1 Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, and 2 Departamento de Gastroenterología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
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ABSTRACT |
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Hepatocyte gap junction
proteins, connexins (Cxs) 26 and 32, are downregulated during
obstructive cholestasis (OC) and lipopolysaccharide hepatocellular
cholestasis (LPS-HC). We investigated rat hepatic Cxs during
ethynylestradiol hepatocellular cholestasis (EE-HC) and choledochocaval
fistula (CCF) and compared them with OC and LPS-HC. Levels
(immunoblotting) and cellular distribution (immunofluorescence) of
Cx26, -32, and -43, as well as macrophage infiltration, were studied in
livers of rats under each condition. Cx26 and -32 were reduced in
LPS-HC, OC, and CCF. However, in EE-HC, Cx26 did not change and Cx32
was increased. Prominent inflammation occurred in LPS-HC, OC, and CCF,
which was associated with increased levels of Cx43 in LPS-HC and OC but
not CCF. No inflammation nor changes in Cx43 levels occurred during
EE-HC. In cultured hepatocytes, dye coupling was reduced by tumor
necrosis factor- and interleukins-1
and -6, whereas reduction
induced by LPS required coculture with Kupffer cells. Thus hepatocyte
gap junctions are downregulated in forms of cholestasis associated with
inflammation, and reduced intercellular communication might be induced
in part by proinflammatory mediators.
obstructive cholestasis; hepatocellular cholestasis; cytokines; macrophages.
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INTRODUCTION |
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GAP JUNCTION CHANNELS ARE permeable to ions and small molecules, providing a direct pathway for cytoplasmic communication between adjacent cells (8). They are constituted by integral membrane proteins, termed connexins (Cxs). In each cell, these proteins assemble into hexameric hemichannels (connexons) that at the plasma membrane dock and register with connexons in adjacent cells to form gap junctional channels (8). Cxs have been named according to the molecular mass deduced from their cloned cDNA (e.g., Cx26, -32, and -43) (5). The expression of different Cxs varies among tissues, and most organs express more than one Cx. While Cx40, -43, and -45 are expressed in rat heart (26), Cx26, -32, and -43 are expressed in rat liver (4). In addition, a single cell type can express more than one Cx, which may be found in the same (38, 57) or different gap junction plaques (19).
In mouse liver, both Cx26 and -32 are expressed by all hepatocytes (38), whereas in rat liver Cx32 is present throughout the acinus but Cx26 is found mainly in periportal hepatocytes (57). Cx43 is expressed by nonparenchymal cells of the liver, including Kupffer cells (KC) (46), stellate cells (18, 46), oval cells (64), cholangiocytes (48), cells of the Glisson capsule, and possibly endothelial cells (4).
Gap junction permeability properties allows for coordinated activities between cells and optimal functioning of different tissues (8). In the liver, gap junctions are necessary for cell-to-cell propagation of hormone-induced Ca2+ waves (34, 44). Moreover, Ca2+ waves are involved in coordinating Ca2+-dependent functions among hepatocytes, such as glycogenolysis (12), glucose secretion (36, 54), and bile secretion (35). Therefore, disruption of this intercellular communicating network due to reduced hepatocyte Cx expression might contribute to liver dysfunction. Accordingly, Cx26 and -32 have been reported to be reduced during obstructive cholestasis (OC) (13, 56), a frequent condition known to cause liver dysfunction. In addition, Cx32 expression has been shown to be reduced in other conditions, including the inflammatory response induced by bacterial lipopolysaccharide (LPS) (9, 17, 55) and ischemia-reperfusion-induced liver injury (16).
It is unknown whether the action of released inflammatory mediators
might be a common mechanism involved in regulating liver gap junctions
during cholestasis. In addition, it is possible that retention of bile
components associated with cholestasis could be contributing to
alteration of Cx expression. To address these questions, first we
compared changes in expression and cellular distribution of hepatic Cxs
in different models of cholestasis. A reduction in the expression of
Cxs by hepatocytes was found only under conditions [LPS, OC, and total
bile retention induced by choledochocaval fistula (CCF)] in which a
marked infiltration of inflammatory cells was present. Second, we
analyzed the effect of inflammatory mediators on gap junction
communication, demonstrating that tumor necrosis factor (TNF)-,
interleukin (IL)-1
, and IL-6 reduced dye coupling between cultured
hepatocytes. Finally, the addition of LPS induced a decrease in dye
coupling between hepatocytes when cocultured with KC, suggesting the
involvement of KC-derived cytokines in the reduction of hepatocyte gap
junction communication.
