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# Role of FAN in Tumor Necrosis Factor-α and Lipopolysaccharide-induced Interleukin-6 Secretion and Lethality in d-Galactosamine-sensitized Mice

Sophie Malagarie-Cazenave, Bruno Ségui, Sophie Lévêque, Virginie Garcia, Stéphane Carpentier, Marie-Françoise Altié, Anne Brouchet, Valérie Gouazé, Nathalie Andrieu-Abadie, Yara Barreira, et al.

► To cite this version:

Sophie Malagarie-Cazenave, Bruno Ségui, Sophie Lévêque, Virginie Garcia, Stéphane Carpentier, et al. Role of FAN in Tumor Necrosis Factor-α and Lipopolysaccharide-induced Interleukin-6 Secretion and Lethality in d-Galactosamine-sensitized Mice. Journal of Biological Chemistry, 2004, 279 (18), pp.18648 - 18655. 10.1074/jbc.m314294200. inserm-04963650

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# Role of FAN in Tumor Necrosis Factor-α and Lipopolysaccharide-induced Interleukin-6 Secretion and Lethality in D-Galactosamine-sensitized Mice*

Received for publication, December 30, 2003, and in revised form, February 6, 2004
Published, JBC Papers in Press, February 25, 2004, DOI 10.1074/jbc.M314294200

Sophie Malagarie-Cazenave‡, Bruno Ségui, Sophie Lévêque, Virginie Garcia, Stéphane Carpentier, Marie-Françoise Altié, Anne Brouchet, Valérie Gouazé, Nathalie Andrieu-Abadie, Yara Barreira§, Hervé Benoist, and Thierry Levade¶

From the INSERM U.466, Laboratoire de Biochimie and the §Animal Facility, Institut Louis Bugnard, Centre Hospitalier Universitaire Rangueil, 31059 Toulouse, France

Tumor necrosis factor (TNF) α-induced neutral sphingomyelinase-mediated generation of ceramide, a bioactive lipid molecule, is transduced by the adaptor protein FAN, which binds to the intracellular region of the CD120a TNFα receptor. FAN-deficient mice do not exhibit any gross abnormality. To further explore the functions of FAN in vivo and because CD120a-deficient mice are resistant to endotoxin-induced liver failure and lethality, we investigated the susceptibility of FAN-deficient animals to lipopolysaccharide (LPS). We show that after D-galactosamine sensitization, FAN-deficient mice were partially resistant to LPS- and TNFα-induced lethality. Although LPS challenge resulted in a hepatic ceramide content lower in mutant mice than in control animals, it triggered similar histological alterations, caspase activation, and DNA fragmentation in the liver. Interestingly, LPS-induced elevation of IL-6 (but not TNFα) serum concentrations was attenuated in FAN-deficient mice. A less pronounced secretion of IL-6 was also observed after LPS or TNFα treatment of cultured peritoneal macrophages and embryonic fibroblasts isolated from FAN-deficient mice, as well as in human fibroblasts expressing a mutated FAN. Finally, we show that D-galactosamine-sensitized IL-6-deficient mice were partially resistant to endotoxin-induced liver apoptosis and lethality. These findings highlight the role of FAN and IL-6 in the inflammatory response initiated by endotoxin, implicating TNFα.

Tumor necrosis factor (TNF)¹ α is a potent cytokine produced by many cell types that exerts pleiotropic functions, mediating inflammation, immunity, and modulation of cell death, differentiation, and survival (1, 2). As a multifunctional proinflammatory cytokine, TNFα has been implicated in the pathogenesis of a number of human disorders, including sepsis, diabetes,

cancer, multiple sclerosis, and inflammatory diseases (3–5). TNFα elicits its biological effects through binding to two cell surface receptors, a 60-kDa receptor (TNFαRSF1A or CD120a) and a 80-kDa receptor (TNFαRSF1B or CD120b), which belong to a superfamily of proteins regulating host defense, organogenesis, immune, and inflammatory responses (5–8). Most TNFα activities appear to be mediated by CD120a. These are signaled through a complex network of intracellular pathways that involve adapter proteins and lead to activation of the caspase cascade or AP-1 and NF-κB transcription factors (1, 6, 7, 9). The crucial role of the TNFα/CD120a system in host defense and immunity is well illustrated by the observation that mice lacking CD120a are highly susceptible to infections by intracellular pathogens but are protected from acute hepatotoxicity induced by lipopolysaccharide (LPS) after sensitization with the liver-specific transcriptional inhibitor D-galactosamine (10, 11).

In the last decade, sphingolipids have emerged as a novel class of biomodulators that can regulate a wide spectrum of cellular responses (12–14). In particular, sphingomyelin metabolites, i.e. ceramide, sphingosine, and sphingosine 1-phosphate, have been shown to signal some TNFα biological effects (for reviews, see Refs. 15 and 16). Engagement of TNFα with CD120a has been reported to activate both acidic and neutral sphingomyelinases, resulting in sphingomyelin degradation and formation of ceramide (17–20). This lipid molecule and/or its metabolites are believed to mediate various TNFα-induced responses, including differentiation of leukemic cells, IL-6 secretion by osteoblasts, and apoptosis of various cell types (16, 19, 21, 22). In endothelial cells, however, TNFα stimulation of sphingosine kinase is accompanied by cell survival (23).

TNFα-triggered activation of neutral sphingomyelinase is mediated by the adapter protein FAN (factor associated with neutral sphingomyelinase activation), which binds a short motif of the intracellular part of CD120a termed the neutral sphingomyelinase domain (24, 25). FAN is a member of the WD repeat family that is required for neutral sphingomyelinase-mediated generation of ceramide following cell stimulation with TNFα (25) as well as CD40 ligand (26) and cannabinoids (27). Cell culture models have demonstrated that FAN regulates TNFα-induced apoptosis (28) yet does not affect other TNFα effects such as activation of mitogen-activated protein kinases or expression of adhesion molecules (28–30). Mice harboring a targeted disruption of the gene encoding FAN exhibit no gross phenotypic abnormalities but display a delayed (possibly TNFα-mediated) cutaneous barrier repair (29).

To further investigate the physiological functions of FAN and based on the role of CD120a in the inflammatory response

* This work was supported by INSERM. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

‡ Recipient of fellowships from Association pour la Recherche sur la Sclérose en Plaques and Fondation pour la Recherche Médicale.

¶ To whom correspondence should be addressed: INSERM U.466, Laboratoire de Biochimie, Institut Louis Bugnard, CHU Rangueil, 1 avenue Jean Poulhès, TSA 50032, 31059 Toulouse cedex 9, France. Tel.: 33-561-32-20-60; Fax: 33-561-32-20-84; E-mail: levade@toulouse.inserm.fr or levade.t@chu-toulouse.fr.

¹ The abbreviations used are: TNF, tumor necrosis factor; IL-6, interleukin-6; LPS, lipopolysaccharide; NF-κB, nuclear factor κB; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
and liver failure, we have analyzed the response of FAN-deficient mice to LPS or TNFα challenges. Here, we show that after sensitization with D-galactosamine, these knockout animals are partially resistant to LPS- and TNFα-induced lethality.

## EXPERIMENTAL PROCEDURES

**Cytokines and Antibodies**—Recombinant human and murine TNFα were purchased from PeproTech (Tebu-Bio, Le Perray-en-Yvelines, France). Anti-CD95 (clone Jo2) was from Pharmingen (BD Biosciences, Le-Pont-de-Claix, France), and anti-β-actin was from Sigma (L'Isle d'Abeau, France).

**Animals and Treatments**—FAN-deficient (FAN −/−) mice were obtained from FAN +/− embryos (kindly provided by Drs. S. Adam and M. Krönke, University of Kiel, Kiel, Germany) (29) that were implanted in C57BL/6 females (in Centre de Développement des Techniques Avancées; CNRS, Orléans, France). Embryo freezing was done exactly after 10 backcrosses into the C57BL/6 strain. The mice were genotyped by PCR using tail DNA and the following primers: Typ4 (sense), 5'-CCT TAG TTA TCT CGG TGC CAG G-3'; Typ4rev (antisense), 5'-GGA AGC CAC ACT GCT ACA CAG G-3'; and Neo1500, 5'-TCG CCT TCT ATC GCC TCC TTG-3'. Each DNA sample was amplified with primers Typ4 and Typ4rev (giving a 1000-bp product in wild-type and heterozygous mice), and, independently, with all three primers (giving either a 1000-bp product in wild-type mice or a 800-bp fragment in FAN −/− mice). FAN +/− mice obtained from heterozygous breeding pairs served as controls. IL-6-deficient mice, maintained in C57BL/6 background (31), were obtained from Drs. M. Thomsen and F. Bayard (Institut Louis Bugnard, Toulouse, France). All of the animals were housed under specific pathogen-free conditions and maintained on a 12:12 h light-dark cycle with lights on at 07.00. The animal studies were ethically approved and carried out in agreement with national and international policies.

At the age of 7–10 weeks, mice (weighing ~20–25 g) were injected intraperitoneally with a single dose of D-galactosamine (20 mg; Sigma) followed by either intraperitoneal injection of LPS (0.1–10 µg; Sal mo n e l a m i n e s o t a; Sigma) or intravenous injection of murine recombi- nant TNFα (1–10 µg/kg of body weight) in a total volume of 0.1 ml of PBS alone or PBS containing 1% bovine serum albumin, respectively. Alternatively, mice received intraperitoneally only LPS (100 µg) or anti-CD95 antibody (0.36 µg/g of body weight) in PBS. The animals were continuously monitored for survival. Any animal that appeared moribund was euthanized to avoid undue pain and suffering. Blood was collected by retro-orbital puncture for the determination of cytokine concentrations and liver enzyme activities. The mice were sacrificed at designated time points for histology and biochemical studies under anesthesia. The right lateral lobe of the liver was preserved for routine histology, and the remainder of the tissue was immediately frozen in liquid nitrogen.

**Histological Analyses**—Liver specimens were rapidly washed with saline and immediately fixed in PBS containing 4% formaldehyde. Five-µm sections were obtained from paraffin-embedded blocks and stained with hematoxylin-eosin. Liver injury was assessed (by a blinded pathologist) by evaluating lobular necrosis, presence of apoptotic bod- ies, hemorrhage (peliosis hepatis), and portal inflammation.

**Detection of DNA Fragmentation**—Frozen liver specimens (~0.2 g) were homogenized with 1 ml of 50 mM Tris-HCl buffer (pH 8.0) contain- ing 10 mM EDTA, 0.2% SDS, and 0.5 mg of proteinase K/ml. After incubation for 1 h at 50 °C, the lysates were centrifuged for 15,000 × g for 5 min. DNA in the supernatants was extracted using phenol/chlo- roform and treated for 10 min at 37 °C with 10 µg of RNase A to eliminate overlapping bands of RNA. The sample equivalents to 25 µg DNA were subjected to 2% agarose gel electrophoresis, stained with ethidium bromide, and visualized by UV illumination.

**Determination of Caspase Activity**—Effector caspase activity was measured on liver lysates using the fluorogenic substrate Ac-Asp- Glu-Val-Asp-aminomethylcoumarin (Bachem, Voisins-Le-Bretonneux, France) as described (32). Lysates were prepared by homogenizing liver specimens (~100 mg) in 1 ml of ice-cold DEVDase buffer (10 mM Hepes-KOH, pH 7.4, 42 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol, 0.5% CHAPS, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml leupeptin) with a Dounce homogenizer. DEVDase activity was determined by incubating at room temperature for 30 min the 300 × g supernatant with 40 µM of the substrate. Fluorescence intensity was recorded on a Jobin-Yvon spectrofluorometer at 351 and 430 nm for the excitation and emission wavelengths, respectively. Alternatively, caspase-3 cleavage was examined by Western blot using a rabbit anti-caspase-3 antibody that recognizes the cleaved forms (Cell Signaling-Ozyme, St Quentin- en-Yvelines, France).

<caption>TABLE I</caption>

Susceptibility of FAN −/− mice to the lethal effects of LPS

The mice received intraperitoneally D-galactosamine (GalN) and/or LPS, and lethality was monitored up to 72 h. ND, not determined. The differences in 75% survival times of the two animal groups were statis- tically studied by actuarial analysis.

<table><thead><tr><th rowspan="2">LPS</th><th rowspan="2">GalN</th><th colspan="2">Dead vs. injected animals</th><th rowspan="2">p</th></tr><tr><th>FAN +/+</th><th>FAN −/−</th></tr><tr><th>µg/mice</th><th>mg/mice</th><th></th><th></th><th></th></tr></thead><tbody><tr><td>0</td><td>20</td><td>ND<sup>a</sup></td><td>0/2</td><td>ND</td></tr><tr><td>0.1</td><td>20</td><td>5/10</td><td>2/7</td><td>&lt;0.01</td></tr><tr><td>1</td><td>20</td><td>7/9</td><td>4/7</td><td>NS<sup>b</sup></td></tr><tr><td>10</td><td>20</td><td>14/18</td><td>4/15</td><td>&lt;0.01</td></tr><tr><td>10</td><td>0</td><td>ND</td><td>0/2</td><td>ND</td></tr><tr><td>100</td><td>0</td><td>0/6</td><td>0/6</td><td>NS</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>FIG. 1. Mice survival after intraperitoneal injection of LPS (10 µg/mice) and D-galactosamine (20 mg). The number of mice is given in the plot. The log rank test indicated a statistically significant differ- ence (*p* < 0.01) in survival between the two animal groups.</figcaption></figure>

**Lipid Concentration Measurements**—The lipids were extracted with chloroform/methanol from the liver lysates prepared for DEVDase as- say. Aliquots of lipid extracts were used for determining total phospho- lipid and sphingomyelin contents by measuring inorganic phosphorus before and after mild alkaline methanolysis (33). The ceramide content was determined using *Escherichia coli* diacylglycerol kinase (kindly provided by Drs. D. Perry and Y. A. Hannun, Charleston, SC) and [γ-³²P]ATP (6 000 Ci/mmol; PerkinElmer Life Sciences) as previously reported (34).

**Cell Culture**—SV40-transformed fibroblasts from wild-type and FAN −/− mice were kindly provided by Dr. S. Adam (Kiel, Germany) and grown in Dulbecco's modified Eagle's medium containing Glutamax and 10% heat-inactivated fetal calf serum (Invitrogen). Human SV40-transformed skin fibroblasts transfected with an empty vector (pcDNA3) or with a plasmid carrying a truncated version (encoding amino acids 703–917) of the FAN cDNA (pcDNA3-ΔFAN) were obtained as described (28). Transfected cells were maintained in the presence of G418 (0.2 mg/ml). Mouse peritoneal macrophages were collected by lavage of the peritoneal cavity with serum-free medium and then cultured in Macro- phage-SFM medium (Invitrogen). Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. Myco- plasma contamination was checked by PCR using appropriate primers.

**IL-6 and TNFα Assay**—Cultured cells were stimulated under serum- free and nontoxic conditions. Unless otherwise stated, culture medium was collected after a 24-h incubation. The cells were harvested to determine viability and protein content (to ensure that wells contained similar cell numbers). IL-6 in serum or extracellular medium was quantified by enzyme immunoassay using a kit (Immunotech, Mar- seille, France) or a set (Pharmingen) for the human and murine cyto- kine, respectively. Murine TNFα was similarly quantified using an EIA set from Pharmingen.

**Other Determinations**—Serum alanine aminotransferase and aspar- tic aminotransferase activities were determined using an Ektachem 950 automatic analyzer. The cell surface expression of CD120a was assessed by flow cytometry using a rat anti-mouse CD120a antibody conjugated to R-phycioerythrin (Serotec, Cergy, France) on a FACSCali- bur (Becton-Dickinson) cytometer. The protein content was determined using bicinchoninic acid.

<footnote>a ND, not determined.</footnote>

<footnote>b NS, not significant.</footnote>
FIG. 2. Induction of apoptosis in livers of D-galactosamine-sensitized wild-type and FAN-deficient mice treated with LPS. Liver specimens were harvested 5-6 h after LPS (10 µg) injection. A, histological analysis of hematoxylin-eosin stained liver sections taken from wild-type (panels a and c) and FAN-deficient (panels b and d) mice treated with (panels c and d) or without (panels a and b) D-galactosamine (GalN) and LPS. Shown are representative sections from at least 11 mice of each genotype (magnification, ×400). B, DEVDase activity in liver specimens. C, Western blot of cleaved forms of caspase-3. D, analysis of DNA fragmentation. Liver genomic DNA was subjected to agarose gel electrophoresis and stained with ethidium bromide. Lane M, DNA marker. Samples from untreated animals showed no laddering but sometimes a smeary pattern (data not shown). E, hepatic content of ceramide in FAN +/+ and FAN -/- mice. Liver concentration of ceramide was determined using the diacylglycerol kinase assay and is expressed as the ratio to liver sphingomyelin concentration. In control mice, concentrations of sphingomyelin and ceramide averaged 4052 and 190 nmol/g of fresh tissue, respectively (i.e. 104 and 4.9 nmol/µmol of lipid phosphorus). The numbers of mice and the *p* values for statistical analysis are indicated in the plots.

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*Statistical Studies*—The results are expressed as the means ± S.D. and are the averages of at least three values/experiment. The mean values were compared using the Student's *t* test. For the animal studies, Kaplan-Meier survival rates were compared using the log rank test. When 75% or 50% survival times were compared, the Student's *t* test was employed.

## RESULTS

D-Galactosamine-sensitized FAN-deficient Mice Partially Resist LPS—Sensitivity of mice to LPS injection after sensitization with D-galactosamine is known to critically depend on functional TNFα/CD120α signaling (10, 11). Because FAN has been reported to transduce some TNFα signals through CD120α, we tested the susceptibility of FAN-deficient mice to titrated doses of LPS. FAN-deficient and wild-type mice were injected intraperitoneally with varying doses of LPS, and survival was monitored. As previously observed, the mice were unsensitive to LPS (100 µg) in the absence of pretreatment with D-galactosamine (Table I). As indicated in Table I and illustrated in Fig. 1, after sensitization with D-galactosamine FAN-deficient animals were more resistant to the lethal effects of low doses (0.1–10 µg) of LPS than their control counterparts.

Because liver injury is viewed as the primary factor responsible for LPS-induced lethality and because hepatic damage is due to apoptosis of hepatocytes (35, 36), we analyzed histologically and biochemically the livers of FAN-deficient and wild-type mice challenged with D-galactosamine and LPS. No significant genotype-dependent differences could be morphologically observed in the extent of parenchymal damage char-

acterized by apoptosis, caspase activation (as assessed by DEVDase activity or, alternatively, immunoblotting for caspase-3), and DNA fragmentation (Fig. 2). Accordingly, serum alanine aminotransferase and aspartic aminotransferase activities were similarly elevated in the two animal groups (data not shown).

However, the ceramide content of the livers from FAN-deficient mice was lower than that found in the livers from control animals (Fig. 2E), although they were still elevated as compared with untreated mice (the ceramide to sphingomyelin ratio reached 166 and 257% in FAN-deficient and wild-type mice, respectively). This finding is consistent with the notion that FAN is required for CD120α-mediated formation of ceramide via activation of neutral sphingomyelinase (29).

D-Galactosamine-sensitized FAN-deficient Mice Are Also Partially Resistant to TNFα—Because TNFα is considered as a central mediator of the LPS-induced liver injury and lethal toxicity (37–41), we next studied the susceptibility of FAN-deficient mice to the toxic effects of murine TNFα after pretreatment with D-galactosamine. As shown in Fig. 3, FAN knockout mice were less sensitive to rather low doses (2.5 to 5 µg/kg) of TNFα than wild-type mice. This partial resistance was overcome by increasing the TNFα dose (Fig. 3). Moreover, a comparable susceptibility of mutant and control mice to the lethal effects of an anti-CD95 agonist antibody was seen (data not shown), indicating that not all death receptor pathways are affected in FAN-deficient animals. As observed for LPS-in-
<figure><img src="image_9.png" /><figcaption>FIG. 3. Mice survival after intraperitoneal injection of D-galactosamine (20 mg) followed by intravenous injection of murine TNFα (1–10 µg/kg of body weight). The numbers of mice are given in the plots. The log rank test indicated a statistically significant difference (*p* < 0.03) in survival between the two animal groups for the 3.5 µg/kg TNFα dose but not for the other doses. However, actuarial analysis of the 75% (as well as 50%) survival times showed a significant difference (*p* < 0.01) between the two animal groups for the 2.5-, 3.5-, and 5-µg/kg TNFα doses.</figcaption></figure>

duced liver injury, hepatic damage (as assessed by histopathology and measurement of serum alanine aminotransferase and aspartic aminotransferase activities), caspase activation, and DNA fragmentation were essentially the same in FAN-deficient and wild-type mice (data not shown).

Impaired IL-6 Secretion in FAN-deficient Mice—The inflammatory response initiated by LPS and mediated by TNFα implicates the production and release of other cytokines such as IL-6 (6, 42). As a potential mediator of TNFα, ceramide has been shown to stimulate IL-6 secretion (e.g. (22, 43)). Because FAN regulates TNFα-induced ceramide generation, we investigated whether IL-6 secretion can be modulated by FAN. First, we measured IL-6 in the serum of FAN-deficient and wild-type mice that were challenged with LPS. Injection of 10 µg LPS in D-galactosamine-sensitized wild-type mice resulted in a robust IL-6 secretion that peaked at about 2.5 h (Fig. 4B). Serum IL-6 levels were markedly lower (up to 2.6-fold) in FAN-deficient mice. When mice were challenged with 100 µg of LPS alone, serum IL-6 levels were also reduced in the mutant mice (Fig. 5B). Of note, serum TNFα levels were similar in the two animal groups (Figs. 4A and 5A). In the serum from animals injected with TNFα, IL-6 levels in mutant mice were 36 ± 5% lower than in wild-type animals (n = 18 mice), where the levels averaged 480 and 790 pg/ml at 2 and 3.5 h, respectively.

Second, IL-6 secretion by cultured murine cells was assessed. Peritoneal macrophages were isolated from FAN-deficient and wild-type mice and treated with LPS. As shown in Fig. 6A, mutant macrophages secreted less IL-6 than their normal counterparts. IL-6 secretion by FAN-deficient resident peritoneal macrophages was also lower than that of control cells after challenge with 50 ng/ml TNFα (IL-6 secretion was 49 ± 32 and 11 ± 20 pg/ml in wild-type and mutant cells (n = 10 mice; *p* < 0.05). A similar difference was noted on thioglycolate-elicited peritoneal macrophages (data not shown). We then tested murine embryonic fibroblasts treated with TNFα. Again, mutant cells secreted less IL-6 than their normal counterparts (Fig. 6B). Interestingly, human and murine TNFα gave comparable results, indicating that these effects are mediated by CD120a because human TNFα specifically binds murine CD120a (6). Of note, mutant and wild-type murine macrophages and fibroblasts expressed at their surface similar levels of CD120a as demonstrated by flow cytometry (data not shown). Finally, IL-6 secretion by human skin fibroblasts overexpressing a trun-

<figure><img src="image_10.png" /><figcaption>FIG. 4. Serum concentrations of TNFα (A) and IL-6 (B) in FAN +/+ and FAN −/− mice after challenge with D-galactosamine (20 mg) and LPS (10 µg). Cytokine concentrations were determined at the indicated times post-injection. Each value corresponds to the mean ± S.D. of 8–23 mice. The *p* values for statistical analyses are indicated in the plot.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>FIG. 5. Serum concentrations of TNFα (A) and IL-6 (B) in FAN +/+ and FAN −/− mice after challenge with LPS (100 µg) alone. The cytokine concentrations were determined at the indicated times post-injection. Each value corresponds to the mean ± S.D. of at least four mice. The *p* values for statistical analyses are indicated in the plot.</figcaption></figure>

cated, dominant-negative form of FAN was found to be lower than that in control fibroblasts (Fig. 6C). However, FAN-deficient and control cells responded similarly to phorbol 12-myristate 13-acetate (Fig. 6). Collectively, these data suggest that FAN-deficient cells, which generate less ceramide in response to TNFα (28, 29), have an impaired ability to secrete IL-6 when challenged with TNFα or LPS.

D-Galactosamine-sensitized IL-6-deficient Mice Are Partially Resistant to LPS—IL-6 is a multifaceted cytokine with both pro- and anti-inflammatory properties (44). To test whether a defective IL-6 secretion could account for a partial protection against endotoxin under D-galactosamine sensitization conditions, IL-6-deficient mice that are homozygous for a disruption in the second exon of IL-6 gene (31) were challenged with LPS.
<figure><img src="image_12.png" /></figure>

FIG. 6. TNFα- and LPS-induced IL-6 production by murine peritoneal macrophages (A), murine embryonic fibroblasts (B), and human skin fibroblasts (C) transfected with an empty vector (pcDNA3) or a construct encoding a truncated FAN (ΔFAN). The cells were grown in serum-free medium and stimulated for 24 h in the absence (Con) or presence of LPS (10 µg/ml), TNFα (50 ng/ml and 10 ng/ml for the human and murine cytokine, respectively), or phorbol 12-myristate 13-acetate (PMA, 200 nM). IL-6 concentration in the culture medium was measured by immunoassay. The determinations were performed in triplicate on the culture medium of cells from at least three different mice or three independent experiments. The *p* values for statistical analyses are indicated in the plots.

As shown in Fig. 7A, these mutant animals were partially protected from the lethal effects of LPS. Whereas serum IL-6 remained undetectable in these mice (Fig. 7C), serum TNFα levels were comparable with those of control mice (Fig. 7B).

Histologically, liver apoptosis was less prominent in IL-6-deficient mice (Fig. 8A). In addition, DEVDase activation was less pronounced (Fig. 8B) and DNA degradation was strongly attenuated (Fig. 8C) in mice lacking IL-6. Accordingly, serum transaminase activities were lower in mutant animals (data not shown). However, of special interest was the finding that hepatic ceramide content was comparably elevated in control and mutant mice (Fig. 8D; see Fig. 2E for comparison). These observations suggest that, in D-galactosamine-sensitized mice, IL-6 deficiency plays a protective role against the (hepato)toxicity of endotoxin without affecting ceramide production.

DISCUSSION

Acute liver failure (or fulminant hepatitis) can occur in several pathological conditions, including viral hepatitis, sepsis, ischemia, metabolic disorders, and poisoning by various toxins or drugs (45). In mice, these situations have often been mimicked by the administration of endotoxin or TNFα following sensitization with D-galactosamine. TNFα is indeed known to play a pivotal role in liver homeostasis and many of these hepatotoxic processes (37–41, 46), although their underlying signaling pathways remain poorly elucidated. Understanding the signal transduction mechanisms of hepatocellular damage and the lethal effects of LPS or TNFα is therefore of crucial

<figure><img src="image_13.png" /></figure>

FIG. 7. Susceptibility of D-galactosamine-sensitized IL-6-deficient mice to LPS. FAN +/+ animals were used as controls. A, survival of mice after intraperitoneal injection of D-galactosamine (20 mg) and LPS (10 µg). The number of mice is given in the plot. The log rank test indicated a statistically significant difference (*p* < 0.01) in survival between the two animal groups. B and C, the serum concentrations of TNFα (B) and IL-6 (C) in control and IL-6-deficient mice 1 h after challenge with D-galactosamine and LPS. The numbers of mice and *p* values for statistical analyses are indicated in the plots.

importance for developing potential appropriate therapies of liver diseases.

This study addressed the specific contribution of FAN, an adapter protein regulating some cytotoxic effects of TNFα, to the pathogenesis of LPS or TNFα-induced lethality. Evidence is provided that mice with a genetic deletion of FAN are partially resistant to these challenges. In addition, mice lacking FAN (or cells derived thereof) produced less ceramide and IL-6 than their control littermates when challenged with endotoxin, suggesting a connection between IL-6 secretion and responsiveness to LPS. As a matter of fact, IL-6-deficient animals were partially resistant to LPS-induced liver damage and lethality.

Numerous reports have documented the production of the bioactive sphingolipid ceramide in response to TNFα, both in cell culture models and *in vivo* (for a recent review, see Ref. 16). With regard to the potential role of ceramide in liver apoptosis, TNFα has been described to trigger ceramide generation in cultured hepatocytes as well as in the liver of D-galactosamine-sensitized mice (47, 48), and exogenous ceramide elicits hepatocyte apoptosis (49). Hepatic ceramide content is also increased after LPS injection (48, 50), a condition previously reported to result in ceramide accumulation in several other tissues (51, 52). Moreover, ceramide levels are elevated in the hepatocyte nuclei following portal vein branch ligation (53).

Accumulation of ceramide or its metabolic derivative GD3 ganglioside has been proposed to mediate the toxic effects of TNFα or LPS based on the resistance of mice lacking acid sphingomyelinase (48, 51). LPS has even been suggested to mimic ceramide (54). The present study indicates that the ceramide produced by neutral sphingomyelinase, which is regulated by FAN, may also participate to the TNFα or LPS toxicity. Whether the generation of this putative sphingolipid
FIG. 8. Induction of apoptosis in liv-
ers of D-galactosamine-sensitized IL-
6-deficient and control mice treated
with LPS. Liver specimens were har-
vested 5-6 h after LPS (10 µg) injection.
A, histological analysis of hematoxylin-
eosin stained liver sections taken from
control (panels a and c) and IL-6-deficient
(panels b and d) mice treated with (panels
c and d) or without (panels a and b) D-
galactosamine (GalN) and LPS. Shown
are representative sections from at least
11 mice of each genotype (magnification,
×250). B, DEVDase activity in liver spec-
imens. C, analysis of liver DNA fragmen-
tation. D, hepatic content of ceramide, as
expressed as the ratio to liver sphingomy-
elin concentration. The numbers of mice
and *p* values for statistical analyses are
indicated in the plots.

mediator in the liver is specifically and uniquely responsible for
hepatocyte apoptosis and subsequent systemic demise after
injection of endotoxin is not yet established. Rather, the obser-
vation that FAN-deficient mice are partially protected from the
lethal effects of LPS and TNFα despite induction of cell death
in the liver suggests that (neutral sphingomyelinase-derived)
ceramide is only part of the signaling pathways leading to
apoptosis in liver. One could postulate that optimal cell death
induction occurs when both acid and neutral sphingomyeli-
nases are being activated. The fact that ceramide was equally
elevated in the liver of control and IL-6-deficient mice would
suggest that ceramide is not sufficient for the induction of
hepatic apoptosis. Another possibility is that ceramide or its
metabolites regulate other pathways, not exclusively in liver,
that eventually lead to animal death or survival. Interestingly,
elevated ceramide levels in mononuclear cells have been found
to correlate with TNFα levels and severity of sepsis (55), indi-
cating that sphingolipids may also regulate some other aspects
of the inflammatory response.

Because of the important role of TNFα in inflammation,
including secretion of interleukins such as IL-6 (6), and be-
cause the cytotoxic effect and IL-6 production elicited by TNFα
are two closely linked responses (56), we evaluated the role of
FAN in the secretion of IL-6. Experimental evidence is pro-
vided here that FAN can indeed regulate IL-6 secretion. Not
only cultured cells isolated from mice lacking FAN but also
cells overexpressing a dominant-negative form of FAN exhib-
ited a defect in IL-6 secretion. This impairment was also no-

ticed when serum cytokine concentration was measured after
LPS challenge. This suggests that the ceramide produced by
neutral sphingomyelinase can stimulate IL-6 secretion. Either
ceramide or its metabolites have been described to enhance
IL-6 gene expression and protein production in different cell
types, including fibroblasts (43, 57), osteoblasts (22, 58, 59),
epithelial cells (60, 61), and human astrocytoma cells (62). In
some instances, the ceramide metabolite sphingosine 1-phos-
phate was proposed as the mediator for cytokine induction (58).
The fact that FAN (and the neutral sphingomyelinase domain
of CD120a) only partially regulates IL-6 secretion does not
disagree with previous observations indicating that the death
domain of CD120a is sufficient for IL-6 gene induction (63). As
for induction of cytotoxicity (28), it is conceivable that the
neutral sphingomyelinase domain and death domain cooperate
to give a full response.

How sphingolipids and FAN regulate IL-6 secretion is still
unclear. TNFα is known to trigger IL-6 production by increased
NF-κB-mediated gene transcription, the activation of NF-κB
likely implicating mitogen-activated protein kinase, and in par-
ticular p38 (42, 64), as well as reactive oxygen species (65).
However, TNFα-induced activation of the extracellular signal-
regulated kinase type kinases (28–30) and p38² appears inde-
pendent of FAN. Whether NF-κB activation and subsequent
IL-6 secretion is modulated by FAN through production of

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<footnote n="2">² S. Malagarie-Cazenave, B. Ségui, and T. Levade, unpublished observations.</footnote>
reactive oxygen species is currently under investigation. Interestingly, LPS has very recently been reported to stimulate neutral sphingomyelinase activity, which is mandatory for induction of inducible nitric-oxide synthase and NF-κB activation (66).

IL-6 is a multifaceted cytokine that, just like TNFα (2, 67), can show a functional duality in liver, promoting either apoptosis and injury or survival and regeneration (44, 68). The action of TNFα and IL-6 on liver as two-edge swords might be related to the activation state of transcription factors (45). On the one hand, IL-6 can confer protection against some liver insults such as CD95 or concanavalin A. Pretreatment of mice with an antibody to IL-6 prior to administration of LPS or enterotoxin and d-galactosamine enhanced mortality, whereas pretreatment with IL-6 reduced death (69, 70). On the other hand, IL-6 acts as a pro-inflammatory cytokine in the context of endotoxemia and acute liver failure. It is well known that IL-6 serum levels correlate with the severity of sepsis (71, 72). LPS failed to induce fever response in IL-6-deficient mice (73). Furthermore, increased sensitivity to LPS in some mutant mice has been related to high circulating levels of IL-6 (74). IL-6 can also enhance TNFα-induced apoptosis of hepatocytes sensitized by actinomycin D (75). However, IL-6-deficient mice appeared equally sensitive to TNFα even after sensitization with d-galactosamine (76). Hence, the contribution of IL-6 to the animal models of acute liver failure represented by the injection of LPS or TNFα to d-galactosamine-sensitized mice is not clear. Our observations on knockout mice argue for a role of IL-6 in the development of the lethal effects of LPS. Interestingly, whereas IL-6-deficient mice displayed less apoptosis in the liver and survived longer than control animals, the hepatic content of ceramide was equally increased. This strengthens the notion that ceramide generation occurs upstream of IL-6 gene expression and that IL-6 may exert pro-inflammatory effects that ultimately contribute to lethality induced by LPS and d-galactosamine. Whether the liver is the only IL-6 target that mediates lethality needs to be determined.

In summary, FAN appears to contribute to the pathogenesis of the fulminant hepatitis triggered by endotoxin, possibly by regulating IL-6 secretion. Whether ceramide is the only mediator of these effects, whether FAN also regulates the production of other cytokines such as IL-8 or granulocyte/macrophage colony-stimulating factor secreted in response to TNFα (64), and whether FAN modulates other LPS-induced responses than those mediated by TNFα and by which molecular mechanisms remain to be elucidated. Clarifying these issues may help developing novel strategies for treatment of diseases characterized by acute liver dysfunction, implicating TNFα.

*Acknowledgments*—We thank Drs. S. Adam and M. Krönke for providing FAN −/− mouse embryos and Drs. M. Thomsen and F. Bayard for IL-6-deficient mice. The assistance of M. A. Bertrand, C. Nevoit, H. Lulka, J. P. Basile, J. C. Thiers, N. Therville, and P. Clavé is acknowledged.

REFERENCES

1. Chen, G., and Goeddel, D. V. (2002) *Science* **296**, 1634–1635

2. Wajant, H., Pfizenmaier, K., and Scheurich, P. (2003) *Cell Death Differ.* **10**, 45–65

3. Tracey, K. J., and Cerami, A. (1993) *Annu. Rev. Cell Biol.* **9**, 317–343

4. McDermott, M. F. (2001) *Cell. Mol. Biol.* **47**, 619–635

5. Aggarwal, B. B. (2003) *Nat Rev Immunol* **3**, 745–756

6. Vandenabeele, P., Declercq, W., Beyaert, R., and Fiers, W. (1995) *Trends Cell Biol.* **5**, 392–399

7. Wallach, D., Varfolomeev, E. E., Malinin, N. L., Goltsev, Y. V., Kovalenko, A. V., and Boldin, M. P. (1999) *Annu. Rev. Immunol.* **17**, 331–367

8. Locksley, R. M., Killeen, N., and Lenardo, M. J. (2001) *Cell* **104**, 487–501

9. Baud, V., and Karin, M. (2001) *Trends Cell Biol.* **11**, 372–377

10. Pfeffer, K., Matsuyama, T., Kundig, T. M., Wakeham, A., Kishihara, K., Shahinian, A., Wiegmann, K., Ohashi, P. S., Kronke, M., and Mak, T. W. (1993) *Cell* **73**, 457–467

11. Rothe, J., Lesslauer, W., Lotscher, H., Lang, Y., Koebel, P., Kontgen, F., Althage, A., Zinkernagel, R., Steinmetz, M., and Bluethmann, H. (1993)

12. Hannun, Y. (1996) *Science* **274**, 1855–1859

13. Kolesnick, R. N., and Krönke, M. (1998) *Annu. Rev. Physiol.* **60**, 643–665

14. Hannun, Y. A., and Obeid, L. M. (2002) *J. Biol. Chem.* **277**, 25847–25850

15. Kolesnick, R., and Golde, D. W. (1994) *Cell* **77**, 325–328

16. Malagarie-Cazenave, S., Andrieu-Abadie, N., Ségui, B., Gouazé, V., Tardy, C., Cuvillier, O., and Levade, T. (2002) *Exp. Rev. Mol. Med.* **2002**, 1–15

17. Kim, M., Linardic, C., Obeid, L., and Hannun, Y. (1991) *J. Biol. Chem.* **266**, 484–489

18. Dressler, K. A., Mathias, S., and Kolesnick, R. N. (1992) *Science* **255**, 1715–1718

19. Obeid, L. M., Linardic, C. M., Karolak, L. A., and Hannun, Y. A. (1993) *Science* **259**, 1769–1771

20. Wiegmann, K., Schütze, S., Machleidt, T., Witte, D., and Krönke, M. (1994) *Cell* **78**, 1005–1015

21. Adam-Klages, S., Schwandner, R., Adam, D., Kreder, D., Bernardo, K., and Krönke, M. (1998) *J. Leukocyte Biol.* **63**, 678–682

22. Shinoda, J., Kozawa, O., Tokuda, H., and Uematsu, T. (1999) *Cell Signal.* **11**, 435–441

23. Xia, P., Wang, L., Gamble, J. R., and Vadas, M. A. (1999) *J. Biol. Chem.* **274**, 34499–34505

24. Adam, D., Wiegmann, K., Adam-Klages, S., Ruff, A., and Krönke, M. (1996) *J. Biol. Chem.* **271**, 14617–14626

25. Adam-Klages, S., Adam, D., Wiegmann, K., Struve, S., Kolanus, W., Schneider-Mergener, J., and Krönke, M. (1996) *Cell* **66**, 937–947

26. Ségui, B., Andrieu-Abadie, N., Adam-Klages, S., Meilhac, O., Kreder, D., Garcia, V., Bruno, A. P., Jaffrézou, J. P., Salvayre, R., Krönke, M., and Levade, T. (1999) *J. Biol. Chem.* **274**, 37251–37258

27. Sanchez, C., Rueda, D., Segui, B., Galve-Roperh, I., Levade, T., and Guzman, M. (2001) *Mol. Pharmacol.* **59**, 955–959

28. Ségui, B., Cuvillier, O., Adam-Klages, S., Garcia, V., Malagarie-Cazenave, S., Lévèque, S., Caspar-Bauguil, S., Coudert, J., Salvayre, R., Krönke, M., and Levade, T. (2001) *J. Clin. Invest.* **108**, 143–151

29. Kreder, D., Krut, O., Adam-Klages, S., Wiegmann, K., Scherer, G., Plitz, T., Jensen, J. M., Proksch, E., Steinmann, J., Pfeffer, K., and Krönke, M. (1999) *EMBO J.* **18**, 2472–2479

30. Lüschen, S., Adam, D., Ussat, S., Kreder, D., Schneider-Brachert, W., Krönke, M., and Adam-Klages, S. (2000) *Biochem. Biophys. Res. Commun.* **274**, 506–512

31. Kopf, M., Baumann, H., Freer, G., Freudenberg, M., Lamers, M., Kishimoto, T., Zinkernagel, R., Bluethmann, H., and Kohler, G. (1994) *Nature* **368**, 339–342

32. Cuvillier, O., Edsall, L., and Spiegel, S. (2000) *J. Biol. Chem.* **275**, 15691–15700

33. Andrieu, N., Salvayre, R., and Levade, T. (1994) *Biochem. J.* **303**, 341–345

34. Bielawska, A., Perry, D. K., and Hannun, Y. A. (2001) *Anal. Biochem.* **298**, 141–150

35. Leist, M., Gantner, F., Bohlinger, I., Tiegs, G., Germann, P. G., and Wendel, A. (1995) *Am. J. Pathol.* **146**, 1220–1234

36. Jaeschke, H., Fisher, M. A., Lawson, J. A., Simmons, C. A., Farhood, A., and Jones, D. A. (1998) *J. Immunol.* **160**, 3480–3486

37. Lehmann, V., Freudenberg, M. A., and Galanos, C. (1987) *J. Exp. Med.* **165**, 657–663

38. Tiegs, G., Wolter, M., and Wendel, A. (1989) *Biochem. Pharmacol* **38**, 627–631

39. Morikawa, A., Sugiyama, T., Kato, Y., Koide, N., Jiang, G. Z., Takahashi, K., Tamada, Y., and Yokochi, T. (1996) *Infect. Immun.* **64**, 734–738

40. Bradham, C. A., Plumpe, J., Manns, M. P., Brenner, D. A., and Trautwein, C. (1998) *Am. J. Physiol.* **275**, G387–G392

41. Nowak, M., Gaines, G. C., Rosenberg, J., Minter, R., Bahjat, F. R., Rectenly, J., MacKay, S. L., Edwards, C. K., 3rd, and Moldawer, L. L. (2000) *Am. J. Physiol.* **278**, R1202–R1209

42. Vanden Berghe, W., Vermeulen, L., De Wilde, G., De Bosscher, K., Boone, E., and Haegeman, G. (2000) *Biochem. Pharmacol.* **60**, 1185–1195

43. Laulederkind, S. J., Bielawska, A., Raghow, R., Hannun, Y. A., and Ballou, L. R. (1995) *J. Exp. Med.* **182**, 599–604

44. Naka, T., Nishimoto, N., and Kishimoto, T. (2002) *Arthritis Res* **4**, (Suppl. 3) 233–242

45. Galun, E., and Axelrod, J. H. (2002) *Biochim. Biophys. Acta* **1592**, 345–358

46. Streetz, K., Leifeld, L., Grundmann, D., Ramakers, J., Eckert, K., Spengler, U., Brenner, D., Manns, M., and Trautwein, C. (2000) *Gastroenterology* **119**, 446–460

47. Osawa, Y., Banno, Y., Nagaki, M., Nozawa, Y., Moriwaki, H., and Nakashima, S. (2001) *Liver* **21**, 309–319

48. Garcia-Ruiz, C., Colell, A., Mari, M., