1 Department of Medicine and Pathology, University of Southern California School of Medicine, Los Angeles 90033; 2 Research Service, Department of Veteran Affairs Medical Center, Sepulveda 91343; 4 Department of Pathology, Harbor-UCLA Medical Center, Torrance, California 90073; 3 Department of Chemistry, University of Minnesota, Duluth, Minnesota 55812; and 5 Departments of Pediatrics and Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032
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ABSTRACT |
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NF-B activation induced
by lipopolysaccharide (LPS) in cultured hepatic macrophages (HM) may be
abrogated by pretreatment of cells with a lipophilic iron chelator,
1,2-dimethyl-3-hydroxypyrid-4-one (L1, deferiprone),
suggesting a role for iron in this molecular event [M. Lin, M.,
R. A. Rippe, O. Niemelä, G. Brittenham, and H. Tsukamoto,
Am. J. Physiol. 272 (Gastrointest. Liver
Physiol. 35): G1355-G1364, 1997]. To ascertain the
relevance in vivo of this hypothesis, HM from an experimental model of
alcoholic liver injury were examined for the relationship between
nuclear factor (NF)-
B activation and iron storage. HM showed a
significant increase in nonheme iron concentration (+70%), accompanied
by enhanced generation of electron paramagnetic resonance-detected
radicals (+200%), NF-
B activation (+100%), and tumor necrosis
factor-
(+150%) and macrophage inflammatory protein-1 (+280%) mRNA
induction. Treatment of the cells ex vivo with L1 normalized all these
parameters. HM content of ferritin protein, ferritin L chain mRNA, and
hemeoxygenase-1 mRNA and splenic content of nonheme iron were
increased, suggesting enhanced heme turnover as a cause of the
increased iron storage and NF-
B activation. To test this
possibility, increased iron content in HM was reproduced in
vitro by phagocytosis of heat-treated red blood cells. Treatment caused
a 40% increase in nonheme iron concentration and accentuated
LPS-induced NF-
B activation twofold. Both effects could be abolished
by pretreatment of cells with zinc protoporphyrin, a hemeoxygenase
inhibitor. To extend this observation, animals were splenectomized
before 9-wk alcohol feeding. Splenectomy resulted in further increments
in HM nonheme iron storage (+60%) and NF-
B activation (+90%) and
mononuclear cell infiltration (+450%), particularly around the
iron-loaded HM in alcohol-fed animals. These results support the
pivotal role of heme-derived iron in priming HM for NF-
B activation
and expression of proinflammatory genes in alcoholic liver injury.
Kupffer cells; nuclear factor-B; chemokines; inflammation; erythrophagocytosis
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INTRODUCTION |
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A GROWING BODY of evidence exists for the effector role
of hepatic macrophages (HM) in liver injury (see Ref. 37 for review). This role appears to be facilitated in part by the release of cytotoxic, proinflammatory, or fibrogenic mediators such as tumor necrosis factor- (TNF-
), interleukin (IL)-6, IL-1, and
transforming growth factor-
(9, 19, 24, 26). Inactivation of HM by treatment with gadolinium chloride (GdCl3) ameliorates
liver injury induced by various hepatoxic agents or conditions,
including alcohol intake (see Ref. 37 for review). TNF-
is believed
to be a key mediator, mediating hepatocellular injury through its
potential direct stimulation of oxidant stress in mitochondria (13) or initiation of inflammation via upregulation of chemokines and adhesion
molecules (41). In fact, administration of antibodies against TNF-
or against soluble TNF-
receptor protects against liver injury in a
manner similar to that of GdCl3 (8). Expression of TNF-
and many other proinflammatory mediators is regulated by a
transcription factor, nuclear factor (NF)-
B (1, 6). For NF-
B to
be translocated to the nucleus to bind to its target genes, the latent
form has to be activated by the removal of the inhibitory protein
(I
B) via phosphorylation and proteolysis of this protein (1).
Oxidative stress is believed to be one of the critical events leading
to the activation signaling of NF-
B (33).
Iron is essential to almost all known cell types (20). Iron not only
participates in numerous vital biological processes but also plays a
central role in oxidative stress as a major catalyst for hydroxyl
radical (· OH) formation via the Fenton reaction (15).
