Precision-cut liver slices in culture as a tool to assess the physiological involvement of Kupffer cells in hepatic metabolism.

Full text

Page 1

BioMed Central
Page 1 of 4
(page number not for citation purposes)
Comparative Hepatology
Open Access
Proceedings
Precision-cut liver slices in culture as a tool to assess the
physiological involvement of Kupffer cells in hepatic metabolism
Audrey M Neyrinck*, Cristina Gomez and Nathalie M Delzenne
Address: Unité de Pharmacocinétique, Métabolisme, Nutrition et Toxicologie, Département des Sciences Pharmaceutiques, Université Catholique
de Louvain, PMNT-UCL 73 avenue Mounier, B-1200 Brussels, Belgium
Email: Audrey M Neyrinck* - Audrey.Neyrinck@pmnt.ucl.ac.be; Cristina Gomez - Audrey.Neyrinck@pmnt.ucl.ac.be;
Nathalie M Delzenne - Delzenne@pmnt.ucl.ac.be
* Corresponding author
Introduction
Hepatic macrophages have the capacity to secrete a tre-
mendous array of molecules, which can be divided into 3
categories – cytokines (TNF-alpha), lipid mediators (pros-
taglandins PGE2) and reactive intermediates (NO·) – in
response to stimulus, such as lipopolysaccharides (LPS)
[1,2]. Such mediators are capable to modulate both the
metabolism and the integrity of hepatocytes in vitro [2].
The physiological role of Kupffer cell in hepatic metabo-
lism regulation has been approached in the present study
by using the original in vitro model of precision-cut liver
slices (PCLS) in culture; this model allows preserving the
liver lobule architecture, by maintaining namely cell
diversity in physiological proportion and cell-cell interac-
tions [3]. First, we established whether non-parenchymal
cells are still viable in rat PCLS and are able to respond to
LPS in vitro; TNF-alpha, PGE2, NOx (reflecting NO·
release) were measured in the incubation medium of
PCLS from rats previously treated with GdCl3 – a specific
inhibitor of Kupffer cell phagocytosis [4] – or NaCl as a
control- in order to evaluate the contribution of Kupffer
cell in mediator release. Moreover, by using the same
model, we have investigated the role of Kupffer cell in the
regulation of lipid synthesis in PCLS, in order to approach
the biochemical mechanism explaining our last results,
which indicate that the inhibition of Kupffer cell by GdCl3
leads to triglycerides accumulation in liver tissue [5].
Methods
Materials
Male Wistar rats weighing 240–280 g were used for the
preparation of PCLS or for isolation of hepatocytes. Most
chemicals of purest grade available were purchased from
Sigma (Filter Service, Belgium), Roche Diagnostics Bel-
gium or Invitrogen™ (Belgium). [1-14C]-acetic acid (spe-
cific activity 60 mCi/mmol) was obtained from
Amersham Pharmacia Biotech Europe (Buckinghamshire,
United Kingdom).
Study of mediator secretion by PCLS in culture
PCLS were prepared from treated with GdCl3 (10 mg/kg
i.v) (Gd+) or NaCl 0.9% (Gd-) 24 h before liver removal
according to a procedure previously described [6] and
were incubated in William's E medium, supplemented
with penicillin (100 IU/ml), streptomycin (100 micro-
grams/ml), glutamine (2 mM), insulin (100 nM) and
bovine serum albumin 0.1 %) containing LPS at 0 – 0.1 –
10 micrograms/ml. Medium was frozen after 2 h and 20 h
of incubation for further analysis. PGE2 and TNF-alpha
concentration were measured in frozen aliquots incuba-
tion medium with immunoassay kits (PGE2 Immu-
noassay, DE0100 and Quantikine rat TNF-alpha
immunoassay, RTA00 from R&D Systems) whereas NOx
(NO2- + NO3-) concentration was measured by the Griess
reaction [7]. ATP content of PCLS was greater than 8
nmol/mg protein in all experiments.
Study of lipid synthesis by PCLS
PCLS were prepared from treated with GdCl3 (10 mg/kg
i.v) (Gd+) or NaCl 0.9% (Gd-) 48 h before liver removal
according to a procedure previously described [6]. PCLS
were incubated as described above. Blood was collected
from vena cava for serum PGE2 measurement. After 2 h of
preincubation, medium was frozen for further analysis
from 11th International Symposium on the Cells of the Hepatic Sinusoid and their Relation to Other Cells
Tucson, Arizona, USA, 25–29 August, 2002
Published: 14 January 2004
Comparative Hepatology 2004, 3(Suppl 1):S45
<supplement> <title> <p>11th International Symposium on the Cells of the Hepatic Sinusoid and their Relation to Other Cells</p> </title> </supplement>
This article is available from: http://www.comparative-hepatology.com/content/3/S1/S45

Page 2

Comparative Hepatology 2004, 3http://www.comparative-hepatology.com/content/3/S1/S45
Page 2 of 4
(page number not for citation purposes)
and PCLS were transferred into fresh medium containing
2 mM [14C]-acetate (0.2 mCi/mmol); after 3 h of incuba-
tion, PCLS were sonicated in 0.5 ml NaCl 0.05 M before
lipid extraction and separation by thin-layer chromatogra-
phy with hexane/ether/acetic acid (80:20:1) [8,9]. Spots
corresponding to triglycerides, phospholipids and choles-
terol were scrapped from the plate and counted in 10 ml
scintillation fluid (Ultima Gold) in a beta counter.
Study of lipid synthesis by isolated hepatocytes in
suspension
The hepatocytes were isolated from fed animals [10],
through perfusion with a buffer containing Liberase™ (35
micrograms/ml). Cells were incubated in the presence
PGE2 (Cayman Chemicals) dissolved in DMSO at 10
micromolar and 2 mM [14C]-acetate (0.2 mCi/mmol) in
the same medium described above. After 15 min of incu-
bation, hepatocytes were pelleted and sonicated in 0.5 ml
NaCl 0.05 M before lipid extraction according to Folch
[8]. The chloroform phase was counted in 10 ml scintilla-
tion fluid (Ultima Gold) in a beta counter.
Statistical analysis
Values are presented as means ± S.E.M. In the study of
mediator secretion by PCLS, statistical analysis was per-
formed by two-way ANOVA with the Tukey's post hoc test
(with SPSS™ statistical software). Other data were ana-
lysed by Student-t test. Statistical significance was set at p
< 0.05.
Results
Validation of the PCLS model to assess the functionality of
Kupffer cells
Unstimulated-PCLS in culture release in the medium sig-
nificant amount of TNF-alpha, PGE2 and NOx (Figure 1)
which level increases with the time of incubation. TNF-
alpha concentration increases significantly in the presence
of LPS; the effect is dependent on the dose of LPS and is
already present after 2 h of incubation. The extent of both
PGE2 and NOx production, is also dependent on LPS con-
centration; the increase in both parameters appears signif-
icantly only after 20 h of incubation. The administration
of GdCl3 1 day before PCLS preparation strongly reduces
the basal production of the 3 mediators measured as well
as upon stimulus by LPS.
Role of Kupffer cell in the physiological regulation of lipid
synthesis in the liver tissue
PCLS obtained from rats treated with GdCl3, 2 days before
experiment, are characterised by a higher incorporation of
[14C]-acetate into lipids (table 1), on one hand, and by a
lower release of PGE2 in the preincubation medium, on
the other hand (PGE2 concentration in the medium after
2 h: 5.4 ± 0.8 nM and 0.9 ± 0.1 nM for Gd- and Gd+ rats
respectively; *p < 0.05, Student t-test). Moreover, PGE2,
measured in the serum obtained from vena cava, was
much lower in animals treated with GdCl3 2 days before
blood sampling (< 1 nM) as compared to control rats
(26.7 ± 12.1 nM). The role of PGE2 in the short-term con-
trol of lipid synthesis was assessed by incubation of iso-
lated hepatocytes in suspension: the addition of PGE2 (10
micromolar) to the culture medium rapidly decreases the
incorporation of [14C]-acetate into total lipids in isolated
hepatocytes after 15 minutes of incubation, total lipids –
expressed in nmol acetate equivalent/mg protein –
reached 1.3 ± 0.2 and only 0.87 ± 0.1 in control condi-
tions (DMSO) and after addition of PGE2 10 micromolar,
respectively (*p < 0.05, Student t-test).
Discussion
Our study demonstrates that GdCl3, a specific inhibitor of
Kupffer cell phagocytosis [4], decreases the rate of LPS-
induced TNF-alpha, PGE2 and NOx production by PCLS;
those 3 molecules are known as typical mediators pro-
duced by macrophages (and namely by Kupffer cells in
culture) upon stimulation by LPS (cytokines, lipid media-
tors and reactive intermediates) [10]. Moreover, we have
shown that PCLS are able, independently of any stimulus
(absence of LPS or any toxic agents) to produce TNF-
alpha, NOx and PGE2. In this case also, the release of
mediators is decreased by previous GdCl3 treatment.
Therefore, we conclude that PCLS from GdCl3-treated rats
– compared to PCLS obtained from control rats – is a con-
venient in vitro system to study the complex Kupffer cell-
hepatocyte interactions retained inside the liver tissue, in
basal conditions or after an inflammatory stimulus. PGE2
Table 1: Effect of Kupffer cell inhibition by GdCl3 on lipid synthesis by PCLS
Acetate equivalent incorporated into lipids
(nmol/mg prot.)
Gd-Gd+
- phospholipids
- triglycerides
- cholesterol
3.7 ± 0.1
2.4 ± 0.6
0.8 ± 0.2
6.1 ± 0.6*
4.0 ± 0.8*
2.0 ± 0.2*
PCLS, prepared from 24 h-fasted rats pretreated with GdCl3 (Gd+) or NaCl (Gd-) 2 days before the experiment, were incubated in medium
containing 2 mM [14C]-acetate for 3 h. Values are means ± S.E.M (n ≥ 4) (*p < 0.05, Student t-test).

Page 3

Comparative Hepatology 2004, 3http://www.comparative-hepatology.com/content/3/S1/S45
Page 3 of 4
(page number not for citation purposes)
merits special attention: when PCLS are prepared from
Gd+ animals, PGE2 release is strongly depressed (mainly
during short time incubation) suggesting that Kupffer
cells are important producers of PGE2 by the liver. Since
GdCl3 treatment leads to a strong depression of PGE2 con-
centration in the serum, we may propose that Kupffer cells
inside the liver constitute an important source of circulat-
ing PGE2, known to exert pleiotropic effects outside the
liver (control of lipolysis, platelet aggregation, gastric acid
secretion, immunoregulation, neurotransmitter release,
contraction-relaxation of smooth muscle) [11]. On the
other hand, we show here that PGE2 release by Kupffer
cells, not only plays a role in the systemic availability of
this prostaglandin, but is also able to exert paracrine effect
on hepatocyte, with relevant metabolic consequences in
the liver tissue: we had previously shown that, in vivo, the
accumulation of lipids in the liver tissue after GdCl3
administration, modifies the histological image of the
liver, revealing steatosis [6]. We have demonstrated in the
results presented here that the inhibition of Kupffer cells
by GdCl3 leads to a higher cholesterol, triglycerides and
phospholipids synthesis by PCLS, a phenomenon related
to a lower PGE2 release. Since PGE2 alone added in the cul-
ture medium of isolated hepatocytes decreases lipid syn-
thesis, we may postulate that the lower PGE2 secretion
after GdCl3 treatment is, at least partly, responsible for a
higher lipid synthesis inside the liver tissue. This is in
favour of a role of Kupffer cell-released PGE2 in the phys-
iological control of lipid synthesis in hepatocytes. By
which mechanism PGE2 could control lipid synthesis in
the liver tissue? The phosphorylation rate of hepatic
enzymes involved in the lipogenesis or cholesterogenesis
pathways (acetyl-CoA synthetase, HMG-CoA-reductase)
could be modulated by PGE2 which is released at concen-
tration sufficient to alter the phosphorylation of specific
proteins inside the hepatocytes [12]. The use of
indomethacin, known to inhibit prostaglandin produc-
tion, in the incubation medium of PCLS could help to elu-
cidate the role of PGE2released by Kupffer cell in the
regulation of hepatic metabolism.
References
1. Decker K: Biologically active products of stimulated liver
macrophages (Kupffer cells). Eur J Biochem 1990, 192:245-261.
2. Laskin DL, Pendino KJ: Macrophages and inflammatory media-
tors in tissue injury. Annu Rev Pharmacol Toxicol 1995, 35:655-677.
3. C Lerche-Langrand, Toutain HJ: Precision-cut liver slices: charac-
teristics and use for in vitro pharmaco-toxicology. Toxicology
2000, 153:221-253.
4. Hardonk MJ, Dijkhuis FWJ, Hulstaert CE, Koudstaal J: Heterogene-
ity of rat liver and spleen macrophages in gadolinium chlo-
ride-induced elimination and repopulation. J Leukoc Biol 1992,
52:296-302.
Effect of Kupffer cell inhibition on mediator secretion (TNF-alpha, PGE2 and NOx) by PCLS, obtained from rats pretreated with GdCl3 (Gd+) or NaCl (Gd-) 24 h before experiment; PCLS were incubated during 2 h and 20 h in the presence of LPS (from left to right, each group of three bars correspond to: 0 micrograms/ml, 0.1 micrograms/ml, 10 micrograms/ml)
Figure 1
Effect of Kupffer cell inhibition on mediator secretion (TNF-alpha, PGE2 and NOx) by PCLS, obtained from rats pretreated
with GdCl3 (Gd+) or NaCl (Gd-) 24 h before experiment; PCLS were incubated during 2 h and 20 h in the presence of LPS
(from left to right, each group of three bars correspond to: 0 micrograms/ml, 0.1 micrograms/ml, 10 micrograms/ml). Values
are means ± S.E.M (n ≥ 3; *p < 0.05 Gd+ vs Gd-, two-way ANOVA; a p < 0.05 LPS 10 micrograms/ml vs LPS 0 micrograms/ml
and b p < 0.05 LPS 10 micrograms/ml vs LPS 0.1 micrograms/ml, two-way ANOVA followed by Tukey's post hoc test).
TNF-alpha (pg/mg prot.)PGE2 (pg/mg prot.)NOx (nmol/mg prot.)
2h
0
200
400
600
800
Gd-Gd+
ab
ab
*
0
200
400
600
Gd- Gd+
*
0
2
4
6
8
Gd- Gd+
20h
0
500
1000
1500
2000
2500
Gd-Gd+
a
a
*
0
2000
4000
6000
8000
10000
12000
14000
16000
Gd- Gd+
*
ab
ab
a
a
0
100
200
300
400
500
600
a
a
*

Page 4

Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral
Comparative Hepatology 2004, 3 http://www.comparative-hepatology.com/content/3/S1/S45
Page 4 of 4
(page number not for citation purposes)
5. Neyrinck AM, Taper HS, Delzenne NM: Gadolinium chloride
does not block the hypertriglyceridemia induced by LPS
administration to rats. In: Cells of the Hepatic sinusoid Volume 8.
Edited by: Wisse E, Knook DL, de Zanger R, Fraser R. Kupffer Cell Foun-
dation, Leiden; 2001:30-33.
Neyrinck AM, Taper HS, Gevers V, Declerck B, Delzenne NM: Inhi-
bition of Kupffer cell activity induces hepatic triglyceride
synthesis in fasted rats, independent of lipopolysaccharide
challenge. J Hepatol 2002, 36:466-473.
Bishop-Bailey D, Larkin SW, Warner TD, Chen G, Mitchell JA: Char-
acterization of the induction of nitric oxide synthase and
cyclo-oxygenase in rat aorta in organ culture. Br J Pharmacol
1997, 121:125-133.
Folch J, Lee M, Sloane-Standley G: A simple method for the isola-
tion and purification of total lipids from animal tissues. J Biol
Chem 1957, 226:497-509.
Liao W, Florèn CH: Upregulation of low density lipoprotein
receptor activity by TNF, a process of TNF-induced lipid syn-
thesis and secretion. Lipids 1994, 29:679-684.
Roland CR, Naziruddin B, Mohanakumar T, Flye MW: Gadolinium
chloride inhibits Kupffer cell nitric oxide synthase (iNOS)
induction. J Leukoc Biol 1996, 60:487-492.
Coleman RA, Smith WL, Narumiya S: International union of phar-
macology classification of prostanoid receptors: properties,
distribution and structure of the receptors and their
subtypes. Pharmacological Reviews 1994, 46:205-229.
Casteleijn E, Kuiper J, Van Rooij HC, Koster JF, Van Berckel TJ: Con-
ditioned media of Kupffer and endothelial cells influence pro-
tein phosphorylation in parenchymal liver cells. Involvement
of prostaglandins. Biochem J 1988, 252:601-605.
6.
7.
8.
9.
10.
11.
12.