SHORT-TERM ETHANOL EXPOSURE INCREASES THE EXPRESSION OF KUPFFER CELL CD14 RECEPTOR AND LIPOPOLYSACCHARIDE BINDING PROTEIN IN RAT LIVER

Tuomo A. Lukkari, Harri A. Järveläinen, Teija Oinonen, Eeva Kettunen and Kai O. Lindros*

Alcohol Research Center, National Public Health Institute, POB 719, 00101 Helsinki, Finland

Received 7 September 1998; in revised form 23 November 1998; accepted 11 January 1999


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Gut-derived endotoxins (lipopolysaccharide, LPS) complexed to LPS-binding protein (LBP) activate liver Kupffer cells via their CD14 receptor. Pro-inflammatory cytokines are released and this is postulated to promote liver injury. We previously demonstrated enhanced expression of CD14 endotoxin receptor after 2 weeks of alcohol administration. A similar result, based on 6 weeks of ethanol treatment, was recently reported and suggested to correlate with alcohol-induced liver injury. To establish whether this occurs prior to or after the initiation of damage, we investigated the temporal effect of continuous ethanol exposure on the expression of CD14 and the associated LBP. In addition, we studied the effect of treatment with gadolinium chloride (GdCl3) that inactivates Kupffer cells and alleviates alcohol-induced liver damage. The amount of CD14 and LBP mRNA, as determined by reverse transcriptase–polymerase chain reaction (RT–PCR), was unchanged 4–8 h after intragastric ethanol administration. However, after 24–48 h of repeated ethanol administration, CD14 and LBP mRNA both increased significantly and reached a level similar to that observed after 6 weeks of ethanol exposure by liquid diet. Immunostaining experiments with ED2 antibody demonstrated that GdCl3 efficiently inactivated Kupffer cells. However, there was no concomitant reduction in the expression of CD14 mRNA, suggesting that compensatory infiltration by ED2-negative, but CD14-positive, macrophages had occurred. Our results demonstrate that soon after the initiation of ethanol exposure, i.e. within 24–48 h, the hepatic expression of both the CD14 receptor and LBP is increased. This suggests that these increases could contribute to the initiation of alcoholic damage rather than being a consequence of the injury.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Increased plasma levels of endotoxins, derived from intestinal Gram-negative bacteria, are frequently seen in alcoholics (Bode et al., 1987Go). Endotoxins are translocated from the gut and an increase in their plasma levels also results from depressed reticuloendothelial phagocytosis (Ali and Nolan, 1967Go; Bjarnason et al., 1984Go). In rats chronically exposed to ethanol by intragastric infusion, a correlation between alcohol-induced liver damage (ALD) and serum endotoxin levels has been reported (Nanji et al., 1993Go). Furthermore, treatment with antibiotics that reduce intestinal Gram-negative bacteria has been shown to alleviate ALD (Adachi et al., 1995Go).

Kupffer cells, the resident macrophages in the liver, are activated by circulating endotoxins to produce pro-inflammatory cytokines, such as tumour necrosis factor (TNF)-{alpha} and interleukin (IL)-1ß. These cells seem to play a critical role in experimental liver damage, since their inactivation with gadolinium chloride (GdCl3 ) has been shown to alleviate or even prevent different forms of liver injury (Tsukamoto and Lin, 1997Go) including that seen after intragastric alcohol feeding (Adachi et al., 1994Go; Koop et al., 1997Go).

Circulating LPS is complexed to LPS-binding protein (LBP), a secretory class I acute phase protein (Schumann et al., 1996Go), which is synthesized by the liver and secreted into the plasma as a 60 kDa glycoprotein (Ramadori et al., 1990Go). Its expression is up-regulated by LPS (Geller et al., 1993Go).

The LPS–LBP complex binds to the CD14 receptor, a 55 kDa myeloid membrane glycoprotein expressed by monocytes and macrophages. The receptor mediates the activation of macrophages by LPS and other noxious stimuli to produce inflammatory cytokines (Schumann et al., 1990Go; Wright et al., 1990Go). The pivotal role of CD14 in mediating the LPS effect was demonstrated by: (a) using CD14-specific monoclonal antibodies (Wright et al., 1990Go); (b) using transgenic mice over-expressing human CD14 receptor (Ferrero et al., 1993Go); (c) using CD14-deficient mice (Haziot et al., 1996Go). CD14 protein expression in human monocytes is up-regulated by LPS (Landmann et al., 1996Go) and the soluble form of the CD14 receptor is elevated in serum of alcoholics (Österreicher et al., 1995Go).