Hydroxyl radical is the most potent radical species that abstracts a
hydrogen atom from biological molecules, leading to deleterious effects
such as DNA damage, protein modification, and lipid peroxidation (3,
17). A lipophilic iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one (L1,
deferiprone) has recently been shown to inhibit NF-B activation in
lipopolysaccharide (LPS)-stimulated cultured HM (24). In fact, the
derivatives of dithiocarbamates, which have been used as specific
inhibitors of NF-
B, function as antioxidants, partly through their
metal chelating capacity (32). Thus these observations suggest that
iron or iron-catalyzed oxidative stress is involved in NF-
B activation.
Both oxidative stress and iron are implicated in the pathogenesis of
alcoholic liver injury (36, 39). Chronic alcohol consumption may cause
an increase in hepatic iron (39), and iron supplementation results in
exacerbation of alcoholic liver injury (36). Iron-mediated potentiation
of alcoholic liver injury is associated with enhanced NF-B
activation, upregulation of NF-
B-responsive chemokine gene
expression, and mononuclear cell infiltration (38), suggesting a
critical role for iron in the NF-
B-mediated inflammatory response in
this experimental model.
Nonetheless, the exact means whereby iron enhances NF-B activation
have not been determined. Increased NF-
B activation might be a
direct effect of iron or an indirect consequence, perhaps as a result
of iron-induced exacerbation of hepatocellular injury. Our previous
studies of cultured HM treated with L1 provided evidence for a direct
regulatory role of iron in NF-
B activation (24), but the pertinence
of these findings in vitro to HM in vivo has not been investigated.
The present study has examined the role of increased iron stores within
HM in enhancing NF-B activation in experimental alcoholic liver
injury. We found that increased iron stores were associated with
intensified NF-
B activation in HM in the model and that both could
be normalized by treatment of the cells ex vivo with L1. In addition,
erythrophagocytosis by cultured HM increased intracellular iron
concentrations and promoted LPS-stimulated NF-
B. Finally,
splenectomy of animals resulted in an incremental increase in HM iron
and NF-
B activation following alcohol feeding.
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METHODS |
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Animals.
Male Wistar rats (~350-375 g body wt) were implanted with
gastrostomy catheters under general anesthesia achieved with ketamine (35 mg) and xylazine (1.5 mg) (19). The animals received continuous intragastric infusion of a high-fat (35% calories as corn oil) or a
low-fat (8% calories as corn oil) diet plus an increasing concentration of ethanol (HFD-alcohol or LFD-alcohol groups) or isocaloric dextrose (HFD-control or LFD-control groups) for 9 wk, as
previously described (19). Splenectomy was also performed on subsets of
HFD-alcohol and HFD-control groups at the time of gastrostomy catheter
implantation to examine the effect of the absence of the spleen on
alcohol-induced alterations in HM iron storage and NF-B activation.
Hepatic macrophage isolation.
After a 9-wk intragastric infusion as described above, HM were isolated
by sequential digestion of the liver with Pronase and type IV
collagenase followed by arabinogalactan gradient centrifugation as
previously described (19). The purity of HM was assessed by
phase-contrast microscopy and latex bead (1.0 µm) phagocytosis (Sigma
Chemical, St. Louis, MO) and always exceeded 85%. The cell viability
examined by the trypan blue exclusion test was always >95%. By our
method, the presence of sinusoidal endothelial cells and hepatic
stellate cells in the HM fraction were <10% and 5%, respectively.
The HM isolated from the animals were subjected to total RNA, nuclear
protein, and cytoplasmic protein extraction, as well as measurement of
iron and ferritin concentration. These cells were also used for an
experiment ex vivo to test effects of L1 on NF-B binding and TNF-
and macrophage inflammatory protein (MIP)-1 mRNA expression.
Nuclear protein extraction and gel mobility shift assay.
