Defending the liver from inflammation

Christian Trautwein

Department of Gastroenterology, Hepatology and Endocrinology, Medizinische

Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.

* To whom correspondence should be sent: Professor Dr. med. C. Trautwein,

Department of Gastroenterology, Hepatology and Endocrinology, Medizinische

Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover

Tel.: +49-511-532-6620, Fax: +49-511-532-5692

Email: Trautwein.Christian@mh-hannover.de

INTRODUCTION:

The liver is involved in different tasks of the body. A very old observation is the

induction of the acute phase response which represents a first line of defense in

order to restrict bacterial growth. Different cytokines have been shown to contribute

to this regulation, however interleukin-6 (IL-6) and tumor necrosis factor (TNF)

have been shown to play a prominent role during this process.

In recent years it became obvious that besides regulating the acute phase

response cytokines like IL-6 and TNF are also involved in regulating different

functions during liver physiology. These include an involvement during liver

regeneration, liver failure, cancer development or glucose metabolism. This article

will cover more specifically the molecular mechanisms of IL-6 and TNF-dependent

signaling during liver regeneration and their role during acute liver failure.

Interleukin-6 dependent signal transduction

Interleukin-6 (IL-6) belongs to a family comprising of IL-6, IL-11, leukaemia

inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotropic factor (CNTF) and

cardiotropin 1 (CT-1). They all need the gp130 molecule for signal transduction

(Taga, et al. 1997, Heinrich et al. 1998). Cytokines of the IL-6 family interact with a

receptor complex on the cell surface. In this complex gp130 is the central molecule

as it is used by several family members for signal transduction. IL-6 first binds the IL-

6 receptor (gp80) and then interacts with gp130. Subsequently dimerisation of two

gp130 molecules activates Janus kinases (Jaks), which phosphorylate specific

tyrosine residues of gp130 and thus activate the SHP2/Erk/Map pathways or the

transcription factors STAT1 and STAT3 (Figure 1) (Taga et al. 1997, Heinrich et al.

1998).

Tumour necrosis factor- (TNF)

TNF signals through two distinct cell surface receptors, TNF-R1 and TNF-R2, of

which TNF-R1 initiates the majority of TNF’s biological activities in hepatocytes.

Binding of TNF to its receptor leads to the release of the inhibitory protein silencer of

death domains (SODD) from TNF-R1’s intracellular domain. This leads to the

recognition of the intracellular TNF-R1 domain by the adapter protein TNF receptor

associated death domain (TRADD), which recruits additional adapter proteins:

receptor-interacting protein (RIP), TNF-R–associated factor 2 (TRAF2), and Fas-

associated death domain (FADD). These proteins then activate distinct signaling

cascades (Figure 2).

FADD recruits procaspase-8 via the so called “death-effector-domain” (DED)

of FADD. Procaspase-8 then becomes activated to caspase-8 via the aggregation of

2 or more procaspase-8 molecules by a self-processing mechanism (Ashkenazi &

Dixit 1998) . Activated caspase-8 has been shown to cleave a cytosolic protein called

p22 Bid to its active form, p15 Bid, which translocates to the mitochondria as an

integral membrane protein (Luo et al. 1998, Li et al. 1998). This effects the

mitochondria in a way that leads to the release of cytochrome c, a 12-kDa protein

which normally functions in the mitochondrial electron transport chain. This process

is accompanied by the so called mitochondrial permeability transition (MPT), an

abrupt increase of permeability of the inner mitochondrial membrane to solute

proteins with a molecular mass of less than 1500 Da (Zoratti et al 1995). After

cytochrome c release, caspases are activated, and the cell undergoes apoptosis.

This occurs through the formation of an “apoptosome”, consisting of cytochrome c,

apoptotic protease activating factor-1 (Apaf-1) and procaspase-9. The apoptosome

then recruits procaspase-3, which is cleaved and activated by the active caspase-9

and released to mediate apoptosis.

RAF2 is upstream of several cascades. It activates cIAP-1 and –2, a mitogen

activated protein kinase kinase kinase (MAPKKK) which ultimately activates c-Jun

NH2-terminal kinase (JNK). Additionally TRAF2 is involved in NF-kB activation. Here

also RIP is required, but it does not need its enzymatic activity (for review see Chen

& Goedell 2002).