In a previous study, we reported a marked up-regulation of Kupffer cell CD14 after 2 weeks of chronic ethanol administration (Järveläinen et al., 1997Go). Recently, Su et al. (1998) reported a similar effect and in addition showed a similar increase in LBP mRNA. In this latter study, ethanol diets differing in their fatty acid composition and pathogenic effect were compared. A correlation between the degree of ethanol-induced liver lesions and the increases in CD14 and LBP was observed. This suggested that the up-regulation of CD14 and LBP expression could be secondary to the initiation of damage. This prompted us to study the time course of the effect of the exposure to ethanol on CD14 and LBP expression. Since treatment with GdCl3 has been found to alleviate alcoholic liver damage, its effect on the hepatic expression of LBP and CD14 was also studied.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Animals
Male rats of the Wistar strain initially weighing 150–180 g and housed in stainless-steel wire cages were used. For short-term ethanol experiments, five groups, each consisting of five animals, were fed standard chow pellets (Rat and Mouse No. 1 Maintenance Diet, Special Diets Service Ltd, Essex, UK) and tap water ad libitum. Ethanol (3.5 g/kg as a 17.5% v/v solution) was intubated at 8 h intervals. Rats were killed at 0, 4, 8, 24 and 48 h after the first ethanol dose and liver samples were collected as described below.

Long-term ethanol administration was undertaken by feeding four groups of rats (n = 7–8) a modified low-carbohydrate form of a commercially available liquid diet (Purina Mills, Richmond, IN, USA) for 6 weeks. The diet contained 17.1% (joules) protein, 43.7% fat (all groups) and either 39.2% carbohydrate (control groups) or 5.2% carbohydrate and 34.2% ethanol (ethanol-fed groups) (Lindros and Järveläinen, 1998Go). Half of the animals were treated to inactivate Kupffer cells by injecting GdCl3 (10 mg/kg in acidified saline) into the tail vein every third day. Controls were injected with acidified saline only.

Collection of liver samples
Rats were anaesthetized with pentobarbital (50 or 60 mg/kg body weight intraperitoneally). The liver was exposed and perfused in situ through the portal vein for 1–2 min. The papilliform lobe was ligated and removed, and liver samples were collected in buffered formalin solution or frozen in liquid nitrogen for RNA isolation and preparation of cryostat sections.

Immunohistochemical staining
For immunohistochemistry, 5 µm cryostat sections were dried for 30 min at room temperature and fixed in absolute acetone for 10 min. Endogenous peroxidase activity was blocked by treating the sections for 10 min in methanol containing 0.3% hydrogen peroxide. Rehydrated sections were incubated overnight at 4°C with monoclonal ED2 antibody (Serotec, Oxford, UK) specific for rat Kupffer cells. For detection, an immunohistochemistry kit (Zymed) was used.

Reverse transcriptase–polymerase chain reaction (RT–PCR)-based mRNA assay
A semiquantitative RT–PCR assay was used to estimate the levels of LBP and CD14 mRNA in liver samples. Total RNA was isolated from liver samples using an RNeasy kit from Qiagen (Hilden, Germany). The yield was determined spectrophotometrically at 260 nm and the integrity from the A260/A280 ratio of the RNA preparation and by RNA electrophoresis in formaldehyde-denatured 1.25% agarose gels. First strand cDNA was produced using Promega's (Madison, WI, USA) reverse transcription system and random hexanucleotide primers according to the manufacturer's instructions.

The assay of CD14 mRNA was based on amplification of a 189 bp product and was performed as described previously (Järveläinen et al., 1997Go). For the LBP PCR reaction, 4 µl of cDNA were amplified in a 100 µl reaction volume containing 50 pmol of both primers, 2 U Taq polymerase, 1xPCR reaction buffer (also containing 1.5 M MgCl2, both from Boehringer Mannheim GmbH, Mannheim, Germany) and 0.2 M of each deoxynucleotide triphosphate (Promega). The reaction mixture was heated for 4 min at 94°C and 22 cycles, consisting of 1 min at 94°C, 1 min at 57°C and 2 min at 72°C each, were run. The last step of the final cycle lasted for 5 min. The oligonucleotide primer sequences used were 5'-GAGGCCTGAGTCTCTCCATCT-3' and 5'-TCTGAGATGGCAAAGTAGACC-3' and these amplified a 552 bp PCR product. The linearity of amplification was validated by varying the amount of RNA, the amount of cDNA, and the number of cycles.

