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Endocrinology Vol. 142, No. 7 2953-2960
Copyright © 2001 by The Endocrine Society

ARTICLES

Retarded Liver Growth in Interleukin-6-Deficient and Tumor Necrosis Factor Receptor-1-Deficient Mice1

Ville Wallenius, Kristina Wallenius, Masanori Hisaoka, Jonas Sandstedt, Claes Ohlsson, Manfred Kopf and John-Olov Jansson

Research Center for Endocrinology and Metabolism, Departments of Internal Medicine (V.W., K.W., J.S., C.O., J.-O.J.) and Pathology (M.H.), Sahlgrenska University Hospital, SE-413 45 Gothenburg, Sweden; and Basel Institute for Immunology (M.K.), CH-4005 Basel, Switzerland

Address all correspondence and requests for reprints to: V. Wallenius, M.D., Ph.D., Research Center for Endocrinology and Metabolism, Gröna Stråket 8, SE-413 45 Gothenburg, Sweden. E-mail: ville.wallenius{at}medic.gu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The liver size in adult mammals is tightly regulated in relation to body weight, but the hormonal control of this is largely unknown. We investigated the roles of interleukin-6 (IL-6) and tumor necrosis factor (TNF) receptor-1 in the regulation of intact liver weight in adult mice. The relative liver wet and dry weights of older adult (5- to 10-month-old) IL-6 knockout (IL-6-/-) mice were decreased by 22–28%, and total contents of DNA and protein were decreased compared with those in age-matched wild-type mice. Weights of other visceral organs were unaffected. Older adult (6- to 8-month-old) TNF receptor-1 knockout (TNFR1-/-) mice displayed decreased relative liver weight. Treatment with a single injection of IL-6 increased liver wet and dry weights in IL-6-/- and wild-type mice, but not TNFR1-/- mice. Treatment with TNF{alpha} enhanced liver weight and DNA synthesis of nonparenchymal liver cells at 24 h in wild-type, but not IL-6-/-, mice. At 48 h, TNF{alpha} induced DNA synthesis in nonparenchymal cells and hepatocytes of both wild-type and IL-6-/- mice. In conclusion, TNF receptor-1 stimulation and IL-6 production are both necessary for normal liver weight gain in older adult mice. The results of TNF{alpha} and IL-6 treatment further indicate that the effects of TNF receptor-1 and IL-6 depend on each other for full stimulation of liver growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH THE regenerative capacity of the liver after loss of liver tissue has been extensively studied in different animal models (1, 2, 3), little is known about the regulation of the size of the intact liver. However, it is clear that the liver weight is set under tight control, because it is essentially constant in relation to body weight throughout life (4, 5, 6, 7).

A number of peptide growth factors have been shown to stimulate the proliferation of hepatocytes in vitro (2, 4, 5, 6, 7), but few have been shown to regulate liver size in vivo. To date GH is the most prominent one, and high levels of GH can induce a relative increase in liver weight compared with body weight in mice (8, 9). Further, the absence of GH (8, 9, 10) or the GH receptor (11, 12) decreases liver weight more than body weight. Nevertheless, GH does not stimulate hepatocyte proliferation in vitro (13), and its mechanism of action on liver growth in vivo has not been elucidated. Insulin-like growth factor I (IGF-I) is the main mediator of GH effects in peripheral organs, but it is probably not important for liver growth in vivo. IGF-I does not induce a substantial liver growth in normal or GH-deficient mice (9, 10). Moreover, a selective depletion of liver-derived IGF-I with retained GH secretion has been reported to result in unchanged (14) or even increased (15) liver weight in mice.

Based on overexpression experiments in vivo in transgenic animals, transforming growth factor-{alpha} (TGF{alpha}) (16) and hepatocyte growth factor (HGF) (17, 18) can increase relative liver weight. The physiological role of endogenous production of these growth factors in regulation of liver growth, however, is unclear. The liver size is normal in TGF{alpha} knockout mice (19), whereas it has been impossible to investigate the effect of loss of endogenous HGF on liver growth due to neonatal lethality in HGF and HGF receptor knockout mice (20, 21).

