Involvement of the TNF and FasL Produced by CD11b Kupffer Cells/Macrophages in CCl4-Induced Acute Hepatic Injury
et al. (2014) Involvement of the TNF and FasL Produced by CD11b Kupffer Cells/
Macrophages in CCl4-Induced Acute Hepatic Injury. PLoS ONE 9(3): e92515. doi:10.1371/journal.pone.0092515
Involvement of the TNF and FasL Produced by CD11b Kupffer Cells/Macrophages in CCl4-Induced Acute Hepatic Injury
Atsushi Sato 0
Hiroyuki Nakashima 0
Masahiro Nakashima 0
Masami Ikarashi 0
Kiyoshi Nishiyama 0
Manabu Kinoshita 0
Shuhji Seki 0
Rajesh Mohanraj, UAE University, Faculty of Medicine & Health Sciences, United Arab Emirates
0 1 Department of Immunology and Microbiology, National Defense Medical College , Namiki, Tokorozawa, Saitama , Japan , 2 Department of Surgery 1, National Defense Medical College , Namiki, Tokorozawa, Saitama , Japan
We previously reported that F4/80+ Kupffer cells are subclassified into CD68+ Kupffer cells with phagocytic and ROS producing capacity, and CD11b+ Kupffer cells with cytokine-producing capacity. Carbon tetrachloride (CCl4)-induced hepatic injury is a well-known chemical-induced hepatocyte injury. In the present study, we investigated the immunological role of Kupffer cells/macrophages in CCl4-induced hepatitis in mice. The immunohistochemical analysis of the liver and the flow cytometry of the liver mononuclear cells showed that clodronate liposome (c-lipo) treatment greatly decreased the spindle-shaped F4/80+ or CD68+ cells, while the oval-shaped F4/80+ CD11b+ cells increased. Notably, severe hepatic injury induced by CCl4 was further aggravated by c-lipo-pretreatment. The population of CD11b+ Kupffer cells/macrophages dramatically increased 24 hour (h) after CCl4 administration, especially in c-lipo-pretreated mice. The CD11b+ Kupffer cells expressed intracellular TNF and surface Fas-ligand (FasL). Furthermore, anti-TNF Ab pretreatment (which decreased the FasL expression of CD11b+ Kupffer cells), anti-FasL Ab pretreatment or gld/gld mice attenuated the liver injury induced by CCl4. CD1d2/2 mouse and cell depletion experiments showed that NKT cells and NK cells were not involved in the hepatic injury. The adoptive transfer and cytotoxic assay against primary cultured hepatocytes confirmed the role of CD11b+ Kupffer cells in CCl4-induced hepatitis. Interestingly, the serum MCP-1 level rapidly increased and peaked at six h after c-lipo pretreatment, suggesting that the MCP-1 produced by c-lipo-phagocytized CD68+ Kupffer cells may recruit CD11b+ macrophages from the periphery and bone marrow. The CD11b+ Kupffer cells producing TNF and FasL thus play a pivotal role in CCl4-induced acute hepatic injury.
Funding: This work was supported by a grant from the National Defense Medical College to S. Seki, JSPS KAKENHI Grant Number 25460508, and Takeda Science
Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Carbon tetrachloride (CCl4) is a highly toxic chemical agent
that induces acute hepatic injury, while chronic administration of
CCl4 induces fibrosis, cirrhosis and carcinogenesis. Although
chronic CCl4 injection models have been extensively studied as
liver fibrosis and cirrhosis models , the acute phase of this
hepatitis has been less characterized. The acute phase of CCl4
hepatic injury may be produced by the formation of reactive
oxygen species (ROS) in the endoplasmic reticulum of hepatocytes
by cytochrome p450 enzymes, which may also induce
mitochondrial dysfunction, including changes in calcium homeostasis,
energy production and the beta-oxidation of fatty acids, all of
which can result in hepatocyte damage [4,6,7]. However, although
a role for Kupffer cells has been suggested [2,810], the immune
mechanism involved in the acute phase of CCl4-induced hepatic
injury has not been extensively examined.
It is now generally accepted that the livers of mice and humans
contain various kinds of innate immune cells . It is well
known that liver NK cells and NKT cells potently produce IFN-c
in response to IL-12 and/or LPS . Interestingly, liver B
cells (mostly B-2 cells) produce IL-12 and IFN-c but not IgM, in
response to LPS (vice versa for spleen B cells) . Furthermore,
these IL-12-producing liver B cells, in contrast to spleen B cells,
phagocytose bacteria and kill them [15,16]. Therefore, these liver
immune cells, including B cells and their cytokines, primarily act
as innate immune effectors against infections and tumors by their
T helper-1 immune response in the liver. However, they also
sometimes induce hepatic injury, septic shock and multi-organ
failure [12,13,17]. In addition, we have recently reported that liver
F4/80+ Kupffer cells/macrophages can be subclassified almost
exclusively into two different subsets; a CD68+ subset with
phagocytic, ROS production and bactericidal capacities, and a
CD11b+ subset with cytokine (TNF and IL-12) production and
antitumor capacities [12,13,18,19].
The hepatic injuries induced by a-galactocylceramide
(aGalCer) or bacterial-DNA motifs (CpG-ODN) are
TNF/FasLdependent hepatitis , and concanavalin-A
(Con-A)induced hepatic injury is a TNF/ROS-dependent hepatitis
[12,13,24]. FasL-expressing NKT cells and ROS-producing
CD68+ Kupffer cells, both activated by the TNF produced by
CD11b+ Kupffer cells [17,2024], are the final effectors in these
hepatitis models. CD11b (complement 3b receptor) is present on
the surface of monocytes/macrophages, granulocytes and NK
cells. CD68 (macrosialin) is also used as a marker of macrophages,
including Kupffer cells, and this antigen is also localized in the
cytosol of CD11b+ macrophages, but it is expressed on the cell
surface upon activation [18,25,26].
Gadolinium chloride (GdCl3) and clodronate liposomes (c-lipo),
are both cytotoxic to Kupffer cells, and have been used to deplete
Kupffer cells in rodents. Some reports have suggested that GdCl3
and c-lipo completely eliminate Kupffer cells based on
immunohistochemical examinations. However, we reported and
demonstrated herein that these agents deplete only CD68+ Kuppfer cells
(resident or fixed), but not CD11b+ Kupffer cells, based on the
flow cytometric analysis of liver mononuclear cells [18,19].
Consistent with our data, Holt et al. also demonstrated that
clipo administration did not eliminate the CD11bhighF4/80low
subset, whereas the other CD11blowF4/80high subset was almost
completely depleted . We consider that the former population
corresponds to CD11b+ Kupffer cells and the latter population
corresponds to CD68+ cells in our studies.
In the present study, we demonstrate by immunohistochemistry,
as well as flow cytometry, that the large and spindle-shaped
CD68+ cells were indeed depleted by c-lipo treatment, whereas the
small and round-shaped CD11b+ population increased.
Furthermore, CD11b+ Kupffer cells play an important role in the acute
phase of CCl4-induced hepatitis as a result of their production of
TNF and FasL, which occurs in an NKT cell-independent
manner. In addition, the c-lipo-phagocytized CD68+ Kupffer cells
were found to produce monocyte chemoattractant protein
(MCP)1, and lead to the subsequent accumulation of CD11b+ Kupffer
cells/macrophages into the liver before CCl4 injection, which
aggravates the hepatic injury induced by CCl4 injection.
Materials and Methods
for animals and the intended procedures of the present study
(Permission number: 12039).
Male C57BL/6 mice (ten weeks of age) and gld/gld (gld) mice
with C57BL/6 background were purchased from Japan SLC
(Hamamatsu, Japan). Because B6 CD1d2/2 mice were not
commercially available, CD1d2/2 mice on a BALB/c
background were purchased from the Jackson Laboratory. Carbon
tetrachloride was purchased from Kanto Chemical Co., Inc.
(Tokyo, Japan). C-lipo was purchased from LKT Laboratories,
Inc. (St. Paul, MN 55130, USA).
Induction of Acute Liver Injury
To induce acute liver injury by CCl4, mice were injected
intraperitoneally with a single dose of CCl4 (0.6 mg/kg in oil).
Control groups received the same volume of vehicle (oil)
Isolation of MNCs, Including Kupffer Cells
The murine livers were removed under deep anesthesia. The
liver MNCs were prepared essentially as described previously .
Briefly, the livers were minced and suspended in HBSS containing
0.05% collagenase (Wako, Osaka, Japan), and then were shaken
for 20 min in a 37uC water bath. Next, the liver specimens were
washed in 1% FBS RPMI 1640 and then filtered through a
stainless steel mesh. After mixing in isotonic 33% Percoll solution
containing heparin, the samples were centrifuged for 15 min at
5006g at room temperature. After removing the supernatant, the
pellets were resuspended in a red blood cell lysis solution and then
were washed twice in 10% FBS RPMI 1640.
For pathological examinations, the mice were euthanized prior
to removal of their livers. The liver was then immersed in 10%
formalin for two days. Slides were prepared from these specimens
and stained with hematoxylin and eosin.
Mice and Reagents
The Ethics Committee of Animal Care and Experimentation,
National Defense Medical College, Japan, approved all requests
Flow Cytometric Analysis
After incubation with Fc-blocker (2.4 G2; BD PharMingen, San
Diego, CA), MNCs were stained with a FITC-conjugated anti-F4/
Figure 1. Immunohistochemical staining of liver Kupffer cells/macrophages for F4/80, CD68 and CD11b expression. Livers were
harvested from mice 36 h after pretreatment with c-lipo or PBS, and liver sections were stained with an anti-F4/80 Ab, anti- CD68 Ab or anti- CD11b
Ab. The data are representative of three mice in each group, with similar results obtained for the three animals. 6200 magnification.
80 Ab (eBioscience, San Diego, CA) , Cy5-conjugated
antiCD11b Ab (eBioscience, San Diego, CA)  or biotin-conjugated
anti-CD68 Ab (FA-11, AbD Serotec, Oxford, UK) [25,26,30] with
PE-streptavidin. The flow cytometric analysis was performed using
an FC500 instrument (Beckman Coulter, Miami, FL).
Intracellular Staining for TNF
MNCs were incubated with BD GolgiStop (0.7 mg/ml, BD
PharMingen) for three h before staining. After incubation with
Fcblocker, the cells were stained with a FITC-conjugated anti-F4/80
Ab and Cy5-conjugated anti-CD11b Ab or a biotin-conjugated
anti-CD68 Ab with Cy5-streptavidin. Subsequently, the cells were
incubated with BD Cytofix/Cytoperm solution (BD Pharmingen)
at 4uC for 20 min and then washed with BD Perm/Wash solution
(BD Pharmingen). Thereafter, the cells were stained with a
PEconjugated anti-TNF mAb (eBioscience) or isotype rat IgG1 Ab
(eBioscience) at 4uC for 20 min and then analyzed using the
Pretreatment with c-lipo
Clodronate (LKT Laboratories, Inc., St. Paul, MN) was
encapsulated into liposomes and 100 mL of a 25 mg/ml
suspension was intraperitoneally injected into the mice to deplete the
CD68+ Kupffer cells 36 h before experiments .
Neutralization of TNF or FasL, or Depletion of NK or NK/
To neutralize TNF, FasL, anti-TNF Ab (0.5
mg/mouse)(MP6TX3,BD PharMingen) or anti-FasL Ab (0.5 mg/mouse)(MFL4,
BD PharMingen) was injected intravenously one hour before and
six h after CCl4 administration. To deplete NK cells or both NK
and NKT cells, an anti-asialo GM1 (AGM1) Ab (50 mg/mouse) or
anti-NK1.1 Ab (PK136; 200 mg/mouse) was injected
intravenously into the mice three days before CCl4 administration [17,21,23].
For the neutralization of FasL in in vitro killing assay, 10 mg/ml
anti-FasL Ab (MFL4, BD PharMingen) were added to the
Measurement of the Alanine Amino Transferase,
Cytokine and MCP-1 Levels
The serum alanine amino transferase (ALT) level was measured
using a DRICHEM 3000V instrument (Fuji Medical Systems,
Tokyo). ELISA kits for TNF (BD Biosciences, San Jose, CA, USA)
and MCP-1 (R&D system, San Jose, CA, USA) were used to
analyze the levels of these cytokines.
Isolation of F4/80+ CD11b+ Kupffer Cells from CCl4
Treated Mice using MACS Sort System
Livers were obtained from mice 14 h after the injection of CCl4,
and minced liver specimens without collagenase treatment in 1%
FBS RPMI 1640 were filtered through a stainless steel mesh.
Thereafter, the liver MNCs were obtained using a 33% Percoll
solution. The MNCs were stained with PE-Cy5 labeled anti-F4/80
antibody following conjugation with anti-PE magnetic beads
(Miltenyi Biotec GmbH). Beads conjugated F4/80+ cells (which
were also positive for PE-Cy5) or F4/802 cells were magnetically
sorted by Super MACS system (Miltenyi Biotec GmbH).
Figure 2. The flow cytometric analysis of Kupffer cells/macrophages. Livers were harvested from mice 36 h after pretreatment with c-lipo or
PBS, and liver MNCs obtained after collagenase digestion of livers were stained for F4/80, CD11b and CD68. The results of a forward scatter (FS)/side
scatter (SS) analysis of the total MNCs are shown (left panels). The mean FS values are also shown (40162.9 vs. 43666.8, n = 5, p,005) (left panels).
The F4/80/CD11b expression is also presented and the F4/80 positive populations are inside of square gate (middle panels). The CD11b/CD68
expression levels of the gated F4/80-positive cells are demonstrated (right panels). F4/80+ CD11b+ cells are shown by red dots and the F4/80+ CD68+
cells are shown by blue dots. **p,0.01, *p,0.05 vs. other groups. The data are representative of five mice in each group, with similar results for the
Figure 3. C-lipo pretreatment aggravates CCl4-induced hepatic injury. Mice pretreated with c-lipo or PBS were injected intraperitoneally
with CCl4 or oil. (A) The serum ALT levels were evaluated at the indicated times after CCl4 stimulation. (B) The influence of CCl4 challenge on the
serum TNF levels. The data are the means 6 SE from 10 mice in each group. **p,0.01 vs. other groups.
Adoptive Transfer Experiments
MACS sorted 56106 F4/80+ cells (PE-Cy5+) or 56106 F4/802
cells were adoptively transferred into normal mice or CD68+
Kupffer cell-depleted (by c-lipo) mice. As an experimental control,
liver 56106 F4/80+ cells from oil-treated mice were transferred
into normal mice. After adoptive transfer, recruitment of
transferred F4/80+ cells into recipient liver was confirmed by
the presence of PE-Cy5 positive cells. The induction of liver injury
after transfer was analysed and compared within each group.
In vitro Cytotoxic Assay Against Primary Cultured
Primary cultured hepatocytes were used as target cells.
Hepatocytes were obtained from 8 week of age B6 mice essentially
described previously . In brief, liver were perfused with
collagenase from portal vein, and dispersed hepatocytes were
suspended in hepatocyte growth medium (HCGM) and seeded
into collagen type I coated 96 well plate (Iwaki, Funabashi, Japan)
with 2.06103 cells/well concentration. After 12 h of incubation,
hepatocytes adhered to the bottom of the plate and medium was
changed by HCGM containing 10 mCi of Na251Cr04/ml and
incubated additional 12 h. The 51Cr labeled hepatocytes were
washed three times with HCGM, and effector cells were added
following 4 h of incubation. F4/80+ cells (which were also positive
for PE-Cy5) or F4/802 cells were obtained by MACS system as
described above. The concentration of effector MNCs were
adjusted to 5.06105 cells/well (250:1) and 2.56105 cells/well
125:1). Culture supernatants were harvested and radio activities
were measured by gamma counter.
The results are expressed as the mean values 6 SE. The
statistical analyses were performed using a GraphPad Prism 5
software package (GraphPad Software, La Jolla, CA). Statistical
evaluations were compared using the standard one-way analysis of
variance followed by the Bonferroni post-hoc test. A value of P,
0.05 was considered to be significant.
Depletion of CD68+ Kupffer Cells and Aggravation of
CCl4- Induced Hepatic Injury by c-lipo Pretreatment
We previously reported that c-lipo or GdCl3 selectively depleted
only CD68+ Kupffer cells, but increased the population of
CD11b+ Kupffer cells/macrophages, as determined by flow
cytometry . Indeed, 36 h after c-lipo treatment, an
immunohistochemical analysis showed that the spindle-shaped CD68+ cells
and F4/80+ cells were greatly decreased in the liver, while the
oval-shaped CD68+ or F4/80+ cells still remained, and the
population of CD11b+ cells appeared to increase (Fig. 1). A flow
cytometric analysis also confirmed that c-lipo treatment
proportionally decreased the liver CD68+ Kupffer cells (41% to 12.8%,
Fig. 2 right panels) but that the liver CD11b+ Kupffer cells/
macrophages increased (37% to 75.7%, Fig. 2 right panels). Of
note, the number of spleen, bone marrow and peripheral blood
CD11b+ monocytes/macrophages did not decrease . A
forward scatter (FS) and side scatter (SS) analysis revealed that
the CD68+ Kupffer cells are relatively large and show a complex
structure, and most of them disappeared following c-lipo treatment
(blue dots, Fig. 2, left panels). In contrast, the CD11b+ Kupffer
cells/macrophages are small and have a simple structure (red dots,
Fig. 2, left panels). The CD11b+ Kupffer cells/macrophages
became larger after c-lipo treatment, as indicated by the FS
analysis (Fig. 2, left panels, FS values; 43666.8 vs 40162.9, n = 5,
p,0.05), suggesting that they were activated after c-lipo treatment.
However, the total number of liver MNCs yielded from the liver
did not significantly change following the c-lipo treatment
(approximately 76106/liver). Thus, the number of CD68+ Kupffer
cells decreased, while the number of CD11b+ Kupffer cells/
macrophages increased, in the liver.
CCl4 injection induced severe hepatic injury, as indicated by the
ALT levels, and the hepatic injury was aggravated in c-lipo
pretreated mice (Fig. 3A). Consistently, the TNF levels in c-lipo
pretreated mice after CCl4 injection were higher than those of
control mice (Fig. 3B).
Figure 4. The effect of c-lipo pretreatment on the population of F4/80+ CD11b+ Kupffer cells after CCl4 injection. The changes in the
population of F4/80-positive Kupffer cells after CCl4 challenge with c-lipo or PBS pretreatment were examined. Mice were intraperitoneally injected
with CCl4 36 h after c-lipo or PBS treatment. As an experimental control, the vehicle oil was intraperitoneally injected. The changes in the total
amount of liver MNCs, the population of F4/80+ cells, and the proportion of each Kupffer cell subset following CCl4 challenge after c-lipo or PBS
pretreatment are shown. The F4/80+ CD11b+ cells are shown by red dots and the F4/80+ CD68+ cells are shown by blue dots. The data are
representative of five mice in each group, with similar results. **p,0.01, *p,0.05 vs. other groups.
The Increase in the Number of F4/80+ CD11b+ Kupffer
Cells After CCl4 Injection in Mice with or without c-lipo
Pretreatment, and the Liver Histopathology
Twenty-four h after CCl4 injection, the population of F4/80+
CD11b+ Kupffer cells was greatly increased compared to that in
control oil-injected mice, and c-lipo-pretreatment further
increased the number of F4/80+ Kupffer cells after CCl4 injection
(Fig. 4). However, the number of CD68+ Kupffer cells was
reduced, especially in c-lipo-pretreated mice, after CCl4 injection.
In addition, the livers of c-lipo-pretreated mice showed more and
larger necrotic areas after CCl4-injection than did the
PBSpretreated control mice (Fig. 5).
The Role of TNF, FasL, NKT Cells and NK Cells in
CCl4induced Hepatic Injury
Since we previously reported that TNF and/or FasL are
involved in the pathogenesis of some experimental models of
hepatitis [17,21,23], we examined the functions of TNF and FasL
in CCl4-induced hepatitis. Pretreatment of mice with a
neutralizing anti-TNF Ab or anti-FasL Ab significantly decreased the
serum ALT levels after CCl4 administration (Fig. 6A). However,
pretreatment with a neutralizing anti-IFN-c Ab did not affect the
serum ALT levels after CCl4 administration (Fig. 6A).
Additionally, liver injury was significantly ameliorated in FasL deficient gld
mice (Fig. 6B). These results suggest that TNF and FasL, but not
IFN-c, are profoundly involved in the CCl4-induced acute hepatic
Since we previously reported that NKT cells are responsible for
the hepatic injury induced by a-GalCer or bacterial DNA motifs
[17,21,23], we next examined the effects of CCl4 on the
CD1d2/2 mice. Since CD1d2/2 mice on a B6 background
were not commercially available, we used CD1d2/2 mice on a
BALB/c background. The results showed that the serum ALT
levels were not significantly different between CD1d2/2 mice
and control mice (Fig. 6C). Next, we examined the effect of CCl4
Figure 5. The effect of c-lipo pretreatment on the liver histopathology after CCl4 injection. The pathological findings of c-lipo- or
PBSpretreated mice 48 h after CCl4 challenge. The liver is shown (6100 magnification; H&E staining). (A) The PBS pretreated mice and (B) c-lipo
pretreated mice. The data are representative of three mice in each group, with similar results observed for all animals.
on mice depleted of NK cells by an anti-AGM1 Ab, or depleted of
NKT cells as well as NK cells (by an anti-NK1.1 Ab), and found
that the serum ALT levels were similar to those in control mice
(Fig. 6D). These results suggest that NK/NKT cells are not
involved in the CCl4-induced acute hepatic injury.
Intracellular TNF Production and FasL Expression of
Kupffer Cells, and the Effects of an anti-TNF Ab
We next examined the intracellular TNF production and
surface FasL expression of liver MNCs 12 h after CCl4 injection.
The F4/80+ CD11b+ Kupffer cells, but not other cells, including
F4/80+ CD68+ Kupffer cells, produced TNF and expressed FasL
(Figs. 7A, B). The staining of F4/80+ CD11b+ Kupffer cells for
TNF was lower at six and 24 h after CCl4 injection compared to
that at 12 h after CCl4 injection (not shown). We further examined
the relationship between the TNF and FasL expression of F4/80+
CD11b+ Kupffer cells. Pretreatment with a neutralizing TNF Ab
dramatically decreased the FasL expression of the F4/80+ CD11b+
Kupffer cells (Fig. 7C). These results suggest that the TNF
produced by F4/80+ CD11b+ Kupffer cells induces their FasL
expression, and that F4/80+ CD11b+ Kupffer cells play a crucial
role in CCl4-induced acute hepatic injury via TNF/FasL.