The PCR-amplified CD14 and LBP products were quantified by anion exchange high-pressure liquid chromatography as described previously (Katz and Dong, 1990Go; Oinonen and Lindros, 1995Go). The amplification products were also run in 20% polyacrylamide Phastgels (Pharmacia LKB Biotechnology, Uppsala, Sweden) using a native buffer system and then stained with Phastgel Silver Kit according to the manufacturer's instructions. The staining revealed that the size of the amplified products corresponded to their expected molecular weights (see Fig. 1Go). The inter-series variation in quantification of PCR products on the chronic alcohol/GdCl3 series was reduced by normalizing them mathematically, as described previously (Lindros et al., 1997Go). The four independent PCR runs used to normalize the data were based on the LBP, CD14, CYP2E1, and ß-actin products. Expression relative to ß-actin mRNA was not used here because alcohol treatment affects ß-actin mRNA expression.



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Fig. 1. Determination of the size of the polymerase chain reaction products of CD14 (A) and lipopolysaccharide-binding receptor (LBP) (B).

Phastgel electrophoresis was used to determine the size of the amplified products for CD14 (189 bp) and LBP (552 bp), which was verified by using appropriate DNA markers. One sample from each experimental group was used (C = control; CG = control + GdCl3; E = ethanol; EG = ethanol + GdCl3.

 
Statistical analyses
Results were analysed by ANOVA followed by the Student–Newman–Keuls or Dunett post-hoc test. P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Effect of short-term ethanol exposure
To establish the time course of the ethanol-induced increase in CD14 and LBP mRNA, the animals were dosed with ethanol solution orally at 8 h intervals, such that ethanol was calculated to be present all through the 48 h treatment period. Rats were killed at 4, 8, 24 and 48 h after the first ethanol dose. Compared to controls, there was no significant effect on either CD14 mRNA or LBP mRNA at 4 or 8 h (Fig. 2Go). At 24 h, a significant (P < 0.05) increase in the level of CD14 mRNA was observed. This increase persisted after 48 h of ethanol exposure. A similar increase was observed after 3 days (results not shown) and after 6 weeks (Fig. 2Go) of ethanol treatment.



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Fig. 2. Effect of duration of ethanol exposure on the expression of CD14 mRNA and lipopolysaccharide-binding receptor (LBP) mRNA in rat liver.

Rats were intubated at 8 h intervals with ethanol (3.5 g/kg body wt). Animals were killed at 0, 4, 8, 24 and 48 h after the first intubation and liver samples were analysed for the relative amount of CD14 mRNA and LBP mRNA as described in the Materials and methods section. Mean ± SEM mRNA values of five samples, with controls arbitrarily set as 1, are given. *Significantly different (P < 0.05) from controls. Results from a separate experiment, where rats were given ethanol by liquid diet for 6 weeks, are shown on the right-hand side of the graph.

 
The time course of the increase in LBP mRNA followed a similar pattern (Fig. 2Go). A slight increase was observed after 24 h; this effect was more obvious and reached statistical significance (P < 0.05 ) after 48 h. Again, a similar increase in LBP mRNA, as seen after 48 h, was also observed after 3 days (results not shown) and after 6 weeks of ethanol treatment (Fig. 2Go).

Effect of long-term administration of ethanol and GdCl3
We used our new modified low-carbohydrate/ ethanol liquid diet (Lindros and Järveläinen, 1998Go) and inactivation of Kupffer cells with GdCl3 to explore the role of LPS-CD14-mediated cytokine activation in liver lesions after prolonged ethanol administration. Animals were treated for 6 weeks with ethanol and GdCl3. The average daily ethanol intake was 12.7 g/kg for ethanol-treated rats and 12.9 g/kg for ethanol plus GdCl3-treated rats. The efficiency of GdCl3 treatment was investigated by staining cryostat liver sections with an antibody to ED2, a marker for macrophages and Kupffer cells. Compared with untreated controls (Fig. 3AGo), ED2 staining was almost completely absent in livers from GdCl3-treated rats (Fig. 3BGo).




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Fig. 3. Effect of GdCl3 treatment on the expression of the Kupffer cell-membrane marker ED2.

Cryostat sections were stained with the ED2 antibody as described in the Materials and methods section. Marked staining of Kupffer cells is seen in a liver from an animal injected with saline (A), in contrast to a liver from an animal treated with GdCl3 (B).

 
Ethanol treatment caused a significant (P < 0.05) increase in CD14 mRNA (Fig. 4Go). However, while GdCl3 significantly alleviated liver changes induced by ethanol (Järveläinen et al., 1998Go), this effect was not accompanied by a down-regulation of CD14 mRNA. The level of LBP mRNA was significantly elevated (P < 0.05) by the 6-week ethanol treatment. The additional treatment with GdCl3 caused a further significant increase.



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Fig. 4. Effect of treatment with ethanol and GdCl3 for 6 weeks on the hepatic expression of CD14 and lipopolysaccharide-binding receptor (LBP) mRNA.

