1 Department of Veterinary Pharmacy, Pharmacology, and Toxicology, Utrecht University, 3584 CM Utrecht; 2 Department of Pharmacology, TNO Pharma, 3704 HE Zeist, The Netherlands; and 3 Pharmacia and Upjohn, Drug Metabolism Research, 20014 Nerviano, Italy
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study focuses on
the importance of direct contact between Kupffer cells (KCs) and
hepatocytes (HCs) during the hepatic inflammatory response using an in
vitro approach. The lipopolysaccharide (LPS)-induced inflammatory
response in monocultures of porcine HCs and KCs were compared with
cocultures prepared either with direct contact between KCs and HCs (DC
cocultures) or without direct contact using cell culture membrane
inserts. Our data show that DC cocultures exhibited the highest
production of tumor necrosis factor (TNF)-, interleukin-6, and
nitric oxide (NO) compared with the other cultures. Immunohistochemical
studies revealed that TNF-
was exclusively produced by KCs, whereas
HCs were responsible for NO production after LPS stimulation.
Biotransformation capacity, as determined by cytochrome
P-450 and UDP glucuronosyl transferase enzyme activities,
was most significantly decreased in DC cocultures. These results
provide evidence that direct contact between KCs and HCs favors the
extensive TNF-
production by KCs but in turn affects HC
functionality and viability. These findings suggest that direct contact
between KCs and HCs plays a key role in the development of a
fulminating hepatic inflammatory response.
lipopolysaccharide; cytokines; biotransformation; nitric oxide
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DURING INFECTION, TRAUMA, OR an inflammatory response, liver functions are significantly altered. At the cellular level, these changes include production of acute phase proteins and a decrease in xenobiotic metabolizing enzyme systems (3, 23). Bacterial products such as endotoxins, including lipopolysaccharides (LPS), are among the agents that are able to induce these changes in liver functions either directly or indirectly via activation of Kupffer cells (KCs). KCs are among the first cells that respond to LPS and are considered to be the primary macrophages involved in the clearance of gut-derived bacteria or bacterial toxins (11). High portal levels of LPS can lead to a pronounced secretion of inflammatory mediators by KCs and ultimately to endotoxin-induced liver injury (14, 37).
KCs are located in the hepatic sinusoids and lie in between or on top
of endothelial cells. However, they do have direct cell contact with
parenchymatous hepatocytes (HCs) through their cytoplasmic extensions
(38). Cellular communication between KCs and HCs is
thought to occur mainly by production of cytokines and excretion of
inflammatory mediators such as eicosanoids, nitric oxide (NO), and/or
reactive oxygen species (ROS) (8). Proinflammatory
cytokines, in particular tumor necrosis factor (TNF)-, interleukin
(IL)-1, and IL-6 have been shown to be early and important mediators of the hepatic inflammatory response. These cytokines induce the synthesis
of acute phase proteins and are also involved in the downregulation of xenobiotic metabolizing enzyme systems such as
cytochrome P-450 (CYP) and UDP glucuronosyl transferase
(UDPGT) (2, 24). Intercellular communication between KCs
and HCs has been studied in various in vitro model systems. Most
studies of intercellular communication are performed in cocultures
consisting of KCs and HCs in direct contact (5, 34).
Alternately, KC-conditioned culture medium was added to HC cultures or
vice versa (5, 27, 34). Such approaches cannot
discriminate between the effect on inflammatory responses concerning
direct contact vs. indirect contact between KCs and HCs.
The present study was designed to dissect the LPS-induced inflammatory response of direct cellular contact between KCs and HCs from indirect contact via signaling molecules. In particular, we focused on the interaction between KCs and HCs in vitro, using separately isolated porcine HCs and KCs. To this end, HCs and KCs either were cultured as monocultures or used in two types of cocultures, either allowing direct contact (DC cocultures) or preventing direct cell-to-cell contact by using semipermeable membrane inserts (MI cocultures). In addition, direct responsiveness of HCs to LPS was examined using ultrapure HC cultures (UHC cultures).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chemicals and drugs.
Powdered Williams' medium E, glutamine, gentamicin, LPS
[Escherichia coli, O111:B4], EDTA,
3,3'-diaminobenzidine,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
brefeldin A, saponin, alkaline phosphatase-conjugated goat
anti-rabbit IgG, testosterone, 11-hydroxytestosterone, 1-naphthol, 1-naphthyl glucuronide, and diethanol were obtained from Sigma Chemical
(St. Louis, MO). Myoclone super plus fetal bovine serum (FBS; endotoxin
<10 EU/ml) was obtained from Life Technologies (Breda, The
Netherlands). Recombinant porcine TNF-
, recombinant porcine IL-6,
monoclonal antibody (MAb) mouse anti-porcine TNF-
(6Clone 9B4,
isotype IgG1), and porcine TNF-
ELISA were obtained from Endogen
(Cambridge, MA). N-acetyl-3,7-dihydroxyphenoxazine (A6550)
and dichlorodihydroxyfluorescein diacetate (H2DCF-DA) were
obtained from Molecular Probes Europe (Leiden, The Netherlands). Percoll was obtained from Pharmacia (Uppsala, Sweden). DAKO provided rabbit anti-mouse IgG/horseradish peroxidase, and rabbit anti-mouse inducible NO synthase (iNOS) was obtained from Cayman Chemical (Ann
Arbor, MI). MAb anti porcine macrophage (CVI-SWNL 517.2) was a kind
gift of Dr. J. M. A. Pol (ID-DLO, Lelystad, The Netherlands).
