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B and c-Jun N-Terminal Kinase and Enhances Chemokine Secretion on Activated Human Hepatic Stellate Cells1
Department of Medicine and Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Stimulation of CD40 with
CD40 ligand (CD40L)-transfected baby hamster kidney cells induces
NF-
B, as demonstrated by the activation of I-B kinase (IKK),
increased NF-B DNA binding, and p65 nuclear translocation.
CD40-activated IKK also phosphorylates a GST-p65 substrate at serine
536 in the transactivation domain 1. Concomitant with the activation of
IKK, CD40L-transfected baby hamster kidney cell treatment strongly
activates c-Jun N-terminal kinase. CD40 activation increases the
secretion of IL-8 and monocyte chemoattractant protein-1 by HSCs 10-
and 2-fold, respectively. Adenovirally delivered dominant negative (dn)
IKK2 and TNFR-associated factor 2dn inhibit IKK-mediated
GST-I-B and GST-p65 phosphorylation, NF-B binding, and IL-8
secretion, whereas IKK1dn and NF-B-inducing kinase dominant negative
do not have inhibitory effects. We conclude that the CD40-CD40L
receptor-ligand pair is involved in a cross-talk between HSCs and
immune effector cells that contributes to the perpetuation of HSC
activation in liver fibrosis through TNFR-associated factor 2- and
IKK2-dependent pathways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD40 is a member of the TNFR superfamily with important functions in
the regulation of humoral and cellular immune responses
(10). The expression of CD40 on a variety of nonhemopoetic
cells, including basal epithelia, mesenchymal cells, and cancer cells,
implies a broader function in vivo. On fibroblasts, activation of CD40
by its ligand (CD40L) induces NF-B activation and cytokine secretion
(11), and may up-regulate the production of extracellular
matrix (12). CD40 is functionally expressed on hepatocytes
during allograft rejection (13) and on hepatocellular
carcinoma (14), but the expression of CD40 in other cell
populations of the liver and its role in liver injury are unknown.
In this study, we evaluated the expression and function of CD40 on
activated human HSCs. CD40 was expressed on culture-activated HSCs and
HSCs activated in vivo. Ligation of CD40 on HSCs induced the activation
of important signaling pathways such as I-B kinase (IKK)/NF-B and
c-Jun N-terminal kinase (JNK) and enhanced IL-8 and monocyte
chemoattractant protein-1 (MCP-1) secretion. IKK2 and TNFR-associated
factor 2 (TRAF2) were the critical transducers of CD40-induced
IKK/NF-B activation and IL-8 secretion. Therefore, CD40 may be an
important mediator of the cross-talk between HSCs and immune effector
cells and contribute to maintaining HSCs in an activated state.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HSCs were isolated by a two-step collagenase perfusion from
surgical specimens of normal human liver. All tissues were obtained
through qualified medical staff, with donor consent and the approval of
the University of North Carolina Ethics Committee. The encapsulated
liver tissue was perfused with calcium-free buffer, followed by
perfusion with buffer containing 1.5 mM calcium and collagenase
(0.3–0.4 mg/ml). The hepatocytes were pelleted, and the supernatant
was further digested with 0.035% pronase (Boehringer Mannheim,
Indianapolis, IN) for 15 min. The suspension was layered on top of an
arabinogalactan (Sigma, St. Louis, MO) two-layer discontinuous density
gradient (1.035 and 1.058 g/ml). The gradient was centrifuged, and HSCs
were recovered from the two interfaces and washed (15).
The purity after 4 days in culture was 
98% as estimated by
autofluorescence. Cells were seeded on uncoated plastic tissue culture
dishes and cultured in DMEM (Life Technologies, Grand Island, NY)
supplemented with 10% FCS and standard antibiotics in 95% air and 5%
CO2 humidified atmosphere at 37°C. Growth
medium was changed daily. The activated HSCs expressed smooth muscle
-actin, glial fibrillar-associated protein, and
synaptophysin.4
Activation of CD40 on HSCs
One day before activating CD40, HSCs were cultured in DMEM
containing 0.5% FBS and 500 IU IFN-. Activation of CD40 was
achieved by incubation with either CD40-transfected baby hamster kidney
cells (BHKCD40L) or mock-transfected baby hamster
kidney cells (BHKptcf) (a gift from Dr. H.