Activation of NF-kB by TNF requires a complex network of kinases. First the

IKK complex interacts with TRAF2 and RIP. Upon activation the IKK kinase

phosphorylates I-kB which results in its degradation and as a consequence NF-kB is

released to the nucleus where target gene transcription starts.

The high molecular weight IKK complex that mediates the phosphorylation of

I-B has been purified and characterized. This complex consists of three tightly

associated I-B kinase (IKK) polypeptides: IKK1 (also called IKK) and IKK2 (IKK)

are the catalytic subunits of the kinase complex and have very similar primary

structures with 52% overall similarity (DiDonato et al. 1997; Karin 1997, Regnier et al.

1997). Moreover, it contains a regulatory subunit called NEMO (NF-B Essential

Modulator), IKK or IKKAP-1 (Rothwarf et al. 1998, Yamaoka et al. 1998). In vitro,

IKK1 and IKK2 can form homo- and heterodimers (Zandi et al. 1998). Both IKK1 and

IKK2 are able to phosphorylate I-B in vitro, but IKK2 has a higher kinase activity in

vitro compared with IKK1 (Dehase et al. 1999, Woronicz et al. 1997, Zandi et al.

1997).

The IKK complex phosphorylates I-kBs at the N-terminal domain at two

conserved serines (S32 and S36 in human I-B). After phosphorylation, the I-Bs

undergo a second post-translational modification: polyubiquitination by a cascade of

enzymatic reactions, mediated by the -TrCP-SCF complex (or the E3IkB ubiquitin

ligase complex). This process is followed by the degradation of I-B proteins by the

proteasome, thus releasing NF-B from its inhibitory I-B-binding partner, so it can

translocate to the nucleus and activate transcription of NF-B-dependent target

genes (Karin 1999, Yamamoto & Gaynor 2004). Since the enzymes that catalyze the

ubiquitination of I-B are constitutively active, the only regulated step in NF-B

activation appears to be in most cases the phosphorylation of I-B molecules.

Role of IL-6 during liver regeneration

Shortly after the STAT transcription factors were identified (Zhong et al. 1994),

it became evident that there is transient IL-6-dependent STAT3 activation after partial

hepatectomy, which is restricted to the first hours as in turn its inhibitor SOCS3 is

immediately induced and thus limits its activity (Cressmann et al. 1995, Campbell et

al. 2001, Trautwein et al. 1996). The ultimate proof for the relevance of IL-6 for liver

regeneration came from experiment with IL-6-/- mice. First experiments published by

Taub´s group demonstrated that these animals had a defect in hepatocyte

proliferation after partial hepatectomy. Significantly more of the IL-6 -/- animals died

compared to wt control mice (Cressmann et al. 1996). The relevance of these

findings was further underlined as the defect in liver regeneration found in TNFR-1 -/-

mice could be reverted by IL-6 injection (Yamada et al. 1997). Through these two

findings the hypothesis was raised that IL-6 is an essential factor involved in driving

the resting hepatocyte into the cell cycle.

Further experiments aimed at better defining the pathways activated by IL-6

that are essential for liver regeneration. The most prominent factor activated by IL-6

in hepatocytes is STAT3. Treatment of IL-6 -/- mice after partial hepatectomy with

stem cell factor restored Stat3 activation and DNA-synthesis (Ren et al. 2003). As

STAT3 knockout mice are embryonal lethal (Takeda et al. 1997) conditional knockout

mice with a hepatocyte-specific knockout for STAT3 were used to study the role of IL-

6/gp130-dependent STAT3 activation during liver regeneration. These animals also

showed impairment in liver regeneration resembling the results of IL-6 -/- animals (Li

et al. 2002). Therefore these results suggested that especially the STAT3 pathway

seems required for liver regeneration following partial hepatectomy. However in

these animals there was strong STAT1 activation, which is normally not found after

partial hepatectomy. STAT1 is known to mediate opposite effects to STAT3.