Origin of primary cells. HCs and KCs were isolated from livers of three castrated male pigs (Great Yorkshire × Dutch landrace) aged ~12 wk and weighing between 28 and 35 kg. The animals were obtained from the University's breeding farm.
Preparation of HC cultures.
The procedure for isolation of porcine HCs was based on Seglen's
method (29), with some modifications as described by
Monshouwer et al. (24). The obtained cell suspension was
diluted in modified Hanks' buffered salt solution (pH 7.65, 4°C, 9.2 mM HEPES, 9.91 g/l Hanks' buffered salt solution without
Ca2+ and Mg2+) and centrifuged at 200 g for 5 min. The supernatant was discarded, and cells were
resuspended with PBS (pH 7.4) to a final volume of 100 ml and
centrifuged for 2 min at 50 g. The supernatant, containing
mainly nonparenchymal cells, was collected for isolation of KCs (see
Preparation of KCs). Pellets containing mainly HCs were
resuspended in Williams' medium E and washed four times for 2 min at
50 g with Williams' medium E. After the final wash step, cells were counted and diluted with Williams' medium E supplemented with 5% (vol/vol) FBS, glutamine (2 mM), and gentamicin (50 µg/ml) to a final concentration of 1 × 106 cells/ml.
Viability of cultures was 95% as assessed by trypan blue dye
exclusion. FACS analysis and light microscopic observations revealed a
purity of 90-95% HCs for HC cultures.
Preparation of UHC cultures. To examine whether HCs responded directly to LPS, HC cultures were further purified up to >99% purity using Percoll, based on the method described by Smedsrød et al. (31) plus an additional purification step using the selective adhesion of KCs. Briefly, 4 ml of the HC suspension (see Preparation of HC cultures) was layered over 15 ml of 60% Percoll in PBS and centrifuged at 400 g for 15 min. Supernatants were discarded, and the pellet containing purified HCs was washed at 100 g for 5 min with Williams' medium E. Cells were incubated on a 10-cm culture dish (Greiner, Alphen a/d Rijn, The Netherlands) for 1 h, resulting in selective attachment to the plate surface of contaminating cells but not of HCs. After 1 h, the unattached HCs were collected, counted, and diluted to a final concentration of 1 × 106 cells/ml. Immunohistochemical and FACS analyses revealed a purity of >99% for these UHC cultures.
Preparation of KCs.
The procedure for KC isolation was based on the method of Smedsrød et
al. (31) with slight modifications. Supernatants (see Preparation of HC cultures) containing mainly nonparenchymal
cells were transferred to four 50-ml Falcon tubes (Micronic, Lelystad, the Netherlands) followed by centrifugation at 50 g in a
swing-out rotor at 4°C for 2 min. This procedure was repeated twice
to discard the remaining HCs. After the final step, supernatants were
centrifuged at 200 g in a swing-out rotor at 4°C for 10 min. Supernatants were discarded, and the resulting pellets were
resuspended in PBS to a final volume of 40 ml. The nonparenchymal cell
suspension was carefully placed on a two-step (60 and 25%) Percoll
gradient and centrifuged for 15 min at 400 g (4°C). After
centrifugation, the cell suspension, which was situated between the two
layers of Percoll, was collected and diluted with PBS. The cell
suspension, containing both KCs and endothelial cells, was centrifuged
at 200 g for 10 min, and the resulting pellet was diluted
with Williams' medium E (without serum) and washed again. Hereafter,
pellets consisting of 50% KCs and 50% endothelial cells were diluted
in Williams' medium E (without serum) to a final concentration of 2 × 106 cells/ml. To separate KCs from endothelial
cells, cells were plated on tissue culture plates or membrane inserts
at 37°C and 5% CO2 for 30 min followed by a single wash
step discarding the nonadherent endothelial cells. Viability of KCs was
95% as determined by trypan blue dye exclusion assay.
Immunohistochemistry and microscopic observations revealed a purity of
85% KCs for KC cultures.
Cell cultures. HCs were cultured at a density of 0.5 × 106 cells/well in 24-well culture dishes (Greiner) using Williams medium E, supplemented with 5% (vol/vol) FBS, glutamine (2 mM), and gentamicin (50 µg/ml). KCs were cultured in Williams' medium E containing 10% FBS, 2 mM glutamine, and 50 µg/ml gentamicin at a density of 0.5 × 106 cells/well in 24-well culture dishes.
DC cocultures consisted of 0.5 × 106 attached KCs in 24-well tissue culture plates with the addition of 0.5 × 106 HCs in direct contact. MI cocultures were prepared by culturing 0.5 × 106 HCs at the bottom of 24-well tissue culture plates and 0.5 × 106 KCs plated on membrane inserts with a pore size of 0.45 µM (Becton Dickinson). For both coculture systems, Williams' medium E supplemented with 5% (vol/vol) FBS, glutamine (2 mM), and gentamicin (50 µg/ml) was used. After an attachment period of 4 h, medium was replaced by fresh medium in all culture types.Experimental design.
After a recovery period of 24 h at 37°C and 5% CO2,
medium was replaced by medium containing 0, 1, or 10 µg/ml LPS.
Because HCs are known to produce various important serum compounds such as LPS-binding protein, medium of KC cultures still contained 10% FBS.
After 2, 4, 8, and 24 h of incubation, tissue culture supernatants
were collected for analysis of cytokines and NO and were stored at
70°C until used. After 24 h, biotransformation capacity of HC
cultures, DC cocultures, and MI cocultures were determined.
Concomitantly, to measure the LPS-induced cytotoxicity, viability of
different cultures was determined by measuring mitochondrial activity
using MTT.
Immunohistochemical analyses.
HC cultures, KC cultures, and DC cocultures were plated on sterile no.
1 coverslips placed in 24-well plates. Intracellular TNF- labeling
was performed as described by Openshaw et al. (26) using a
specific MAb for porcine TNF-
. Cells were stimulated with various
concentrations of LPS in the presence of 10 µg/ml brefeldin A to
inactivate the Golgi apparatus, resulting in an intracellular TNF-
assemblage. After 4 h of stimulation, cells were fixed with 2%
paraformaldehyde in PBS containing 10 µg/ml brefeldin A. Fixed cells
were washed three times with PBS containing 1% BSA and 0.5% saponin
(pH 7.4). Cells were labeled for 45 min with a MAb directed against
porcine TNF-
. After being washed three times for 5 min with PBS (1%
BSA and saponin) cells were incubated for another 45 min with rabbit
anti-mouse FITC (Molecular Probes Europe). Cells were washed and
immediately used for fluorescence microscopy.
TNF- bioassay.
TNF-
concentrations in cell culture supernatants were measured with
a cytotoxicity assay using a porcine kidney cell line (PK-15) according
to the method of Bertoni et al. (4). Cytotoxicity was
determined by measuring the decrease in mitochondrial activity using
MTT. Before the experiments, the PK-15 bioassay was validated by
comparison with a specific ELISA for porcine TNF-
(results not shown).
IL-6 bioassay. Porcine IL-6 was measured with a murine hybridoma B9 cell line in 96-well plates according to the method of Helle et al. (16). Proliferation was determined by measuring the increase in mitochondrial activity using MTT. Samples were titrated in threefold dilutions, and as positive control threefold dilutions of recombinant porcine IL-6 were used. Absorbance was measured at 590 nm. IL-6 in tissue culture supernatants was quantified by comparison of calculated EC50 values from supernatants with EC50 values from the recombinant porcine IL-6 standard curve. Experiments were performed in duplicate for each sample from cells isolated from each pig.
NO measurement.
After 24 h of incubation with or without LPS, samples were
collected and stored at 70°C until analysis. The NO production was
determined by measuring the amount of nitrite in culture supernatants according to the Griess reaction (12).
Western blot analysis for iNOS.
After 24 h incubation with or without LPS, Western blot analysis
of iNOS expression in KC cultures, HC cultures, and DC cocultures was
performed. After the cells were washed once in PBS, protein for Western
blotting was prepared from 8 × 106 KCs, HCs, or DC
coculture using a rubber policeman. Cells were centrifuged at 200 g and resuspended in 200 µl PBS. After homogenization by
repeated freezing (180°C) and thawing (37°C), samples were centrifuged at 13,000 rpm for 2 min in an Eppendorf centrifuge. Supernatants were collected, and the protein contents were determined using the method of Lowry. Total protein (10 µg) was loaded onto an
SDS/polyacrylamide (8%) gel and blotted on to nitrocellulose membranes
after electrophoresis. Membranes were blocked for 30 min by
Tris-buffered saline (10 mM Tris · HCl, 150 mM NaCl, pH 8)
containing 1% (wt/vol) BSA and 0.3% (vol/vol) Tween 20. The primary
antibody rabbit anti-mouse iNOS, which strongly cross-reacts with
porcine iNOS, was diluted 1:1,000 in Tris-buffered saline (1% BSA,
0.3% Tween 20), and blots were incubated for 1 h at room temperature. After 6 washes with Tris-buffered saline for 5 min, the
secondary antibody (pig anti-rabbit alkaline phosphatase) was added in
a 1:5,000 dilution in Tris-buffered saline (1% BSA and 0.3% Tween
20). Blots were incubated for 1 h, and, after being washed 6 times, blots were stained using nitroblue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate.
Biotransformation enzyme assays.
After 24 h incubation of cultures in medium with or without LPS,
HC functionality was determined by measuring 6- and
2
-testosterone hydroxylation rates (both marker activities for
CYP3A) and glucuronidation of 1-naphthol (marker for UDPGT enzyme activity).
Measurement of ROS. For the measurement of intracellular ROS production, oxidation of the fluorescent probe H2DCF-DA was measured following the method of Trayner et al. (33) with some slight modifications. Briefly, KCs and HCs were plated in 96-well plates at a density of 1 × 104 cells/well. Cocultures consisted of 1 × 104 KCs and 1 × 104 HCs per well. After incubation overnight at 37°C and 5% CO2, medium was removed and cells were washed with 100 µl/well Krebs-Ringer-buffered glucose (KRBG) (10 mM glucose, 10 mM HEPES, 140 mM NaCl, 4.86 mM KCl, 1.8 mM CaCl2, and 1 mM MgCl2, pH 7.4). Cells were loaded with 10 µM H2DCF-DA for 30 min at 37°C and 5% CO2. After 30 min, cells were washed with medium followed by addition of medium with or without various concentrations of LPS. After 4 h of incubation at 37°C and 5% CO2, fluorescence was determined by using a Cytofluor model 2300 microplate fluorometer (Millipore, Bedford, MA) using an excitation wavelength of 485 nm and emission wavelength of 538 nm. Experiments were performed in quadruplicate for cells derived from each pig, and values were normalized to control incubations for each culture type and depicted as relative signal intensity.
Extracellular release of ROS was determined using a horseradish peroxidase substrate A6550, which becomes highly fluorescent on horseradish peroxidase-catalyzed H2O2 oxidation. The experiments were performed as described by Mohanty et al. (22). Briefly, KCs and HCs were plated in 96-well plates at a density of 1 × 104 cells/well. Cocultures consisted of 1 × 104 KCs and 1 × 104 HCs per well. After incubation overnight at 37°C and 5% CO2, cells were washed with KRBG and incubated with 100 µl/well of 10 µM A6550 and 1 U/ml horseradish peroxidase in KRBG with various doses of LPS. After 4 h incubation at 37°C and 5% CO2, fluorescence was determined using a Cytofluor model 2300 microplate fluorometer and an excitation wavelength of 590 nm and emission wavelength of 645 nm. Experiments were performed in quadruplicate, and values were quantified by comparison with a standard curve of H2O2 for each experiment.Viability. After 24 h incubation with or without LPS, cell viability was assessed in all cultures by measuring mitochondrial activity using 3,3'-diaminobenzidine and MTT. Briefly, cells were incubated with 0.5 ml MTT (0.5 mg/ml) in Williams' medium E. After 4 h of incubation, medium was discarded and cells were lysed with 0.5% SDS in isopropanol. After lysis, absorbances were measured at 590 nm.
Statistics. Unless stated otherwise, data are expressed as means ± SE or SD and evaluated using a two-way ANOVA followed by Dunnett's test for comparison between two groups. P < 0.01 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proinflammatory cytokine production after stimulation with LPS.
Production of TNF- and IL-6 was measured after incubation of HC and
KC cultures and MI and DC cocultures in medium with or without LPS. A
time course study showed that maximal cytokine levels were observed
after 4 h for TNF-
and 24 h incubation for IL-6,
respectively (data not shown). Maximal TNF-
and IL-6 levels as
measured in different culture types are depicted in Fig.
1. Stimulation of MI cocultures and KC
cultures with LPS resulted in comparable levels of both TNF-
and
IL-6. However, in DC cocultures the amount of TNF-
was significantly
increased up to 10-fold after stimulation with LPS (Fig.
1A). The effect of direct cell-to-cell contact was even more
pronounced on IL-6 levels in DC cocultures, and an increase of 100- to
1,000-fold in IL-6 was observed compared with other culture types (Fig.
1B). Exposure of HC cultures to various concentrations of
LPS resulted in the release of small amounts of TNF-
and IL-6 (Fig.
1). In LPS-stimulated UHC cultures, this response was even less
pronounced and no significant release of TNF-
or IL-6 could be
measured (results not shown).
|
|
Release of NO and expression of iNOS.
LPS addition to HC cultures and both cocultures resulted in a
dose-dependent formation of nitrite as measured by Griess reaction (Fig. 3A). NO production was
significantly increased in DC cocultures as well as MI cocultures
during a 24-h incubation period with LPS. Nitrite levels in
supernatants of DC cocultures were already significantly increased when
cells were exposed to 10 ng/ml LPS (Fig. 3B). No significant
differences were found with regard to nitrite formation in UHC cultures
compared with HC cultures. Exposure of both cultures to 10 µg/ml LPS
for 24 h resulted in concentrations up to 12 µM nitrite in
culture supernatants for both culture types (Fig. 3B). In KC
cultures, no significant nitrite levels could be measured (Fig.
3A).
|
|
Production of extracellular and intracellular ROS.
Stimulation of different cultures with LPS resulted in a dose-dependent
release of H2O2 as measured extracellularly
during 4 h incubation (Fig.
5A). This release was
significantly higher in DC cocultures and HC cultures compared with KC
cultures. However, only minor differences were observed between the
LPS-induced release of ROS in HC cultures and DC cocultures.
|
Biotransformation capacity of HC cultures and cocultures.
Testosterone hydroxylation rates (CYP3A) and naphthol glucuronidation
rates (UDPGT) were measured to determine HC functionality in different
cultures. CYP3A and UDPGT enzyme activities after 24 h stimulation
with LPS are shown in Table 1. Compared
with HC cultures, both cocultures showed a significant decrease in testosterone hydroxylation rate during control incubations, but this
decrease was most abundant in DC cocultures. After stimulation of DC
cocultures with LPS, CYP3A activity was almost completely suppressed,
whereas in MI cocultures CYP3A activity was still 50-60% of
control levels when stimulated with 1 or 10 µg/ml LPS.
|
Viability.
To determine the LPS-induced cytotoxicity in the various culture types,
viability was assessed using MTT after 24 h incubation with or
without LPS. Although LPS stimulation in all cultures decreased
viability, this decrease was most apparent in DC cocultures (Fig.
6).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the intact liver, HCs are in direct contact with KCs. The
present experiments were designed to assess whether direct contact between KCs and HCs is of influence for the outcome of an LPS-induced inflammatory response. When comparing the production of TNF- and
IL-6 in the different culture types, direct contact between KCs and HCs
seems to be essential, because an ~10- and 500-fold increase of
TNF-
and IL-6, respectively, could be observed in DC cocultures.
Results from intracellular TNF-
labeling indicated that TNF-
in
these cultures is exclusively produced by KCs and not by HCs.
Furthermore, these results show that the increased TNF-
production
is due to an increased activation of KCs in DC cocultures and not the
result of a decreased viability of KCs in KC cultures. Although
cytokine levels in KC cultures were markedly lower than those observed
in cocultures, incubation of KC cultures with 10 µg/ml LPS still
resulted in an abundant cytokine response of 3,300 pg/ml TNF-
and
3,100 U/ml IL-6. Although LPS-induced cytokine expression in cocultures
or KC cultures has been extensively studied (21, 36), in
this study we have shown that direct contact between KCs and HCs
significantly increases TNF-
production by porcine KCs.
The LPS-induced NO release in monocultures vs. cocultures is in
agreement with results published by the group of Billiar et al.
(5, 6). However, our data show that NO production in porcine liver cell cultures is due to an LPS-induced iNOS expression only by HCs and not KCs. Evidence is accumulating that there are considerable species differences in iNOS expression and regulation in
the liver. For instance, in mouse and rat macrophages iNOS expression
can be induced by LPS, whereas in human monocytes and macrophages it
has been difficult to demonstrate iNOS expression (1, 9).
The latter observation is in accordance with our study, in which iNOS
expression could not be demonstrated in KCs, indicating that porcine
KCs more closely resemble human macrophages. In addition, both rat and
mouse HCs have been shown to express high levels of iNOS in response to
TNF-, IL-1
, and interferon-
as a single stimulus, whereas
human HCs responded to LPS alone without exposure to additional stimuli
(25). In our study, exposure of porcine UHC cultures to
LPS resulted in significant ROS and NO productions, whereas LPS
exposure did not result in increased levels of TNF-
and/or IL-6 in
these cultures. These results suggest that in porcine HCs LPS alone,
without further involvement of KCs and/or cytokines, initiates NO
production. Together with the observation that in porcine KCs no iNOS
expression could be demonstrated, these findings suggest that the
regulation and expression of iNOS in porcine liver closely resemble the
human liver physiology.
The relatively high NO concentrations measured in MI cocultures suggest
that NO production could be further induced by soluble factors capable
of transporting through the highly porous membrane of the inserts and
is not dependent on direct cell-to-cell contact. Several studies have
shown that TNF- is at least in a synergistic way able to induce iNOS
expression in various cell types (7, 13, 30, 32). Compared
with HC cultures, MI cocultures show an increased production of
TNF-
. Time course studies performed with these cultures reveal an
increased production of TNF-
preceding the NO production, suggesting
that increased TNF-
levels could be involved in the increased NO
production, as observed in MI cocultures.
LPS stimulation of DC cocultures resulted in significant cytotoxicity,
whereas the cell viability in the other culture systems remained less
affected. It seems unlikely that the observed cytotoxicity was mediated
by release of extracellular ROS since LPS stimulation did not result in
a significant difference between ROS formation in HC cultures and DC
cocultures. However, the observed cell death in DC cocultures could be
mediated by the excessive amounts of TNF- produced in DC cocultures
on LPS stimulation. Various studies in vivo as well as in vitro have
shown that TNF-
is involved in induction of direct hepatotoxicity
through apoptosis (15, 17, 20). Although the
significance of TNF-
production in the observed cell death seems
apparent, several studies have shown that KC-mediated cytotoxicity
against HCs or hepatoma cells can occur through production of NO
(5, 28). Furthermore, Kurose et al. (19)
showed that the increased production of NO by KCs was
CD-18/intracellular adhesion molecule-1-dependent and correlated with
an increased oxidative activation of nuclear factor-
B.
An important functional parameter of HCs is their biotransformation
capacity. Although our results show that KCs strongly suppress the
activity of at least the enzymes CYP3A and UDPGT, even in the absence
of LPS, the exact mechanism underlying this process is still not clear.
These results are in agreement with studies performed by Simmons and
co-workers (34, 35), who showed that cocultures of KCs
and HCs in direct cell-to-cell contact resulted in a decreased protein
synthesis by HCs. Furthermore, they showed that the decrease in HC
protein synthesis was NO dependent (5, 7). With regard to
CYP, evidence is accumulating that both NO and proinflammatory
cytokines are important mediators involved in the regulation of CYP
activity (18, 24). The observed decrease in
biotransformation capacity after stimulation with LPS may result from
an increased production of NO and/or proinflammatory cytokines as
observed in DC cocultures. However, a decrease in biotransformation
capacity of HCs was already observed during control incubations in DC
cocultures as well as MI cocultures, whereas no significant TNF-,
IL-6, or NO could be detected in the culture medium. This implies that
alternate KC-derived signaling molecules, capable of transporting
through the membrane insert, might contribute to the observed decrease
in biotransformation capacity.
Results obtained from MI and DC cocultures clearly demonstrate that different types of cocultures consisting of KCs and HCs can lead to disparate results. Cocultures from KCs and HCs separated by cell culture membrane inserts showed comparable proinflammatory cytokine responses as observed for KC cultures. In contrast, DC cocultures with direct cell-to-cell contact showed an increased cytokine production. Initial experiments had shown that the high-density membranes used in the experiments did not limit the diffusion of cytokines or other signaling molecules through the membrane (results not shown). However, the distance between KCs and HCs in these cocultures could be of importance for the distribution of reactive oxygen and/or nitrogen species. Since these small size molecular mediators have a short half-life, they will not reach the target sites if the two cell types are clearly separated. Therefore, these small reactive mediators are less effective in MI cocultures.
The increased proinflammatory cytokine production in DC cocultures clearly suggests involvement of mechanisms via direct cell-to-cell contact. Whether these mechanisms include adhesion molecules and/or short- life small-size molecular mediators remains to be elucidated. The present results provide evidence that the degree of the LPS-induced cytokine response is mediated by direct cell-to-cell contact between KCs and HCs. At the same time, DC cocultures reflect the mutual interaction between HCs and KCs in vivo more closely. To understand the mechanisms involved in the imposed LPS response in DC cocultures, future experiments should be directed to unravel all contributing signaling molecules. The insight into these mechanisms is essential for the identification of molecular targets in the treatment of a fulminating hepatic inflammatory response or endotoxin-induced liver injury.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Rob Bleumink for his technical assistance and Dr. B. J. Blaauboer for his stimulating discussions during the preparation of this article.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: K. H. N. Hoebe, Faculty of Veterinary Medicine, Dept. of Veterinary Pharmacy, Pharmacology, and Toxicology, Yalelaan 16, 3584 CM Utrecht, The Netherlands (E-mail: j.fink{at}vfft.vet.uu.nl).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 January 2000; accepted in final form 16 October 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Albina, JE.
On the expression of nitric oxide synthase by human macrophages. Why no NO?
J Leukoc Biol
58:
643-649,
1995[Abstract].
2.
Andus, T,
Geiger T,
Hirano T,
Kishimoto T,
and
Heinrich PC.
Action of recombinant human interleukin 6, interleukin 1 beta and tumor necrosis factor alpha on the mRNA induction of acute-phase proteins.
Eur J Immunol
18:
739-746,
1988[ISI][Medline].
3.
Baumann, H,
and
Gauldie J.
The acute phase response.
Immunol Today
15:
74-80,
1994[ISI][Medline].
4.
Bertoni, G,
Kuhnert P,
Peterhans E,
and
Pauli U.
Improved bioassay for the detection of porcine tumor necrosis factor using a homologous cell line: PK(15).
J Immunol Methods
160:
267-271,
1993[ISI][Medline].
5.
Billiar, TR,
Curran RD,
Ferrari FK,
Williams DL,
and
Simmons RL.
Kupffer cell:hepatocyte cocultures release nitric oxide in response to bacterial endotoxin.
J Surg Res
48:
349-353,
1990[ISI][Medline].
6.
Curran, RD,
Billiar TR,
Stuehr DJ,
Hofmann K,
and
Simmons RL.
Hepatocytes produce nitrogen oxides from L-arginine in response to inflammatory products of Kupffer cells.
J Exp Med
170:
1769-1774,
1989[Abstract].
7.
Curran, RD,
Ferrari FK,
Kispert PH,
Stadler J,
Stuehr DJ,
Simmons RL,
and
Billiar TR.
Nitric oxide and nitric oxide-generating compounds inhibit hepatocyte protein synthesis.
FASEB J
5:
2085-2092,
1991
8.
Decker, K.
The response of liver macrophages to inflammatory stimulation.
Keio J Med
47:
1-9,
1998[Medline].
9.
Denis, M.
Human monocytes/macrophages: NO or no NO?
J Leukoc Biol
55:
682-684,
1994[Abstract].
10.
Dominguez, J,
Ezquerra A,
Alonso F,
McCullough K,
Summerfield A,
Bianchi A,
Zwart RJ,
Kim YB,
Blecha F,
Eicher S,
Murtaugh M,
Pampusch M,
and
Burger K.
Porcine myelomonocytic markers: summary of the Second International Swine CD Workshop.
Vet Immunol Immunopathol
60:
329-341,
1998[ISI][Medline].
11.
Freudenberg, MA,
Freudenberg N,
and
Galanos C.
Time course of cellular distribution of endotoxin in liver, lungs and kidneys of rats.
Br J Exp Pathol
63:
56-65,
1982[ISI][Medline].
12.
Green, LC,
Wagner DA,
Glogowski J,
Skipper PL,
Wishnok JS,
and
Tannenbaum SR.
Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids.
Anal Biochem
126:
131-138,
1982[ISI][Medline].
13.
Harbrecht, BG,
Di Silvio M,
Demetris AJ,
Simmons RL,
and
Billiar TR.
Tumor necrosis factor-alpha regulates in vivo nitric oxide synthesis and induces liver injury during endotoxemia.
Hepatology
20:
1055-1060,
1994[ISI][Medline].
14.
Hartung, T,
Sauer A,
Hermann C,
Brockhaus F,
and
Wendel A.
Overactivation of the immune system by translocated bacteria and bacterial products.
Scand J Gastroenterol Suppl
222:
98-99,
1997.
15.
Hatano, E,
Bradham CA,
Stark A,
Iimuro Y,
Lemasters JJ,
and
Brenner DA.
The mitochondrial permeability transition augments Fas-induced apoptosis in mouse hepatocytes.
J Biol Chem
275:
11814-11823,
2000
16.
Helle, M,
Boeije L,
and
Aarden LA.
Functional discrimination between interleukin 6 and interleukin 1.
Eur J Immunol
18:
1535-1540,
1988[ISI][Medline].
17.
Jaeschke, H,
Fisher MA,
Lawson JA,
Simmons CA,
Farhood A,
and
Jones DA.
Activation of caspase 3 (CPP32)-like proteases is essential for TNF-alpha-induced hepatic parenchymal cell apoptosis and neutrophil-mediated necrosis in a murine endotoxin shock model.
J Immunol
160:
3480-3486,
1998
18.
Khatsenko, OG,
Barteneva NS,
de la Maza LM,
and
Kikkawa Y.
Role of nitric oxide in the inhibition of cytochrome P450 in the liver of mice infected with Chlamydia trachomatis.
Biochem Pharmacol
55:
1835-1842,
1998[ISI][Medline].
19.
Kurose, I,
Saito H,
Miura S,
Ebinuma H,
Higuchi H,
Watanabe N,
Zeki S,
Nakamura T,
Takaishi M,
and
Ishii H.
CD18/ICAM-1-dependent oxidative NF-kappaB activation leading to nitric oxide production in rat Kupffer cells cocultured with syngeneic hepatoma cells.
J Clin Invest
99:
867-878,
1997
20.
Leist, M,
Gantner F,
Jilg S,
and
Wendel A.
Activation of the 55 kDa TNF receptor is necessary and sufficient for TNF-induced liver failure, hepatocyte apoptosis, and nitrite release.
J Immunol
154:
1307-1316,
1995
21.
Lichtman, SN,
Wang J,
and
Lemasters JJ.
LPS receptor CD14 participates in release of TNF-alpha in RAW 264.7 and peritoneal cells but not in Kupffer cells.
Am J Physiol Gastrointest Liver Physiol
275:
G39-G46,
1998
22.
Mohanty, JG,
Jaffe JS,
Schulman ES,
and
Raible DG.
A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative.
J Immunol Methods
202:
133-141,
1997[ISI][Medline].
23.
Monshouwer, M,
Witkamp RF,
Nijmeijer SM,
Van Leengoed LA,
Vernooy HC,
Verheijden JH,
and
Van Miert AS.
A lipopolysaccharide-induced acute phase response in the pig is associated with a decrease in hepatic cytochrome P450-mediated drug metabolism.
J Vet Pharmacol Ther
19:
382-388,
1996[ISI][Medline].
24.
Monshouwer, M,
Witkamp RF,
Nijmeijer SM,
Van Amsterdam JG,
and
Van Miert AS.
Suppression of cytochrome P450- and UDP glucuronosyl transferase-dependent enzyme activities by proinflammatory cytokines and possible role of nitric oxide in primary cultures of pig hepatocytes.