Induction of Hepatic Injury in Mice by the Adoptive
Transfer of CD11b+ Kupffer Cells from mice Injected with
We previously reported that the hepatic MNCs obtained
without collagenase digestion contain many CD11b+ Kupffer cells
but few CD68+ Kupffer cells . Consistently, the MACS-sorted
liver F4/80+ cells without collagenase digestion from
CCl4injected mice were primarily F4/80+ CD11b+ Kupffer cells
(85%, Fig. 8A). A major proportion of the F4/80- CD11b high
cells were Gr1-positive neutrophils (62%, Fig. 8A and not shown)
and the CD11b- F4/802 cells were lymphocytes (30%, Fig. 8A
and not shown). Moreover, MACS sorted F4/80+ CD11b+
Kupffer cells were adoptively transferred into normal mice or
CD68+ cell depleted mice, and serum ALTs were examined. The
hepatic injury induced by transferred F4/80+ CD11b+ Kupffer
cells was stronger than that induced by transferred F4/802 cells
(Fig. 8B). Moreover, when F4/80+ CD11b+ Kupffer cells from
CCl4-injected mice were transferred into mice depleted of CD68+
Kupffer cells (36 h after c-lipo injection), a more severe hepatic
injury was evoked than that in mice without c-lipo pretreatment
(Fig. 8B). These results raise the possibility that resident CD68+
Kupffer cells may normally inhibit the function of CD11b+
Kupffer cells/macrophages. However, it should be noted that the
transfer of liver F4/802 cells also induced a substantial hepatic
injury in clodronate-pretreated mice (Fig. 8B), suggesting that the
activated neutrophils contained in the F4/802 cells may also have
hepatotoxicity under in vivo condition. Flow cytometric analysis of
recipient liver MNCs at 1.5 h after the adoptive transfer without
additional staining showed that 4.5% were positive for PE-Cy5 in
mice transferred with F4/80+ cells (Fig. S1, left panel) and few
(0.6%) were postive in mice transferred with F4/802 cells
(nonspecific staining) (Fig. S1, middle panel). FS/SS analysis verified
that such positive cells (in left panel) were confirmed to be
macrophages (Fig. S1, right panel). Furthermore, MACS sorted
F4/80+ CD11b+ Kupffer cells from CCl4-injected mice showed
cytotoxicity against primary cultured hepatocytes in vitro, and this
cytotoxicity was effectively blocked by neutralization of FasL,
whereas F4/802 cells did not showed the killing activity in vitro
C-lipo Treatment of Mice Before CCl4 Administration
Increases the Serum Level of MCP-1
To elucidate the mechanism by which CD11b Kupffer cells are
increased by c-lipo pretreatment, the serum MCP-1 levels were
monitored after c-lipo injection. MCP-1 is a major chemokine,
and is a ligand for CC-chemokine receptor 2 (CCR2). Intriguingly,
the serum MCP-1 levels rapidly increased and peaked at six h after
c-lipo injection after c-lipo treatment (Fig. 9A). Furthermore, the
MCP-1 levels did not increase any more in the c-lipo-pretreated
mice after CCl4 injection (Fig. 9B). These results suggest that the
CD68 Kupffer cells are activated after phagocytosing c-lipo, and
that they produced MCP-1 and thereafter underwent apoptosis
due to the cytotoxicity of clodronate. Our results also suggest that
MCP-1 plays a critical role in the recruitment and activation of
CD11b Kupffer cells from the periphery or bone marrow.
To confirm the presence of MCP-1-producing CD68 cells, liver
MNCs were harvested from mice one hour after CCl4
administration, and CD68+ cells were purified by the MACS system from
liver MNCs obtained from liver specimens with collagenase
treatment, and F4/80+ CD11b+ cells (F4/80+ cells from mice
depleted of CD68 cells by c-lipo) were also obtained by the MACS
Figure 6. The role of TNF, FasL, NKT cells and NK cells in CCl4-induced hepatic injury. (A) The serum ALT levels after CCl4 administration in
mice pretreated with an anti-TNF Ab, IFN-c or anti-FasL Ab, (B) in FasL deficient gld mice, (C) in NKT cell-deficient CD1d2/2 mice, and (D) in mice
pretreated with an anti-AGM1 Ab or anti-NK1.1 Ab at the indicated time points. The data are the means 6 SE from 10 mice in each group. **p,0.01,
*p,0.05 vs. other groups.
system. Both subsets were cultured in vitro for the indicated amount
of time. The results showed that CD68 cells produced a substantial
amount of MCP-1 beyond 12 h after culture, but CD11b cells did
not produce any MCP-1 (Fig. 9C). We also found that, in mice
depleted of CD68 Kupffer cells injected with viable Staphylococcus
aureus, the serum MCP-1 levels (peak at 3 h) were greatly reduced
compared to those in control mice, and that CD68 Kupffer
celldepleted liver MNCs cultured with bacteria in vitro did not
produce any MCP-1 . However, it was considered possible
that the MCP-1 produced by CD68 Kupffer cells at 24 h after
CCl4 administration may not be involved in the recruitment of
CD11b macrophages/Kupffer cells or hepatic injury.
In the current study, we explored a unique immunological
mechanism of CCl4-induced acute hepatic injury in mice. Namely,
CD11b+ Kupffer cells produce TNF, as well as FasL, and induce
hepatic injury, in which IFN-c, NK cells and NKT cells are not
involved. Furthermore, c-lipo pretreatment to deplete CD68+
Kupffer cells promoted MCP-1 production from the CD68+
Kupffer cells, presumably before they underwent apoptosis, which
led to the accumulation and activation of CD11b+ Kupffer cells,
and markedly aggravated the hepatic injury following CCl4
injection. Both CpG-ODN and a-GalCer-mediated hepatitis are
TNF/FasL/Fas pathway-dependent, and the final effectors in
Figure 7. The intracellular TNF production and FasL expression of Kupffer cells. (A) The expression of intra-cellular TNF and (B) FasL
expression in the F4/80+ CD11b+ cells or F4/80+ CD68+ cells was examined 12 h after the CCl4 injection. (C) The effect of pretreatment with a
neutralizing TNF Ab on the FasL expression of F4/80+ CD11b+ cells. The data are representative of five mice in each experiment, with similar results
obtained for each mouse. **p,0.01 vs. other groups.
these types of hepatitis are FasL-expressing NKT cells activated by
the TNF produced by CD11b+ Kupffer cells/macrophages
[17,21,23,34]. However, the final immune effectors in
CCl4induced hepatitis are CD11b+ Kupffer cells/macrophages, which
themselves have FasL expression.
We recently demonstrated that CD68+ Kupffer cells are fixed
Kupffer cells and cannot be harvested unless collagenase treatment
of liver tissues is carried out, whereas CD11b+ Kupffer cells are
easily obtained without collagenase treatment from liver specimens
. In addition, although CD68+ Kupffer cells are mainly
located in the mid-zonal region between the portal vein and the
central vein, CD11b+ Kupffer cells are equally distributed in the
liver tissues . Therefore, it was suggested that CD68+ cells are
resident Kupffer cells, and that CD11b+ cells may be recruited
from the periphery or bone marrow to the inflamed liver [18,19].
Furthermore, the functions of these cell subsets are quite different:
CD68+ Kupffer cells have phagocytic, ROS-producing and
bactericidal activities, while CD11b+ Kupffer cells have cytokine
(IL-12 and TNF)-producing capacity and are involved in
antitumor immunity [18,19]. Our present results also confirmed
by immunohistochemistry, as well as flow cytometry, that c-lipo
depletes CD68+ Kupffer cells, but increases the number of
CD11b+ Kupffer cells. Furthermore, resident CD68+ Kupffer
cells are radio-resistant, whereas CD11b+ Kupffer
cells/macrophages are radio-sensitive [19,27] which were reconstituted by the
transfer of bone marrow cells . Therefore, they are distinct
types of macrophages, and most of the monocytes/macrophages in
the spleen and peripheral blood are CD11b+ cells, while CD68+
Kupffer cells predominate in the liver. Although intracellular
CD68 was present in the cytosol of CD11b+ Kupffer cells and may
be expressed on the cell surface of the cells upon activation ,
the intra-cellular CD68 expression was still much lower than that
Figure 9. The serum MCP-1 levels after c-lipo treatment and the MCP-1 production from CD68+ Kupffer cells after CCl4 injection
with/without c-lipo pretreatment. (A) There was an increase in the serum MCP-1 levels in mice early after c-lipo injection. The mice were i.p.
injected with c-lipo or PBS, the sera were obtained at the indicated time points, and the MCP-1 levels were measured (n = 3 in each group). (B) The
serum MCP-1 levels did not increase after CCl4 injection in mice pretreated with c-lipo. The mice pretreated with c-lipo or PBS were injected
intraperitoneally with CCl4 or oil, and sera were obtained at the indicated time points to measure the MCP-1 levels (n = 3 in each group).(C) The ex
vivo production of MCP-1 from the liver CD68+ Kupffer cells from CCl4-injected mice. One hour after the injection of CCl4, the liver MNCs were
harvested from the liver by collagenase treatment, and CD68+ cells were obtained by magnetic beads (MACS system). F4/80+ CD11b+ cells obtained
by F4/80 magnetic beads from the liver MNCs from c-lipo treated mice. Both purified subsets were cultured in vitro for the indicated amounts of time.
The data are the means 6 SE from three independent experiments. **p,0.01, *p,0.05 vs. other groups.
of CD68+ Kupffer cells, as revealed by flow cytometry (our
In contrast to the hepatic injury induced by either a-GalCer or
CpG-ODN, CD68+ Kupffer cells and their ROS production
induced by the TNF produced by CD11b Kupffer cells/
macrophages are the final effectors in Con-A-induced hepatitis
[12,13,24]. In all of these types of hepatitis, the TNF was produced
by CD11b+ Kupffer cells in the early period (at 1 h) after the
injection of reagents [17,2023]. However, the serum TNF levels
did not start to increase until 12 h after CCl4 injection, and the
intracellular production of TNF in CD11b+ Kupffer cells reached
a maximum at 12 h after CCl4 injection, suggesting that TNF may
be released from CD11b+ Kupffer cells into the systemic
circulation beyond 12 h after CCl4 injection. These results imply
that, although CD11b+ Kupffer cells are effectors involved in the
hepatic injury induced by CCl4, CD11b+ Kupffer cells may
produce TNF and FasL in order to reject the hepatocytes
chemically-damaged by CCl4 to ensure the early termination of
hepatic injury. Karlmark et al. also pointed out the marked
infiltration of F4/80+ CD11b+ Gr1+ macrophages in the acute
phase of CCl4 hepatic injury, and demonstrated the indispensable
role of CCR2 (MCP-1 ligand) for their recruitment to the liver .
It is plausible that the CD11b+ Kupffer cells in our study may be
identical to the population reported in their study.
It was previously reported that CCR2-deficient mice showed a
dramatic reduction in macrophage accumulation in both the
peritoneal cavity and liver upon exposure to inflammatory stimuli
[2,35,36]. MCP-1 is a major ligand of CCR2 , and is thus an
important chemokine that recruits monocytes/macrophages to
inflamed organs and tissues. CD68 Kupffer cells may be activated
by phagocytosing c-lipo, and may produce MCP-1 before
undergoing apoptosis. Although CD68 Kupffer cell-depleted mice
did not show an increase in MCP-1 after CCl4 injection, the
hepatic injury, as well as the CD11b+ cell recruitment, was
enhanced 24 h after CCl4 injection. These results suggest that,
although increased MCP-1 in the early phase (6 h after
c-lipopretreatment) may be required to increase and prime CD11b
Kupffer cells/macrophages in the liver before CCl4
administration, other cytokines, including TNF, may be critical for further
recruitment of CD11b+ cells into the liver and for the resultant
hepatic injury after CCl4 injection.
The time course of the serum MCP-1 levels after the injection of
c-lipo or bacteria  suggests an important point. When CD11b
Kupffer cells are activated and produce TNF in vivo due to
exposure to LPS, CpG-ODN and a-GalCer or whole bacteria, the
serum TNF levels usually peak at 1 h after administration
[17,22,23,37], which is much earlier than the peaks of serum
MCP-1 after bacteria or c-lipo injection (3 h or 6 h, respectively).
This may be because the production of MCP-1 from CD68
Kupffer cells requires the digestion of c-lipo or bacteria
(phagolysosomal formation) , whereas CD11b Kupffer cells
may rapidly respond to these ligands via TLR-4, 9, or CD1d.
However, further investigations of both CD68+ Kupffer cells
and CD11b+ Kupffer cells/macrophages and their mutual
interactions, as well as their interactions with other liver
leukocytes, are required for understanding the chronic
inflammation and fibrosis induced by CCl4.
Figure S1 The recruitment of adoptive transferred F4/
80+ cells into liver. Liver MNCs were isolated from CCl4
treated mice and stained with PE-Cy5 labeled anti-F4/80
antibody. F4/80+ and F4/802 cells were separated using
antiPE magnetic beads and MACS sorting device. Sorted F4/80+ cells
labeled with PE-Cy5, and F4/802 cells without labelling, were
adoptively transferred into normal mice. Then, liver MNCs
obtained from each recipient mouse at 1.5 h after adoptive
transfer were analyzed by flow cytometry without additional
staining. PE-Cy5 (F4/80) positive cells were counted and depicted
in red area and dots. Data are representative of three mice in each
group, with similar results.
We thank A. Kotani for technical assistance and help.
Conceived and designed the experiments: SS. Performed the experiments:
AS HN. Analyzed the data: HN MN MI. Contributed reagents/materials/
analysis tools: MK KN. Wrote the paper: AS HN SS.
1. Galligani L , Lonati-Galligani M , Fuller GC ( 1979 ) Collagen synthesis in explant cultures of normal and CCl4-treated mouse liver . Toxicol Appl Pharmacol 48 : 131 - 137 .
2. Karlmark KR , Weiskirchen R , Zimmermann HW , Gassler N , Ginhoux F , et al. ( 2009 ) Hepatic recruitment of the inflammatory Gr1+ monocyte subset upon liver injury promotes hepatic fibrosis . Hepatology 50 : 261 - 274 .
3. Perez Tamayo R ( 1983 ) Is cirrhosis of the liver experimentally produced by CCl4 and adequate model of human cirrhosis? Hepatology 3 : 112 - 120 .
4. Recknagel RO , Glende EAJ , Dolak JA , Waller RL ( 1989 ) Mechanisms of carbon tetrachloride toxicity . Pharmacol Ther 43 : 139 - 154 .
5. Unakar NJ ( 1966 ) p-Hydroxypropiophenone vs CCL4-induced injury . Arch Pathol 82 : 170 - 177 .
6. Brattin WJ , Glende EAJ , Recknagel RO ( 1985 ) Pathological mechanisms in carbon tetrachloride hepatotoxicity . J Free Radic Biol Med 1 : 27 - 38 .
7. Clawson GA ( 1989 ) Mechanisms of carbon tetrachloride hepatotoxicity . Pathol Immunopathol Res 8 : 104 - 112 .
8. Louis H , Van Laethem JL , Wu W , Quertinmont E , Degraef C , et al. ( 1998 ) Interleukin-10 controls neutrophilic infiltration, hepatocyte proliferation, and liver fibrosis induced by carbon tetrachloride in mice . Hepatology 28 : 1607 - 1615 .
9. Liu C , Tao Q , Sun M , Wu JZ , Yang W , et al. Kupffer cells are associated with apoptosis, inflammation and fibrotic effects in hepatic fibrosis in rats . Lab Invest 90 : 1805 - 1816 .
10. Yang Y , Harvey SA , Gandhi CR ( 2003 ) Kupffer cells are a major source of increased platelet activating factor in the CCl4-induced cirrhotic rat liver . J Hepatol 39 : 200 - 207 .
11. Seki S , Habu Y , Kawamura T , Takeda K , Dobashi H , et al. ( 2000 ) The liver as a crucial organ in the first line of host defense: the roles of Kupffer cells , natural killer (NK) cells and NK1 .1 Ag + T cells in T helper 1 immune responses . Immunol Rev 174 : 35 - 46 .
12. Seki S , Nakashima H , Kinoshita M ( 2012 ) The Liver as a Pivotal Innate Immune Organ . Immuno-Gastroenterology 1: 76 - 89 .
13. Seki S , Nakashima H , Nakashima M , Kinoshita M ( 2011 ) Antitumor immunity produced by the liver Kupffer cells , NK cells, NKT cells, and CD8 CD122 T cells . Clin Dev Immunol 2011 : 868345 .
14. Matsumoto A , Kinoshita M , Ono S , Tsujimoto H , Majima T , et al. ( 2006 ) Cooperative IFN-gamma production of mouse liver B cells and natural killer cells stimulated with lipopolysaccharide . J Hepatol 45 : 290 - 298 .