Mean ± SEM mRNA values of six to eight samples, with controls arbitrarily set as 1, are given. The amount of CD14 and LBP mRNA was determined by reverse transcriptase–polymerase chain reaction as described in the Materials and methods section. Significant differences from controls: *P < 0.05 and **P < 0.01; significant difference from the ethanol grouponly: #P < 0.001.

 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
This study demonstrates that, within 24–48 h of initiation of ethanol exposure, the expression of both CD14 and LBP mRNA in the liver is significantly increased. Thus the increase occurs much earlier than any signs of damage due to chronic alcohol exposure can be observed. No effect was observed 4 or 8 h after ethanol challenge, although the administered dose of ethanol (3.5 g/kg body wt) causes marked intoxication, which is accompanied by a marked release of stress hormones such as corticosterone (Ellis, 1966Go). By comparison, within 6–8 h of acute challenge with LPS (3 mg/kg), we observed a dramatic increase in the amount of both LBP (40-fold) and CD14 (8-fold) mRNA (data not shown). This suggests that an intoxicating dose of ethanol does not per se cause transcriptional (or at least a pretranslational) activation of these genes.

Previous studies on CD14 expression in rodents (Matsuura et al., 1994Go; Schumann et al., 1996Go; Järveläinen et al., 1997Go; Su et al., 1998Go) and in human monocytes (Landmann et al., 1996Go) have demonstrated that corresponding changes occur at the protein level, indicating the functional importance of changes at the mRNA level. The increases in the expression of CD14 and LBP seen in this study both after short- and long-term ethanol exposure are in concordance with earlier reports (Järveläinen et al., 1997Go; Su et al., 1998Go), and indicate that exposure to ethanol causes a persistent, albeit moderate, effect. Alcohol abuse is often associated with an increased level of circulating endotoxins (Bode et al., 1987Go). The potential pathogenic action of endotoxins may thus be permanently enhanced via the increased expression of CD14 and LBP, and may sensitize the Kupffer cells to stimulate their production of pro-inflammatory cytokines. In fact, when Kupffer cells isolated from ethanol-pretreated animals are challenged with LPS, they liberate increased amounts of TNF-{alpha}, suggesting their increased sensitivity (Batey et al., 1998Go). A similar sensitization effect has been observed in experiments in vivo (Pennington et al., 1997Go). Such an effect could indeed be mediated via an increased expression of CD14 receptor on Kupffer cells. Chronic ethanol treatment was found not to affect the distribution of mononuclear cells (Batey et al., 1998Go), suggesting that ethanol-induced sensitization is hardly due to a change in the macrophage population of the liver. Our own observations, based on immunostaining of liver sections with the Kupffer cell-specific ED2 antibody, also indicate that ethanol treatment by itself does not affect the number of Kupffer cells.

Our finding that the GdCl3 injections did not significantly change hepatic CD14 mRNA expression, despite almost complete disappearance of ED2-positive cells, suggests that chronic GdCl3 treatment causes a compensatory recruitment of CD14-positive macrophages. Indeed, there is evidence that GdCl3 treatment may not reduce the number of phagocytically active cells, but may alter their acinar distribution and favour cells with an ED2-negative phenotype (Rai et al., 1996Go). Our data are consistent with these observations. Consequently, our data suggest that GdCl3 alleviates ALD (Adachi et al., 1994Go; Koop et al., 1997Go; Järveläinen et al., 1998Go) by mechanisms unrelated to CD14 or LBP expression.

LBP is an acute phase protein, and such proteins are frequently subject to sensitive regulation. Thus pro-inflammatory cytokines increase the expression of LBP (Shumann et al., 1996). GdCl3 treatment has been shown to increase the expression of pro-inflammatory cytokines (TNF-{alpha} and IL-6), but to reduce the expression of anti-inflammatory IL-10 (Rai et al., 1996Go; Rüttinger et al., 1996Go), effects that may explain why GdCl3 increases the expression of LBP.

In conclusion, our data demonstrate that even a short exposure to ethanol leads to a significant up-regulation of the hepatic expression of both the CD14 receptor and LBP in liver, suggesting that when the serum levels of bacterial endotoxins are elevated, these events could contribute to the initiation of alcohol-induced liver damage rather than being consequences of the injury.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The expert technical assistance by Gunilla Rönnholm is gratefully acknowledged. This study was supported by a grant from the Oscar Öflund Foundation.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
* Author to whom correspondence should be addressed. Back

Note added in proof. During the preparation of this manuscript a paper (Enomoto et al., 1998Go) appeared describing a similar increase in the expression of CD14 and LBP at 24 h after ethanol challenge as reported by us in the present communication.


    REFERENCES
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 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
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