Tumor necrosis factor {alpha} is not required for WY14,643-induced cell proliferation

Jeffrey W. Lawrence,1, Gordon K. Wollenberg and John G. DeLuca

Department of Safety Assessment, Merck Research Laboratories, Merck and Co. Inc., Sumneytown Pike, WP45A-201, West Point, PA 19486, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been proposed that the cytokine tumor necrosis factor {alpha} (TNF{alpha}) stimulates peroxisome proliferator-induced hepatic cell proliferation. To test this hypothesis, induction of peroxisome proliferation and hepatocyte proliferation were compared in wild-type C57Bl/6 and TNF{alpha} knockout mice. Animals were dosed with either vehicle or 100 mg/kg/day WY14,643 by oral gavage for 4 days. Liver to brain weight ratios increased in both wild-type and TNF{alpha} knockout animals after WY14,643 administration. In addition, WY14,643-treated wild-type C57Bl/6 and TNF{alpha} knockout mice displayed marked hepatic induction of fatty acyl-CoA oxidase activity (~8-fold) and mRNA content (~5-fold). Electron microscopic examination confirmed increased numbers of peroxisomes in hepatocytes in both mouse models. Moreover, WY14,643 markedly induced hepatic cell proliferation (~15-fold) in both wild-type C57Bl/6 and TNF{alpha} knockout mice as measured by bromodeoxyuridine incorporation into hepatocyte nuclei. In addition, a 50% decrease in TNF{alpha} mRNA was observed in wild-type mice after treatment with WY14,643. These results suggest that the hepatocellular proliferation induced after peroxisome proliferator treatment occurs independently of TNF{alpha} signaling.

Abbreviations: BrdU, bromodeoxyuridine; FACO, fatty acyl-CoA oxidase; FAM, 6-carboxyfluorescein; 4F1G, 4% neutral buffered formalin and 1% gluteraldehyde; IL-6, interleukin-6; LPS, lipopolysaccharide; PPAR{alpha}, peroxisome proliferator-activated receptor {alpha}; TEM, transmission electron microscopy; TNF{alpha}, tumor necrosis factor {alpha}; TNF R, TNF receptor.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peroxisome proliferators are a diverse group of chemicals that include plasticizers, herbicides and pharmaceutical hypolipidemic agents (1). Upon administration of these agents to rodents characteristic changes occur in the liver that include hepatomegaly, an increase in the size and number of peroxisomes, hepatic cell proliferation and the induction of hepatocarcinogenesis after long-term administration (2). It is thought that the hepatocarcinogenesis is related to the increased oxidative stress resulting from increased oxidative metabolism and/or the increased cell proliferation observed after peroxisome proliferator treatment (3,4).

Peroxisome proliferators mediate their effects through interaction with a nuclear ligand-dependent transcription factor called peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) (5). PPAR{alpha} knockout mice lack the characteristic responses to peroxisome proliferators, including hepatomegaly, proliferation of peroxisomes, increased hepatic cell proliferation and hepatocarcinogensis (6,7).

Previous in vitro and in vivo studies have shown that tumor necrosis factor {alpha} (TNF{alpha}) is involved in regenerative growth after partial hepatectomy. For example, treatment of rats with an anti-TNF{alpha} antibody prevented the mitogenic response after partial hepatectomy (8). In addition, mice lacking the type 1 TNF receptor (TNF R) were deficient in the regenerative response after partial hepatectomy (9) and liver regeneration seen after CCl4-induced hepatic necrosis (10). When TNF{alpha} was incubated with murine hepatocytes in culture, a mitogenic response was observed (11). These studies demonstrated that TNF{alpha} plays a role in hepatocellular proliferation and liver mass homeostasis.

