IL-1beta mediates induction of hepatic type 1 plasminogen activator inhibitor in response to local tissue injury

Taiichiro Seki1,2, Annette M. Healy2, Daniel S. Fletcher3, Toshinori Noguchi1, and Thomas D. Gelehrter2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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)-1beta 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-1beta -deficient mice. In contrast, turpentine increased plasma PAI-1 and hepatic PAI-1 mRNA accumulation in wild-type mice but not in IL-1beta -deficient mice. Intraperitoneal injection of murine IL-1beta rapidly increased plasma PAI-1 and hepatic PAI-1 mRNA in both wild-type and IL-1beta -deficient mice. These results suggest that IL-1beta 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha (TNF-alpha ), 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, alpha 2-macroglobulin and alpha 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-1beta gene has been genetically inactivated by homologous recombination (IL-1beta -/- mice) (38). We report here that IL-1beta 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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-1beta -/- 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-1beta null mice subsequently became unavailable, homozygous IL-1beta -/- 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-1beta +/+ littermates served as wild-type controls.

Sterile abscesses were evoked by subcutaneous injection of 100 µl of turpentine (commercial grade, steam distilled wood turpentine, Sunneyside, Wheeling, IL) into a hindlimb (38). For intraperitoneal injections, pyrogen-free saline containing 0.1% endotoxin-free BSA (<1.2 ng endotoxin/mg protein; Behringer Diagnostics, La Jolla, CA) as carrier protein was used as injection vehicle in all cases except where noted. Mice were given an intraperitoneal injection of 0.5 ml vehicle alone or vehicle containing 50 or 200 µg LPS (E. coli 0111:B4, Difco Laboratories, Detroit, MI), 22.7 ng IL-1beta (2.2 × 107 U/mg; Genzyme, Cambridge, MA), or 500 ng TNF-alpha (1.3 × 108 U/mg, Genzyme). At various times after injection, mice were euthanized with CO2, and heparinized (50 U/ml) blood was obtained by cardiac puncture. Plasma was prepared by centrifugation of blood at 1,000 g for 15 min and stored at -70°C until assayed. Livers and lungs were harvested, and one-half of each type of tissue was frozen immediately in liquid nitrogen; tissues were stored at -70°C until used for RNA isolation. The remaining half of each tissue was fixed in 4% paraformaldehyde and stored at room temperature in 70% ethanol until used for in situ hybridization.

Immunologic assays. ELISAs were developed for murine IL-1beta and IL-6 using antibodies as described by Molineaux et al. (24). Monoclonal antibody to murine IL-1beta was kindly provided by Dr. D. Chapman (Washington University, St. Louis, MO). The ELISA for TNF-alpha was devised using a monoclonal antibody to murine TNF-alpha (Celltech PLC, Slough, UK) and a polyclonal antibody to murine TNF-alpha (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 [alpha -32P]dCTP (Amersham).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To establish the role of IL-1beta in the AP regulation of hepatic PAI-1, we investigated its regulation in both wild-type mice and in mice in which the IL-1beta gene had been genetically inactivated by homologous recombination (38). In our initial set of experiments (Figs. 1-3), wild-type (129SvEv) and IL-1beta -/- (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-alpha after LPS treatment in both wild-type and IL-1beta -/- 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-1beta was modestly increased by LPS in wild-type mice and, as expected, was undetectable in IL-1beta -/- mice (Fig. 1A). In contrast, IL-1beta and TNF-alpha antigen were undetectable in the plasma of either wild-type or IL-1beta -/- 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-1beta -/- mice (data not shown). These results are consistent with previously published reports (8, 13, 38).


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Fig. 1.   Induction of plasma cytokine concentrations by lipopolysaccharide (LPS) in wild-type and interleukin (IL)-1beta -/- mice. Wild-type and IL-1beta -/- mice were treated with an intraperitoneal injection of 200 µg of LPS, and plasma samples were obtained at the times indicated. IL-1beta (A), IL-6 (B), and tumor necrosis factor-alpha (TNF-alpha ; C) concentrations were determined by ELISA. Three animals were used at each time point for each condition. Each time point represents mean ± SE. Absence of error bars for a given data point in this and subsequent figures indicates that SE is smaller than size of symbol. Note the differences in scale of the y-axes. Apparent presence of IL-1beta in IL-1beta -/- mice at 16 h (A) reflects detectable IL-1beta in only 1 of 3 animals and may indicate a mistake in genotyping this mouse.