Rapid liver growth can be induced pharmacologically by treatment with so-called primary liver growth promoters, i.e. a variety of structurally different compounds, many of which cause proliferation of peroxisomes via binding to the peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) (22). There is no decrease in liver weight in mice with a deleted PPAR{alpha} gene, and therefore, there is no evidence that endogenous PPAR{alpha} ligands are necessary for maintaining normal liver growth (23).

The aim of the present study was to identify endogenous factors of importance for growth of the intact liver. Interleukin-6 (IL-6) is a pleiotropic and multifunctional cytokine that is a mediator of hepatic acute phase reaction (24). As it has been reported that IL-6 is essential for liver regeneration (25, 26, 27), we studied the effect of loss of IL-6 on growth of intact liver in IL-6-/- mice. We also studied the effect of IL-6 replacement in IL-6 knockout mice. Because tumor necrosis factor-{alpha} (TNF{alpha}) enhances IL-6 via TNF receptor-1 (28) and because TNF receptor-1 knockout (TNFR1-/-) mice have decreased liver regeneration (29), we also investigated the effect of TNF receptor-1 depletion on intact liver weight. To investigate the interrelation between TNF receptor-1 stimulation and IL-6 in regulation of liver growth, we studied the effects of TNF{alpha} treatment on IL-6-/- mice and of IL-6 treatment on TNFR1-/- mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
IL-6-/- mice were generated as described by Kopf et al. (30), and TNFR1-/- mice were generated as described by Pfeffer et al. (31). To reduce genetic heterogeneity, the IL-6-/- and TNFR1-/- genotypes were moved onto the C57BL/6 background by eight and nine successive backcrosses, respectively. The resulting strains of mice consist genetically of more than 99.5% C57BL/6. Normal C57BL/6 mice from B&K Universal (Sollentuna, Sweden) were used as wild-type controls for both IL-6-/- and TNFR1-/- mice. The animals were maintained under standardized environmental conditions, i.e. 24-26 C, 50–60% relative humidity, artificial lighting between 0500–1900 h, and water and pelleted food ad libitum. In all experiments the total body weight of each animal was measured before the liver was excised and weighed. The relative liver weight was calculated as a percentage of the liver weight per total body weight. All procedures involving the mice were conducted in accordance with protocols approved by the institution and the local ethical committee on animal care.

Measurements of DNA synthesis and contents of DNA, protein, and triglycerides
DNA synthesis was measured as 5'-bromo-2'-deoxyuridine (BrdU) incorporation. After IL-6 and TNF{alpha} treatment, BrdU was given in ip injections at 2, 6, and 22 h (24-h group) or at 2, 6, 22, 35, and 46 h (48-h group) at a dose of 100 nmol/g BW. Labeled cells were detected on cryosections using the BrdU Labeling and Detection Kit II (Roche, Basel, Switzerland). The number of cells undergoing mitosis was calculated by counting the numbers of BrdU-labeled hepatocytes and nonparenchymal cells per 1000 hepatocytes. The estimation that hepatocytes account for 60% of the total number of cells in the liver (22) was used to calculate the total number of nonparenchymal cells. The results are expressed as the percentage of BrdU-labeled hepatocytes and nonparenchymal cells in relation to the total number of the respective cell type. For measurement of total DNA, pieces of frozen liver tissue were weighed and homogenized in 2 M LiCl and 0.05 M Tris, pH 7.5. The DNA-binding fluorochrome dye Hoechst 33258 was added at a concentration of 1:200. The DNA concentration was measured using a DyNA Quant 200 Fluorometer (Amersham Pharmacia Biotech, Uppsala, Sweden) by comparing the samples to a standard curve. Protein concentrations were determined using the DC Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA) according to the manufacturer’s instructions. BSA (fraction V; Sigma, St. Louis, MO) was used for preparation of a standard curve. Absorbance was then measured with an enzyme-linked immunosorbent assay spectrophotometer at a wavelength of 650 nm. Triglycerides were extracted from homogenized liver tissue by the Bligh-Dyer method. One milliliter of liver homogenate (~100 mg liver) in double distilled water was added to 3.75 ml methanol-chloroform (2:1). After centrifugation the infranatant was reextracted with 4.75 ml methanol-chloroform-H2O (2:1:0.8). The supernatants were pooled, and 2.5 ml chloroform and 2.5 ml H2O were added. After centrifugation the infranatant was recovered and evaporated under N2 and resolved in 95% ethanol. The concentration of triglycerides was measured using the TG MPR2 GPO-PAP enzymatic colorimetric test (Roche Molecular Biochemicals, Mannheim, Germany) and a Shimadzu spectrophotometer. The concentrations of DNA, protein, and triglycerides were multiplied by total liver weight to get the total contents of these cellular components per liver.