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MATERIALS AND METHODS |
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Reagents.
Rabbit anti-mouse IgG antibody, goat anti-rabbit antibody conjugated to
alkaline phosphatase, leupeptin, pepstatin A, di-isopropyl fluorophosphate, phenylmethylsulfonyl fluoride,
1,4-diazabicyclo[2.2.2]octane, EDTA, fish gelatin, IgG-free BSA, LPS
(E. coli serotype 0127:B8), 17-ethynylestradiol (EE),
N
-nitro-L-arginine
(L-NNA), typeVI collagenase, Percoll, 1,2-propylene glycol, Lucifer yellow CH, dexamethasone-21-acetate, insulin from bovine pancreas, mouse recombinant TNF-
, mouse recombinant IL-1
, and mouse recombinant IL-6 were from Sigma (St. Louis, MO). Goat anti-mouse IgG antibody conjugated to rhodamine and goat anti-rabbit IgG antibody conjugated to FITC were obtained from Organon (Durham, NC). Gelvatol airvol 205 was obtained from Air Product and Chemical (Allentown, PA). Goat serum, FCS, Waymouth medium, Lebovitz medium (L-15), F-12 nutrient mixture, penicillin, streptomycin, and insulin (bovine pancreas) were obtained from GIBCO-BRL (Grand Island, NY).
Monoclonal anti-macrophage ED1 antibody was obtained from Serotec
Biosourse International (Camarillo, CA). Previously characterized antibodies that recognize Cx26, -32, or -43 (4, 13, 62) were used. These antibodies were kindly provided by Dr. Elliot Hertzberg, Department of Neuroscience, Albert Einstein College of
Medicine (New York, NY).
Purification of F(ab)2 fragments.
F(ab)2 fragments were obtained by means of a commercial kit
(Pierce, Rockford, IL). IgGs were isolated from the serum, and then
F(ab)2 fragments were obtained by enzymatic digestion and were purified using an affinity column according to the manufacturer's instructions. Briefly, for IgG purification serum was diluted (1:1) in
union buffer (0.1 M sodium acetate), pH 5, and then centrifuged at
1,000 g for 20 min. The diluted serum supernatant was
applied over a G protein column previously equilibrated with union
buffer. The column was washed with 6-10 volumes of union buffer
until the absorbancy measured at 260 nm reached the baseline. IgGs were eluted with 3-5 volumes of 75-mM glycine · HCl buffer (pH
2.6). The elutes were collected in 1-ml fractions, and the pH of each one was neutralized immediately by the addition of 500 mM Tris (pH
7.6). The absorbancy of each fraction was measured at 280 nm, and those
with the highest value were pooled and dialyzed in semipermeable
membranes of 12,000 molecular weight exclusion (Spectrapor 4;
Spectrum Medical industries, Terminal Annex, LA) against 0.1 M
NH4HCO3. Samples were concentrated using a
speed vac concentrator (Integrated Speed VAC System IS100; Savant
Instruments, Farmingdale, NY). Ten milligrams of IgGs were dissolved in
1 ml of digestion buffer (20 nM cysteine · HCl in phosphate
buffer, pH 10). Papain coupled to Sepharose (0.5 ml) was washed twice with 4 ml digestion buffer, resuspended in 0.5 ml digestion buffer, and
added to 1 ml IgG solution. The reaction mixture was incubated at
37°C for 5 h or overnight under constant shaking. The digestion products were recovered, mixed with 1.5 ml union buffer, and then passed through a union buffer-equilibrated column of protein A coupled
to Sepharose. The column was washed with 6 ml of union buffer, and the
collected elute that contained F(ab)2 fragments was
dialyzed as mentioned above. Finally, F(ab)2 fragments were suspended in 500 µl of 1% BSA suspended in PBS and frozen at
80°C until they were used. The reactivity of anti-Cx43 IgG
F(ab)2 fragments was tested by immunofluorescence in rat
heart cryosections.
