1 Department of Veterans Affairs Medical Center, Ann Arbor, Michigan 48109; Departments of 2 Medicine, 3 Surgery, and 4 Pathology, University of Michigan, Ann Arbor, 48109-0666; and 5 North Shore University Hospital/New York University School of Medicine, Manhasset, New York 11030
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ABSTRACT |
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Upregulation of CD14 in Kupffer cells has
been implicated in the pathogenesis of several forms of liver injury,
including alcoholic liver disease. However, it remains unclear whether
CD14 mediates lipopolysaccharide (LPS) signaling in this specialized liver macrophage population. In this series of experiments, we determined the role of CD14 in LPS activation of Kupffer cells by using
several complementary approaches. First, we isolated Kupffer cells from
human livers and studied the effects of anti-CD14 antibodies on LPS
activation of these cells. Kupffer cells were incubated with increasing
concentrations of LPS in the presence and absence of recombinant human
LPS binding protein (LBP). With increasing concentrations of LPS, human
Kupffer cell tumor necrosis factor- (TNF-
) production (a marker
for Kupffer cell activation) increased in a dose-dependent manner in
the presence and absence of LBP. In the presence of anti-human CD14
antibodies, the production of TNF-
was significantly diminished.
Second, we compared LPS activation of Kupffer cells isolated from
wild-type and CD14 knockout mice. Kupffer cells from CD14 knockout mice
produced significantly less TNF-
in response to the same amount of
LPS. Together, these data strongly support a critical role for CD14 in
Kupffer cell responses to LPS.
liver; endotoxin; tumor necrosis factor; macrophages
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INTRODUCTION |
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KUPFFER CELLS, THE
RESIDENT macrophages of the liver, represent the largest
population of fixed macrophages within the body. Located in the
sinusoidal space, Kupffer cells are in contact with portal blood
draining the intestinal tract and are thus exposed to bacteria and
bacterial products, such as lipopolysaccharide (LPS), which traverse
the intestinal barrier. Kupffer cells play a key role in clearing LPS,
as demonstrated by experiments that show that the majority of injected
125I-labeled LPS can be localized to the Kupffer cells
within 30 min of injection (18). Kupffer cell interactions
with LPS, however, are not limited to clearance. Kupffer cells, when
exposed to LPS, are activated and can produce a spectrum of cytokines
and reactive oxygen intermediates, including the proinflammatory
cytokine tumor necrosis factor- (TNF-
) (2-4).
LPS is a potent stimulator of Kupffer cell TNF-
production, and this
pathway has been implicated in the pathogenesis of many types of liver
injury, including alcohol-induced liver injury (32). In
human alcoholic hepatitis, elevations of TNF-
are associated with a
worse prognosis (7). Treatment of severe alcoholic
hepatitis with corticosteroids has been shown to be beneficial,
suggesting that immune over-activation may be contributing to the
pathogenesis of this disease (5, 20). In rodent models of
alcoholic hepatitis, the pathogenesis of immune activation has been
well studied. Experimental evidence supporting an important role for
TNF-
in mediating liver injury has been provided by in vivo studies
using anti-TNF-
antibodies and TNF receptor 1 knockout mice
(13, 35). A role for Kupffer cell activation by endogenous
LPS is further supported by studies showing significantly less liver
injury in the presence of antibiotics, lactobacillus, or gadolinium
chloride (1, 2, 22).
Despite its importance, the mechanism by which LPS activates Kupffer cells is not clearly understood. In peripheral blood monocytes, LPS activation is mediated through LPS binding protein (LBP) and CD14. In serum, LPS binds to LBP, a 60-kDa glycoprotein produced predominantly by the liver and secreted in serum (25, 33). LBP catalyzes the transfer of LPS to cell surface receptors such as membrane CD14 (9). In the presence of LBP, markedly less LPS is needed to activate peripheral blood monocytes. The LBP and CD14 pathway is critical for interactions at the low concentrations of LPS found under physiological conditions (30). Multiple lines of evidence suggest that Kupffer cells differ significantly from peripheral blood monocytes in their interactions with LPS (15). Unlike peripheral blood monocytes, Kupffer cells have relatively low baseline expression of CD14 (3, 29, 36). Furthermore, Kupffer cells and other macrophages can interact with LPS in a serum-independent fashion (4, 16). This has led many other studies (4, 16) to suggest that Kupffer cell activation by LPS is mediated not through the LBP/CD14 pathway but through some other less-characterized pathway. To directly address this question, we studied the LPS response of human Kupffer cells to LPS in the presence of neutralizing monoclonal anti-CD14 antibodies. In addition, we compared the response of murine Kupffer cells isolated from mice that lack CD14 to the response of their wild-type controls that possess an intact CD14 receptor.