IL-6 and TNF-{alpha} treatment
Human recombinant IL-6 (Chemicon International, Temecula, CA) was given in one ip injection at a dose of 0.8 µg/g BW to 11- to 17-month-old female IL-6-/- and to 3-month-old wild-type and TNFR1-/- mice. Murine or human recombinant TNF{alpha} (R & D Systems, Inc., Minneapolis, MN) was given in one ip injection at a dose of 0.1 µg/g BW to 3-month-old IL-6-/- and wild-type female mice. Control animals received injections of the vehicle [0.01 M PBS and 0.1% BSA (fraction V; <0.1 ng endotoxin/mg; Sigma)]. Both cytokines were dissolved in 0.01 M PBS and 0.1% BSA immediately before treatment. The mice were killed 24 or 48 h after treatment, and liver tissue was weighed and immediately frozen in liquid nitrogen or was formalin fixed.

Measurement of liver dry weights
Frozen pieces of liver were weighed and vacuum dried in a Leybold-Heraeus Lyovac GT2 vacuum drier (Heraeus, Hanau, Germany) for approximately 48 h and then weighed again to calculate water content. Relative liver dry weights were calculated as (liver sample dry weight/liver sample wet weight) x (total liver wet weight/body weight).

Measurements of liver and plasma IL-6
IL-6 in plasma was measured using an enzyme-linked immunosorbent assay (R & D Systems, Inc., Minneapolis, MN). For measurement of liver IL-6, approximately 100 mg liver tissue were homogenized and diluted in the calibrator diluent of the assay. The least detectable value of the assay was 15.6 pg/ml. Subsequently, IL-6 was measured in the samples according to the manufacturer’s instructions.

Histology and measurement of apoptosis
Microscopic examination was performed using conventional hematoxylin-eosin sections of liver tissue. The rate of apoptosis was evaluated using the ApopTag In Situ Apoptosis Detection Kit (Appligene Oncor Lifescreen, Montreal, Canada) according to the manufacturer’s instructions. The extent of apoptotic DNA fragmentation was also analyzed by autoradiography of size-fractionated DNA from the liver labeled at the 3'-ends by [33P]dideoxy-ATP as described by Billig et al. (32).

GH treatment
The GH treatment started when the animals were 2 months old. Human GH (Genotropin, Pharmacia-Upjohn, Stockholm, Sweden) was dissolved in 0.9% saline and given as a continuous sc infusion (3.5 µg/g BW·day) for 3 months by osmotic minipumps (model 2004, Alza Corp., Palo Alto, CA) implanted on the back of the mice. Control animals received 0.9% saline administered in the same way. The minipumps were replaced three times during the treatment period. The animals were killed at 5 months of age.

Statistical analysis
Values are given as the mean and SEM or as the SE of the estimates. Comparisons between two groups were made using Student’s t test. Comparisons between more than two groups were made using one-way ANOVA, followed by Student’s t test with Bonferroni’s correction. P < 0.05 was considered significant. Linear regression was used for analysis of the relation between body weight and liver weight in wild-type and IL-6-/- mice (Fig. 1Go, C and D). The slopes for the analyses of the two groups were then compared by testing the null hypothesis, that a certain increase in body weight results in a similar increase in liver weight in both wild-type and IL-6-/- mice. The slopes were compared using a t test.