Animal treatments. Male Sprague-Dawley rats (180-200 g body wt) from the Animal Institute of the Pontificia Universidad Católica de Chile were used. Rats were subjected to hepatocellular cholestasis by the administration of LPS (LPS-HC) or EE (EE-HC) or were subjected to OC by ligation of the common bile duct. A separate group of animals was subjected to total bile retention without obstruction by a CCF, as previously described (22).
Rats fasted overnight were anaesthetized at 8:00 AM with ether, and a single dose of LPS dissolved in sterile saline was administered (2 mg/kg body wt) through the right femoral vein. Within the first 12 h after LPS administration, 10-15% of the rats died and were not included in these studies. A separate group of rats was treated with EE dissolved in 1,2-propylene glycol. A daily (9:00 AM) dose of EE was administered subcutaneously (5 mg/kg body wt). To induce OC or CCF, the common bile duct was double ligated and transected or cannulated close to the bifurcation with a PE-50 tube and secured with a double silk ligature. The cannula with free-flowing bile was looped outside the abdominal cavity by crossing the incision, tunneled subcutaneously to the neck of the rat, inserted ~1.0 cm into the jugular vein, and secured with double silk sutures. Patency of the common bile duct ligation was assessed by confirming the dilation of the bile duct before each rat was killed. CCF patency was examined before performing the experiments by measuring bile flow rates of the jugular ends and the common bile duct. Rats with CCF in which dilation of the extrahepatic bile duct proximal to the site of cannulation was apparent and/or with low bile flow rates were excluded. Control (no intervention) and adequate sham-injected or -operated groups of animals were included in each protocol. The liver was obtained under ether anesthesia, and thereafter animals died without pain due to hypovolemia.Immunofluorescence.
For immunofluorescence, 6-µm-thick frozen sections of rat liver
applied onto poly-L-lysine (Sigma) -coated glass coverslips were fixed and permeabilized in 70% ethanol at 20°C for 20 min. Samples were incubated with blocking solution (5 mM EDTA, 1% gelatin, 1% normal goat serum, and 0.05% BSA in PBS) for 1 h at room
temperature to reduce nonspecific binding of primary antibodies.
Samples were then incubated overnight at 4°C with the anti-Cx26,
-Cx32, and -Cx43 [antiserum or purified F(ab)2 fragments]
antibodies or for 30 min at room temperature with the
anti-monocyte/macrophage ED1 antibody diluted appropriately in blocking
solution. After three 15-min washes with PBS, sections were incubated
with a secondary antibody coupled to FITC for 30 min at room
temperature, washed three times with PBS for 10 min, and mounted in
Gelvatol airvol 205 with 200 mg/ml
1,4-diazabicyclo[2.2.2]octane. In double immunolabeling studies,
liver sections were first incubated overnight at 4°C with anti-Cx43
antibody and then were incubated with the ED1 antibody for 3 h at
room temperature. After three 15-min washes with PBS, sections were
incubated with both goat anti-rabbit IgG and goat anti-mouse IgG
secondary antibodies coupled to FITC and rhodamine, respectively.
Fluorescence was viewed under a xenon arc lamp on a Nikon Labophot-2
equipped with epifluorescent illumination and photographed using T-Max
400 film (Kodak). Specificity of immunoreactivity was assessed by
replacing the primary antibody with preimmune serum or the preimmune
serum IgG F(ab)2 fragments when appropriate.
Western blot analysis.