To examine the NF-B binding, nuclear proteins of the HM were
extracted using the method of Dignam and co-workers (10). The extract
(5 µg) was incubated in a reaction mixture [20 mM HEPES, pH
7.6, 20% glycerol, 200 µg/ml poly(dI-dC)] on ice in the
absence or presence of a 500-fold molar excess of unlabeled double-stranded oligonucleotide with the
B consensus sequence (top
strand: 5'-GCAGAGGGGACTTTCCGGA-3', bottom strand:
5'-GTCTGCCAAAGTCCCCTCTG-3') (2). One of the four NF-
B
response elements in the TNF-
promoter was also used as a probe for
the assay (6). After a 10-min incubation, 1-2 ng of
32P-labeled double-strand
B oligonucleotide were added
and the incubation continued for an additional 20 min. The reaction
mixture (a total volume of 20 µl) was then resolved on a 6%
nondenaturing polyacrylamide gel using 0.4× Tris-borate-EDTA. The
gel was dried and subjected to autoradiography at
80°C. For
the supershift assays, 1.5 µg (1.5 µl) of antibodies against p50 or
p65 (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the
reaction mixture for an additional 30-min incubation at room
temperature subsequent to the 20-min incubation with
32P-labeled NF-
B probe. The competitive displacement of
the shifted bands and the supershift assays were previously performed
by us on HM nuclear extracts to support the specificity of NF-
B
binding detected by this method (24).
Measurements of nonheme iron and ferritin concentrations. The nonheme iron concentration in HM and spleen was measured by a bathophenanthroline sulfonate-thioglycolic acid chromogen assay (35). The ferritin content in HM was determined by a rat-specific ferritin ELISA assay (RAMCO Laboratories, Houston, TX).
RNA extraction and RT-PCR.
Total RNA from freshly isolated HM was extracted using a
guanidinium-phenol-chloroform method (5). The integrity and equal loading of RNA samples were assessed by running a minigel of the samples and examining ethidium bromide staining of 18S and 28S rRNA.
Because the numbers of HM isolated from the animal model were limited
and multiple parameters needed to be examined on the cells, the use of
a large number of the cells to extract sufficient RNA for Northern blot
analysis or RNase protection assay was difficult. For this reason,
semiquantitative RT-PCR analysis was performed on a small quantity of
RNA to assess changes in the mRNA levels for cytokines, ferritin
subunits, hemeoxygenase-1, and -actin. Three micrograms of total RNA
were reverse transcribed into cDNA with 600 units of Moloney murine
leukemia virus reverse transcriptase and oligo(dT)15 (GIBCO
BRL, Grand Island, NY) as a primer at 37°C for 60 min. To provide
semiquantitative assessments on the mRNA level of NF-
B-responsive
genes in the HM, a portion of the synthesized cDNA for rat TNF-
(12)
or MIP-1 (11) was amplified by 25-35 cycles of PCR using a set of
gene-specific primers (Table
1). To assess the iron
homeostasis in HM, a portion of the synthesized cDNA was amplified by
using specific PCR primers for ferritin L chain, H chain, or
hemeoxygenase-1 (Table 1) (22, 27, 34). As a housekeeping gene,
-actin was amplified by PCR at the same time (29). The linear ranges
of PCR amplification for all the above genes were assessed in
preliminary experiments, and the appropriate cycles were determined.
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RNA electrophoretic mobility shift assay.
Binding activities of iron regulatory proteins (IRP), which mediate
posttranscriptional regulation of ferritin expression, were examined by
RNA electrophoretic mobility shift assay of HM cytoplasmic extracts.
The RNA probe is a 108-base sense transcript prepared with SP6 RNA
polymerase from Sma I-digested rat ferritin L-gene 66 (pseudogene), which was subcloned into the Sac I-Hind III site of pGEM 2. The plasmid pGEM-iron-responsive element (IRE) was
kindly provided by Dr. Elizabeth A. Leibold (Human Molecular Biology
and Genetics, University of Utah) and contains the sequence corresponding to the IRE of rat ferritin L chain encompassing 38 bases
of the 5'-flanking sequence and 70 bases of the 5'-UTR. A
transcription reaction was performed in the presence of 1 µg of
linearized template (Sma I digested), 60 µCi
[-32P]CTP (3,000 Ci/mmol; New Life Science
Products, Boston, MA), 0.5 mM each NTP (except CTP), 10 units of SP6
RNA polymerase (Boehringer Mannheim, Indianapolis, IN), and 20 units of
RNase inhibitor in a total volume of 20 µl. After 20 min of
incubation at 37°C, the reaction was stopped by heating at 65°C
for 15 min and incubated another 15 min at 37°C following addition
of DNase 1 (20 units). The labeled RNA was separated from the
nonincorporated ribonucleotide triphosphates on a 1-ml column (Nuctrap
probe purification column, Stratagene, La Jolla, CA), and 45 µl of
1× STE (0.1 M NaCl, 10 mM Tris·HCl, 1 mM EDTA, pH
8.0) were added to the eluent. The cytoplasmic extract was prepared by
lysis in the ice-cold buffer of 10 mM HEPES (pH 7.6), 40 mM KCl,
3 mM MgCl2, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 2 µg/ml aprotinin, 5% glycerol, and 0.2% Nonidet P-40. After removal of the
nuclei, the cytoplasmic fraction was centrifuged at 10,000 g,
and the supernatant was stored in aliquots at
80°C. A
binding reaction was carried out as previously described (23),
using 20 µg of cytoplasmic extract and 104 cpm of
32P-labeled IRE probe in a buffer of 10 mM HEPES (pH 7.6),
3 mM MgCl2, 1 mM dithiothreitol, 40 mM KCl, and 5%
glycerol in a total volume of 15-25 µl. After incubation at
25°C for 30 min, 1 unit of RNase T1 (Boehringer Mannheim) was added
and incubated for 10 min followed by an additional 10-min incubation
with heparin (5 mg/ml) (Sigma). This mixture was subjected to
electrophoresis on 5% nondenaturing polyacrylamide gel
(acrylamide-to-methylene bisacrylamide ratio of 60:1). The gel was
dried and analyzed by autoradiography at
80°C.
Treatment of HM with L1 ex vivo.
To further test the association between NF-B activity and TNF-
and MIP-1 mRNA expression by HM, isolated HM from HFD-alcohol and
HFD-control rats were incubated in serum-free RPMI 1640 with or without
L1 (100 µM) for 18 h at 37°C. The cells were then washed with
cold PBS and subjected to RNA/nuclear protein extraction as described above.
Effects of erythrophagocytosis on NF-B binding by
cultured HM.
This experiment was performed to examine whether an increased nonheme
iron concentration produced by erythrophagocytosis affected NF-
B
binding in cultured HM. Heat-denatured red blood cells (RBC) were used
for erythrophagocytosis. Blood was collected from a male Wistar rat
with a heparinized syringe, and RBC were washed three times with normal
saline by centrifugation at 1,400 rpm for 4 min at 4°C. RBC were
then suspended in 5 vol of ACD-B solution (Baxter
Healthcare) and incubated at 40°C for 15 min. After incubation, heat-treated RBC were washed twice in normal saline, resuspended in
normal saline, and kept on ice until they were used. Heat-treated RBC
were added to cultured HM from normal male Wistar rats at the ratio of
10:1 (RBC to HM), and the cells were cultured in RPMI 1640 with 5% FCS
for 2 h to allow phagocytosis of RBC. The nonadhering RBC were then
removed by gentle washing with PBS, and noninternalized RBC were
hypotonically lysed by exposure to cold water for 20 s. After another
18-h incubation, the cells were exposed to LPS (1 ng/ml) for 1 h and
subjected to nuclear protein extraction for NF-
B gel shift assay.
For inhibition of hemeoxygenase activity, zinc protoporphyrin (10 µM)
was added to the culture 30 min before the phagocytosis period.
Electron paramagnetic resonance spectra of Kupffer cells.
Freshly isolated Kupffer cells (107 cells/ml) from
alcohol-fed and control animals were suspended in 5-10 mM glucose
in PBS. To this mixture, 50 mM
-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and
0.1% (vol/vol) DMSO were added and the samples were immediately
transferred to bottom-sealed Pasteur pipettes. The elecron paramagnetic
resonance (EPR) spectra were recorded at room temperature in a Bruker
ECS 106 spectrometer operating at 9.8 GHz. Instrument conditions were
as follows: modulation frequency, 100 kHz; time constant, 1.3 s; sweep
scan, 18 G/min; modulation amplitude, 0.9 G; and microwave power, 20 mW. The spectra were compared with simulated ones obtained by using the
published hyperfine splitting constants and the simulation program from
Oklahoma Research Center. Different simulation spectra were run with
one species at a time until the best match was found in terms of line
width, g values, number of lines, g shifts, and peak heights.
Statistical analysis. Numerical results were presented as means ± SD. The significance of the difference between the groups was assessed by Student's t-test.
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RESULTS |
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NF-B activation and cytokine expression by freshly
isolated HM.
NF-
B binding was examined in nuclear extracts of HM isolated from
four groups of the rats intragastrically fed with a high-fat or low-fat
diet plus ethanol (HFD-alcohol or LFD-alcohol) or isocaloric dextrose
(HFD-control or LFD-control) as shown in Fig.
1. There was a twofold increase in NF-
B
binding activity in HM from HFD-alcohol compared with the cells from
the pair-fed control (HFD-control) rats. No increase was noted for
NF-
B binding in HM from LFD-alcohol rats who received the identical
amount of ethanol with the isocaloric low-fat diet. Because
centrilobular liver necrosis and inflammation were evident in
HFD-alcohol but not in LFD-alcohol rats, these results indicate that
NF-
B activation in HM in the model was not due to ethanol
consumption per se but to conditions associated with progressive
alcoholic liver injury. The specificity of NF-
B binding was
supported by complete displacement of the shifted bands by competition
with unlabeled probe in excess (data not shown) as previously reported
by us (24).
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Expression of mRNA for NF-B-responsive genes such as
TNF-
and MIP-1.
Semiquantitative analysis of the TNF-
mRNA level by RT-PCR indicated
a 150% increase in HM from HFD-alcohol compared with the cells from
the pair-fed control rats (Fig. 2),
confirming our previous finding (19). MIP-1 mRNA expression was
increased by ~280% in HFD-alcohol rats.
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Effects of iron chelator L1 on NF-B binding activity
and cytokine gene expression.
To test the association between NF-
B activation and induction of
TNF-
and MIP-1 mRNA expression, the isolated HM from HFD-alcohol and
HFD-control groups were incubated overnight (18 h) in serum-free RPMI
1640 with or without L1 (100 µM), the iron-chelating agent, which has
been previously shown to effectively block NF-
B activation in HM
(24). Nuclear proteins and total RNA were extracted from these cells
for the NF-
B gel mobility shift assay and cytokine RT-PCR. The
treatment with L1 caused complete blockade of NF-
B activation by the
HFD-alcohol HM, whereas the constitutive NF-
B binding in HFD-control
HM was not affected by L1 (Fig. 3).
Coordinately, semiquantitative analysis of TNF-
and MIP-1 mRNA
expression showed apparent suppression with L1 in HFD-alcohol but not
in HFD-control rats (Fig. 4). These results
support an association between NF-
B activation and TNF-
and MIP-1
mRNA induction by HM in experimental alcoholic liver injury and provide
evidence for a critical role of iron in these molecular events.
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Nonheme iron concentration and EPR spectrum.
Nonheme iron concentration was determined in HM from HFD-alcohol and
HFD-control groups before and after the L1 treatment. A significant
70% increase in the nonheme iron level was noted in HFD-alcohol
compared with HFD-control rats (Table 2).
The L1 treatment resulted in a significant reduction of nonheme iron concentration in the HFD-alcohol cells but not in the HFD-control cells
(Table 2). There was no significant increase in the nonheme iron
content in HM from LFD-alcohol compared with LFD-control or HFD-control
groups. Because changes in intracellular iron are usually associated
with the production of oxygen radicals, we evaluated the presence of
oxygen radicals in these cells by EPR in conjunction with the spin
trapping technique (Fig. 5). The EPR
signals of HM from HFD-control were low, and the signal-to-noise ratio
of this spectrum was far from desirable. However, the ratio increased
with the number of the cells and decreased or was abolished if the
cells were not viable (data not shown). The signals are low because the
steady-state concentration of free radicals in normal living cells is
low (below the nanomolar range). Nevertheless, the spectrum
demonstrates a signal attributable to a composite of methyl, hydroxyl,
and superoxide anion adduct signals in a ratio of 1:1.3:1,
respectively. These signals, in addition to the signal of ethyl-POBN
adduct (-hydroxyethyl-POBN), were also observed in the spectrum
obtained with HM from HFD-alcohol animals (Fig. 5, C and
D), albeit of higher intensity (3-fold).
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Ferritin and heme oxygenase-I expression.
To assess a homeostatic response to the observed increase in the
cellular iron levels, the ferritin protein content, ferritin L chain,
and ferritin H chain mRNA levels were semiquantitatively examined in
HM. The ferritin protein content was increased by 50% in HM from
HFD-alcohol compared with HFD-control rats (Table 2). The ferritin L
chain mRNA level was increased by twofold, whereas no change was noted
for ferritin H chain mRNA expression (Fig.
6). Thus these results demonstrate that HM
in alcoholic liver injury have the increased iron storage and
consequent upregulation of the storage protein expression. Notably,
mRNA expression of hemeoxygenase-1, a pivotal enzyme for the breakdown
of heme, was increased by twofold in the HFD-alcohol HM (Fig.
7), suggesting that enhanced heme
metabolism via increased erythrophagocytosis contributes to the
increased iron storage in HM in alcoholic liver injury.
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Iron response element binding by HM in alcoholic liver injury.
The size of the intracellular pool of iron is regulated by the
cytoplasmic proteins, iron regulatory proteins (IRP-1 and IRP-2). IRP
bind to IRE within transferrin receptor and ferritin transcripts to
coordinately mediate transferrin receptor mRNA stability and ferritin
mRNA translational efficiency to control the intracellular "free"
iron level. We analyzed the binding of IRP of HM cytoplasmic extracts
to examine whether the observed increases in the iron and ferritin
content in the HFD-alcohol HM were associated with suppressed IRE
binding. Surprisingly, no changes in IRE binding were observed (Fig.
8).
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Effects of erythrophagocytosis on NF-B activation by
cultured HM.
Because the aforementioned data suggested erythrophagocytosis as a
possible mechanism contributing to the increased iron storage in HM, we
examined the effects of phagocytosis of heat-treated RBC by cultured HM
to test whether the increase in storage iron produced by this technique
affects NF-
B activation. To allow the recovery of the HM from
phagocytosis-associated stimulation, the cells were incubated for an
additional 18 h and examined for NF-
B binding with or without LPS
stimulation. The erythrophagocytosis procedure resulted in a 40%
increase in the nonheme iron content. Under basal conditions, these
cells did not show a significant increase in NF-
B binding. In
contrast, LPS-stimulated NF-
B activation was intensified by twofold
(Fig. 9A) in the HM that had
been iron loaded by phagocytosis of RBC. Furthermore, the
pretreatment of the cells with zinc protoporphyrin before
erythrophagocytosis completely abrogated the increase in LPS-mediated
NF-
B binding (Fig. 9A), suggesting that the observed effect
of erythrophagocytosis is dependent on hemeoxygenase activity. The
specificity of NF-
B binding was supported by the supershift assays
with antibodies against p50 and p65 as shown in Fig. 9B.
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Effects of splenectomy on HM iron storage and NF-B
activation in alcohol-fed animals.
To further extend the above observation in vitro to conditions in vivo,
we examined effects of splenectomy on HM iron storage and NF-
B
activation in the HFD-alcohol and HFD-control rats. Our hypothesis was
that splenectomy, by removing the iron storage site provided by splenic
macrophages, would further increase the iron content in HM and
consequently promote NF-
B activation in the cells, leading to
enhanced inflammation in the livers of the HFD-alcohol rats. Our
results demonstrate that HM from the splenectomized HFD-alcohol animal
showed further increases in iron storage (1.98 ± 0.17 vs. 1.34 ± 0.21 µg/107 cells, P < 0.05) and NF-
B
binding (+90%) (Fig. 10, A and
C) compared with HFD-alcohol without splenectomy. In addition,
the morphometric analysis of liver histology demonstrated that the
number of mononuclear inflammatory cells in the liver was increased
fivefold in the splenectomized HFD-alcohol compared with the
HFD-alcohol animal without splenectomy (Fig. 10B). In
particular, the inflammatory cells were found in close association with
HM with higher iron storage (Fig. 10D). HM from the
splenectomized HFD-control animal also showed a similar increase in the
nonheme iron content compared with HFD-control animals (1.48 ± 0.19 vs. 0.98 ± 0.13 µg/107 cells, P < 0.05), and
NF-
B binding was mildly increased in some of the former animals
(data not shown). Nonetheless, the liver histology of these animals
showed no apparent inflammatory response.
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DISCUSSION |
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The results of the present study support the pivotal role of increased
intracellular iron content in NF-B activation by HM in alcoholic
liver injury. First, the increased intracellular iron concentration and
NF-
B binding in HM were effectively and simultaneously normalized by
iron chelation ex vivo. This ex vivo approach has obvious limitations
in recapitulating the in vivo changes without introducing
potentially confounding effects that may result from the isolation and
incubation procedures. Nevertheless, the results obtained from the ex
vivo experiment are still considered as the supporting evidence for the
proposed role of iron. Second, the simultaneous increments in splenic
iron concentration and in the expression of heme oxygenase-I in HM
suggested that increased RBC uptake and enhanced heme catabolism
contributed to the increased intracellular in HM of the alcohol-fed
animals. In fact, the reproduction of this hypothetical mechanism in
cultured HM by erythrophagocytosis (with or without the use of a
hemeoxygenase inhibitor) confirmed that increased intracellular iron
promoted LPS-induced NF-
B activation in a hemeoxygenase-dependent
manner. To test this hypothesis in vivo, HM heme catabolism was further
augmented in the alcohol-fed animals by splenectomy, a procedure that,
by removing splenic macrophages, increases RBC uptake by HM. Indeed,
the splenectomy resulted in an incremental increase in the nonheme iron
content of HM, further promoted NF-
B binding, and caused a fivefold
increase in the number of mononuclear inflammatory cells infiltrated
into the livers of alcohol-fed animals.
Our results indicate that increases in intracellular iron prime HM for
activation of NF-B but do not identify the specific iron pool
involved. It should be emphasized that, in our experiments, the HM were
exposed to constant and relatively high concentrations of the iron
chelator L1, conditions under which this bidentate chelator could bind
iron effectively. With L1 in excess, three molecules of the chelator
are available to occupy the six coordination sites of each atom of
iron. In these experiments, we did not examine the effects of lower
concentrations of the chelator. Other investigators have reported that
L1 may potentiate iron-catalyzed oxidative damage in liver cells at low
ratios of L1 to iron (7) and, in clinical use, may worsen hepatic
fibrosis (30).
NF-B activation by HM was found only in the HFD-alcohol group.
Expression of TNF-
is under the transcriptional regulation of
NF-
B (6). MIP-1, a C-C chemokine for neutrophils and mononuclear cells, is induced with LPS and TNF-
(11), presumably through NF-
B
activation (14). In fact, both NF-
B activation and the induced
TNF-
and MIP-1 mRNA expression by HM from HFD-alcohol animals were
abrogated by ex vivo treatment with the iron chelator (L1), as
previously demonstrated for NF-
B activation and TNF-
expression
in cholestatic liver injury or under in vitro LPS stimulation (24).
Thus these results support not only the role of iron in NF-
B
activation but also the importance of NF-
B in TNF-
and MIP-1
expression by HM.
Our in vitro erythrophagocytosis experiment was crucial for
demonstrating the link between the increment in intracellular iron in
HM resulting from increased heme catabolism and accentuated NF-B
activation in response to LPS. After a series of preliminary experiments, the erythrophagositosis technique described here was shown
to achieve an ~30-40% increase in the nonheme iron
concentration. This iron loading did not affect NF-
B under the basal
condition but accentuated LPS-stimulated NF-
B activation (Fig.
9A). It was important to allow the cells to rest for a
sufficient time after erythrophagocytosis, since the phagocytotic
process itself is known to be followed immediately by downregulation of
macrophage functions (25). Another critical factor was the number of
phagocytosed erythrocytes by one macrophage. Phagocytosis of more than
1.5 opsonized erythrocytes per Kupffer cell was shown to lead to cell cytotoxicity (21). Thus suppressed macrophage functions in patients with iron overload (40) or phagocytosis of a large number of erythrocytes (25) may be related to cell cytotoxicity. Our method of
using the erythrocyte ratio of 10:1 (erythrocytes to Kupffer cells) and
washing and lysing the erythrocytes after the 2-h incubation resulted
in a phagocytosis ratio of ~1:1.
The splenectomy experiment further extended the observation made in the
in vitro phagocytosis experiment. Removal of the spleen indeed caused a
further increase in HM iron concentration in alcohol-fed animals and
accentuated NF-B activation and inflammatory response. The same
procedure resulted in a similar magnitude of increase in the HM iron
content of the pair-fed control animals (HFD-control). In fact, some
but not all of these animals showed mildly increased NF-
B binding
(data not shown). However, histologically, the livers were normal
without any sign of inflammation. These results suggest that the
increased iron concentration was not sufficient enough to induce
inflammation in the control animals but primed the HM in the
alcohol-fed animals for NF-
B activation, cytokine expression, and
inflammation. These data corroborate with our in vitro finding that
erythrophagocytosis did not enhance NF-
B activation by cultured HM
under basal conditions but did so after LPS stimulation. An iron
chelator blocks LPS-mediated NF-
B activation and TNF-
and IL-6
expression by cultured peritoneal macrophages (4) and Kupffer cells
(24). Collectively, these results provide evidence for a regulatory
role of iron or iron-catalyzed oxidative stress in NF-
B activation.
Our in vivo and in vitro findings support this notion and a novel
hypothesis that the increased intracellular iron in HM primes the cells
for NF-
B activation.
The mechanism for the increase in intracellular iron stores in HM in
the liver injury induced by alcohol has not been studied in detail. The
effects of alcohol and alcohol-induced liver disease on RBC production
and survival are complex. Overall, the observed increase in iron both
within HM and in the spleen in the HFD-alcohol group (Table 2) is
likely to reflect a combination of increased RBC uptake
(erythophgocytosis) by macrophages associated with a decreased RBC life
span and impaired mobilization of iron from macrophages. In particular,
acetaldehyde, a metabolite of ethanol, may form adducts with hemoglobin
in ethanol-consuming subjects, and the epitopes may be expressed on the
surface of the RBC, which can be recognized by circulating antibodies
against them (28). This facilitates Fc receptor-mediated phagocytosis
of the opsonized cells by macrophages. In addition, modification of RBC
membrane with malondialdehyde promotes phagocytosis of the cells by
macrophages, which appears to involve both IgG-dependent and
IgG-independent mechanisms (16). In alcoholic liver injury, many other
reactive molecules are produced, including hydroxyethyl radicals (31) and lipid aldehydes produced via lipid peroxidation (18). Thus these
reactive substances may readily modify erythrocytes, decreasing RBC
survival and resulting in increased erythrophagocytosis by macrophages.
In addition, alcohol liver injury may cause the impairment of iron
release by erythrophagocytosed macrophages as shown for chronic
inflammation models (21). If the increased iron content by
erythrophagocytosis is the underlying mechanism for the enhanced NF-B activation in HM, then selective and targeted iron chelation in
macrophages may provide a potential therapeutic or preventive modality
for suppression of NF-
B-mediated cytotoxic or proinflammatory responses.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Drs. Widong Yin, Thai-Van Pham, and Hong-yan Li for their excellent technical assistance and Rosy Macias for preparation of the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported by National Institutes of Health Grants R37-AA-06603 (H. Tsukamoto) and R01-HL-61219 and R01-DK-49108 (G. Brittenham), Molecular Biology and Tissue Culture Core Facilities of USC Research Center for Liver Disease Grant P30-DK-48522, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Disease Grant P50-AA-11999, and by the Medical Research Service of Department of Veterans Affairs (H. Tsukamoto).
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: H. Tsukamoto, 1333 San Pablo St., MMR-412, Los Angeles, CA 90033-1034 (E-mail: htsukamo{at}hsc.usc.edu).
Received 5 March 1999; accepted in final form 1 September 1999.
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