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  		Figure 1. A and B, Relative liver weights (percent liver weight/body weight) in young adult (2-month-old) and older (5- to 10-month-old) female and male IL-6-/- and wild-type mice. C and D, Absolute liver weights in wild-type and IL-6-/- mice of both genders, analyzed with regression analysis for the relation between liver and body weight. IL-6-/- depicts mice with IL-6 gene knockout. There were 6–10 mice in each group in A and 7 or 8 mice in each group in B. There were 64 mice in each group in C and 72 mice in each group in D. ###, P < 0.001 (vs. wild-type mice of corresponding age). {dagger}{dagger}{dagger}, P < 0.001 (vs. young adult IL-6-/- mice).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Relative liver weights in young and older IL-6-/- and wild-type mice
The liver weight in relation to body weight (relative liver weight = % liver weight/body weight) in young (2-month-old) adult female mice was similar in wild-type and IL-6-depleted animals (Fig. 1AGo). In contrast, the relative liver weight in the older female (5- to 10-month-old) IL-6-/- mice was decreased by 26–28% compared with those in both age-matched wild-type mice and young female (2-month-old) IL-6-/- mice (Fig. 1AGo). Similar to the females, relative liver weight was reduced in older IL-6-/- males by 22–28% compared with older wild-type mice and young IL-6-/- mice (Fig. 1BGo). The relative weights of the spleen, heart, and kidneys were not different between IL-6-/- and wild-type mice (data not shown). Thus, the lack of IL-6 caused a specific reduction in relative liver weight in older, but not in young adult, IL-6-/- mice of both sexes.

The relation between body weight and absolute liver weight in wild-type and IL-6-/- mice was further analyzed by linear regression (Fig. 1Go, C and D). The regression coefficients for the wild-type and IL-6-/- mice were 0.05 (SE = 0.003) and 0.03 (SE = 0.004) g liver weight/g BW, respectively. The null hypothesis that the slopes for the linear regression analyses shown in Fig. 1Go, C and D, were not different between wild-type and IL-6-/- mice was tested and rejected (P < 0.001). This finding indicated that the liver growth rate in relation to body weight gain was different in older IL-6-/- mice compared with wild-type controls.

Histological examination did not reveal any morphological differences or pathological changes in the livers of 5- to 6-month-old IL-6-/- and wild-type mice (not shown). The rate of apoptosis in intact livers, as assessed by both the in situ terminal deoxynucleotidyltransferase method and DNA laddering, was not increased in the livers of the IL-6-/- mice compared with those of the wild-type mice (data not shown).

Effects of IL-6 knockout on hepatic content of DNA, protein, and triglycerides
To examine the composition of livers we measured total hepatic contents of DNA, protein, and triglycerides. Total DNA and protein contents were decreased by 18–20% in the livers of older (5- to 10-month-old) male IL-6-/- mice compared with those of corresponding wild-type mice (Table 1Go), showing that the decrease in liver weight in older IL-6-/- mice is not only due to decreased water content. The total triglyceride content was not significantly changed in IL-6-/- mice compared with wild-type controls (Table 1Go).


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  		Table 1. DNA, protein, and triglyceride contents in livers of older adult male IL-6-/- and wild-type mice

 
IL-6 treatment in older IL-6-/- mice
IL-6 treatment partly reversed the age-dependent reduction in relative liver wet weight in older adult female IL-6-/- mice (Fig. 2AGo). Liver dry weights were also decreased in older IL-6-/- mice and were increased by IL-6 replacement (Fig. 2BGo), indicating that the effects on wet weight were not caused by changes in water retention. There was also a tendency for increased total hepatic protein content after IL-6 treatment (43.2 ± 2.3 vs. 48.0 ± 2.0, vehicle vs. IL-6 treatment), but this effect was not statistically significant (P = 0.14). There was no effect of IL-6 on DNA synthesis measured by BrdU incorporation (data not shown). This finding was supported by measurement of total hepatic content of DNA, which was not increased after IL-6 treatment in the older adult IL-6-/- mice compared with that after vehicle treatment (not shown). There was no change in the total hepatic content of triglycerides after IL-6 treatment (not shown).



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  		Figure 2. A, Liver wet weights after IL-6 treatment in older adult (11- to 17-month-old) female IL-6-/- mice. Livers were collected and weighed 24 h after a single injection of human recombinant IL-6 at a dose of 0.8 µg/g BW. Data were pooled from two separate experiments. B, Liver dry weights after IL-6 treatment in older adult (11-month-old) female IL-6-/- mice. There were six or seven mice in each group in A and three or four in B. {dagger}{dagger}, P < 0.01 (vs. young IL-6-/- mice). *, P < 0.05; ** P < 0.01 (vs. corresponding vehicle-treated mice).