Therefore this experimental setting has major problems to solve the role of STAT3

during liver regeneration.

Blindenbacher et al. (2003) performed a careful study in IL-6 -/- mice to better

define the role of IL-6 during liver regeneration. They tested if IL-6 has a direct impact

on hepatocyte proliferation or body homeostasis. By using intravenous or

subcutaneous IL-6 injection the authors found that the role of IL-6 seems not to be

directly involved in stimulating hepatocyte proliferation, but in maintaining body

homeostasis in order to allow normal liver regeneration. These results were further

confirmed in conditional knockout animals for gp130. These animals showed normal

liver regeneration compared to wt animals (Wüstefeld et al. 2003). However after

LPS-injection – mimicking bacterial infection – more of the gp130 -/- animals died

compared to controls and showed impaired hepatocyte proliferation. Taken together,

the work of these groups indicate that IL-6/gp130 is involved in contributing to liver

regeneration through mechanism that are not directly related to cell cycle control.

At present the pathways which are relevant to mediate this effect are not

completely understood. However in recent years several reports demonstrate that IL-

6 activates anti-apoptotic pathways also in hepatocytes. Earlier experiments by

Kovalovich et al. demonstrated that IL-6 can activate BcL-xL expression and also a

role for activating Akt has been suggested (Kovalich et al. 2001, Streetz et al. 2003).

Therefore these results indicate that IL-6/gp130 might be relevant to directly protect

hepatocytes during cell cycle progression.

Additionally, IL-6 induces pathways involved in mediating immune-dependent

mechanisms. IL-6 via STAT3 is the major cytokine to induce the acute phase

response (APR) in the liver. The APR is also involved in the regulation of other

pathophysiological mechanisms e.g. macrophage activation, interaction with the

complement system (Strey et al. 2003). Besides controlling APR expression, IL-6

contributes to the regulation of the TH1/TH2 response (Betz et al. 1998). Therefore

these IL-6 dependent tasks could also be relevant in contributing to body

homeostasis after partial hepatectomy.

Role of TNF during liver regeneration

NF-B was first identified in the liver as a factor that is rapidly activated within 30

minutes after PH (Cressmann et al. 1994). The importance of NF-B and TNF

signalling was further confirmed by the fact that liver regeneration is defective in

TNF- receptor1 knockout mice that do not show hepatic NF-B activation after PH

(Yamada et al. 1997).

The question remained if NF-B is able to directly promote hepatocyte

proliferation in this model. NF-B has been shown to be able to directly stimulate the

transcription of genes that encode G1-phase cyclins, and a B-site is present within

the cyclin D1 promoter (Guttridge et al. 1999, Hinz et al. 1999). Additionally,

experiments using an adenovirus of non-degradable I-kB superrepressor, which

blocks NF-kB activation, indicated that NF-kB activation after partial hepatectomy is

required for liver regeneration. Animals treated with the virus showed a lack of

hepatocyte proliferation and increased apoptosis (Limuro et al. 1998).

In contrast, Chaisson et al. used transgenic mice that expressed the non-

degradable I-kB superrepressor specifically in hepatocytes, but only 60% of the

hepatocytes expressed the transgene. These mice – in contrast to the adenovirus

experiments - showed normal hepatocyte proliferation after PH (Chaisson et al.

2002). However, both systems, which were used to block NF-kB activation, have

some experimental problems. Therefore at present it is unclear which level of NF-kB

activation is required to allow normal liver regeneration after partial hepatectomy.

TNF also triggers Junkinase (JNK) activity and c-Jun activation during liver

regeneration (Diehl et al. 1994, Westwick et al. 1995). Both factors are essential for

cell cycle progression after partial hepatectomy. Inhibition of JNK activity results in

reduced hepatocyte proliferation and Go/G1 transition of hepatocytes. However no

impact on apoptosis was observed (Schwabe et al. 2003). Conditional knockout mice

for c-Jun have a severe phenotype after partial hepatectomy as half of the mice die,

showed impaired regeneration, increased cell death and lipid accumulation in

hepatocytes (Behrens et al. 2002). Together these results demonstrate that JNK/c-

Jun activation is crucial to stimulate liver regeneration after partial hepatectomy.