A current hypothesis suggests that TNF{alpha} mediates the mitogenic response induced by peroxisome proliferators (12). According to this hypothesis, peroxisome proliferator-activated Kupffer cells would synthesize and secrete TNF{alpha}, which stimulates neighboring hepatocytes to proliferate. Rose et al. (13) demonstrated that peroxisome proliferators could activate Kupffer cells in vivo and in vitro. Inactivation of Kupffer cells with either dietary glycine or methylpalmitate prevented peroxisome proliferator-induced cell proliferation in rats (14, 15). In addition, TNF{alpha} mRNA was observed to be increased 2- to 2.5-fold by WY14,643 treatment in rats (12,15). Treatment of rats with anti-TNF{alpha} polyclonal antibody also prevented WY14,643-induced cell proliferation (12). In an attempt to directly test the requirement for TNF{alpha} in peroxisome proliferator-induced cell proliferation, wild-type C57Bl/6 and TNF{alpha} knockout mice, a genetically engineered mouse line that has had the gene for TNF{alpha} disrupted, were treated with WY14,643 for 4 days and hepatic peroxisome induction and cell proliferation were assessed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental design
C57Bl/6 wild-type male mice were purchased from Charles River Laboratories (Raleigh, NC) and were 10 weeks old and weighed between 24 and 26 g. TNF{alpha} knockout male mice (16) were obtained from Dr George Kollias (Hellenic Pasteur Institute, Athens, Greece). The knockout mice used in the study were ~29 weeks old and weighed between 32 and 42 g. The phenotype of the six founder knockout mice was confirmed by demonstrating the absence of an elevation in plasma TNF{alpha} concentrations 90 min after lipopolysaccharide (LPS) treatment (data not shown). All mice were individually housed in plastic boxes in a climate controlled room. PMI Certified Rodent Chow and water were available ad libitum. Ten mice of both genetic backgrounds were implanted s.c. with osmotic minipumps containing 50 mg/ml bromodeoxyuridine (BrdU) and five mice from each background were dosed for 4 days with either vehicle (0.5% methylcellulose) or 100 mg/kg/day WY14,643 (Chemsyn Science Laboratories, Lenexa, KS). This dose of WY14,643 has been shown to stimulate cell proliferation and lead to tumor formation in both rats and mice (17). At necropsy, terminal body weights, liver weights and brain weights were determined. A section from the left lateral lobe of the liver from each animal was fixed in 4% neutral buffered formalin and 1% gluteraldehyde (4F1G) for transmission electron microscopy (TEM), 10% formalin overnight followed by 70% ethanol for immunohistochemistry or frozen at –70°C for fatty acyl-CoA oxidase (FACO) activity and mRNA analysis. All animal care and treatment procedures were approved by the Merck Research Laboratories Institutional Animal Care and Use Committee.

Cell proliferation analysis
BrdU was detected immunohistochemically by staining formalin-fixed wax-embedded sections of liver by an indirect avidin–biotinylated peroxidase technique using rat monoclonal anti-BrdU IgG (Accurate, Westbury, NY) as previously described (18). Briefly, tissue sections were post-fixed in zinc-buffered formalin and then incubated in 0.04% pepsin (Sigma, St Louis, MO) in 0.1 N HCl for 25 min at room temperature. The sections were then denatured in 4 M HCl and neutralized in a solution of 0.1 M borax, pH 8.5. Antibody binding and detection were then performed using diaminobenzadine tetrahydrochloride as the chromogen, which stains brown. Digital images were obtained using the Bioquant/TCW capture software (R&M Biometrics, Nashville, TN) and randomly scanning 20 fields throughout sections of the right lateral lobe from each animal in each dose group (40x objective) as previously described (19,20). The images were then subject to analysis with CHRIS software (Sverdrup Technologies, Fort Walton Beach, FL) to determine the total number of hepatocyte nuclei and the number of nuclei that stained positive for BrdU and the percent labeling index calculated for each animal.

Electron microscopy
4F1G-fixed 1 mm3 sections of liver were post-fixed in 2% osmium tetroxide and then embedded in epoxy resin. Ultrathin sections were cut on a Reichert Ultracut S ultramicrotome, stained with uranyl acetate and lead citrate and viewed on a Philips CM12 transmission electron microscope.

FACO activity
FACO activity was assayed by monitoring the evolution of H2O2 according to the procedure of Poosch and Yamazaki (21) using lauryl-CoA as the substrate and 1 mM hydroxyphenylacetic acid as the indicator. Incubations were carried out at 37°C and stopped with 2 mM KCN in carbonate buffer after 10 min. All samples were assayed in duplicate with a corresponding blank (lacking lauryl-CoA) subtracted. Results were converted to nmol product by comparison with a H2O2 standard curve and normalized to mg protein.

RNA analysis
Total RNA was isolated by a combination of Triazol (Gibco, Rockville, MD) extraction and solid phase extraction using a kit from Qiagen (Valencia, CA). Briefly, 100 mg of liver tissue was homogenized in 1 ml of Triazol. To a 0.3 ml aliquot, 60 µl of chloroform was added and the mixture was centrifuged at 10 000 g for 15 min at 4°C. The supernatant was removed and processed using a Qiagen RNeasy RNA isolation kit. The RNA was quantified by spectrophotometic determination at 260 nm. cDNA synthesis was performed in 25 µl with 0.1 µg total RNA with a Taqman RT kit (PE Applied Biosystems, Foster City, CA) at 25°C for 10 min, 48°C for 30 min and 95°C for 5 min. After reverse transcription, a 3 µl aliquot was transferred into each of two 25 µl Taqman amplification reactions containing primer/probe sets for 18S rRNA (6-carboxy-4',5'-dichloro-2',7'-dimethylfluorescein tagged) and either mouse FACO or TNF{alpha} [6-carboxyfluorescein (FAM) tagged] and amplified at 50°C for 2 min and 95°C for 10 min and at 95°C for 15 s and 60°C for 1 min for 40 cycles on a ABI Prism 7700 Sequence Detection System following the manufacturer's instructions (PE Applied Biosystems). The specific sequences of the primers and probes were

FACO: F primer, 5'-GAG TGA GCT GCC TGA GCT TCA; R primer, 5'-AAG CTA TGG TCG TAA CCG A; probe, 5'-TAMRA-CCC TCA CAG CTG GGC TGA AGG CT-FAM

TNF{alpha}: F primer, 5'-AGG AAT GAG AAG AGG CTG AGA CAT; R primer, 5'-CCT TGA CCG TCT TCT CCG GT; probe, 5'-TAMRA-CCG CCT GGA GTT CTG GAA GCC C-FAM

TNF{alpha} mRNA levels were not assessed in TNF{alpha} knockout animals due to the design of the primer/probe sets.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To directly test the requirement for TNF{alpha} in peroxisome proliferator-induced cell proliferation, measures of peroxisome proliferation and cell proliferation were assessed in wild-type and TNF{alpha} knockout mice after treatment with 100 mg/kg/day WY14,643 for 4 days. Both wild-type C56Bl/6 and TNF{alpha} knockout mice showed similar levels of hepatomegaly after WY14,643 treatment (Figure 1Go). Relative liver weights appeared lower in untreated TNF{alpha} knockout mice compared with untreated wild-type mice. Histological assessment demonstrated that both wild-type and TNF{alpha} knockout mice displayed hepatocellular hypertrophy after WY14,643 treatment (data not shown). In addition, FACO activity and FACO mRNA were increased to similar levels in both wild-type and TNF{alpha} knockout mice (Figure 2A and BGo). Peroxisome proliferation was confirmed by ultrastructural examination of liver sections that showed that both wild-type and TNF{alpha} knockout mice given WY14,643 had increases in the size and number of hepatic peroxisomes relative to their respective controls (Figure 3Go).



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Fig. 1. Effects of WY14,643 on hepatomegaly induction in wild-type C57Bl/6 and TNF{alpha} knockout mice. Wild-type (+/+) or TNF{alpha} knockout (–/–) mice were dosed with 100 mg/kg/day WY14,643 for 4 days. Livers were removed and weighed. Data are expressed as mean percent brain weights ± SEM.

 


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Fig. 2. Effects of WY14,643 on FACO activity and mRNA induction in wild-type C57Bl/6 and TNF{alpha} knockout mice. Wild-type (+/+) or TNF{alpha} knockout (–/–) mice were dosed with 100 mg/kg/day WY14,643 for 4 days. (A) Livers were homogenized and FACO activity levels assessed as described in Materials and methods. Data are expressed as means ± SEM. (B) Total RNA was isolated and FACO mRNA was quantified by real time RT–PCR as described in Materials and methods. Data are expressed as means ± SEM relative to wild-type control induction.

 


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Fig. 3. Effects of WY14,643 on peroxisome proliferation induction in wild-type C57Bl/6 and TNF{alpha} knockout mice. Wild-type or TNF{alpha} knockout mice were dosed with 100 mg/kg/day WY14,643 for 4 days. Liver sections were removed, fixed and processed for TEM analysis as described in Materials and methods.

 
To measure cell proliferation, mice were implanted with BrdU-containing minipumps and hepatocytes that underwent S phase DNA synthesis were identified by immunohistochemical detection of BrdU incorporation into nuclei. Photomicrographs demonstrated that a greater number of hepatocytes had undergone DNA synthesis after treatment with WY14,643 in both wild-type and TNF{alpha} knockout mice than in the respective control mice (Figure 4Go). Both untreated wild-type and TNF{alpha} knockout mice had low basal hepatocyte labeling indices (0.6 and 0.8%, respectively). WY14,643 treatment induced a 15-fold increase in the labeling index in both wild-type and TNF{alpha} knockout mice (Figure 5Go). Consistent with the increased hepatocellular proliferation stimulated by WY14,643, two of five wild-type and three of five TNF{alpha} knockout mice treated with WY14,643 had a slight increase in the number of mitotic hepatocytes (data not shown).



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Fig. 4. Immunohistochemical localization of BrdU incorporation in nuclei in liver sections from wild-type C57Bl/6 and TNF{alpha} knockout mice treated with 100 mg/kg/day WY14,643 for 4 days. Livers were removed, fixed and then BrdU was detected as described in Materials and methods.

 


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Fig. 5. Effects of WY14,643 on hepatocellular proliferation in wild-type C57Bl/6 and TNF{alpha} knockout mice. Wild-type (+/+) or TNF{alpha} knockout (–/–) mice were dosed with 100 mg/kg/day WY14,643 for 4 days. The percent labeling index was calculated as described in Materials and methods. Data are expressed as means ± SEM.

 
To determine if synthesis of TNF{alpha} was stimulated by WY14,643 treatment in wild-type mice, TNF{alpha} mRNA levels were measured in the livers of these mice. Other groups have reported that hepatic TNF{alpha} mRNA levels were increased after WY14,643 treatment of rats (12), however, in our study TNF{alpha} mRNA levels decreased with WY14,643 treatment in wild-type mice (Figure 6Go).



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Fig. 6. Effects of WY14,643 on TNF{alpha} mRNA expression in wild-type C57Bl/6 mice. Wild-type mice were dosed with 100 mg/kg/day WY14,643 for 4 days. Total RNA was isolated and TNF{alpha} mRNA was quantified by real time RT–PCR as described in Materials and methods. Data are expressed as means ± SEM.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we have tested the hypothesis that TNF{alpha} is required for the hepatocellular proliferation observed after peroxisome proliferator treatment. The basis for this hypothesis is the reported inhibitory effects of anti-TNF{alpha} antibodies on WY14,643-induced hepatocellular proliferation in rats (12). In an attempt to directly test this hypothesis, we treated wild-type C57Bl/6 and TNF{alpha} knockout mice with WY14,643 for 4 days. The markers of peroxisome proliferation that were examined, including FACO activity, FACO mRNA and liver enlargement, were increased to equivalent levels by WY14,643 in both wild-type and TNF{alpha} knockout mice. Likewise, morphological evaluation demonstrated increases in the size and number of peroxisomes in hepatocytes from mice of both genotypes. In addition, hepatocyte proliferative responses of both wild-type and TNF{alpha} knockout mice were similarly increased by WY14,643 treatment. Lastly, we observed a decrease in TNF{alpha} mRNA levels in wild-type mice after WY14,643 treatment. These data suggest that TNF{alpha} is not involved in the pleiotropic response of rodent liver to peroxisome proliferators.

TNF{alpha} knockout mice had 23% smaller livers compared with wild-type mice. This is consistent with the reported role of TNF{alpha} in liver growth (8,9). Still, the liver weight changes that occurred after WY14,643 treatment resulted in relative liver weights that were equivalent to those in wild-type mice treated with WY14,643. The liver enlargement could be due to both increased cell size and cell number.

Our data demonstrate that peroxisome proliferator-induced hepatic mitogenesis can occur independently of TNF{alpha} and that TNF{alpha} may not be required for this event. This is supported by the fact that even though TNF{alpha} knockout mice have no detectable levels of TNF{alpha}, even after LPS treatment (data not shown), they still respond with marked hepatocellular proliferation to WY14,643 treatment. Moreover, in wild-type mice we observed a decrease in TNF{alpha} mRNA after treatment with WY14,643, in direct contrast to results reported in the literature for WY14,643-treated rats (12). The studies reported by Bojes et al. differed from our studies in several ways. In the previous studies a rat model was used with a polyclonal antibody to neutralize TNF{alpha} activity in vivo and cell proliferation was monitored only over the first 24 h. It is possible that treatment with anti-TNF{alpha} antibody simply delayed the induction of cell proliferation, making it appear as if the antibody blocked the cell proliferation response, when, in fact, it might have occurred with prolonged treatment. Alternatively, the absence of TNF{alpha} during in utero and neonatal development may have led to the elaboration of compensatory pathways that are either minor or non-existent in normal animals. This possibility could be tested by the generation of conditional TNF{alpha} knockouts or with longer term neutralizing antibody studies.

In studies examining the role of TNF{alpha} in acetaminophen toxicity a paradox was observed between studies using TNF{alpha} knockout mice and studies that neutralized TNF{alpha} with anti-TNF{alpha} antibodies. Neutralizing TNF{alpha} with an antibody protected wild-type mice from acetaminophen toxicity whereas acetaminophen toxicity was exaggerated in TNF{alpha} knockout mice (22). In other models of regenerative hyperplasia, such as occurs after CCl4-induced hepatic necrosis, a clear role for TNF{alpha} was demonstrated using TNF R1 knockout mice (10). TNF R1 knockout mice demonstrated severely limited hepatocyte DNA synthesis compared with either TNF R2 knockout or wild-type control mice after treatment with CCl4, even though equivalent liver injury was observed in both models. In partial hepatectomy models of liver regeneration, TNF R1 knockout mice displayed a marked reduction in hepatocellular proliferation (9). Anti-TNF{alpha} antibodies also inhibited cell proliferation after partial hepatectomy in rats (8), consistent with the findings from the TNF R1 knockout mice. Thus, while some inconsistency in response between TNF{alpha} antibody and TNF{alpha}-deficient models for hepatotoxicity end points is apparent, these two models have consistent results regarding mitogenic end points. Our data suggest that even though TNF{alpha} is important for some types of hepatic mitogenesis, like those observed after partial hepatectomy and CCl4 treatment, the mechanism of hepatocellular proliferation after peroxisome proliferator treatment appears to be different. This suggestion is consistent with reports that interleukin-6 (IL-6)-deficient mice also elicit a cell proliferation response after peroxisome proliferator treatment that is equivalent to wild-type mice (23). IL-6 knockout mice also have deficiencies in hepatic regeneration after partial hepatectomy (24). Furthermore, adding exogenous IL-6 relieves the deficiency in liver regeneration found in TNF R1 knockout mice (9). Anderson et al. (25) and Corton (personal communication) have found that mice with defects in TNF R1 or TNF R2 or both also retain the mitogenic response after peroxisome proliferator treatment. These studies are consistent with our findings and confirm the lack of a requirement for TNF{alpha} in peroxisome proliferator-induced hepatic cell proliferation.

In summary, we have shown that mice that lack TNF{alpha} have equivalent peroxisome and cell proliferation induction in response to peroxisome proliferator treatment, indicating that in knockout animals TNF{alpha} is not required for peroxisome proliferator-induced hepatic mitogenesis. These data also suggest that the mitogenic response observed after peroxisome proliferator treatment may occur via a mechanism distinct from that of liver regeneration after partial hepatectomy or CCl4 treatment.


    Notes
 
1 To whom correspondence should be addressedEmail: jeff_lawrence{at}merck.com Back


    Acknowledgments
 
The authors gratefully acknowledge the technical assistance of David Alberts, Gary Dysart, John Frank, Brenda Givler, Karen MacNaul, Carol McCoy and Marcia Pitzenberger. We also thank Drs David Moller and George Kollias for help in obtaining the TNF{alpha} knockout mice. We are grateful to Dr Chris Corton for his permission to cite his group's work as a personal communication. In addition, we thank Drs Karen Richards and Tom Rushmore for the use of equipment and Dr Scott Grossman for helpful suggestions.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 24, 2000; revised November 16, 2000; accepted November 20, 2000.