 
Relative liver weights in young and older adult TNFR1-/- and wild-type mice
As several studies have indicated that IL-6 is a mediator of TNF{alpha} effects (25, 29, 33) and as liver weights were decreased in IL-6-/- mice, we investigated liver weights in young and older adult TNFR1-/- mice. In the older (6- to 8-month-old) TNFR1-/- mice, relative liver weights were decreased by approximately 11% in both females (Fig. 3AGo) and males (Fig. 3BGo) compared with those in age-matched wild-type mice. The older TNFR1-/- females had lower relative liver weights than the young TNFR1-/- mice (Fig. 3AGo). In young (2-month-old) mice of both genders, there was no significant difference between TNFR1-/- and wild-type mice (Fig. 3Go, A and B). To determine why relative weights of TNFR1-/- livers were less decreased than those of IL-6-/- livers, we measured basal IL-6 levels in liver homogenate and plasma of TNFR1-/- and wild-type mice. In both groups IL-6 levels were below the detection limit of the assay in both liver homogenate and plasma.



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  		Figure 3. A and B, The effect of TNF receptor-1 gene knockout on the relative liver weights (percent liver weight/body weight) in young adult (2-month-old) and older (5–8 month-old) female and male TNFR1-/- and wild-type mice. There were 4–10 mice in each group in A and 5–10 mice in each group in B. #, P < 0.05; ###, P < 0.001 (vs. older wild-type mice). {dagger}, P < 0.05 (vs. young TNFR1-/- mice).

 
IL-6 treatment to young wild-type and TNFR1-/- mice
To investigate the possible interrelation between the decreases in liver weight caused by IL-6-/- and TNFR1-/-, we next compared the effects of IL-6 in TNFR1-/- and wild-type mice. Liver weights of young wild-type mice, but not those of TNFR1-/- mice, were increased by a single injection of IL-6 (Fig. 4AGo). Liver dry weights were increased by IL-6 treatment in wild-type, but not TNFR1-/-, mice (Fig. 4BGo). There was, however, no increase in DNA synthesis measured by BrdU incorporation in response to this acute IL-6 treatment in either wild-type or TNFR1-/- mice (data not shown), in line with the findings that a single injection of IL-6 had no such effect in IL-6-/- mice (see above). Body weights were not affected by IL-6 treatment in any group of mice.



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  		Figure 4. A, Liver wet weights after IL-6 treatment to young (3-month-old) wild-type and TNFR1-/- mice. Livers were collected 24 or 48 h after treatment. Values for vehicle-treated control mice at 24 and 48 h were pooled. B, Liver dry weights after IL-6 treatment in young (3-month-old) wild-type and TNFR1-/- mice. There were three to five mice in each group. *, P < 0.05; **, P < 0.01 (vs. corresponding vehicle-treated mice). ##, P < 0.01; ###, P < 0.001 (vs. corresponding wild-type mice).

 
Liver growth in response to TNF-{alpha} treatment in IL-6-/- and wild-type mice
As both IL-6-/- and TNFR1-/- mice had small livers (see above), we compared responsiveness to TNF{alpha} in IL-6-/- and wild-type mice. TNF{alpha} has been shown to cause proliferation of both nonparenchymal and parenchymal liver cells, depending on the dose and time after administration (22). Treatment with TNF{alpha} increased the relative liver weight at 24 h (Fig. 5AGo) and BrdU incorporation in nonparenchymal cells of wild-type female mice at 24 and 48 h (Fig. 5BGo). In IL-6-/- mice, BrdU incorporation in response to TNF{alpha} in nonparenchymal cells was delayed compared with that in wild-type mice and was not present until 48 h after treatment (Fig. 5BGo). There was a tendency for increased liver weight in the IL-6-/- mice at 48 h after TNF{alpha} treatment, but this change was not statistically significant (P = 0.15). Hepatocyte BrdU incorporation was increased at 48 h in both wild-type and IL-6-/- mice. The finding that TNF{alpha} enhances DNA synthesis first in nonparenchymal cells and then in hepatocytes is in line with previous studies (22). Hepatic morphology was unchanged by TNF{alpha} treatment (not shown).