Levels of Cx32 and -43 were determined by immunoblot, as previously
described (4). Cx26 levels were analyzed in alkali residue
plasma membranes, prepared as described by Fallon et al. (13). Briefly, tissue samples were pulverized in a chilled
mortar (60°C), suspended in a solution containing phosphatase (100 mM NaF and 100 mM sodium pyrophosphate) and protease (500 µg/ml
leupeptin, 40 µg/ml aprotinin, 2 mg/ml soybean trypsin inhibitor, 1 mg/ml benzamidine, 1 mg/ml
-amino caproic acid, 3 mM
phenylmethylsulfonyl fluoride, and 20 mM EDTA) inhibitors, and
sonicated for 10 s (Microson ultrasonic cell disrupter; Heat
Systems). Protein content was determined by the Bradford method
(7) obtained from Bio-Rad (Hercules, CA). Samples were
resuspended in a final concentration of 1× Laemmli gel sample buffer
(29) and stored at
80°C.
Preparation of acute cultures of rat hepatocytes. Primary cultures of adult rat hepatocytes were prepared by using a collagenase perfusion technique described by Berry and Friend (3). Cells were resuspended in Waymouth medium supplemented with 10% FCS, 100 U/ml penicillin, 100 g/ml streptomycin, and 0.5 g/ml insulin. Cell viability was between 85 and 90% as assessed by cell exclusion to Trypan blue. Cells (1.5 × 106/3 ml culture medium) were preplated in 60-mm Primaria plates (Becton Dickinson, Oxnard, CA) for 90 min. The medium was then replaced by fresh L-15 medium without bicarbonate supplemented with 10% FCS, 100 U/ml of penicillin, and 100 g/ml streptomycin. At this point either LPS or different cytokines were added to the culture medium, and unless otherwise mentioned functional studies were carried out within the following 16 h.
Cocultures of hepatocytes with KC. KC were isolated using a double Percoll gradient, as previously described (52). Isolated KC were resuspended in Waymouth medium, and 2 × 106 cells/3 ml of medium were preplated in 60-mm Primaria plates. Thirty minutes later, cells were washed three times with fresh medium to discard nonadherent cells. Cultures of KC showed ~95% purity as assayed by the number of cells reactive to the anti-ED1 (monocyte/macrophage) antibody. Then 106 hepatocytes were seeded onto plates containing KC, and 90 min later the Waymouth medium was replaced by L-15 medium without bicarbonate supplemented with 10% FCS, 100 units penicillin, and 100 µg/ml streptomycin.
Cell coupling. To evaluate the functional state of gap junctions, the culture medium was replaced with F-12 nutrient mixture containing 10 mM HEPES. The cell-to-cell transfer of Lucifer yellow (5% in 150 mM LiCl) microinjected into a single cell of hepatocyte triplets or larger cell clusters was tested, as previously described (12). To determine if dye transfer occurred, cells were observed for 30 s on a Nikon Diaphot microscope equipped with a xenon arc lamp illumination and a Nikon B filter block (excitation wavelength 450-490 nm; emission wavelength >520 nm). Positive cell coupling was defined as the transfer of dye to one or more adjacent cells. Each data point was obtained from 4 independent experiments in which a minimum of 20 cells were microinjected. Coupling was expressed as the percentage of cells that showed dye coupling (incidence of dye coupling).
Macrophage quantification in the liver. In four separate experiments, monocyte/macrophages were detected in liver sections by immunofluorescence with the anti-ED1 antibody. In each animal, cells were counted by a blinded observer using a stereological method (61) in microphotographs taken from five different fields at ×100 magnification (20 fields/group).
Statistical analysis. Mean differences were tested with the nonparametric Kruskal-Wallis analysis. If a significant F value was obtained, means were compared with the Bonferroni-Dunn multiple comparisons test. A value of P < 0.05 was considered significant.
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RESULTS |
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Inflammatory response induced by cholestasis. The time course and mechanism of cholestasis in each experimental group differed. Although a sublethal dose of LPS induces an acute and transient effect (several hours), the administration of EE induces an effect that lasts for several days and is accompanied by the deterioration of the rats due to anorexia (20). Although CCF does not correspond precisely to a cholestatic state, the levels of serum bile components are similar to those seen in OC, but without an increase in biliary pressure. This, in part, allows separation of the effects of biliary obstruction from those of serum bile overload (22).