Via FADD, TNF can trigger apoptosis via caspase 8 activation. Fas can use

the same pathway. However in contrast to TNF, hepatocytes are more sensitive to

Fas-induced apoptosis as the counterbalancing effect of NF-kB activation is missing

(Galle et al. 1995). During liver regeneration after partial hepatectomy, hepatocytes

are less sensitive to Fas-induced apoptosis. Additionally, Fas-stimulation enhances

hepatocyte proliferation indicating that the FADD/caspase 8 pathway during liver

regeneration induces pro-proliferative effects (Desbarats & Newell 2000).

TNF in hepatocyte injury and acute hepatic failure

Although very different agents can cause hepatocyte injury and fulminant

hepatic failure (FHF), a lot of studies in patients and animal models have strongly

implicated that soluble cell death cytokines such as TNF and Fas ligand (FasL) -

another member of the TNF superfamily - are involved in the induction of apoptosis

and in triggering destruction of the liver, which ultimately leads to hepatic failure.

TNF was originally identified by its capacity to induce hemorrhagic necrosis in

mice tumors (Carswell et al. 1975), but severe side effects led to a failure of its use

as a systemic anticancer chemotherapeutic agent (Kimura et al. 1987, Feinberg et al.

1998). A very prominent effect was the direct cytotoxic role of TNF for human

hepatocytes, resulting in increased levels of serum transaminases and bilirubin.

Since then, many clinical studies have underlined the crucial role of TNF in fulminant

hepatic failure and other liver diseases. TNF participates in many forms of hepatic

pathology, including ischemia/reperfusion injury, alcoholic and viral hepatitis, and

injury through hepatotoxins (Colletti et al. 1990, Felver et al. 1990, Gonzales-Amoro

et al. 1994, Leist et al. 1997). Exogenous TNF induces fulminant liver failure and

hepatocyte apoptosis in combination with other toxins (Leist et al. 1997). TNF serum

levels are clearly elevated in patients with FHF (Muto et al. 1998). In another study, it

was shown that serum TNF levels were significantly higher in patients who died than

in patients who survived (Bird et al. 1990).

We also analysed in more detail the role of TNF in fulminant hepatic failure.

Serum TNF, TNF-R1 and TNF-R2 levels were markedly increased in patients with

fulminant hepatic failure and these changes directly correlated with disease activity.

In explanted livers of patients with FHF, infiltrating mononuclear cells expressed high

amounts of TNF and hepatocytes overexpressed TNF-R1. Moreover, the number of

apoptotic hepatocytes was significantly increased in livers from FHF-patients, and

there was a strong correlation with TNF- expression (Streetz et al. 2000). Thus, it is

very likely that the TNF- system is involved in the pathogenesis of FHF in humans,

and its significance has also been shown in several animal models of hepatic failure,

e.g. in the endotoxin/D-galactosamine (GalN) and the concavalin A (ConA) model

(Pfeffer et al. 1993, Ganter et al. 1995).

First described in 1989 (Trauth et al. 1989), the interaction between Fas

receptor and FasL has become a well characterized extracellular system triggering

apoptosis (Krammer. et al 1999, Galle & Krammer 1998). Hepatocytes constitutively

express Fas (APO-1/CD95). A single-dose of an activating anti-Fas-antibody can

lead to apoptosis and cell death in parenchymal and non-parenchymal liver cells

(Ogasawara et al. 1993, Bait et al. 2000) and there is god evidence that this

mechanism is also important during liver fulminant hepatic failure.

IL-6: a protective cytokine in the context of liver failure?

In terms of apoptosis, a lot of experiments showed a role for gp130 in

promoting antiapoptotic effects in different cell types. Activation of STAT3 in B cells

and human myeloma cells causes activation of antiapoptotic genes such as bcl-2 and

bcl-xl and protects these cells from Fas dependent apoptosis (Catlett-Falcone et al.