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  		Figure 5. A, Relative liver weights (percent liver weight/body weight) in young (3-month-old) IL-6-/- and wild-type female mice 24 and 48 h after a single injection of murine TNF{alpha}. B, Accumulated DNA synthesis 24 and 48 h after TNF{alpha} treatment was estimated as BrdU-positive (BrdU+) nonparenchymal liver cells (NPC) and hepatocytes in IL-6-/- and wild-type mice. There were three or four mice in each group. ##, P < 0.01 (vs. corresponding TNF{alpha}-treated wild-type mice). *, P < 0.05; **, P < 0.01 (vs. vehicle-treated wild-type mice).

 
Liver growth in response to human and mouse TNF{alpha} treatment
We investigated whether TNF{alpha} stimulates liver growth via type 1 (p55) or type 2 (p75) TNF receptors. Murine and human TNF{alpha} have different specificities for the murine TNF receptor subtypes. Human TNF{alpha} binds only to murine TNF receptor-1, whereas murine TNF{alpha} binds to both type 1 and type 2 receptors (34). The effects of human and murine TNF{alpha} in wild-type mice were similar, as liver weights were increased by 14–15% at 8 h and by 30–33% at 16 h after both treatments (Table 2Go). Thus, stimulation of the murine TNF receptor-1 by human TNF{alpha} was sufficient to induce liver growth, indicating that the effect of TNF{alpha} on liver growth is exerted via type 1, rather than type 2, TNF receptors.


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  		Table 2. The effects of human vs. murine TNF{alpha} on liver growth in wild-type mice

 
Liver growth in response to GH treatment in older IL-6-/- and wild-type mice
GH is an important regulator of liver size in mice, as demonstrated by the relative decrease in liver weight in GH-deficient dwarf mice (8) and the relative increase in liver weight in GH transgenic giant mice (9). In both examples liver weight is changed more than body weight, indicating that GH has a specific effect on relative liver weight. To test the hypothesis that the effect of GH on liver weight is not dependent on IL-6, we treated adult male IL-6-/- and wild-type mice with GH by continuous infusion in osmotic minipumps for 3 months. The relative liver weights were increased by 12–16% in both groups after GH treatment compared with those in vehicle-treated controls (Table 3Go). The relative liver weights were lower in the control IL-6-/- compared with the control wild-type mice (-23.4 ± 1.4%; P < 0.001; Table 3Go), and a similar difference remained after GH treatment (-20.6 ± 5.3%; P < 0.01).


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  		Table 3. The effect of 3-month GH treatment on liver weight in wild-type and IL-6-/- mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results indicate that endogenous IL-6 regulates liver growth in an age-dependent and organ-specific manner. Compared with wild-type mice, relative liver weight was reduced in older IL-6-/- mice. The effect of IL-6 depletion on relative liver weight was also demonstrated by the regression analysis of liver weight vs. body weight in IL-6-/- and wild-type mice. Liver dry weight as well as total contents of DNA and protein were reduced in IL-6-/- mice, indicating that the decrease in wet weight is not only due to loss of water. The lack of IL-6 did not influence the liver weight of young (2- to 3-month-old) IL-6-/- mice. Liver weights were also reduced in older adult TNFR1-/- mice. Thus, the constancy of relative liver weight (4, 5, 6, 7) throughout life in rodents seems to be dependent on the presence of TNF receptor-1 and IL-6. The stimulatory effect of high doses of GH on liver growth was not dependent on IL-6.

The regulation of liver weight by IL-6 was supported by the partial recovery of both wet and dry liver weight in older IL-6-/- mice treated with a single injection of IL-6. The single IL-6 injection did not, however, reverse the decrease in the total liver content of DNA in IL-6-/- mice and did not increase DNA synthesis measured by BrdU incorporation, which has been reported by other investigators (25). One possible explanation is that IL-6 induces cell division selectively of the large pool of hepatocytes that are polyploid or multinuclear in the intact liver. These hepatocytes do not need DNA synthesis to go through cell division. It is possible that more sustained IL-6 treatment could stimulate DNA synthesis and that the decrease in liver DNA content in IL-6-/- mice could be reversed at least in part. After partial hepatectomy, liver weight seems to increase before DNA synthesis occurs (26, 27, 35), showing that liver weight can change in part independently of changes in DNA synthesis.

The present finding that liver weight was increased only transiently at 24 h after TNF{alpha} treatment in wild-type mice and seemed to decrease at 48 h could be explained by decreased water retention. The stimulatory effect of TNF{alpha} on hepatocyte and nonparenchymal cell DNA synthesis at 48 h in conjunction with the decreased liver size in TNF receptor-1 knockout mice suggest, however, that prolonged TNF{alpha} treatment would cause a more sustained increase in liver weight.