15. Nakashima M , Kinoshita M , Nakashima H , Habu Y , Miyazaki H , et al. ( 2012 ) Pivotal advance: characterization of mouse liver phagocytic B cells in innate immunity . J Leukoc Biol 91 : 537 - 546 .
16. Cancro MP ( 2012 ) Editorial: phagocytic B cells: deja vu all over again ? J Leukoc Biol 91 : 519 - 521 .
17. Nakashima H , Ogawa Y , Shono S , Kinoshita M , Nakashima M , et al. ( 2013 ) Activation of CD11b+ Kupffer cells/macrophages as a common cause for exacerbation of TNF/Fas-ligand-dependent hepatitis in hypercholesterolemic mice . PLOS One 8 : e49339 .
18. Kinoshita M , Uchida T , Sato A , Nakashima M , Nakashima H , et al. ( 2010 ) Characterization of two F4/80-positive Kupffer cell subsets by their function and phenotype in mice . J Hepatol 53 : 903 - 910 .
19. Ikarashi M , Nakashima H , Kinoshita M , Sato A , Nakashima M , et al. ( 2013 ) Distinct development and functions of resident and recruited liver Kupffer cells/ macrophages . J Leukoc Biol 94 : 1325 - 1336 .
20. Nakagawa R , Nagafune I , Tazunoki Y , Ehara H , Tomura H , et al. ( 2001 ) Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by alpha-galactosylceramide in mice . J Immunol 166 : 6578 - 6584 .
21. Inui T , Nakagawa R , Ohkura S , Habu Y , Koike Y , et al. ( 2002 ) Age-associated augmentation of the synthetic ligand- mediated function of mouse NK1.1 ag(+) T cells: their cytokine production and hepatotoxicity in vivo and in vitro . J Immunol 169 : 6127 - 6132 .
22. Inui T , Nakashima H , Habu Y , Nakagawa R , Fukasawa M , et al. ( 2005 ) Neutralization of tumor necrosis factor abrogates hepatic failure induced by alpha-galactosylceramide without attenuating its antitumor effect in aged mice . J Hepatol 43 : 670 - 678 .
23. Kawabata T , Kinoshita M , Inatsu A , Habu Y , Nakashima H , et al. ( 2008 ) Functional alterations of liver innate immunity of mice with aging in response to CpG-oligodeoxynucleotide . Hepatology 48 : 1586 - 1597 .
24. Nakashima H , Kinoshita M , Nakashima M , Habu Y , Shono S , et al. ( 2008 ) Superoxide produced by Kupffer cells is an essential effector in concanavalin Ainduced hepatitis in mice . Hepatology 48 : 1979 - 1988 .
25. Rabinowitz SS , Gordon S ( 1991 ) Macrosialin, a macrophage-restricted membrane sialoprotein differentially glycosylated in response to inflammatory stimuli . J Exp Med 174 : 827 - 836 .
26. Ramprasad MP , Terpstra V , Kondratenko N , Quehenberger O , Steinberg D ( 1996 ) Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein . Proc Natl Acad Sci U S A 93 : 14833 - 14838 .
27. Holt MP , Cheng L , Ju C ( 2008 ) Identification and characterization of infiltrating macrophages in acetaminophen-induced liver injury . J Leukoc Biol 84 : 1410 - 1421 .
28. Austyn JM , Gordon S ( 1981 ) F4/80, a monoclonal antibody directed specifically against the mouse macrophage . Eur J Immunol 11 : 805 - 815 .
29. Sanchez-Madrid F , Simon P , Thompson S , Springer TA ( 1983 ) Mapping of antigenic and functional epitopes on the alpha- and beta-subunits of two related mouse glycoproteins involved in cell interactions , LFA-1 and Mac-1. J Exp Med 158 : 586 - 602 .
30. Smith MJ , Koch GL ( 1987 ) Differential expression of murine macrophage surface glycoprotein antigens in intracellular membranes . J Cell Sci 87 (Pt 1): 113 - 119 .
31. Yamamoto T , Naito M , Moriyama H , Umezu H , Matsuo H , et al. ( 1996 ) Repopulation of murine Kupffer cells after intravenous administration of liposome-encapsulated dichloromethylene diphosphonate . Am J Pathol 149 : 1271 - 1286 .
32. Naito M , Nagai H , Kawano S , Umezu H , Zhu H , et al. ( 1996 ) Liposomeencapsulated dichloromethylene diphosphonate induces macrophage apoptosis in vivo and in vitro . J Leukoc Biol 60 : 337 - 344 .
33. Van Rooijen N , Kors N , vd Ende M , Dijkstra CD ( 1990 ) Depletion and repopulation of macrophages in spleen and liver of rat after intravenous treatment with liposome-encapsulated dichloromethylene diphosphonate . Cell Tissue Res 260 : 215 - 222 .
34. Liu ZX , Govindarajan S , Kaplowitz N ( 2004 ) Innate immune system plays a critical role in determining the progression and severity of acetaminophen hepatotoxicity . Gastroenterology 127 : 1760 - 1774 .
35. Dambach DM , Watson LM , Gray KR , Durham SK , Laskin DL ( 2002 ) Role of CCR2 in macrophage migration into the liver during acetaminophen-induced hepatotoxicity in the mouse . Hepatology 35 : 1093 - 1103 .
36. Kuziel WA , Morgan SJ , Dawson TC , Griffin S , Smithies O , et al. ( 1997 ) Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2 . Proc Natl Acad Sci U S A 94 : 12053 - 12058 .
37. Shono S , Habu Y , Nakashima M , Sato A , Nakashima H , et al. ( 2011 ) The immunologic outcome of enhanced function of mouse liver lymphocytes and Kupffer cells by high-fat and high-cholesterol diet . Shock 36 : 484 - 493 .