KC proliferation and infiltration of monocytes occurs during different forms of liver inflammation. To determine the magnitude of macrophage infiltration in the livers of the different models of cholestasis, cells reactive to the anti-ED1 antibody (macrophage marker) were counted in liver sections. LPS-HC, OC, and CCF, but not EE-HC, induced a recruitment of inflammatory cells to the liver (Table 1). Although the number of ED1-positive cells did not change after 8 h of LPS treatment, a significant increase occurred after 16 (75%) and 24 h (110%) (Table 1). In rats subjected to 3, 7, or 14 days OC, a progressive increase (38, 65, and 88%, respectively) in the number of anti-ED1-reactive cells was observed. Similarly, a 36 and 75% increase of anti-ED1-positive cells occurred in the liver of rats subjected to CCF for 2 and 6 days, respectively (Table 1). On the contrary, no significant changes in the number of anti-ED1-reactive cells occurred in rats with either EE-HC or undergoing a sham procedure (Table 1).
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LPS-HC, OC, and CCF, but not EE-HC, reduce Cx26 and -32 in the
liver.
Although the available anti-Cx26 antibody detects Cx26 in
alkali-insoluble membrane fractions, the anti-Cx32 and anti-Cx43 antibodies react well with their respective antigens present in total
cell homogenates (4, 13). Thus aliquots of
alkali-insoluble membranes (Cx26) or total liver homogenates (Cx32 and
-43) from control rats, sham-treated rats, and rats subjected to
LPS-HC, OC, CCF, or EE-HC were analyzed by immunoblotting. Within each time studied, the relative levels of Cx26 and -32 were drastically reduced in rats with LPS-HC and OC (Figs.
1, A and B,
respectively). During OC, there is high pressure in the biliary tree
and the plasma levels of bile components are markedly increased. To
elucidate whether the overload of bile components is sufficient to
induce the changes in Cx expression observed in OC, rats were subjected to total bile retention without bile duct obstruction by a CCF. In this
group of rats, levels of both Cx26 and -32 were reduced (Fig.
1C), as seen in the liver of rats treated with LPS or with OC. In contrast, in EE-HC, the levels of Cx32 increased to about threefold, whereas Cx26 levels remained unchanged (Fig. 1D).
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Gap junction communication between cultured hepatocytes is reduced
by TNF-, IL-1
, and IL-6 but not by LPS.
Since the presence of liver inflammation during cholestasis was
associated with a reduced expression of Cxs by hepatocytes, it is
possible that inflammatory cytokines may mediate this effect. Therefore, the effect of LPS and different cytokines on gap junctional communication was analyzed in primary cultures of adult rat
hepatocytes. The study of agents that reduce gap junctional
communication between hepatocytes within a time course of several hours
is limited by the fact that adult rat hepatocytes lose their gap
junctions within 8-12 h of culture in serum-containing medium
(47, 53). Alternatively, it is known that several
compounds permit maintained expression of Cx32 and gap junctional
communication, such as glucocorticoids (43), dimethyl
sulfoxide (27, 63), and cAMP (28, 47, 58).
However, since glucocorticoids (50), DMSO
(11), and cAMP (32) also block or
significantly attenuate the inflammatory response, they would not be
optimal for studying the effect of proinflammatory agents on hepatocyte
gap junctions. Prolonged intracellular acidification maintains
gap junctional communication between cultured hepatocytes for at least
24 h, presumably by blocking cell trafficking of plasma
membrane proteins (49), and thus offering the opportunity
to test the effect of compounds that might reduce gap junctional
communication. Hence, adult rat hepatocytes were cultured in L-15
medium without bicarbonate in an atmosphere with 5% CO2
(extracellular pH 6.4). Gap junction communication between hepatocytes
was evaluated by the cell-to-cell transfer of the fluorescent dye
Lucifer yellow in controls and in cultures treated with LPS, TNF-
,
IL-1
, or IL-6. Up to 24 h of culture, control hepatocytes
maintained the same spherical shape seen soon after seeding and the
incidence of dye coupling remained between 90-95% (Figs.
6, A-D, and
7, A and B). A
wide concentration range of LPS (0.1-10.0 µg/ml) had no effect
on hepatocyte dye coupling after 16 h (Fig. 6A) or
24 h of incubation (not shown). However, 16 h after the
addition of increasing concentrations (0.1-10.0 ng/ml) of TNF-
,
IL-1
, and IL-6, there was a significant reduction in dye coupling
(40-50%) (Fig. 6B). The reduction in dye coupling
induced by TNF-
(10.0 ng/ml) was not accompanied by changes in
relative levels of Cx26 and -32 (Fig. 6B, inset). This was also true for IL-1
and -6 (not shown). Treatment with LPS
or with cytokines did not alter cell viability (90-95%) as assessed by the cellular exclusion of Trypan blue. In time course studies, the maximal reduction of dye coupling induced by IL-1
(10 ng/ml) occurred earlier (8 h) than that induced by TNF-
or IL-6 (16 h) (Fig. 6C). All cytokines achieved maximal reduction of
dye coupling by 16 h of treatment (Fig. 6C).
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LPS reduces gap junctional communication between hepatocytes
maintained in coculture with KC.
Since LPS did not alter hepatocyte dye coupling, but macrophages are a
major source of cytokines when activated by LPS (23), we
tested if LPS modifies gap junctional communication between hepatocytes
that are cocultured with KC at a ratio of 1:2 (Fig. 8). After 16 h in coculture, the
incidence of dye coupling between hepatocytes was similar (95%) to
that found in cultures of pure hepatocytes (Fig. 8). However, the
addition of LPS (10 µg/ml) to cocultures reduced hepatocyte coupling
to ~35% (Fig. 8). This effect was prevented by 30-min pretreatment
with dexamethasone (10 µM) but not by L-NNA (10 mM) (Fig.
8). The addition of LPS to hepatocyte-KC cocultures did not affect the
hepatocyte viability (90-95%), as assessed by the cellular
exclusion of Trypan blue. Transfer of Lucifer yellow from hepatocytes
to KC or KC to hepatocytes was not detected in either control or
LPS-treated cocultures. In control experiments, neither dexamethasone
nor L-NNA affected the incidence of dye coupling between
hepatocytes in coculture with KC (Fig. 8).
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DISCUSSION |
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Cholestasis is a consequence of many pathological conditions resulting in different degrees of liver dysfunction. In the present study, we found that reduced Cx expression by hepatocytes occurs when cholestasis is associated with an inflammatory response (LPS-HC, OC, and CCF). Furthermore, we demonstrated that putative inflammatory mediators presumably released by KC reduced gap junctional communication between hepatocytes.
The reduction in levels of Cx26 and -32 observed during OC is consistent with that shown in previous reports (13, 56). The downregulation of Cxs in hepatocytes of rats subjected to total bile retention induced by a CCF suggests that the plasma overload of bile components may play an important role in the downregulation of Cxs in OC. In fact, during CCF, plasma bile components including bilirubin and bile acids reach levels comparable with those found in rats subjected to OC (22). The toxic concentrations of bile components that are reached in OC and CCF induce cellular necrosis, promoting inflammation (22). Consistently, in these two models of cholestasis, we observed a prominent infiltration of ED1-positive cells, comparable with that found in LPS-HC, where Cx26 and -32 were also drastically reduced. Therefore, it is probable that the inflammatory process associated with OC, CCF, and LPS-HC plays a primary role in downregulating Cx26 and -32 in hepatocytes during cholestasis. This agrees with the fact that no inflammation occurred in EE-HC, in which the expression of Cxs in hepatocytes remained unchanged (Cx26) or even increased (Cx32). In addition to inflammation, the enhanced hepatocyte proliferation induced by bile salts in CCF (2), LPS-HC (15, 60), and OC (42), but not EE (14, 39), might have further contributed to reduction of both hepatic Cx26 and -32, since these Cxs are drastically reduced in hepatocytes before the onset of DNA synthesis (10, 37).