Engelmann, Institute of Immunology, Munich, Germany) as control for
various times (5–60 min). This method achieves a strong activation of
CD40 due to the high degree of CD40 aggregation by trimeric
membrane-bound CD40L, which is known to be critical for CD40 activation
(16, 17). To avoid BHKCD40Lor
BHKptcf from attaching to HSCs, HSCs were gently
washed with PBS three to four times after the stimulation period. To
demonstrate the specificity of CD40 activation, HSCs were stimulated
with BHKCD40L in the presence of the blocking
anti-CD40L Ab 5c8 (obtained from the American Tissue Culture
Collection, Manassas, VA) in various experiments.
Adenoviral infection
Adenoviruses expressing dominant negative (dn) IKK1,
IKK2dn, TRAF2dn, NF-B-inducing kinase (NIK)dn, and green fluorescent
protein (GFP) were previously described (18, 19, 20, 21, 22).
Infection of HSCs at a multiplicity of infection of 400 resulted in a
transduction rate of at least 80% for all adenoviruses, as tested by
immunofluorescent staining. For all subsequent experiments, HSCs were
infected with recombinant adenoviruses with a multiplicity of infection
of 400 in DMEM containing 2% FBS. After 24 h, the medium was
changed and HSCs were cultured for further 24 h and then incubated
with either BHKCD40L or
BHKptcf. Adenovirus 5 (Ad5) GFP was included as a
control to ensure adequate adenoviral gene transfer in each experiment,
as checked by fluorescent microscopy.
RT-PCR analysis
For the detection of CD40 mRNA, HSCs were cultured for 72 h
in the presence or absence of 500 IU/ml human IFN- (R&D Systems,
Minneapolis, MN) at 500 IU/ml. RNA was isolated by the TRIzol method
(Life Technologies), according to the manufacturer’s instructions. One
microgram of RNA was reverse transcribed using
dT15-oligonucleotide and Moloney murine leukemia
virus reverse transcriptase (Perkin-Elmer/Applied Biosystems, Foster
City, CA) in 25 µl. One microliter of the reverse-transcriptase
reaction was subjected to PCR to measure the mRNA of ubiquitin and
CD40. CD40 was amplified for 35 cycles using
5'-AATCTAGATGCCGCCTGGTCTCACCTCG sense and
3'-AAAAGCTTGCCAACTGCCTGTTTGCCCACG antisense primers at 1 µM in a
50 µl PCR containing 1.5 mM MgCl2, 50 mM KCl,
and 10 mM Tris, pH 8.3. Ubiquitin was amplified for 30 cycles using
5'-GGAAGACCATCACCCTCGAAG sense and 3'-CCAGCACCACATTCATC antisense
primers at a concentration of 1 µM in a 50 µl PCR containing 1.5 mM
MgCl2, 50 mM KCl, and 10 mM Tris (pH
8.3).
Flow cytometric analysis
The expression of CD40 was assessed by flow cytometric analysis.
HSCs were cultured for 72 h in the presence or absence of 500
IU/ml IFN-. Following detachment by PBS containing 2 mM EDTA, the
cells were stained either with anti-CD40 mAb G28-5 or an
isotype-matched irrelevant control Ab, as previously described
(16). After extensive washing, the cells were incubated
with FITC-labeled goat anti-mouse IgG and fixed in 3%
paraformaldehyde. In an additional experiment, CD40 was detected by
staining HSCs with a polyclonal anti-CD40 rabbit serum
(23) and a FITC-labeled goat anti-rabbit IgG secondary
Ab. Viable cells were gated, and 5000 cells were analyzed using a
FACScan instrument (BD Biosciences, Franklin Lakes, NJ).
Immunohistochemistry
Liver specimens were obtained according to the guidelines of the
University of North Carolina Ethics Committee from patients with stage
IV liver fibrosis secondary to chronic hepatitis C and primary
sclerosing cholangitis who underwent surgical liver resection for liver
transplantation. Normal liver specimens were obtained from patients who
underwent liver resection for fibronodular hyperplasia. CD40 and smooth
muscle -actin expression was detected by immunostaining using the
Dako Envision system (Dako, Carpenteria, CA), according to the
manufacturer’s instructions. Briefly, endogenous peroxidase was
blocked with peroxidase-blocking agent, and sections were incubated
with anti-CD40 mouse mAb G28-5 at a concentration of 50 µg/ml or
anti-smooth muscle -actin Ab (Dako) at 42.5 µg/ml for 10 min
at room temperature in 1% BSA in PBS. After two 3-min washes in PBS,
sections were incubated with labeled polymer for 10 min at room
temperature. Sections were washed twice with PBS, incubated with
3.3-diaminobenzidine substrate chromogen for 8 min, washed with water,
incubated with diaminobenzidine enhancer (Innovex Biosciences,
Richmond, CA) for 5 min, and washed with water before
counterstaining with hematoxylin. As negative controls, all specimens
were incubated with an irrelevant isotype-matched control Ab under
identical conditions.
Western blotting
Whole cell extracts of human activated HSCs were prepared by
lysing the cells in Triton lysis buffer (20 mM Tris (pH 7.4), 137 mM
NaCl, 2 mM EDTA, 10% glycerol, and 1% Triton X-100) containing
protease inhibitors (40 µg/ml bestatin, 0.5 mM pefabloc, 700 ng/ml
pepstatin A, 2 µg/ml aprotinin, and 0.5 µg/ml leupeptin; all from
Roche, Indianapolis, IN) and phosphatase inhibitors (20 mM
-glycerophosphate, 10 mM 4-nitrophenylphosphate, and 50 µM sodium
vanadate; all from Sigma). Protein concentration of the extracts was
determined by the Bradford method, and 25 µg was loaded onto 10%
SDS-acrylamide gels. The gels were then blotted onto nitrocellulose
membranes. After confirming equal loading by Ponceau S staining, the
membranes were blocked with 20 mM Tris (pH 7.4) containing 5% milk
powder, 137 mM NaCl, and 0.05% Tween 20. For the detection of
hemagglutinin (HA)- or Flag-tagged proteins, nitrocellulose membranes
were incubated in blocking buffer containing anti-HA (Roche) or
anti-Flag Ab (Eastman Kodak, New Haven, CT) at a dilution of 1/1000
for 1 h. After extensive washing, the membranes were incubated
with blocking buffer containing HRP-conjugated goat anti-mouse Ab
(Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1/1000 for
45 min. Proteins were detected by ECL chemoluminescence (Amersham,
Arlington Heights, IL).
Electrophoretic mobility shift assay
HSCs were incubated with BHKCD40L or
BHKptcf for 60 min. Nuclear extracts were
prepared as described (24). Briefly, the cells were
swollen in buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM
MgCl2, 0.5 mM DTT) containing protease and
phosphatase inhibitors for 15 min on ice and lysed in 10% Nonidet
P-40. After centrifugation, nuclei were lysed in buffer C (10 mM HEPES
(pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.5 mM DTT, and 0.5% Nonidet P-40)
containing protease and phosphatase inhibitors. Protein concentrations
were determined by Bradford assay. Five micrograms of protein were
incubated with 100 pg of a 32P-labeled probe
containing the NF-B consensus site in buffer containing 10 mM HEPES
(pH 7.8), 2 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.1 mM
EDTA, and 20% glycerol in the presence of single-stranded
oligonucleotide (25 µg/ml) and poly(dI/dC) (25 µg/ml) for 15 min at
room temperature. For supershift analysis and competition assays,
extracts were preincubated for 15 min with Abs to p50 or p65 (Santa
Cruz Biotechnology) and 10 ng unlabeled probe, respectively.
Kinase assays
IKK and JNK assays were performed as previously described, with
slight modifications (19). Briefly, cells were lysed in
Triton lysis buffer containing protease and phosphatase inhibitors
after treatment with BHKCD40L or
BHKptcf for various times. For IKK assays, 300
µg protein was immunoprecipitated with 2 µl anti-IKK (a gift
from F. Mercurio, Signal Pharmaceuticals, San Diego, CA) for 2 h,
followed by 20 µl protein A/G agarose (Santa Cruz Biotechnology) for
1 h. The kinase reaction was performed for 30 min at 30°C using
either GST-I-B 1–54(1–54) or various GST-p65 substrates (a gift from H.
Sakurai, Tanabe Seiyaku, Osaka, Japan). For JNK assays, 25 µg protein
was incubated with 1 µl GST-c-Jun bound to reduced glutathione beads,
washed, and subjected to a kinase reaction for 30 min at 30°C.
Supernatant from the kinase reactions was analyzed on 10%
SDS-acrylamide gels. Equal substrate loading was confirmed by Coomassie
blue staining. Gels were exposed to X-OMAT or Biomax film (Eastman
Kodak). Quantification was performed by PhosphorImager analysis
(Molecular Dynamics, Sunnyvale, CA).
ELISA for IL-8 and MCP-1
HSCs were cultured in the presence of IFN- for 24 h and
stimulated with BHKCD40L or
BHKptcf for 60 min. HSCs were washed twice with
PBS to remove nonattached BHKCD40L or
BHKptcf, and cocultured with
BHKCD40L or BHKptcf
(approximate ratio, 1:1) for further 18 h in DMEM containing 1%
FBS and 500 IU IFN-. Supernatants were collected, and a sandwich
ELISA for IL-8 and MCP-1 (R&D Systems) was performed using 1/5
dilutions according to the manufacturer’s instructions. All samples
were measured as duplicates. IL-8/MCP-1 concentrations from wells
containing only BHKCD40L or
BHKptcf were subtracted from all samples that
were incubated with BHKCD40L or
BHKptcf.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HSCs were stained for CD40 and subjected to flow cytometric
analysis. The majority of activated HSCs expressed CD40 at a moderate
level when compared with an isotype-matched control Ab (Fig. 1
A). Pretreatment with IFN-
led to a 5-fold increase of CD40 expression. To confirm these results,
mRNA was isolated from HSCs cultured in the presence or absence of
IFN-, and RT-PCR for CD40 mRNA was performed. CD40 mRNA was detected
in untreated activated HSCs, and its expression was increased 2-fold by
pretreatment with IFN- (Fig. 1B). To extend this
observation to HSCs activated in vivo, we investigated the expression
of CD40 in human liver tissue. Strong staining for CD40 was detected in
patients with liver cirrhosis secondary to hepatitis C (Fig. 2A) and primary sclerosing
cholangitis (data not shown). The pattern of the staining was largely
perisinusoidal, and comparison of parallel sections stained with Abs
either to smooth muscle -actin or CD40 indicated that activated HSCs
are the major CD40-expressing cell population in cirrhotic liver (Fig. 2B). Hepatocytes and sinusoidal endothelial cells showed
weaker staining for CD40, confirming data from previous studies
demonstrating that these cell populations express CD40 under
inflammatory conditions such as chronic rejection and acute liver
failure, respectively (13, 25). No staining was observed
when sections were stained with an isotype-matched control Ab (Fig. 2C). Furthermore, sections from normal livers showed no
hepatic CD40 expression, suggesting that quiescent HSCs do not express
CD40 (Fig. 2D).
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B in HSCs
Because CD40 activates NF-B in fibroblasts (11),
we tested the influence of CD40 activation on NF-B in activated
HSCs. For that purpose, HSCs were treated with the CD40L-transfected
cell line BHKCD40L or mock-transfected
BHKptcf for 60 min and stained for p65. HSCs
treated with BHKptcf (Fig. 3A) and untreated HSCs (data
not shown) showed a cytoplasmic staining for p65, whereas
BHKCD40L (Fig. 3B) as well as
TNF--treated HSCs (Fig. 3C) displayed a nuclear staining
pattern for p65, indicating that CD40 activation induces NF-B
activation and p65 translocation. To confirm these results, we
performed an EMSA for NF-B. Treatment with
BHKCD40L resulted in a 2.5-fold induction of
NF-B activation in comparison with HSCs treated with
BHKptcf (Fig. 3D). Supershift analysis
demonstrated that the p50/p65 heterodimer was the main component of the
NF-B-binding activity. Specificity of the interaction was
demonstrated by competition with excess unlabeled probe. Incubation
with a CD40L Ab that blocks the interaction between CD40 and CD40L
almost completely abolished the induction of NF-B-binding activity
by BHKCD40L (Fig. 3E).
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CD40 induces the activation of IKKs in B cells (26),
resulting in the release of NF-B and its nuclear translocation. To
evaluate whether IKK was involved in the CD40-mediated activation of
NF-B in HSCs, we monitored the phosphorylation of I-B by
immunoprecipitated IKK in an in vitro kinase assay. Induction of IKK
activity was observed over a period of 15–120 min after CD40
activation by BHKCD40L and peaked at 15 min after
stimulation with a 13.5-fold increase in GST-I-B phosphorylation
(Fig. 4A). No activation of
IKK was seen when HSCs were incubated with
BHKptcf (Fig. 4B). The activation of
IKK by CD40 was almost completely blocked by pretreating the cells with
the blocking anti-CD40L Ab 5c8 (Fig. 4C). Because
phosphorylation of p65 is believed to be a second mechanism to regulate
the transcriptional activity of NF-B (27) and has been
demonstrated in response to TNF- (28, 29, 30), we assessed
the effect of CD40L on p65 phosphorylation. Using a GST-p65 354–551(354–551)
substrate in the kinase reaction, we demonstrated that CD40-activated
IKK phosphorylates p65 in vitro with kinetics that were comparable with
the phosphorylation of GST-I-B (Fig. 4D). To analyze
whether phosphorylation of p65 occurred at one of the previously
described sites, we used a mutated form of GST-p65 354–551(354–551) with a
serine to alanine substitution at position 536 termed GST-p65 (536A).
Only a minimal phosphorylation of this substrate was detected,
demonstrating that serine 536 is the main phosphorylation site of p65
(Fig. 4D). We found no evidence for C-terminal
phosphorylation of p65 by the IKK complex, as demonstrated by the use
of a GST-p65 1–305(1–305) substrate (Fig. 4D). CD40L-induced
phosphorylation of GST-p65 354–551(354–551) was blocked by treating HSCs with
anti-CD40L (Fig. 4E).
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B activation in HSCs
To investigate the pathways involved in the activation of NF-B
by CD40, we infected HSCs with adenoviruses containing
dominant-negative forms of IKK1, IKK2, TRAF2, and NIK. Infection with
these viruses led to a strong expression of the transgenes (Fig. 5A, bottom panel)
and a transduction rate of at least 80%, as determined by
immunofluorescent staining (data not shown). Infection with Ad5IKK2dn
and Ad5TRAF2dn significantly inhibited CD40L-induced GST-I-B 1–54(1–54)
and GST-p65 354–551(354–551) phosphorylation (Fig. 5A
top and middle panels). In both cases, the
inhibition was not complete, which reflects the remaining uninfected
HSCs. In contrast, Ad5IKK1dn, Ad5NIKdn, and the GFP-expressing control
virus Ad5GFP did not significantly decrease the phosphorylation of
GST-I-B 1–54(1–54), although the transgenes were highly expressed. The
phosphorylation of GST-p65 354–551(354–551) was similarly inhibited by
Ad5IKK2dn and Ad5TRAF2dn, but not by Ad5NIKdn, Ad5IKK1dn, or Ad5GFP.
Thus, the signal transduction through CD40 involves both TRAF2 and
IKK2, but bypasses the mitogen-activated protein kinase kinase NIK. To
assess the functionality of Ad5NIKdn, HSCs and primary hepatocytes were
infected with Ad5NIKdn and treated with TNF-. Whereas
TNF--induced IKK activation was not blocked by NIKdn in HSCs (Fig. 5B), IKK activity was completely blocked in primary
hepatocytes (Fig. 5C), demonstrating that HSCs possess a
cellular signaling machinery that may circumvent NIK in
cytokine-induced NF-B signaling. Consistent with the results from
the IKK assay, Ad5IKK2dn and Ad5TRAF2dn partially inhibited
NF-B-binding activity, whereas Ad5IKK1dn and Ad5NIKdn only had minor
effect on NF-B-binding activity (Fig. 5B).
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JNK is a second important signaling pathway induced by CD40, and
often occurs concomitantly with IKK activation. We evaluated the
CD40-induced activation of JNK in HSCs by an in vitro kinase assay. JNK
was strongly activated by CD40 activation, with kinetics that were
quite similar to IKK (Fig. 6A). Phosphorylation of
GST-c-Jun was observed as early as 15 min after treatment with
BHKCD40L and peaked at 30 min, with a 36-fold
induction. GST-c-Jun phosphorylation decreased after 120 min. No
significant induction of phosphorylation was observed in HSCs treated
with BHKptcf (Fig. 6B). Treatment with
anti-CD40L Ab completely blocked induction of GST-c-Jun
phosphorylation, demonstrating the specificity of this induction (Fig. 6C). HSCs infected with Ad5TRAF2dn displayed a 60%
reduction of GST-c-Jun phosphorylation, confirming a key role of TRAF2
for the induction of JNK by CD40 (Fig. 6D). Infection with
Ad5IKK1dn, Ad5IKK2dn, Ad5NIKdn, and Ad5GFP had no influence on
GST-c-Jun phosphorylation.
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Chemokines are up-regulated in liver fibrosis and are responsible
for the recruitment of leukocytes and HSCs to the site of injury. To
test whether CD40 induced chemokine secretion in HSCs, we performed an
ELISA for IL-8 and MCP-1. Stimulation of HSCs with
BHKCD40L led to a 10-fold induction of IL-8
secretion and a 2-fold induction of MCP-1 secretion after 18 h in
comparison with BHKptcf-stimulated HSCs
(Fig. 7). The weaker induction of MCP-1
may be explained by the already high baseline MCP-1 secretion of
unstimulated HSCs. Because both IL-8 and MCP-1 genes contain NF-B
and AP-1 binding sites in their promoter, we tested whether inhibition
of these pathways would block the IL-8 and MCP-1 secretion. Ad5IKK2dn
and Ad5TRAF2dn, which both had inhibited IKK/NF-B activation in
HSCs, partially inhibited IL-8 secretion. Inhibition by AdTRAF2dn was
stronger, which may reflect the combined inhibition of IKK and JNK.
Ad5NIKdn and Ad5IKK1dn showed no inhibition of IL-8 secretion in
comparison with Ad5GFP. The induction of MCP-1 secretion was only
affected by Ad5TRAF2dn.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in
different animal models (31, 32). Understanding the
regulation of this cytokine network may therefore be crucial for the
development of future antifibrotic therapies.
In this study, we determined the potential role of CD40, a member of
the TNF superfamily, on human HSCs. The expression of CD40 has been
demonstrated on a variety of mesenchymal cells, and is often increased
under inflammatory conditions. We found a high expression of CD40 not
only on HSCs that were activated in vitro, but also on activated HSCs
in vivo. Our data showed that IFN- strongly up-regulated the
expression of CD40 in vitro, and the high levels of IFN- in liver
fibrosis (33) suggest that this mechanism may account for
the strong expression of CD40 in the fibrotic liver. Our results imply
that CD40 is a important regulator of the inflammatory process in liver
fibrosis. The proinflammatory role of CD40 is supported by two lines of
evidence: 1) CD40 potently activates IKK and JNK in HSCs, and 2) CD40
induces the secretion of chemokines in activated HSCs. Thus, HSCs may
fulfill a novel role in the regulation of immune response in the liver
through CD40.
NF-B is a major pathway that is activated by CD40 and other members
of the TNFR family and has important functions in the regulation of
inflammation. In HSCs, NF-B is up-regulated upon activation,
regulates the expression of proinflammatory genes, and protects
TNF--mediated apoptosis (34, 35). Our study
demonstrates that CD40 induced the activation of IKK and NF-B in
HSCs. Accordingly, CD40 also stimulated the secretion of the
NF-B-dependent chemokines IL-8 and MCP-1. Whereas IL-8 levels were
strongly increased by CD40, the increase of MCP-1 secretion was lower.
The low increase of MCP-1 levels was due to the already high baseline
levels of MCP-1. To address whether CD40 might also up-regulate Th1
cytokines in HSCs, we determined IL-12 levels in HSCs by ELISA. No
secretion of IL-12 (p70) was detected in unstimulated and
CD40-stimulated HSCs, although a weak up-regulation of IL-12 (p40) was
detectable by PCR (data not shown). It therefore seems that HSCs are
not a major source for IL-12 in the liver. Our data showing that TRAF2
and IKK2, but not IKK1, are critical mediators of CD40-induced NF-B
activation are consistent with previous studies that analyzed the role
of TRAF2 and IKK1/IKK2 in CD40- and TNF--induced NF-B activation.
Mice with inactivated TRAF2 showed profound defects in CD40-mediated
NF-B activation (36). The critical role of IKK2, but
not IKK1, in TNF-- and IL-1-mediated NF-B activation was
demonstrated in fibroblasts from mice with inactivated IKK1 and IKK2
(37). Our study extends this predominant role of IKK2 to
CD40-induced NF-B activation. We did not address the function of
TRAF6 in CD40-mediated NF-B activation in HSCs, but TRAF6 seems
another necessary component of this pathway, as demonstrated in mice
with inactivated TRAF6 (38). Surprisingly, NIKdn did not
block the activation of IKK, NF-B-binding activity, and chemokine
secretion in HSCs. Although NIK has been shown to be an important
mediator of NF-B activation, it may not be an essential signaling
component of the CD40-induced NF-B pathway, as demonstrated by a
recent study that found cell-specific differences in the requirement
for NIK in CD40-mediated NF-B activation (39). Our
finding that NIK is also not required for TNF--mediated IKK
activation further underlines the specific signaling requirements in
HSCs and defines an important difference between signaling requirements
of hepatocytes and HSCs.
CD40-induced IKK was capable of phosphorylating a GST-p65 substrate.
The phosphorylation of p65 is believed to be an important mechanism for
the up-regulation of the transcriptional activity of NF-B,
independent of its binding activity. The increased transcriptional
activity after phosphorylation is explained by an enhanced binding of
basal transcription factors such as transcription factor IIB and
TATA-binding protein and transcriptional cofactors to C-terminal
transactivation domains of p65 (40). In our study,
phosphorylation of p65 occurred mainly at position 536, as determined
by the use of different GST-p65 peptides. This residue is highly
conserved among species and has been shown to be phosphorylated upon
TNF- stimulation (30). Previous studies have described
TNF--induced phosphorylation at different residues of p65 inside and
outside the transactivation domain (41, 42). The
contribution of different p65 phosphorylation sites to the
up-regulation of p65 transcription and the possible role of multiple
phosphorylation are largely unknown. Our data demonstrate that IKK2 is
the main mediator of p65 phosphorylation in HSCs. It has been described
that IKK2 mediates p65 phosphorylation (28, 30), but other
studies found that p65 phosphorylation may be mediated by rIKK1
(28, 30) and can occur in the absence of IKK2
(43). These results imply that there is probably a role
for IKK1 or other kinases in p65 phosphorylation dependent on cell type
and stimulus. Altogether, our data suggest that CD40 may induce NF-B
activation via IKK through phosphorylation of both I-B and
p65.
Our data have important implications for the role of CD40 in hepatic
fibrogenesis. We suggest that CD40 is up-regulated on HSCs in vivo in
an inflammatory environment by the action of cytokines, e.g., IFN-.
The activation of CD40 may be mediated by liver-infiltrating
lymphocytes, which are known to express high levels of CD40L
(13), resulting in further amplification of the
inflammatory reaction. These proinflammatory effects may be partially
mediated by an enhanced secretion of IL-8 and MCP-1 after CD40
activation. MCP-1 has been shown to be highly expressed in HSCs and to
mediate the majority of monocyte chemotactic activity of HSCs
(44). IL-8, in contrast, has not been previously
demonstrated to be secreted by HSCs. However, the rat IL-8 homologue
cytokine-induced neutrophil chemoattractant has been shown to be
increased during rat HSC activation and in models of liver disease
(45). As a CXC chemokine, IL-8 is likely to play a role in
the chemotaxis of neutrophils to the liver. This is suggested by the
fact that IL-8 is known to correlate with disease activity in human
alcoholic liver fibrosis, which typically is accompanied by
neutrophilic infiltration (46). We have not assessed the
role of CD40 in protection from apoptosis in this study. It is known
that NF-B protects HSCs from TNF--mediated apoptosis
(35). CD40 up-regulates important antiapoptotic proteins
such as A20 and cellular inhibitor of apoptosis protein 2 in a
NF-B-dependent manner (47, 48) and is therefore likely
to exert cytoprotective effects in HSCs. The potent activation of JNK
by CD40 probably also has a strong impact on HSCs. Currently, the
functions of JNK in HSCs are largely unknown. Possibly, CD40 enhances
the secretion of cytokines and chemokines with AP-1 binding sites via
JNK. Recent experiments in our laboratory have shown an important role
for JNK in the up-regulation of HSC proliferation (manuscript in
preparation, B.S.), suggesting that CD40 may induce HSC proliferation
via JNK.
In conclusion, our study presents evidence for a new role of HSCs in regulating the immune response of the liver through CD40. In HSCs, CD40 activates major proinflammatory pathways through TRAF2 and IKK2, but not NIK. Therefore, CD40 may represent a target for antiinflammatory therapy in liver fibrosis. Blocking the CD40/CD40L pathway has been shown to be highly effective in organ transplantation, including the liver (49, 50), and in this and other settings may have beneficial effects by inhibiting fibrosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David A. Brenner, University of North Carolina, Department of Medicine, CB #7038, Chapel Hill, NC 27599. E-mail address: dab{at}med.unc.edu
3 Abbreviations used in this paper: HSC, hepatic stellate cell; Ad5, adenovirus 5; CD40L, CD40 ligand; BHKCD40L, CD40L-transfected baby hamster kidney cells; BHKptcf, mock-transfected baby hamster kidney cells; dn, dominant negative; GFP, green fluorescent protein; HA, hemagglutinin; IKK, I-B kinase; JNK, c-Jun N-terminal kinase; MCP, monocyte chemoattractant protein; NIK, NF-B-inducing kinase; TRAF, TNFR-associated factor.
4 B. Schnabl, J. C. Olsen, and D. A. Brenner. Telomere shortening induces apoptosis in activated human hepatic stellate cells. Submitted for publication.
Received for publication December 26, 2000. Accepted for publication March 21, 2001.
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