Pronase destroys the lipopolysaccharide receptor CD14 on
Kupffer cells
Kenichi
Ikejima1,2,
Nobuyuki
Enomoto1,
Vitor
Seabra1,
Ayako
Ikejima2,
David A.
Brenner2, and
Ronald G.
Thurman1
1 Laboratory of Hepatobiology
and Toxicology, Department of Pharmacology and
2 Division of Digestive Diseases
and Nutrition, Department of Medicine, University of North
Carolina, Chapel Hill, North Carolina 27599-7365
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ABSTRACT |
CD14 is a lipopolysaccharide (LPS) receptor
distributed largely in macrophages, monocytes, and neutrophils;
however, the role of CD14 in activation of Kupffer cells by LPS remains
controversial. The purpose of this study was to determine if different
methods used to isolate Kupffer cells affect CD14. Kupffer cells were isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and differential centrifugation using Percoll gradients and
cultured for 24 h before experiments. CD14 mRNA was detected by RT-PCR
from Kupffer cell total RNA as well as from peritoneal macrophages.
Western blotting showed that Kupffer cells prepared with collagenase
possess CD14; however, it was absent in cells obtained by
collagenase-Pronase perfusion. Intracellular calcium in Kupffer cells
prepared with collagenase was increased transiently to levels around
300 nM by addition of LPS with 5% rat serum, which contains LPS
binding protein. This increase in intracellular calcium was totally
serum dependent. Moreover, LPS-induced increases in intracellular
calcium in Kupffer cells were blunted significantly (40% of controls)
when cells were treated with phosphatidylinositol-specific phospholipase C, which cleaves CD14 from the plasma membrane. However,
intracellular calcium did not increase when LPS was added to cells
prepared by collagenase-Pronase perfusion even in the presence of
serum. These cells were viable, however, because ATP increased
intracellular calcium to the same levels as cells prepared with
collagenase perfusion. Tumor necrosis factor-
(TNF-
) mRNA was
increased in Kupffer cells prepared with collagenase perfusion 1 h
after addition of LPS, an effect potentiated over twofold by serum;
however, serum did not increase TNF-
mRNA in cells isolated via
collagenase-Pronase perfusion. Moreover, treatment with Pronase rapidly
decreased CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer
cells. These findings indicate that Pronase cleaves CD14 from Kupffer
cells, whereas collagenase perfusion does not, providing an explanation
for why Kupffer cells do not exhibit a CD14-mediated pathway when
prepared with procedures using Pronase. It is concluded that Kupffer
cells indeed contain a functional CD14 LPS receptor when prepared gently.
macrophages; endotoxin receptor; proteases; scavenger
receptor
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INTRODUCTION |
GUT-DERIVED ENDOTOXIN plays a pivotal role in the
development of alcohol-induced liver injury using the chronic
enteral ethanol feeding model in the rat
(Tsukamoto-French). For example, Adachi et al. (2)
demonstrated that elimination of endotoxin by gut sterilization with
enteral antibiotics prevented early alcohol-induced liver injury.
Moreover, Nanji et al. (18) demonstrated that suppression of bacterial
overgrowth using lactobacillus also prevented liver injury in a similar
model by decreasing endotoxin levels in portal blood. Furthermore,
activation of Kupffer cells by gut-derived endotoxin is an essential
step for the development of early alcohol-induced liver injury.
Activation of Kupffer cells leads to the production of cytotoxic
mediators such as tumor necrosis factor-
(TNF-
), interleukin-1
and -6, eicosanoids, oxygen radicals, and nitric oxide. Indeed,
inactivation of Kupffer cells by
GdCl3 treatment (1) or with an
antibody for TNF-
(12) prevented early alcohol-induced liver injury
in the Tsukamoto-French model. However, the precise receptors by which
Kupffer cells are activated by endotoxin remain unclear.
Lipopolysaccharide (LPS), a component of the outer wall of
gram-negative bacteria, is a major endotoxin (19), and macrophages and
neutrophils contain several different LPS receptors on their cell
surface. CD14 is a functional LPS receptor that regulates cytokine
production in macrophages (26). It is a phosphatidylinositol-anchored protein that is easily cleaved from cells by
phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane bound
CD14 does not recognize LPS alone at low concentrations but rather
binds LPS and LPS binding protein (LBP), which is present in serum,
forming a LBP-LPS-mCD14 ternary complex (6, 26). Because of the
requirement for LBP, CD14-mediated pathways are serum dependent;
however, it is not clear whether CD14 plays a role in cytokine and
eicosanoid production by Kupffer cells. Several recent reports support
the hypothesis that CD14 is important for the development of liver
injury caused by acute (4) and chronic alcohol (23) and cholestasis
(24). On the other hand, activation of NF-
B (3) and TNF-
production (16) by LPS in isolated Kupffer cells has been reported to
be serum independent. Together, these studies lead to the idea that the
response to LPS in isolated Kupffer cells is not dependent on CD14.
Therefore, the purpose of this study was to test the hypothesis that
methodological considerations might explain this apparent paradox.
Specifically, it was demonstrated here that preparation of Kupffer
cells with Pronase cleaved CD14 from the Kupffer cell membrane.
Preliminary accounts of this work have appeared elsewhere (13).
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MATERIALS AND METHODS |
Isolation and culture of rat Kupffer cells.
Female Sprague-Dawley rats weighing between 200 and 250 g were used for
all experiments. All animals were given humane care in compliance with
National Institutes of Health and Institutional Guidelines. Here,
Kupffer cells were isolated from the rat liver by collagenase perfusion
and differential centrifugation using Percoll (Pharmacia, Uppsala,
Sweden) as described elsewhere with slight modifications, the standard
procedure in this laboratory. Briefly, the liver was perfused in situ
through the portal vein with Ca2+-
and Mg2+-free Hanks' balanced
salt solution (HBSS) containing 0.5 mM EGTA at 37°C for 5 min at a
flow rate of 26 ml/min. Subsequently, perfusion was with HBSS
containing 0.025% collagenase IV (Sigma Chemical, St. Louis, MO) at
37°C for 5 min (collagenase perfusion). Because the groups who
report CD14 independence used Pronase (3, 16), 0.02% Pronase
(protease, Sigma Chemical) was added to the perfusate (collagenase-Pronase perfusion) to make preparations for comparison. After digestion, the liver was excised and cut into small pieces in the
collagenase buffer previously described. The suspension was filtered
through nylon gauze, and the filtrate was centrifuged twice at 50 g at 4°C for 3 min to remove
parenchymal cells. The nonparenchymal cell fraction was washed with
buffer and centrifuged on a density cushion of Percoll at 1,000 g for 15 min to obtain the Kupffer
cell fraction, followed by washing with buffer again. The viability of
isolated Kupffer cells by trypan blue exclusion was >90%. Cells were
plated in plastic culture dishes (Corning, Corning, NY) or onto 25-mm
glass coverslips at a concentration of 5 × 105 cells/coverslip and cultured
in RPMI 1640 medium (GIBCO Laboratories Life Technologies, Grand
Island, NY) supplemented with 25 mM HEPES, 10% fetal bovine serum
(FBS), and antibiotics (100 U/ml of penicillin G and 100 µg/ml of
streptomycin sulfate). To increase purity, nonadherent cells were
removed by exchanging culture medium 1 h after plating. More than 95%
of adherent cells phagocytosed latex beads, indicating that they were
Kupffer cells. Cells were cultured at 37°C in 5%
CO2 for 24 h before experiments
unless otherwise noted.
Preparation of rat peritoneal macrophages.
Rats were given an intraperitoneal injection of casein hydrolysate (5%
wt/vol, 75 ml/kg) 3 days before cell preparation, and peritoneal cells
were collected by lavaging the abdominal cavity with 30 ml HBSS. The
cell suspension was passed through sterile gauze and centrifuged at 500 g for 7 min at 4°C to obtain a
cell pellet. Cells were washed twice with HBSS and cultured in plastic culture dishes in RPMI 1640 medium containing 10% FBS, 25 mM HEPES, and antibiotics. Nonadherent cells were removed by exchanging the
medium 1 h after plating.
Culture of RAW 264.7 cells.
RAW 264.7 cells, a mouse macrophage cell line, were obtained from
American Type Culture Collection (Rockville, MD) and were cultured in
DMEM (GIBCO Laboratories Life Technologies) containing 10% FBS and
antibiotics at 37°C in 5%
CO2. Medium was exchanged with
fresh medium without FBS before experiments.
RNA preparation, RT-PCR, and Northern blotting.
Total RNA from cultured cells was prepared using TRIzol reagent (Life
Technologies, Grand Island, NY). Concentration and integrity of RNA
were determined by measuring absorbance at 260 nm and electrophoresis on 1% agarose gels.
For RT-PCR, 1 µg of total RNA was reverse transcribed using Moloney
murine leukemia virus reverse transcriptase (GIBCO Laboratories Life
Technologies) and an oligo(dT)16
primer. The cDNA (1 µl) obtained was amplified using Taq DNA
polymerase, and specific forward and reverse primers were used as
follows: for CD14 forward (5'-GTG CTC CTG CCC AGT GAA AGA
T-3') and reverse (5'-GAT CTG TCT GAC AAC CCT GAG
T-3'), yielding a 267-bp product size (5); for Kupffer cell
receptor (KCR) forward (5'-ATG AAG GAG GCG GAA CTG AAC-3')
and reverse (5'-TCA GCT CTG GTC CGT TCT GGC-3'), yielding a
product size of 1653 bp (11); for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward (5'-TGA AGG TCG GAG TCA ACG GAT TTG
GT-3') and reverse (5'-CAT GTG GGC CAT GAG GTC CAC
CAC-3'), yielding a product size of 983 bp (25). The enzyme and
dNTPs were added to the reaction mixture after a 4-min denaturation period; thereafter, 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s followed by final extension at 72°C for 7 min were performed using GeneAmp PCR System 9600 (Perkin Elmer, Norwalk, CT). The size and amount of PCR products were verified by
electrophoresis in 1% agarose gels.
The steady-state levels of mRNA for CD14 and TNF-
were detected by
Northern blot hybridization. Total RNA (5-10 µg) was
electrophoresed in formaldehyde-denaturing 1% agarose gels and
transferred onto nylon membranes. Membranes were prehybridized in 50%
formamide, 5× saline-sodium citrate (SSC), 1× Denhardts'
solution, and 10 µg/ml salmon sperm DNA at 42°C and hybridized
with random prime-labeled probe for CD14 or TNF-
at 42°C
overnight. Membranes were then washed with 2× SSC, 0.1% SDS at
50°C for 30 min, and 0.1× SSC, 0.1% SDS at 55°C for 30 min, and autoradiography was performed using X-OMAT films.
Subsequently, membranes were stripped by washing with 0.1× SSC
and 0.1% SDS at 65°C and reprobed for the housekeeping gene GAPDH.
Western blotting for CD14.
Protein extracts were prepared from cultured RAW 264.7 cells or Kupffer
cells by homogenizing in a cell lysis buffer containing 50 mM Tris, pH
8.0, 150 mM NaCl, 1 mM EDTA, 0.02% sodium azide, 1 µg/ml aprotinin,
100 µg/ml phenylmethylsulfonyl fluoride, and 1% Triton X-100.
Protein concentration was determined spectrophotometrically using a
Bradford assay kit (Bio-Rad, Hercules, CA). Then, 25 µg of protein
(RAW 264.7 cells) or 5 µg of protein (Kupffer cells) were separated
by 10% SDS-PAGE and transferred onto polyvinylidene difluoride
membranes. Membranes were blocked by Tris-buffered saline-Tween
containing 5% skim milk, incubated with a mouse anti-rat myeloid cell
ED-9 antibody (MCA 620; Serotec, Oxford, UK), which recognizes CD14 (5,
24), or a goat anti-mouse CD14 polyclonal antibody (Santa Cruz
Biotechnology, Santa Cruz, CA). Subsequently, membranes were blotted
with a horseradish peroxidase (HRP)-conjugated secondary antibody (rat
anti-mouse IgG-HRP or anti-goat IgG-HRP). A chemiluminescence substrate
(enhanced chemiluminescence reagent) was used for detection of specific
bands. No band was detectable with a Western blot of primary
antibodies leading to the conclusion that there is no cross-link
between the primary antibody and denatured Ig potentially present in
Kupffer cell membranes.
Measurement of intracellular
Ca2+.
Intracellular Ca2+ in individual
Kupffer cells was measured using the fluorescent Ca2+
indicator dye fura 2 and a microspectrofluorometer (Photon Technology International, South Brunswick, NJ) interfaced with an inverted microscope (Diaphot, Nikon, Japan) as reported previously (14). Kupffer
cells cultured on coverslips were incubated in modified Hanks' buffer
[(in mM) 115 NaCl, 5 KCl, 0.3 Na2HPO4,
0.4 KH2PO4, 5.6 glucose, 0.8 MgSO4, 1.26 CaCl2, 15 HEPES, pH 7.4]
containing 5 µM fura 2-acetoxymethyl ester (Molecular Probes, Eugene,
OR), and 0.03% Pluronic F-127 (BASF Wyandotte, Wyandotte, MI) at room temperature for 60 min. Coverslips plated with Kupffer cells were rinsed and placed in chambers with buffer at room temperature. Changes
in fluorescence intensity of fura 2 at excitation wavelengths of 340 and 380 nm and emission at 510 nm were monitored in individual Kupffer
cells. Each value was corrected by subtracting the system dark noise
and autofluorescence, assessed by quenching fura 2 fluorescence with
Mn2+ as described previously (8).
Intracellular Ca2+ concentration
([Ca2+]i) was determined from the equation
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where
F0/Fs is the ratio of fluorescent
intensities evoked by 380-nm light from fura 2 pentapotassium salt
loaded in cells using a buffer containing 3 mM EGTA and 1 µM
ionomycin
([Ca2+]min)
or 10 mM Ca2+ and 1 µM ionomycin
([Ca2+]max).
R is the ratio of fluorescent intensities at excitation wavelengths of
340 and 380 nm, and Rmax and
Rmin are values of R at
[Ca2+]max
and
[Ca2+]min,
respectively. The values of these constants were determined at the end
of each experiment, and a dissociation constant
(Kd) of 135 nM
was used (7).
Statistical analysis.
All results are expressed as means ± SE. Statistical differences
between means were determined using Student's
t-test.
P < 0.05 was selected before the
study to reflect significance.
 |
RESULTS |
Kupffer cells prepared with collagenase but not with
collagenase-Pronase contain CD14 protein.
To clarify whether Kupffer cells contain CD14 mRNA, cells prepared with
collagenase alone were probed for CD14 mRNA by RT-PCR (Fig.
1A).
Isolated Kupffer cells as well as peritoneal macrophages contained CD14
mRNA 3 and 24 h after plating. RT-PCR for KCR (11), which is a Kupffer
cell-specific glycoprotein receptor of unknown function, showed that
total RNA from isolated Kupffer cells contained this specific mRNA. To
check the integrity of CD14 mRNA, total Kupffer cell RNA was analyzed
by Northern blot hybridization (Fig. 1B). Indeed, isolated Kupffer cells
contained mRNA for CD14, indicating that Kupffer cells expressed CD14
mRNA constitutively. Next, to confirm whether Kupffer cells contain
CD14 protein, protein extracts from isolated Kupffer cells were
analyzed by Western blotting using a mouse anti-rat ED9 antibody, which
recognizes rat CD14 (Fig.1C).
Kupffer cells prepared by collagenase perfusion expressed CD14, which
is a 55-kDa protein, both 3 and 24 h after incubation (Fig.
1C, lanes
1 and 2). However,
specific bands for CD14 were absent when cells were prepared by
collagenase-Pronase perfusion (Fig.
1C, lanes
3 and 4).

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Fig. 1.
Kupffer cells contain CD14. A: 1 µg
of total RNA from Kupffer cells or peritoneal macrophages isolated from
rat 3 and 24 h after incubation were analyzed by RT-PCR for CD14,
Kupffer cell receptor (KCR), and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). Blank control without RNA was also performed to
exclude nonspecific amplification. PCR products were electrophoresed in
1% agarose gel and stained with ethidium bromide to detect specific
bands. X174/Hae III marker was used to determine size of PCR
products. B: 10 µg of total
Kupffer cell RNA were analyzed by Northern blot hybridization for CD14.
Membranes were reprobed for the housekeeping gene GAPDH.
C: protein extracts from Kupffer cells
isolated by collagenase perfusion or collagenase-Pronase perfusion were
analyzed by Western blotting for CD14. Mouse anti-rat ED9 antibody was
used to detect rat CD14. Data are representative photographs of 3 individual experiments.
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LPS-induced increases in intracellular
Ca2+ in isolated
Kupffer cells.
LPS increases intracellular Ca2+
in isolated Kupffer cells in the presence of rat serum, which contains
LBP (26). Here, this serum dependence of intracellular
Ca2+ was compared in cells
prepared using collagenase or collagenase-Pronase (Fig.
2). LPS (10 µg/ml) added in the absence
of serum did not increase intracellular
Ca2+ in Kupffer cells prepared by
collagenase perfusion (Fig. 2A). However, when LPS was added with 5% rat serum, intracellular
Ca2+ was increased in cells
isolated by collagenase perfusion with maximal levels reaching values
around 300 nM (Fig. 2B). In sharp contrast, LPS did not increase intracellular
Ca2+ in cells prepared by
collagenase-Pronase perfusion even in the presence of serum (Fig.
2C). However, ATP (100 µM)
increased intracellular Ca2+ to
similar levels irrespective of the method of preparation, proving that
cells were viable and functional.

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Fig. 2.
Differences in lipopolysaccharide (LPS)-induced increases in
intracellular Ca2+ in Kupffer
cells isolated by collagenase or collagenase-Pronase perfusion.
A: intracellular
Ca2+ in isolated Kupffer cells was
measured microfluorometrically using fura 2. LPS (10 µg/ml) was added
to cells isolated by collagenase perfusion in absence of rat serum
followed by ATP (100 µM). B: LPS in
5% rat serum was added to cells isolated by collagenase perfusion
followed by ATP. C: LPS in 5% rat
serum was added to Kupffer cells isolated by collagenase-Pronase
perfusion followed by ATP. Data are representative traces of 4 individual experiments.
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Because it is known that PI-PLC cleaves CD14 from the plasma membrane,
the effect of PI-PLC on LPS-induced increases in intracellular Ca2+ was evaluated (Fig.
3). Maximal increases in intracellular
Ca2+ due to LPS reached values
around 230 nM in the presense of rat serum in cells prepared with
collagenase. However, LPS-induced increases in intracellular
Ca2+ were largely blunted when
cells were preincubated with PI-PLC (0.5 U/ml), with maximal levels
reaching only about 40% of control values. ATP, however, increased
intracellular Ca2+ in
PI-PLC-treated cells to levels similar to untreated controls, indicating that PI-PLC did not affect cell viability.

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Fig. 3.
Effect of phosphatidylinositol-specific phospholipase C (PI-PLC) on
LPS-induced increases in intracellular
Ca2+ concentration in isolated
Kupffer cells. Experimental design is as described in Fig. 2. PI-PLC
(0.5 U/ml) was added to Kupffer cells for 60 min before measurement of
intracellular Ca2+. Values are
means ± SE of maximal increases after stimulation in 5 individual
experiments. * P < 0.001 for comparison with control by Student's
t-test.
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LPS-induced increases in TNF-
mRNA in isolated
Kupffer cells.
Serum dependency of LPS-induced increases in TNF-
mRNA in isolated
Kupffer cells was evaluated by Northern blot analysis (Fig.
4). Rat serum per se did not increase
TNF-
mRNA. However, in the presence of LPS (1 ng/ml), rat serum
(0.1-1%) increased TNF-
mRNA in Kupffer cells isolated by
collagenase perfusion in a dose-dependent manner. Next, the dose
dependency of LPS in the induction of TNF-
mRNA in Kupffer cells was
studied (Fig. 5A).
Serum-dependent increases in TNF-
mRNA were observed clearly when
cells prepared with collagenase were treated with low doses of LPS (1 ng/ml; Fig. 5B). However, TNF-
mRNA was increased even in the absence of serum when higher doses of
LPS (10 ng/ml) were used. In contrast, others have shown (16) that
TNF-
production is serum independent when cells were prepared with
collagenase-Pronase perfusion, a finding confirmed here (Fig.
5C).

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Fig. 4.
Serum dependency of LPS-induced increases in tumor necrosis factor-
(TNF- ) mRNA in Kupffer cells isolated by collagenase perfusion.
Kupffer cells isolated by collagenase perfusion were cultured 24 h
before experiments. Cells were incubated with 1 ng/ml LPS in 0-1%
rat serum for 1 h, and total RNA was prepared. Total RNA (5 µg) was
analyzed by Northern blot hybridization for TNF- . Subsequently,
membranes were reprobed for the housekeeping gene GAPDH. Typical
experiment was repeated 3 times.
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Fig. 5.
Dose dependency of LPS on TNF- mRNA in Kupffer cells isolated by
collagenase or collagenase-Pronase perfusion.
A: experimental design is as in Fig. 3
except cells were incubated with 1-10 ng/ml LPS with
and/or without 1% rat serum for 1 h.
B: ratio of TNF- to GAPDH from
densitometry of data in A was plotted.
, Data without serum; , data with serum.
C: Kupffer cells isolated by
collagenase-Pronase perfusion were incubated with LPS (1 ng/ml) with
and/or without 1% rat serum for 1 h. Ratio of TNF- to GAPDH
was calculated based on densitometric data. Data represent percent
increase due to serum over values with LPS obtained in absence of
serum. GAPDH is the housekeeping gene. Representative data were
repeated 3 times.
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Effect of polyinosinic acid on LPS-induced increases in
TNF-
mRNA in isolated Kupffer cells.
To determine if the serum-independent induction of TNF-
mRNA by LPS
involved the scavenger receptor, Kupffer cells were pretreated with
polyinosinic acid, a scavenger receptor inhibitor. Figure 6 is a representative Northern blot for
TNF-
from cells prepared with collagenase alone. The increase in
TNF-
mRNA 1 h after LPS (10 ng/ml) in the absence of serum was
blunted by polyinosinic acid to about one-half the levels of untreated
controls.

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Fig. 6.
Effect of polyinosinic acid on LPS-induced increases in TNF- mRNA in
Kupffer cells. Experimental design is as in Fig. 3. Kupffer cells
isolated by collagenase perfusion were preincubated with polyinosinic
acid (10 µg/ml), an inhibitor of the scavenger receptor, for 5 min
followed by addition of LPS (10 ng/ml) for 1 h. Representative Northern
blot of TNF- from 3 individual experiments is shown. GAPDH is the
housekeeping gene.
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Effect of Pronase on CD14 expression in RAW 264.7 cells and Kupffer
cells.
To determine if Pronase can indeed remove CD14 from macrophages, whole
cell protein extracts from a mouse macrophage cell line (RAW 264.7) or
rat Kupffer cells were analyzed by Western blotting (Fig.
7A). As
expected, untreated RAW 264.7 cells expressed CD14; however, CD14
progressively disappeared in a time-dependent manner when cells were
incubated with 0.02% Pronase for 0.5-10 min (Fig.
7B,
left). Similar results were obtained
when Kupffer cells were incubated with 0.02% Pronase for 5-10 min
at 37°C (Fig. 7B,
right).

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Fig. 7.
Effect of Pronase on CD14 protein levels in RAW 264.7 cells and Kupffer
cells. RAW 264.7 cells or Kupffer cells were incubated for various
times (0.5-10 min) with 0.02% Pronase before preparation of
protein extracts. Whole cell protein extracts (RAW 264.7 cells, 50 µg; Kupffer cells, 5 µg) were analyzed by Western blotting using
mouse anti-rat ED9 antibody or a goat anti-mouse polyclonal antibody.
Representative photograph (A) and
densitometrical analysis (B) of
specific bands (55 kDa) from 3 individual experiments with each cell
type are shown.
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 |
DISCUSSION |
Role of CD14 in Kupffer cells is paradoxical.
Because CD14 has been described as a putative LPS receptor on
macrophages, many reports have suggested that it participates in the
pathophysiology of endotoxin shock (17) and organ damage (20). However,
the role of CD14 in Kupffer cells has been controversial. It has been
reported that CD14 protein levels were increased in liver macrophages 4 days after bile duct ligation (24). It has also been shown that acute
ethanol increased both CD14 mRNA and protein (4). Furthermore, CD14 and
LBP mRNA were increased in livers following chronic intragastric
ethanol delivery to rats (23). These observations support the
hypothesis that CD14 plays a pivotal role in the development of
endotoxin-related liver injury. On the other hand, Bellezzo et al. (3)
reported that NF-
B activation by LPS, which is important in the
transcriptional upregulation of inflammatory cytokines such as TNF-
,
was serum independent in isolated Kupffer cells. Furthermore, it has
been reported that TNF-
production by isolated Kupffer cells was
serum independent and that endocytosis played a pivotal role in TNF-
synthesis (16). These reports suggested that Kupffer cells in the
normal liver lack CD14-mediated pathways. In this study it was
demonstrated that differences in cell preparation methods are critical
for detection of CD14 (see below).
Pronase cleaves CD14 from Kupffer cells.
Seglen (21) first described the isolation of liver parenchymal cells
using collagenase, which gently digests liver so that it is easy to
obtain fragile hepatocytes. Subsequently, Knook and Sleyster (15)
reported isolation of Kupffer and endothelial cells from liver using
Pronase E, which destroyed hepatocytes allowing the nonparenchymal cell
fraction to be purified. More recently, Pronase has been used by many
investigators because the nonparenchymal cell yield is much higher
compared with preparations using collagenase alone. Our laboratory,
however, has avoided use of Pronase because of the possibility that it
affects membrane receptor proteins. Specifically, the voltage-dependent
Ca2+ channel in Kupffer cells described Hijioka et al. (10)
was shown in preliminary studies to be depolarized only when Kupffer cells were prepared gently with collagenase alone (unpublished observation). This led us to hypothesize that Pronase may
affect other membrane receptors such as CD14 on Kupffer cells. To test this hypothesis, two different isolation methods were compared. Indeed,
CD14 protein was preserved on Kupffer cells isolated by collagenase
perfusion alone; however, it disappeared totally when cells were
prepared with collagenase-Pronase (Fig.
1C). Strikingly, low doses of
Pronase (0.02%) destroyed almost 90% of CD14 on RAW 264.7 macrophages
and Kupffer cells in only 5-10 min (Fig. 7). Serum dependency
of induction of TNF-
mRNA due to LPS also disappeared completely
when Kupffer cells were prepared with Pronase (Fig. 5C). These findings are consistent
with the hypothesis that Pronase cleaves CD14 from the cell surface.
Obviously, the CD14-mediated pathway is not present when Kupffer cells
are prepared using Pronase.
Kupffer cells constitutively express CD14 as a functional LPS
receptor.
It is also not clear whether Kupffer cells express CD14 under normal
conditions. Although CD14 in liver macrophages is increased in
pathological conditions (i.e., LPS injection, cholestasis, and
alcoholic liver injury), one possible explanation was that recruitment
of monocytes-macrophages, which express CD14, is involved in the
mechanism by which CD14 is upregulated in the liver. This idea was
supported by the fact that CD14 was not detected in untreated controls
in a study evaluating cholestasis (24). However, here it was
demonstrated that Kupffer cells indeed express CD14 in the absence of
pathology (Fig. 1). Kupffer cells expressed mRNA for CD14 without any
stimulation (Fig. 1, A and
B). Furthermore, CD14 protein was
detectable by Western blot analysis in protein extracts from Kupffer
cells prepared by collagenase perfusion alone (Fig.
1C). Taken together, it is concluded
that Kupffer cells express both CD14 mRNA and protein constitutively
under normal conditions. Whether the use of Pronase explains the lack of positive results in other laboratories is not clear.
Although CD14 is important in LPS-induced cytokine production in other
types of macrophages, it was uncertain whether the CD14-mediated
pathway is involved in cytokine production in Kupffer cells. To address
this question, serum dependency of induction of TNF-
mRNA by LPS was
investigated. Rat serum was used here to determine CD14-mediated
pathways because it contains LBP, which forms a complex with LPS that
is recognized by CD14 (26). As expected, increases in TNF-
mRNA
levels by low doses of LPS were dependent on serum when Kupffer cells
were prepared with collagenase alone (Figs. 4 and
5A), supporting the hypothesis that
the CD14-mediated pathway is involved in the induction of TNF-
mRNA
in Kupffer cells. Furthermore, increases in intracellular
Ca2+ by LPS in Kupffer cells
isolated by collagenase perfusion were also serum dependent (Fig. 2).
This LPS-induced increase in intracellular Ca2+ most likely occurs via CD14,
since PI-PLC, which cleaves CD14 from the cell surface, largely
(>50%) prevented this increase (Fig. 3). However, the possibility
that receptors other than CD14 are affected cannot be excluded
completely. Moreover, Pronase completely prevented the increase in
intracellular Ca2+ due to LPS
(Fig. 2), supporting the hypothesis that Pronase efficiently removes
CD14 from the cell surface.
CD14-independent induction of TNF-
in Kupffer cells:
role for scavenger receptor.
Although CD14 plays an important role in TNF-
production due to LPS,
there are several different receptors that recognize LPS. In this study
serum-independent induction of TNF-
mRNA was also observed when the
dose of LPS was increased (Fig. 5, A
and B). The ratio of TNF-
mRNA
induction in serum vs. no serum decreased from over 2.0 to around 1.4 as LPS was increased from 1 to 10 ng/ml. It has been reported that
scavenger receptors on macrophages are involved in the recognition and
clearance of LPS (9). Polyinosinic acid, a scavenger receptor
inhibitor, blunted LPS-induced increases in TNF-
mRNA to
approximately one-half of control levels (Fig. 6), indicating that the
scavenger receptor participates in TNF-
production in Kupffer cells.
Bellezzo et al. (3) reported serum- and CD14-independent activation of
NF-
B due to LPS in Kupffer cells prepared using Pronase. Moreover,
Lichtman et al. (16) reported that TNF-
production was serum
independent in Kupffer cells prepared using Pronase. These findings are
consistent with the idea that LPS induces TNF-
via scavenger
receptors in the absence of CD14. On the other hand, Hampton et al. (9)
reported that scavenger receptors are not involved in TNF-
secretion. Importantly, it is obvious that Kupffer cells prepared using
Pronase, which cleaves CD14 from the cell surface, do not demonstrate
CD14-mediated production of TNF-
.
In conclusion, Kupffer cells play an important role in the
pathophysiology of the liver including alcohol-induced injury. Because
endotoxin is one of the most important stimulants of Kupffer cells,
CD14 most likely plays a pivotal role in the activation of this cell.
It is concluded that the method of isolation is important in studies of
the function of this receptor and its signaling pathways.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by a grant from the National
Institutes of Health.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. G. Thurman,
Laboratory of Hepatobiology and Toxicology, Dept. of Pharmacology, CB
7365, Mary Ellen Jones Bldg., Univ. of North Carolina at Chapel Hill,
Chapel Hill, NC 27599-7365 (E-mail:
thurman{at}med.unc.edu).
Received 16 June 1998; accepted in final form 23 November 1998.
 |
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