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MATERIALS AND METHODS |
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Reagents. LPS from Escherichia coli (055:B5) and phosphatidylinositol-specific phospholipase C (PIPLC) were purchased from Sigma (St. Louis, MO), and pronase was obtained from Boerhinger-Mannheim (Indianapolis, IN). Anti-human CD14 antibody MY4 was obtained from Coulter Immunology (Hialeah, FL), and LeuM3 was obtained from Becton, Dickinson Immunocytometry Systems (San Jose, CA).
Animals. Mice studied included 6- to 12-wk-old female CD14 knockout, back-crossed 10 times onto the BALB/c background (10, 11) and age- and/or sex-matched control BALB/c mice (Jackson Laboratory, Bar Harbor, ME). All animals received humane care in compliance with the National Institutes of Health criteria for care of laboratory animals.
Recombinant LBP. Recombinant human LBP was obtained from LBP-transfected Chinese hamster ovary (CHO) cells as previously described (21). Recombinant rat LBP was produced using a baculovirus expression system as previously described (27). Bioactivity of the rat recombinant LBP in mouse cells was demonstrated using RAW 267.4 cells (27). Because of difficulty in maintaining the bioactivity of the purified protein due to rapid degradation, we used in these experiments supernatants (at a concentration of 3% of total volume) from either human LBP-transfected CHO cells or recombinant baculovirus-infected Sf9 cells. The concentration of the human LBP in 3% total volume was ~3 µg/ml as measured by an LBP ELISA previously described by Myc et al. (21).
Isolation and culture of Kupffer cells.
Human Kupffer cells were isolated from normal liver tissue obtained
from fresh surgical hepatectomy specimens with the assistance of the
University of Michigan Tissue Procurement Core. We used the standard
technique of pronase digestion previously described for human Kupffer
cells (12) followed by differential centrifugation using
Percoll (Pharmacia, Uppsala, Sweden) (26). This research protocol was reviewed and approved by the University of Michigan Medical Institutional Review Board. Briefly, the liver was excised and
minced before incubation with Gey's balanced salt solution (GBSS)-pronase solution with continuous stirring at 37°C for 60 min.
DNase (0.8 µg/ml) was added to prevent cell clumping. The liver
slurry was filtered through gauze mesh, washed with culture media, and
centrifuged two times at 600 g for 5 min. Cells were resuspended in PBS with DNase (0.8 µg/ml). Cells were further purified using a discontinuous Percoll gradient of 25 and 50% Percoll
as described in detail by Pertoft and Smedsrod (23). Purified nonparenchymal cells were washed and cultured in media containing Williams E medium supplemented with 100,000 U/l penicillin, 100 mg/l streptomycin, 15 mM HEPES, and 106 M insulin.
Kupffer cells were enriched by differential adherence to tissue culture
plates. Cells (4.0 × 105 cells/well in a 96-well
plate) were plated in tissue culture plates at 37°C for one-half hour
before washing and incubating in tissue culture media containing 5%
FCS overnight. These cells were ~80% pure for Kupffer cells as
estimated by their ability to ingest latex beads. The remaining cells
are mainly endothelial and stellate cells. Cell viability was always
>90% as assessed by trypan blue. All experiments were subsequently
performed after washing the cells three times with serum-free media.
For each experiment, Kupffer cells were isolated from one individual
liver. The amount of liver used was variable and dependent on the
amount of excess liver tissue available from each surgical operation.
Inner salt assay. Cell viability was assessed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI) per the manufacturer's instructions. This assay utilized the novel tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrzolium, inner salt (MTS) and the electron coupling reagent phenazine ethosulfate.
Western blots. Kupffer cells were lysed with a lysis buffer containing 1% IGEPAL, 5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin. Total protein was measured with the BCA protein assay method (Pierce, Rockford, IL). Total protein (12 µg) was loaded into each lane. Cell extracts were separated by SDS-PAGE using a 10-12.5% gel under denaturing conditions using the methods of Laemmli (14). Transfer was carried out electrophoretically by the methods of Towbin et al. (31) to nitrocellulose (Schleicher & Schuell, Keene, NH). The membrane was probed with a primary antibody followed by a horseradish peroxidase-linked secondary antibody. Detection was carried out with the enhanced chemiluminescence Western blotting kit (Amersham, Little Chalfont, UK).
ELISA.
Human TNF- was measured by sandwich ELISA using antibodies and
standards obtained from Pharmingen (San Diego, CA). Mouse TNF-
levels were measured with a rat TNF ELISA kit (Biosource, Camarillo,
CA) per manufacturer's instructions.
Statistical analysis. Data were analyzed using ANOVA and two-tailed Student's t-test when the data had a normal distribution (StatView; Abacus Concepts/SAS Institute, Cary, NC). Statistical significance was assigned at a P value of <0.05. All the figures are graphed with the means ± SE.
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RESULTS |
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LBP augment human Kupffer cell TNF- production in response to
LPS.
Isolated Kupffer cells were incubated with varying concentrations of
LPS (0, 1, or 10 ng/ml) in the presence of either serum-free media or
serum-free media with recombinant human LBP. After 6 h at 37°C,
the supernatant was collected and human TNF-
levels were measured
using ELISA. Without LPS, isolated Kupffer cells produce little
detectable TNF-
. After incubation with LPS, Kupffer cells produced
increasing concentrations of TNF-
in response to increasing
concentration of LPS in the presence and absence of LBP. However, at
low concentrations of LPS (1 ng/ml), the presence of LBP significantly
augmented the production of TNF-
(Fig.
1) (P < 0.001 for control vs.
LBP).
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Inhibition of human Kupffer cell response to LPS by PIPLC.
To indirectly assess the role of CD14 in mediating LPS activation of
human Kupffer cells, we utilized the enzyme PIPLC. PIPLC cleaves
glycosyl phosphatidyl inositol (GPI)-anchored proteins such as CD14,
releasing them from the cell membrane. We analyzed Kupffer cell
responses to LPS with and without enzyme treatment. Isolated human
Kupffer cells were preincubated with PIPLC (500 mU/ml) or serum-free
media for 1 h at 37°C before stimulation with LPS. Subsequently,
the Kupffer cells were washed and incubated with increasing
concentrations of LPS (0, 1, 10 ng/ml) in serum-free media for an
additional 6 h. Supernatants were assayed for TNF-. Minimal
TNF-
was detected in isolated Kupffer cells without LPS in both the
PIPLC and control groups. However, preincubation of Kupffer cells with
PIPLC significantly decreased the amount of TNF-
produced in
response to 1 or 10 ng/ml of LPS (P < 0.001; Fig.
2), suggesting the importance of CD14 in
mediating Kupffer cell response to LPS.
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Anti-CD14 antibodies inhibit LPS-mediated TNF- production in
Kupffer cells.
The effect of the monoclonal anti-human CD14 antibodies (6,
36) MY4 (previously shown to block CD14-mediated function) and
LeuM3 (a CD14-specific nonblocking isotype control monoclonal antibody)
on Kupffer cell production of TNF-
in response to LPS was analyzed.
Kupffer cells were incubated with either MY4 (10 µg/ml), LeuM3 (10 µg/ml), or serum-free media for 1 h at 37°C before wash with
serum-free media. Cells were then incubated with increasing
concentrations of LPS (0, 1, and 10 ng/ml) in the presence of the same
antibodies or serum-free media for 6 h before assay of the
supernatant for TNF-
. As presented in Fig.
3, increasing concentrations of TNF-
were noted in the supernatant of cells treated with increasing
concentrations of LPS. This increase in TNF-
was blocked by the
addition of MY4 but not by the addition of LeuM3. Decrease in TNF-
production in the MY4 groups was not due to loss of cell viability; MTS
assay of the cells after incubation with MY4, LeuM3, or media showed
equal viability in all groups (data not shown). Furthermore, the effect
of MY4 is similar in the presence or absence of recombinant human LBP
(Fig. 4).
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Kupffer cells isolated from CD14 knockout mice respond to LPS in a
LBP/CD14-dependent manner.
Kupffer cells were isolated from CD14 knockout mice or the control mice
(BALB/c). As expected, CD14 expression was not found on isolated
Kupffer cells in CD14 knockout mice in contrast to control BALB/c mice
(Fig. 5). We then examined the response
of Kupffer cells to LPS in vitro. Isolated Kupffer cells from both control and CD14 knockout mice produced no detectable TNF- in the
absence of LPS. Kupffer cells from control mice produced increasing amounts of TNF-
in the presence of increasing LPS (1 and 10 ng/ml) (Fig. 6). CD14 knockout mice produced
little or no TNF-
at either LPS concentration (Fig. 6). In the
presence of LBP, Kupffer cells from control mice produced significantly
(P < 0.005) more TNF-
in response to LPS (1 ng/ml) than
in the absence of LBP (Fig. 7). Under
identical conditions, Kupffer cells from the CD14-deficient mice
produced little or no TNF-
compared with wild-type animals; however,
significantly (P < 0.0001) more TNF-
was produced in the
presence of LBP compared with its absence. (Fig. 7).
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DISCUSSION |
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In this article, we demonstrated that the secretion of TNF- by
human Kupffer cells in response to low concentrations of LPS is
mediated via membrane CD14. In the presence of neutralizing antibody to
human CD14 or after pretreatment of cells with an enzyme (PIPLC) that
removes CD14 from the surface by cleavage of its GPI anchor, LPS
activation is significantly inhibited. Consistent with these results,
Kupffer cells from CD14 knockout mice also show little or no
sensitivity to LPS. Previous studies (4, 16) casting doubt
on the role of both LBP and CD14 in Kupffer cell activation in rats and
mice have been inferred from results showing that serum was not
absolutely necessary for activation by LPS compared with elicited
peritoneal macrophages or the mouse macrophage cell line RAW 264.7. Because serum contains many LPS binding factors in addition to LBP
(such as soluble CD14 and high-density lipoprotein), which could affect
cellular responses to LPS, we chose to perform all of our experiments
in the presence or absence of recombinant LBP. We have shown that,
although Kupffer cells can interact with LPS at concentrations of 10 ng/ml in the absence of recombinant LBP at low concentrations of LPS (1 ng/ml, levels found in the serum of septic patients), the presence of
recombinant LBP significantly (>10-fold) augmented the response.
Therefore, although LBP is not critical for responses to LPS at high
concentrations, it is required for the Kupffer cell response at
physiologically relevant low concentrations of LPS. This is consistent
with the concept that LBP facilitates the transfer of LPS to its
receptors. The fact that LBP is not critical for the response at higher
LPS concentrations does not preclude the need for membrane CD14. In fact, our results show that neutralizing antibodies to CD14 and cleavage of CD14 receptors block responses to LPS both in the presence
and absence of LBP. Our in vitro studies with isolated human and rodent
Kupffer cells are consistent with the concept that Kupffer cells
represent a major source of TNF-
during endotoxemia. In vivo studies
(24) using a rabbit endotoxemia model and neutralizing anti-rabbit CD14 antibodies show diminished TNF-
production in response to LPS. Similarly, in CD14 knockout mice, a lack of TNF-
production is also seen after LPS injection (10).
Despite the relatively low expression of CD14 on Kupffer cells, this receptor pathway appears to be important in mediating LPS responses. Although we have shown that activation of Kupffer cells by LPS utilizes the LBP/CD14 pathway in a manner similar to peripheral blood monocytes, we have not specifically addressed how Kupffer cells differ from peripheral blood monocytes. Multiple studies including our own (19, 28, 29) have shown that Kupffer cells have low baseline levels of CD14 but can be induced to upregulate CD14 expression with different stimuli, including LPS injection and alcohol-induced liver injury. Takai et al. (29) demonstrated 5- to 40-fold increase in mRNA for CD14 in Kupffer cells isolated after in vivo LPS injection and after in vitro exposure to LPS. Consistent with this finding, CD14 expression in vivo as measured with immunohistochemical staining was minimal in normal livers but increased after LPS injection, peaking at 6 h after intraperitoneal LPS injection (20 µg/mouse) (19). In contrast to Kupffer cells, peritoneal macrophages do not show these marked increases in CD14 mRNA. Rather, peritoneal macrophages have a higher baseline level of CD14 mRNA than Kupffer cells (19, 29). Similarly, peripheral blood monocytes have high baseline levels of CD14 expression that is refractory to further increases (3, 36).
In addition to LPS administration, marked increases in CD14 expression
on Kupffer cells are seen after chronic intragastric feeding with
ethanol as opposed to isocaloric dextrose (28). The
physiological significance of these elevations in Kupffer cell CD14 is
not clear, but we suspect that such increases sensitize Kupffer cells
to LPS. Transgenic mice that overexpress CD14 are exquisitely sensitive
to LPS (8). Thus upregulation of Kupffer cell CD14 in
multiple disease models, such as common bile duct ligation and
alcoholic hepatitis, may cause the observed liver injury by sensitizing
Kupffer cells to the effects of endogenous LPS. Consistent with this
hypothesis is a recent report showing decreased liver injury in CD14
knockout mice given ethanol compared with the CD14 wild-type animals
(34). CD14 wild-type animals had an increase in
liver-to-body weight ratio, serum alanine aminotransferase, and increases in steatosis and necrosis histologically when
intragastrically fed ethanol compared with dextrose control diets.
These changes due to ethanol were blunted in CD14 knockout animals fed
an identical diet. An associated increase in TNF- mRNA levels was
seen in the CD14 wild-type animals fed ethanol diets, which was not
seen in ethanol-fed CD14-deficient animals. These studies and our own support the speculation that blocking CD14 may have a benefit in
diseases characterized by overexpression of Kupffer cell CD14.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants DK-53296 (to G. L. Su), GM-54911 and GM-60205 (to S. C. Wang), and AI-23859 (to S. M. Goyert) and a Veterans Affairs Merit Award (to G. L. Su).
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. L. Su, University of Michigan Medical Center, 1510C MSRB I, Box 0666, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0666 (E-mail: gsu{at}umich.edu).
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.
October 17, 2001;10.1152/ajpgi.00253.2001
Received 14 June 2001; accepted in final form 10 October 2001.
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