.
4-5), a B10.RIII background was used
for both the wild-type and IL-1
/
mice. A lower but
still maximally effective (11) dose of LPS (50 vs. 200 µg) was used
in these experiments. As shown in Fig. 4, systemic toxicity (using LPS)
caused a dramatic increase in plasma PAI-1 antigen, hepatic PAI-1 mRNA
accumulation, and pulmonary PAI-1 mRNA accumulation in both wild-type
and IL-1
/
mice. In contrast, local tissue injury
(using turpentine) caused a 12-fold increase in plasma PAI-1 antigen
and a 6-fold increase in hepatic PAI-1 mRNA accumulation in wild-type
mice but had no effect in the IL-1
-deficient mice. Injection of
turpentine had little effect on pulmonary PAI-1 mRNA accumulation in
wild-type or IL-1
-deficient mice. Wild-type and IL-1
/
mice injected with vehicle alone showed little
induction of PAI-1 mRNA and antigen levels (data not shown). Together,
these results demonstrate, for the first time, that IL-1
is a
necessary mediator of the induction of PAI-1 in response to local
tissue injury.
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To confirm the role of IL-1 in the AP induction of PAI-1 expression,
we have examined the effect of intraperitoneal injection of 22.7 ng
(500 units) exogenous IL-1
in both wild-type and IL-1
/
mice. As shown in Fig. 5,
IL-1
dramatically induces the accumulation of plasma PAI-1 antigen
and hepatic PAI-1 mRNA; this induction peaked at 2 h and then rapidly
declined. In each case, the magnitude of the induction was similar in
both wild-type and IL-1
/
mice. Intraperitoneal
injection of 500 ng TNF-
induced the accumulation of plasma PAI-1
antigen and hepatic PAI-1 mRNA in IL-1
/
mice with a
time course similar to that induced by IL-1
(Fig. 5). Thus IL-1
is not required for induction of PAI-1 expression by TNF-
.
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To determine which cell type(s) expresses the PAI-1 gene in the liver
during the AP response, we performed in situ hybridization using a
mouse PAI-1 mRNA-specific DIG-labeled riboprobe (Fig. 6). PAI-1 mRNA-specific signals were
observed in the hepatocytes of both LPS-treated wild-type and
IL-1-deficient mice (Fig. 6, A and
B). No PAI-1 mRNA was detected after
hybridization with the sense PAI-1 riboprobe (Fig.
6C). Significant hybridization to
PAI-1 mRNA was also observed in the hepatocytes of turpentine-injected wild-type mice (Fig. 6D) but not in
IL-1
-deficient mice (Fig. 6E).
Again, no PAI-1 mRNA was detected after hybridization with the sense
PAI-1 riboprobe (Fig. 6F). To
demonstrate more precisely the cellular localization of PAI-1 mRNA
signals in the liver, higher-magnification in situ hybridization
studies are shown in Fig. 7. PAI-1
mRNA-specific signals were clearly observed in the hepatocytes of both
LPS- and turpentine-injected wild-type mice (Fig. 7,
A, B,
and D) but not in the hepatocytes of
turpentine-treated IL-1
/
mice (Fig.
7C). Rarely, central vein
endothelial cells and sinusoidal endothelial cells in the sections
obtained from turpentine-treated wild-type mice showed PAI-1 signals
(data not shown). These in situ hybridization studies are the first
demonstration that parenchymal hepatocytes express the PAI-1 gene
during the AP response to local tissue injury.
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DISCUSSION |
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PAI-1 is an important component of the hepatic AP response in mice and
humans. We have previously reported that IL-1 is a potent inducer of
PAI-1 gene expression in AML12 mouse hepatocytes (31). We have taken
advantage of the availability of IL-1-deficient mice (38) to analyze
the role of IL-1
in the AP regulation of hepatic PAI-1 gene
expression in vivo in response to systemic toxicity and local tissue
injury. Although mice genetically deficient in IL-1
production have
been used to study various immune and AP responses (1, 8-10, 19,
32), they have not previously been used to study PAI-1 gene expression.
The time course of induction of plasma PAI-1 protein and hepatic PAI-1
mRNA by LPS, a model of systemic toxicity, was essentially the same in
both wild-type and IL-1-deficient mice. LPS also dramatically
induced PAI-1 mRNA in lung in both wild-type and IL-1
-deficient
mice. These results are consistent with the hypothesis that the effects
of LPS are mediated primarily by TNF-
(13). LPS and TNF-
both
strongly induce PAI-1 mRNA in mouse liver in vivo. In C3H/HeJ mice,
which exhibit defective TNF-
release in response to LPS, the LPS
induction of hepatic PAI-1 mRNA was markedly attenuated (30). By in
situ hybridization, LPS has been reported to induce PAI-1 gene
expression in hepatocytes and to a lesser extent in hepatic sinusoidal
endothelial cells (11). TNF-
also induced PAI-1 mRNA accumulation in
hepatocytes, and the effects of LPS were attenuated by anti-TNF-
antibodies and agents that decrease TNF-
synthesis (11).
In contrast, local tissue injury, induced by sterile abscess formation
following subcutaneous turpentine injection, induced PAI-1 protein and
hepatic PAI-1 mRNA in wild-type mice but failed to induce PAI-1 in
IL-1-deficient mice. Turpentine did not induce PAI-1 mRNA in lungs
of either wild-type or IL-1
-deficient mice. These results
demonstrate, for the first time, that IL-1
is not required for the
induction of hepatic PAI-1 gene expression in response to systemic
toxicity but is an absolutely essential cytokine for the response to
local tissue injury. IL-1
, which has biological effects similar to
IL-1
, apparently cannot compensate for the lack of IL-1
. These
results are consistent with a previous report that the fever, lethargy,
anorexia, and weight loss induced by turpentine were not observed in
IL-1 type I receptor-deficient mice (22).
Direct intraperitoneal injection of IL-1 dramatically induced
hepatic PAI-1 mRNA accumulation and plasma PAI-1 antigen in both
wild-type and IL-1
-deficient mice (Fig. 5). Direct injection of
TNF-
also induced hepatic PAI-1 mRNA accumulation and plasma PAI-1
antigen in IL-1
-deficient mice, suggesting that IL-1
is not
required for PAI-1 induction by TNF-
(Fig. 5).
The rapid induction of PAI-1 following intraperitoneal injection of
IL-1 is similar to that observed in vitro and probably reflects the
transient, high concentration of IL-1
in the portal circulation.
Although plasma IL-1
concentrations are below the level of detection
after subcutaneous injection of turpentine, the slower, sustained
increase in PAI-1 mRNA induced by turpentine suggests that hepatocytes
may be very sensitive to the actions of low concentrations of IL-1
.
Local tissue concentrations of cytokines are undoubtedly the
physiologically relevant ones but are not directly measurable.
Therefore, the use of animals genetically deficient in IL-1
production is critical to demonstrate unequivocally the essential role
of IL-1
in the AP regulation of hepatic PAI-1 by local tissue injury.
Direct injection of IL-6 caused a very slight and transient increase in
plasma PAI-1 antigen but had no effect on PAI-1 mRNA accumulation in
either liver or lung (data not shown), suggesting that IL-1 does not
induce PAI-1 through the action of IL-6. These data are consistent with
our observations in AML12 mouse hepatocytes in tissue culture, in which
both IL-1 and TNF-
are potent inducers of PAI-1 gene transcription
but IL-6 was without effect (31).
In turpentine-treated wild-type mice, the time course and magnitude of the induction of plasma PAI-1 protein were very similar to those of liver PAI-1 mRNA in both sets of experiments. These data suggest that the liver may be a major source of plasma PAI-1 during the AP response to local tissue injury. The lung, which is rich in endothelial cells, does not appear to be contributing to plasma PAI-1 protein in this setting, although it clearly does in systemic toxicity induced by LPS.
The source of plasma PAI-1 under different physiological or
pathological conditions is not known; however, endothelial cells, smooth muscle cells, hepatocytes, and adipocytes have all been proposed
as a significant source (4, 11, 23, 29, 35). We have performed in situ
hybridization to determine the cell type(s) responsible for the
production of PAI-1 in the liver during the AP response. These studies
confirm that LPS induces PAI-1 expression in hepatocytes (11), and, as
expected, this was observed in both wild-type and IL-1-deficient
mice. Our studies also show, for the first time, that local tissue
injury induced by turpentine causes increased PAI-1 gene expression in
hepatocytes in wild-type mice. PAI-1 gene expression was also observed,
though rarely, in central vein endothelial cells and in sinusoidal
endothelial cells. In IL-1
-deficient mice, however, no hepatocyte or
endothelial cell expression of PAI-1 was observed.
Together, these data establish that IL-1 is a critical mediator of
hepatic PAI-1 gene expression in response to local tissue injury and
that hepatocytes may be an important source of plasma PAI-1 during this response.
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ACKNOWLEDGEMENTS |
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We thank Drs. Hui Zheng and John Mudgett (Merck Research Laboratories)
for providing the IL-1-deficient mice and Professor Paul J. Declerck
(Katholieke Universiteit, Leuven, Belgium) for providing the anti-mouse
PAI-1 monoclonal antibodies and for helpful suggestions regarding the
assay. We also thank Tara E. Siok for technical assistance, Eric
Gelehrter and Dave Siemieniak for help with Fig. 6, and Joanne Heaton
for helpful discussions.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants DK-46010 and CA-22729, a short-term fellowship from the Japan Society for the Promotion of Science (to T. D. Gelehrter), and a grant (10044216) from the program Grants-in Aid for International Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (to T. Seki).
Present address of A. M. Healy: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109-0642.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. D. Gelehrter, Dept. of Human Genetics, The Univ. of Michigan Medical School, Medical Science II M4708, Ann Arbor, MI 48109-0618 (E-mail: tdgum{at}umich.edu).
Received 15 December 1998; accepted in final form 15 July 1999.
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