Toxicol Appl Pharmacol
137:
237-244,
1996[ISI][Medline].
25.
Nussler, AK,
Di Silvio M,
Liu ZZ,
Geller DA,
Freeswick P,
Dorko K,
Bartoli F,
and
Billiar TR.
Further characterization and comparison of inducible nitric oxide synthase in mouse, rat, and human hepatocytes.
Hepatology
21:
1552-1560,
1995[ISI][Medline].
26.
Openshaw, P,
Murphy EE,
Hosken NA,
Maino V,
Davis K,
Murphy K,
and
O'Garra A.
Heterogeneity of intracellular cytokine synthesis at the single-cell level in polarized T helper 1 and T helper 2 populations.
J Exp Med
182:
1357-1367,
1995[Abstract].
27.
Rizzardini, M,
Zappone M,
Villa P,
Gnocchi P,
Sironi M,
Diomede L,
Meazza C,
Monshouwer M,
and
Cantoni L.
Kupffer cell depletion partially prevents hepatic heme oxygenase 1 messenger RNA accumulation in systemic inflammation in mice: role of interleukin 1.
Hepatology
27:
703-710,
1998[ISI][Medline].
28.
Saito, H,
Kurose I,
Ebinuma H,
Fukumura D,
Higuchi H,
Atsukawa K,
Tada S,
Kimura H,
Yonei Y,
Masuda T,
Miura S,
and
Ishii H.
Kupffer cell-mediated cytotoxicity against hepatoma cells occurs through production of nitric oxide and adhesion via ICAM-1/CD18.
Int Immunol
8:
1165-1172,
1996[Abstract].
29.
Seglen, PO.
Preparation of isolated rat liver cells.
Methods Cell Biol
13:
29-83,
1976[Medline].
30.
Shiratori, Y,
Ohmura K,
Hikiba Y,
Matsumura M,
Nagura T,
Okano K,
Kamii K,
and
Omata M.
Hepatocyte nitric oxide production is induced by Kupffer cells.
Dig Dis Sci
43:
1737-1745,
1998[ISI][Medline].
31.
Smedsrød, B,
Pertoft H,
Eggertsen G,
and
Sundstrom C.
Functional and morphological characterization of cultures of Kupffer cells and liver endothelial cells prepared by means of density separation in Percoll, and selective substrate adherence.
Cell Tissue Res
241:
639-649,
1985[ISI][Medline].
32.
Taylor, BS,
Alarcon LH,
and
Billiar TR.
Inducible nitric oxide synthase in the liver: regulation and function.
Biochemistry (Mosc)
63:
766-781,
1998[ISI][Medline].
33.
Trayner, ID,
Rayner AP,
Freeman GE,
and
Farzaneh F.
Quantitative multiwell myeloid differentiation assay using dichlorodihydrofluorescein diacetate (H2DCF-DA) or dihydrorhodamine 123 (H2R123).
J Immunol Methods
186:
275-284,
1995[ISI][Medline].
34.
West, MA,
Billiar TR,
Curran RD,
Hyland BJ,
and
Simmons RL.
Evidence that rat Kupffer cells stimulate and inhibit hepatocyte protein synthesis in vitro by different mechanisms.
Gastroenterology
96:
1572-1582,
1989[ISI][Medline].
35.
West, MA,
Billiar TR,
Hyland BJ,
Jordan ML,
and
Simmons RL.
Regulation of Kupffer cell-mediated alterations in hepatocyte protein synthesis in vitro.
Curr Surg
44:
467-469,
1987[Medline].
36.
Wheeler, MD,
and
Thurman RG.
Production of superoxide and TNF-alpha from alveolar macrophages is blunted by glycine.
Am J Physiol Lung Cell Mol Physiol
277:
L952-L959,
1999
37.
Winwood, PJ,
and
Arthur MJ.
Kupffer cells: their activation and role in animal models of liver injury and human liver disease.
Semin Liver Dis
13:
50-59,
1993[ISI][Medline].
38.
Wisse, E.
On the fine structure and function of rat liver Kupffer cells.
In: The Reticuloendothelial System, edited by Carr I,
and Dames WT.. New York: Plenum, 1980, p. 360-379.
39.
Wortelboer, HM,
de Kruif CA,
van Iersel AA,
Falke HE,
Noordhoek J,
and
Blaauboer BJ.
Acid reaction products of indole-3-carbinol and their effects on cytochrome P450 and phase II enzymes in rat and monkey hepatocytes.
Biochem Pharmacol
43:
1439-1447,
1992[ISI][Medline].
40.
Wortelboer, HM,
de Kruif CA,
van Iersel AA,
Falke HE,
Noordhoek J,
and
Blaauboer BJ.
The isoenzyme pattern of cytochrome P450 in rat hepatocytes in primary culture, comparing different enzyme activities in microsomal incubations and in intact monolayers.
Biochem Pharmacol
40:
2525-2534,
1990[ISI][Medline].