Acute alcohol administration to mice induces hepatic sinusoidal endothelial cell dysfunction

International Hepatology communica~ns Int Hepatol Commun 2 (1994) 81-86

Acute alcohol administration to mice induces hepatic sinusoidal endothelial cell dysfunction Ion V. DeaciuP*,

John J. Spitzer”, Judd E. Shellitob, Nympha B. D’Souzab

Departments of”Physiology and bMedicine, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans. LA 70112, USA

(Received 20 October 1993;accepted 12 November 1993)

Abstract

Ethanol administration to mice (4.4 g kg-’ body wt., i.p.) induced a marked increase (125%) in plasma hyaluronan (HA) concentration associated with a significant decrease (46%) in HA uptake by the isolated, perfused liver. Similar changes were obtained after i.v. administration of Escherichiu colt’ lipopolysaccaharide (1 mg kg-’ body wt.), a strong activator of Kupffer cells. Since: (a) hepatic HA uptake is predominantly a function of the sinusoidal endothelial cells, reflecting their functional state, and (b) liver is a major site of HA clearance from the blood, we conclude that alcohol alters sinusoidal endothelial cell functions, including HA endocytosis. Changes in Kupffer cell functional state may be a primary event underlying the effects of both ethanol and LPS on sinusoidal endothelial cells. Key words: Plasma hyaluronan; thelial cell; Ethanol;

Hyaluronan

uptake; Perfused mouse liver; Sinusoidal endo-

Lipopolysaccharide

1. Introduction The classical viewpoint that alcohol effects on the liver are mainly due to its metabolism by the hepatocyte has been challenged by experimental data, demonstrating that nonparenchymal liver cells, i.e., Kupffer, Ito, and sinusoidal endothelial cells (SEC), are also affected by alcohol, in spite of their low capacity to metabolize the drug. The concept that alcohol action on the hepatocyte can be partly mediated by nonparenchymal cells has gradually gained recognition. Among nonparenchymal liver cells, the Kupffer cell has been most intensively studied with respect to alcohol effects. Kupffer cell functions such as phagocytosis [1,2], and secretion of mediators (e.g., cytokines [3]) or microbicidal agents (e.g., superoxide anion [4]) are altered by *Corresponding

author.

092%4346J94BO7.00 0 1994 Elsevier Science B.V. All rights reserved SSDJ 0928-4346(93)E0057-W

82

I. V. Deaciuc et al. lint Hepatol Commun 2 (1994) 686

alcohol. Fewer studies, however, have dealt with the effects of alcohol on other nonparenchymal liver cells such as SEC and Ito cells. This can be attributed to the lack of adequate, measurable parameters to assess the functional state of these cells. It is likely that, for the same reason, studies of alcohol effects on SEC were limited to ultrastructural observations [S-8]. Functional corollaries to ultrastructural changes were rather inferred than measured. In previous studies [9-l 11,we have shown that: (a) HA uptake by the hepatic SEC can be readily measured in the isolated, perfused liver, displaying rates higher than the ones obtained on the isolated SEC; (b) HA uptake by the isolated, perfused liver can be taken as an index of SEC functional state; (c) variations in HA uptake by the isolated, perfused liver correlate with changes in plasma HA concentration in a manner suggesting a cause-effect relationship; and (d) Kupffer cells modulate the capacity of SEC to take up HA. With this in mind, in the present study we have made an attempt to determine whether alcohol affects the functional state of SEC, as reflected by HA uptake. We have measured changes in both plasma HA levels and HA uptake by the isolated, perfused liver of mice after acute alcohol administration. The data show that acute alcohol administration to mice alters the capacity of the liver to take up HA, leading to an increase in plasma HA level.

2. Materials and methods 2.1. Animals and their treatment Male, BALBlC mice, weighing 24-27 g (6-7 weeks old) were purchased from Hilltop Lab Animals, Inc. (Scottdale, PA), and maintained on standard mouse chow with water ad libitum at least 5-6 days before experimentation. Alcohol was injected intraperitoneally as 20% (v/v) solution in sterile saline, in a dose of 4.4 g kg-’ body wt. Escherichia coli lipopolysaccharide (LPS, 026:B6, Difco Laboratories, Detroit, MI) was given into the pennile vein as a suspension in sterile saline, filtered through 0.22~pm filters (Millipore Corp., Bedford, MA), in a dose of 1 mg kg-’ body wt., 30 min after alcohol or saline administration. Control animals were given sterile saline instead of alcohol or LPS. Three h after LPS or saline administration, the animals were anesthetized with Na-pentabarbital (NembutalR, 60 mg kg-’ body wt.), the abdominal cavity was first opened and blood collected from the inferior vena cava with heparinized syringes. Plasma was obtained after blood centrifugation at 13,000 x g for 1.5 min and used for HA assay. 2.2. Liver perfusion This was performed in a non-recirculating system, using Krebs-Ringer bicarbonate buffer as perfusion medium. The perfusion medium was continuously gassed with O&O, (19: 1) at 34°C and pumped into the liver through the portal vein at a rate of 2.5 ml min-‘. HA (from rooster comb, 1M,1.3 x 106, Sigma Chemical Co., St. Louis, MO) was infused into the perfusate entering the portal vein with the aid of a computerized infusion pump, at a rate of 0.5 ml min-’ to give a final perfusate concentration of 300-350 ng ml-‘. Previous experiments from our laboratory have established that

I. V. Deaciuc et al. IInt Hepatol Commun 2 (1994) 81-84

83

this HA concentration was saturating with respect to the hepatic HA uptake rate. HA infusion was started 15 min after portal vein cannulation in all groups and lasted for 18 min. Influent and eflluent samples were collected with the aid of a fraction colector set at 4-min intervals and used for HA assay. At the end of perfusion, the liver was excised, trimmed of non-hepatic tissue, blotted on a filter paper and weighed. 2.3. Hyaluronan assay This was performed on 100~~1aliquots of plasma or perfusion medium with the aid of a radiometric kit purchased from Kabi Pharmacia (Piscataway, NJ). 2.4. Statistics One-way analysis of variance in combination with Student-Newman-Keuls test was used to assess the statistical significance of the results. A difference at a P less than, or equal to, 0.05 was considered statistically significant. During all experimental procedures the animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23. 1985).

3. Results

As shown in Fig. 1, alcohol markedly increased plasma HA levels (125%, i.e., from 248.5 + 21.0 ng ml-’ in saline-injected animals, to 560.3 _+31.4, same units, P < 0.001, n = 4 in each group). Alcohol doses of 1.5 and 3.0 g kg-l body wt., also

-

600

$

500

-5

400

*

*

7

*

+

+

EtOH

EtOH +

0

.-c k’

100

I -

0

LPS

LPS Fig. 1. Plasma hyaluronan (HA) concentration in saline- (C), lipopolysaccharide (LPS)-, alcohol (EtOH)and EtOH + LPS-treated mice. Plotted are means 2 SE (vertical bars) for four individuals in each group. *Indicates a difference at a P < 0.001 vs. saline-treated (C) group.

I. V. Deaciuc et al. lint Hepatol Commun 2 (1994) 81-86

84

.1, 20. .

6

ad Y c, 16. 35

4,‘Z8- I-1 -$5

$

12. 4-

0, 10

I11

I

1

-4

I A

1

11,

L.1

.L +: ++

v----f\. 14

18

22

+

26

30

Minutes of perfusion Fig. 2. Hyaluronan (HA) uptake by the perfused livers, isolated from saline-, alcohol (EtOH)-, lipopolysaccharide (LPS)-, and EtOH + LPS-treated mice. Plotted points are mean + 0.5 SE (vertical bars) for four individuals in a group. * and ** indicate a difference at a P < 0.001 vs. saline- and EtOH-treated groups, respectively. 0, saline; l, EtOH; A, LPS; A, EtOH + LPS.

increased plasma HA, although to a lesser extent than the dose used in this study (data not shown). A similar effect was seen after LPS or alcohol + LPS administration (539.3 ? 19.1 ng ml-‘, and 559.1 4 31.3, respectively; P < 0.001 vs. corresponding controls; n = 4 in each group). Hepatic HA uptake ranged between 17.2 and 18.8 ,ug g-’ liver wet wt. h-l in control animals (Fig. 2). In the perfused livers, isolated from alcohol- or LPS-treated mice, HA uptake was inhibited by 46% and 82%, respectively (Fig. 2; P < 0.001, vs. corresponding controls, n = 4 in each group). In the livers isolated from alcohol + LPStreated rats, an inhibition of 95% of HA uptake was observed. Such a decrease did not reach statistical significance when compared to LPS-treated mice but it was significantly lower (P c 0.001) than the rate seen in the group treated with alcohol alone.

4. Discussion Most of the studies on the effect of alcohol on the hepatic SEC have dealt with ultrastructural changes. Thus, it has been shown that a single dose of alcohol induces changes in the fenestration pattern, including the size and the number of fenestrae, in the rat [7], and swelling of the SEC in the mouse [8]. Chronic alcohol feeding of rats [5] and baboons [12] also affects the size and the number of fenestrae. To our knowledge, only one study correlated ultrastructural changes induced by either acute or chronic alcohol treatment with a functional parameter of SEC [ 131.In the cited study the authors measured the uptake of formaldehyde-treated serum [12SI]albuminby the perfused liver, isolated from 4-week alcohol-fed rats, and found it to be decreased. The denatured albumin is specifically taken up by the SEC in the perfused liver [14].

I. V. Deaciuc et al. lint Hepatol Commun2 (1994) 81-86

85

In the present study, we provide evidence that acute alcohol administration to mice impairs an important function of the hepatic SEC, namely HA uptake. HA uptake by the liver is primarily a function of the SEC [ 151.Impaired HA uptake was associated with a marked increase in plasma HA levels. Since the liver has a major quantitative contribution to HA clearance from the blood [ 10,161, increased HA plasma levels can be ascribed to an impairment of hepatic HA uptake. The data presented in this study are in agreement with clinical observations showing that alcoholic hepatitis in humans is associated with an increased level of plasma HA [ 17-191. Although no mechanism for such an increase has been described, we now believe that impairment of hepatic SEC capacity to take up HA may be the primary cause of increased plasma HA levels. Our assumption is based on the relationship between these two parameters, described in this and previous studies [lo]. The mechanism by which alcohol impairs SEC function remains unclear. Interestingly, the effect of alcohol on plasma HA level and hepatic HA uptake was similar to that exerted by E. coli LPS. The effects of LPS presented in this study confirm earlier results from our [lo] and other laboratories [20]. LPS is one of the strongest Kupffer cell activators [21]. Upon activation, Kupffer cell secretes a variety of mediators which could affect SEC functional state. Superoxide anion and/or its derivatives are plausible candidates for such a role. This assumption is supported by three lines of evidence. First, elimination of Kupffer cells by GdCl, or liposome-encapsulated dichloromethylene diphosphonate suppresses the inhibitory effect of LPS on hepatic HA uptake by the isolated, perfused liver [I 11. Second, acute alcohol administration to rats markedly enhances superoxide anion production by subsequently isolated, perfused liver [4], and activates Kupffer cells in the mouse liver [8]. Third, SEC alteration during liver preservation, reflected by an increased leakage of purine nucleoside phosphorylase (EC 2.4.2.11, a marker of SEC damage, can be prevented by decreasing superoxide anion production in the preservation fluid upon reperfusion [22]. Experimental work is in progress in our laboratory to elucidate the mechanism of alcohol-induced impairment of hepatic SEC function. In conclusion, our data demonstrate that alcohol-induced ultrastructural changes of the hepatic SEC are parallelled by alterations of a specific function of these cells.

Acknowledgements This work was supported by NIAAA grants AA 08845 and 07282.

References [l] Nolan JP, Leibovits AI, Vladutiu AO. Influence of alcohol on Kupffer cell function and possible significance in liver injury. In: Liehr H, Grun M, editors. The Reticuioendothelial System and the Pathogenesis of Liver Disease. Elsevier/North Holland, Biomedical Press, 1980:125-136. [2] D’Souza NB, Bagby GJ, Lang CH, Deaciuc IV, Spitzer JJ. Ethanol alters the metabolic response of isolated, perfused rat liver to a phagocytic stimulus. Ale Clin Exp Res 1993;17:147-l 54.

86

I. V. Deaciuc et al. lht Hepatol Commun 2 (1994) 81-86

[3] D’Souza NB, Bagby GJ, Nelson S, Lang CH, Spitzer JJ. Acute alcohol infusion suppresses endotoxin-

induced serum tumor necrosis factor. Ale Clin Exp Res 1989;13:295-298. [4] Bautista A, Spitzer JJ. Acute alcohol intoxication stimulates superoxide anion production by in situ perfused rat liver. Hepatology 1992;15:892-898. [5] Fraser R, Bowler LM, Day WA. Damage of rat liver sinusosidal endothelium by alcohol. Pathology 1980;12:371-376. [6] Charels K, De Zanger RB, Van Bossuyt H, van der Smissen P, Wisse E. Influence of acute alcohol administration on endothelial fenestrae of rat livers: an in vivo and in vitro scanning electron microscopic study. In: Kirn A, Knook DL, Wisse E, editors. Cells of the Hepatic Sinusoid. Leiden, The Netherlands: Kupffer Cell Foundation, 1986;1:497-502. [7] Mori T, Okanoue T, Sawa Y, et al. Effect of ethanol on sinusoidal endothelial fenestration of rat liver _ in vivo and in vitro study. In: Wisse E, Knook DL, McCuskey RS, editors. Cells of the Hepatic Sinusoid. Leiden, The Netherlands: Kupffer Cell Foundation, 1991;3:469%471. [8] Eguchi H, McCuskey PA, McCuskey RS. Kupffer cell activity and hepatic microvascular events after acute ethanol ingestion in mice. Hepatology 1991;13:751-757. [9] Deaciuc IV, Bagby GJ, Lang CH, Spitzer JJ. Hyaluronic acid uptake by the isolated, perfused rat liver: an index of hepatic sinusoidal endothelial cell function. Hepatology 1993;17:266-272. [lo] Deaciuc IV, Bagby GJ, Lang CH, Skrepnik N, Spitzer JJ. Gram-negative bacterial lipopolysaccharide impairs hyaluronan clearance in vivo and its uptake by the isolated, perfused rat liver. Hepatology 1993;18:173-178. [ll] Deaciuc IV, Bagby GJ, Niessman MR, Skrepnik N, Spitzer JJ. Modulation of hepatic sinusoidal endothelial cell function by Kupffer cell: an example of intercellular communication in the liver. Hepatology 1994; in press. [12] Mak KM, Lieber CS. Alterations in endothelial fenestrations in liver sinusosids of baboons fed alcohol: a scanning electron microscopy study. Hepatology 1984;4:386391. [13] Rees GM, Miller JA, Casey CA, Sorrel1 MF, Tuma DJ. Chronic alcohol administration impairs liver endothelial cell function. Hepatology, 1992;16:28 (Abstr. 256). [14] Blomhoff R, Eskild W, Berg T. Endocytosis of formaldehyde-treated serum albumin via scavanger pathway in liver endothelial cells. Biochem J 1984;218:81-86. [IS] Eriksson S, Fraser JRE, Laurent TC, Pertoft H, Smedsrod B. Endothelial cells are a site of uptake and degradation of hyaluronic acid in the liver. Exp Cell Res 1983;144:223-228. [16] Engstrom-Laurent A, Hellstrom S. The role of liver and kidneys in the removal of circulating hyaluronan. An experimental study in the rat. Connect Tissue Res 1990;24:219-224. [17] Gibson PR, Fraser JRE, Brown TJ, et al. Hemodynamic and liver function predictors of serum hyaluronan in alcoholic liver disease. Hepatology 1992;15:10541059. [18] Engstrom-Laurent A, Loof L, Nyberg A, Schroder T. Increased serum levels of hyaluronate in liver disease. Hepatology 1985;5:638&642. [19] Frebourg T, Delpech B, Bercoff E, et al. Serum hyaluronate in liver diseases: study by enzymoimmunological assay. Hepatology 1986;6:3922395. [20] Fraser JRE, Pertoft H, Alston-Smith J, Laurent TC. Uptake of hyaluronan in hepatic endothelial cells is not directly affected by endotoxin and associated cytokines. Exp Cell Res 1991;197:8-11. [21] Decker K. Biologically active products of stimulated liver macrophages (Kupffer cells). Eur J Biochem 1990;192:245-261. [22] Rao PN, Walsh TR, Makowka L, et al. Inhibition of free radical generation and improved survival by protection of the hepatic microvascular endothelium by targeted erythrocytes in orthotopic liver transplantation. Transplantation 1990;49:1055-1059.