The mechanism of the decreased liver weight caused by chronic IL-6 depletion remains to be determined. In this study there were no detectable differences in apoptosis, measured by the in situ terminal deoxynucleotidyltransferase method and DNA laddering, between IL-6-/- and wild-type mice. However, a relatively large decrease in liver mass, such as seen in the present study, could be caused by a minute increase in hepatic apoptosis (36) that may be undetectable with the methods we used or by a decrease in mitotic rate over a long period.

The reason why liver weights were decreased only in older adult IL-6-/- and TNFR1-/- mice is unknown. However, there are other examples of age-associated variability in liver weight in mice. For instance, TGF{alpha}-overexpressing transgenic mice had enlarged livers in young, but not older, animals (16). Moreover, 8-month-old male PPAR{alpha} gene knockout mice exhibited an increased relative liver weight and developed steatosis, whereas females and younger males did not (23).

Although the present results suggest a stimulatory effect of endogenous IL-6 on liver growth, the effect of supraphysiological levels of IL-6 is not clear. Mice that overexpress IL-6 alone do not display an increased proliferation of hepatocytes. This could reflect a primary lack of effect by IL-6 or a down-regulation of the sensitivity to IL-6 as an adaptation of these animals to the chronic IL-6 overexpression. In contrast, double transgenic mice that overexpress both IL-6 and soluble IL-6 receptor, display hepatocellular hyperplasia (37, 38). This effect, however, may reflect an unspecific stimulation of the signal transducer gp130 in liver cells by the IL-6/soluble IL-6 receptor complex. It is well known that gp130 is a cellular mediator for several cytokine receptors other than the IL-6 receptor (24).

In the present experiments one injection of human TNF{alpha} induced an increase in liver weight and DNA synthesis, first in nonparenchymal cells and then in hepatocytes, which is in line with the findings of other investigators (22). Our finding that intact liver weight is decreased in both IL-6-/- and TNFR1-/- mice suggests that both stimulation of TNF receptor-1 and the presence of IL-6 are of importance for growth of the intact liver. This is supported by our finding that both murine and human TNF{alpha}, the latter acting only via receptor-1, induced liver growth in wild-type mice. The relative liver weights were less decreased in TNFR1-/- mice than in IL-6-/- mice. One possible explanation for this may be that apoptosis is decreased in the livers of TNFR1-/- mice, as it is well known that TNF receptor-1 can also mediate apoptotic signals in hepatocytes (39).

IL-6 seems to mediate stimulatory effects of TNF{alpha} on liver regeneration (25, 29, 33). Our present results suggest that growth of the intact liver, induced by TNF{alpha}, is partly dependent on IL-6. The TNF{alpha}-induced increases in liver weight and DNA synthesis of nonparenchymal cells were attenuated in IL-6-/- mice, whereas the increase in hepatocyte DNA synthesis was not changed in IL-6-/- mice. The assumption that TNF receptor-1 mediated mechanisms, other than IL-6 production, stimulate liver growth might be supported by the finding that IL-6 could not enhance liver weight in TNFR1-/- mice. A simple interpretation of our results is that stimulation of liver growth involves the actions of IL-6 as well as other factors that are induced by TNF{alpha} via TNF receptor-1. Overall, our data indicate that TNF receptor-1 stimulation and IL-6 are mutually dependent on each other to exert a full stimulatory effect on growth of the intact liver.


   Acknowledgments
 
We thank Martin Gellerstedt for help with the statistical analyses, and Petra Strand, Ted Fjällman, and Karin Karlsson for valuable technical assistance. We also thank Tak Mak and Anders Örn for providing the TNFR1-/- mice, and Håkan Billig for advice regarding the apoptosis measurements.


   Footnotes
 
1 This work was supported by grants from the Swedish Medical Research Council (9894), the Bergvall Foundation, Novo Nordisk Pharma, the Assar Gabrielsson Foundation, the Swedish Medical Society, the Gothenburg Medical Society, and the Swedish Society for Medical Research. The Basel Institute for Immunology has been founded and is supported by Hoffman-LaRoche Inc. Back

Received January 13, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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