In rats with LPS-HC or OC, colocalization of ED1 and Cx43 was frequently found, indicating that macrophages contributed to the increased levels of Cx43. Consistently, in rats subjected to EE-HC the levels of Cx43 did not change nor did the number of ED1/Cx43-positive cells. The expression of Cx43 by inflammatory cells has been previously observed in foam cells of atherosclerotic lesions (41), thioglycolated- or LPS-elicited peritoneal macrophages (1, 25), KC and macrophages infiltrating the liver after acute CCl4-induced intoxication (46), and inflammatory cells in human inflammatory renal diseases (24). In OC, however, the increased levels of Cx43 can also be partly attributed to that detected in proliferating epithelial cells of bile ducts (51).
The expression of Cx43 in KC of control rats was low; however, after 16 h of LPS-HC there was dramatic increase in relative levels of Cx43 (800%), which was proportionally greater than the rise in the number of ED1-positive cells (75%). These findings strongly suggest that the expression of Cx43 was induced in macrophages. This is further supported by the fact that Cx43 is normally not present in circulating monocytes (41). However, this induction was transient, because after 24 h of LPS-HC, there was a significant decrease in levels of Cx43 and the number of infiltrating macrophages remained high. A transient expression of Cx43 could explain the low levels of Cx43 detected in the liver of rats with a CCF, despite the increased number of ED1-positive cells.
Consistent with previous reports, a strong inflammatory response
occurred in the liver of rats with LPS-HC (40). Since
hepatocytes express the LPS receptor CD14 (30), and LPS
induces the production of TNF- in KC-free hepatocyte cultures
(45), we hypothesized that LPS would induce a reduction in
hepatocyte gap junction communication. Nevertheless, a wide
concentration range of LPS had no effect on hepatocyte dye coupling,
which is opposed to previous work reporting that LPS inhibits gap
junction communication between astrocytes (6). However, we
have shown that TNF-
, IL-1
, or IL-6 reduced hepatocyte dye
coupling to ~40%. This is consistent with the reduction in dye
coupling reported in immortalized fetal mouse hepatocytes treated under
similar conditions (55). Moreover, the involvement of
inflammatory mediators in the reduction of gap junction communication
is further substantiated by the decrease in dye coupling we observed in
hepatocytes cocultured with LPS-activated KC. Inhibition of hepatocyte
gap junctional communication by TNF-
and by LPS (in mixed
hepatocyte-KC cultures) was significantly prevented by dexamethasone
but not by blocking the production of nitric oxide. In light of these
results, downstream pathways involving nuclear factor-
B
transcriptional activation, but not nitric oxide, might be involved in
the cytokine-induced cell uncoupling. Nonetheless, we cannot rule out
that dexamethasone may have prevented the effect of cytokines by
enhancing the expression of Cx26 and -32 in hepatocytes, as previously
observed by others (43).
The present work has shown that during cholestasis an inflammatory
response induced by an overload of bile components or by LPS produces
downregulation of hepatocyte gap junction proteins Cx26 and -32. This
could disrupt the communication network between hepatocytes, altering
gap junction-dependent functions such as nerve stimulation- and
hormone-induced glucose secretion (36, 54),
vasopressin-induced glycogenolysis (12), and bile
secretion (35). Furthermore, our findings provide evidence
that cytokines released during the inflammatory response are involved
in reducing hepatocyte gap junction communication, possibly through a
nuclear factor-B pathway but independent of nitric oxide. Finally,
macrophage Cx43 expression might be necessary to transiently coordinate
responses of these inflammatory cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. Michael Nathanson for his thoughtful comments.
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FOOTNOTES |
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This work was partially supported by Fondo Nacional parz el Desarrolo de la Ciencia y Tecnología Grants 8990008 (to J. C. Sáez) and 1000563 (to L. Accatino) and by Grants 12/96 and 2/97 for residents of the school of medicine of the Pontificia Universidad Católica de Chile (to H. González).
The data in this paper are from a thesis submitted in partial fulfillment of the requirements for the Degree of Doctor in Medical Sciences (H. González) at the Pontificia Universidad Católica de Chile.
Address for reprint requests and other correspondence: J. C. Sáez, Dept. de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile (E-mail jsaez{at}genes.bio.puc.cl).
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.
First published January 16, 2002;10.1152/ajpgi.00298.2001
Received 11 July 2001; accepted in final form 8 January 2002.
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