1999). Similar results were found in T cells. STAT3 deficient T-cells were severely

impaired in IL-6 induced proliferation which was due to the profound defect in IL-6

mediated prevention of apoptosis. In hepatocytes, IL-6 protects from transforming

growth factor- (TGF-) induced apoptosis by blocking TGF- induced activation of

caspase-3 via rapid tyrosine phosphorylation of phosphatidylinositol 3 kinase (PI 3

kinase) which constitutively activated the protein kinase Akt (Chen et al. 1999).

In humans, there is strong evidence that IL-6 is directly involved in the

pathogenesis of different diseases, including multiple myeloma and congestive heart

disease (Ludwig et al. 1991, Tsutamato et al. 1998). Recently, we analysed the

potential role of IL-6 in the development of acute and chronic liver injury in humans

and examined the pathophysiological basis in animal models. We found a direct

correlation of IL-6 expression in serum and liver tissue with disease progression in

FHF patients. Additionally, we could show abolished acute phase response and an

increased susceptibility to LPS-induced liver injury in mice deficient for functional

gp130 in hepatocytes (Streetz et al. 2003). Therefore, IL-6 withholds a protective

function in hepatic failure.

Role of IL-6 in the Concabavalin A model

Concanavalin A (Con A) is a leptin with high affinity towards the hepatic sinus

(Tiegs et al. 1992). Accumulation of Con A in the hepatic sinus results in the

activation of liver natural killer T (NKT) cells, i.e. NK 1.1 CD4+ CD8- TCR+ and

NK1.1. CD4- CD8- TCR+ , that are essential to trigger the early phase of Con A-

induced liver injury (Takeda et al. 2000, Kaneko et al. 2000). Consecutively CD4-

positive and polymorphonuclear cells are attracted to the hepatic sinus and trigger an

increase of cytokines like TNF, IL2, IFN IL-6, GM-CSF and IL-1 (Trautwein et al.

1998). Con A-induced liver damage resembles liver injury in humans i.e. autoimmune

or viral hepatitis. Therefore this model might be ideal to potentially identify molecular

mechnaims that result in new treatment options als in humans.

TNF and IFN have direct implications for the induction of liver cell injury, as

anti-TNF and anti-IFN antibodies protect from Con A-induced liver injury (Gantner

et al. 1995, Küsters et al. 1996) and IFN and TNF -/- mice are resistant to Con A

induced liver cell damage.

Early results demonstrated that IL-6 might be protective in this model as

treatment of the animals with this cytokine protected from Con A-induced liver injury

(Mizuhara et al. 1994). Our recent experiments further characterised the molecular

mechanisms that are important to confer liver protection. Interestingly, IL-6-

dependent signaling in hepatocytes was essential to protect the animals from liver

injury. Further dissection of the intracellular gp130-dependent pathways in

hepatocytes showed that STAT3 activation directly confers liver protection. Especially

the activation of the acute phase response and the chemokine KC seems to be

involved in order to block Con A-induced liver failure (Klein et al. 2005).

Figure Legends:

Figure 1. Interleukin-6-dependent signaling

On the cell surface Interleukin-6 (IL-6) first interacts with the IL-6 receptor

(IL-6R)/gp80. This complex interacts with gp130 molecules and in turn triggers

intracellular dimerisation. Receptor-bound Janus kinases (JAKs: Jak1/2/Tyk2)

became activated and phosphorylate tyrosines as the intracellular part of gp130. The

phosphorylated tyrosines are essential to activate downstream pathways. While

phosphorylation of the second tyrosine is important to trigger the Ras/Map pathway

via SH2-domain containing protein tyrosine phosphatase 2 (Shp2), the four distal

tyrosines are essential to activate Stat transcription factors.

Figure 2. TNF-dependent signal transduction

Engagement of TNF with its cognate receptor TNF-R1 results in the release of SODD

and formation of a receptor-proximal complex containing the important adapter

proteins TRADD, TRAF2, RIP, and FADD. These adapter proteins in turn recruit

additional key pathway-specific enzymes (for example, caspase-8 and IKK2) to the

TNF-R1 complex, where they become activated and initiate downstream events

leading to apoptosis via caspase 8, NF- B activation involving the IKK-complex, and

Junkinase (JNK) activation.

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