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-1beta -/- 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-1beta -/- mice (Fig. 2A).


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Fig. 2.   Induction of type 1 plasminogen activator inhibitor (PAI-1) antigen and PAI-1 mRNA accumulation in liver and lung by LPS and turpentine in wild-type and IL-1beta -/- mice. Wild-type and IL-1beta -/- mice were treated with either 1) intraperitoneal injection of 200 µg of LPS or 2) subcutaneous injection of 100 µl of turpentine in 1 hindlimb. Plasma and tissue samples were obtained at the times indicated. A: PAI-1 antigen concentration in plasma was determined by ELISA. B and C: total RNA was isolated from liver (B) and lung (C), and RNase protection analysis was performed. Amount of PAI-1 mRNA was normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and is presented as the ratio of experimental to control. Three animals were used at each time point for each condition. Each time point represents mean ± SE.

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-1beta -/- mice (Fig. 2C) and induced hepatic PAI-1 mRNA ~15-fold in both wild-type and IL-1beta -/- mice (Fig. 2B). In contrast, turpentine induced hepatic PAI-1 mRNA ~10-fold in wild-type mice but not at all in IL-1beta -/- mice (Fig. 2B). Turpentine failed to induce expression of PAI-1 mRNA in the lungs of either wild-type or IL-1beta -/- 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-1beta -/- mice. SAA mRNA accumulation was induced by LPS in both wild-type and IL-1beta -/- 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-1beta -/- mice (Fig. 3B). Together, these results suggest that the AP response to local tissue injury is dependent on IL-1beta and that IL-1beta is a critical inducer of hepatic PAI-1 gene expression during the AP response.


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Fig. 3.   Induction of hepatic serum amyloid A (SAA) mRNA accumulation by LPS (A) and turpentine (B). Northern blot analysis was performed on hepatic total RNA isolated from wild-type and IL-1beta -/- mice treated with LPS or turpentine. Amount of SAA mRNA was normalized to the amount of GAPDH mRNA and is presented as the ratio of experimental to control. Each time point represents mean ± SE.

Because the IL-1beta 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-1beta -/- mice. In this set of experiments (Figs. 4-5), a B10.RIII background was used for both the wild-type and IL-1beta -/- 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-1beta -/- 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-1beta -deficient mice. Injection of turpentine had little effect on pulmonary PAI-1 mRNA accumulation in wild-type or IL-1beta -deficient mice. Wild-type and IL-1beta -/- 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-1beta is a necessary mediator of the induction of PAI-1 in response to local tissue injury.


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Fig. 4.   Induction of PAI-1 antigen and PAI-1 mRNA accumulation in liver and lung by LPS and turpentine. B10.RIII wild-type and IL-1beta -/- mice were treated with either 1) intraperitoneal injection of 50 µg of LPS or 2) subcutaneous injection of 100 µl of turpentine in 1 hindlimb. A: PAI-1 antigen concentration in plasma. B and C: total RNA isolated from liver (B) and lung (C). Experiments were carried out as described in Fig. 2. Four to five animals were used at each time point for each condition.

To confirm the role of IL-1beta in the AP induction of PAI-1 expression, we have examined the effect of intraperitoneal injection of 22.7 ng (500 units) exogenous IL-1beta in both wild-type and IL-1beta -/- mice. As shown in Fig. 5, IL-1beta 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-1beta -/- mice. Intraperitoneal injection of 500 ng TNF-alpha induced the accumulation of plasma PAI-1 antigen and hepatic PAI-1 mRNA in IL-1beta -/- mice with a time course similar to that induced by IL-1beta (Fig. 5). Thus IL-1beta is not required for induction of PAI-1 expression by TNF-alpha .


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Fig. 5.   Induction of PAI-1 antigen and hepatic PAI-1 mRNA accumulation by IL-1beta and TNF-alpha . Wild-type and IL-1beta -/- mice were treated with an intraperitoneal injection of 22.7 ng of IL-1beta , and IL-1beta -/- mice were injected with 500 ng of TNF-alpha . A: PAI-1 antigen concentration in plasma. B: total RNA isolated from liver. Experiments were carried out as described in Fig. 2, with 4-5 animals used at each time point for each condition.

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-1beta -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-1beta -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-1beta -/- 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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Fig. 6.   Cellular localization of hepatic PAI-1 mRNA in the liver of mice treated with LPS and turpentine. B10.RIII wild-type or IL-1beta -/- mice were injected with 50 µg of LPS or 100 µl of turpentine as described in EXPERIMENTAL PROCEDURES. Livers were harvested at 16 h after injection and fixed in 4% paraformaldehyde. Histological sections (5 µm) from paraffin-embedded liver samples were hybridized with digoxigenin-labeled antisense/sense mouse PAI-1 riboprobe as described in experimental procedures. A-C: liver sections from LPS-treated wild-type (A and C) or IL-1beta -/- (B) mice were hybridized with sense (C) or antisense (A and B) PAI-1 riboprobe. D-F: liver sections from turpentine-treated wild-type (D and F) or IL-1beta -/- (E) mice were hybridized with sense (F) or antisense (D and E) PAI-1 riboprobe. Sections were counterstained with hematoxylin. Bar (F) = 50 µm.



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Fig. 7.   Cellular localization of hepatic PAI-1 mRNA in the liver of mice treated with turpentine. These sections are from the same samples as described in Fig. 6 but with higher magnification. A-C: liver sections from turpentine-treated wild-type (A and B) or IL-1beta -/- (C) mice were hybridized with antisense PAI-1 riboprobe. D: liver section from LPS-treated wild-type mouse hybridized with antisense PAI-1 riboprobe for comparison with turpentine-treated sections. Sections were counterstained with hematoxylin. Typical PAI-1 mRNA-expressing hepatocytes are indicated by arrows. Bar (C) = 20 µm.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-1beta -deficient mice (38) to analyze the role of IL-1beta 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-1beta 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-1beta -deficient mice. LPS also dramatically induced PAI-1 mRNA in lung in both wild-type and IL-1beta -deficient mice. These results are consistent with the hypothesis that the effects of LPS are mediated primarily by TNF-alpha (13). LPS and TNF-alpha both strongly induce PAI-1 mRNA in mouse liver in vivo. In C3H/HeJ mice, which exhibit defective TNF-alpha 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-alpha also induced PAI-1 mRNA accumulation in hepatocytes, and the effects of LPS were attenuated by anti-TNF-alpha antibodies and agents that decrease TNF-alpha 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-1beta -deficient mice. Turpentine did not induce PAI-1 mRNA in lungs of either wild-type or IL-1beta -deficient mice. These results demonstrate, for the first time, that IL-1beta 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-1alpha , which has biological effects similar to IL-1beta , apparently cannot compensate for the lack of IL-1beta . 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-1beta dramatically induced hepatic PAI-1 mRNA accumulation and plasma PAI-1 antigen in both wild-type and IL-1beta -deficient mice (Fig. 5). Direct injection of TNF-alpha also induced hepatic PAI-1 mRNA accumulation and plasma PAI-1 antigen in IL-1beta -deficient mice, suggesting that IL-1beta is not required for PAI-1 induction by TNF-alpha (Fig. 5).

The rapid induction of PAI-1 following intraperitoneal injection of IL-1beta is similar to that observed in vitro and probably reflects the transient, high concentration of IL-1beta in the portal circulation. Although plasma IL-1beta 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-1beta . 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-1beta production is critical to demonstrate unequivocally the essential role of IL-1beta 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-1beta 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-alpha 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-1beta -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-1beta -deficient mice, however, no hepatocyte or endothelial cell expression of PAI-1 was observed.

Together, these data establish that IL-1beta 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.


    ACKNOWLEDGEMENTS

We thank Drs. Hui Zheng and John Mudgett (Merck Research Laboratories) for providing the IL-1beta -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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 277(4):G801-G809
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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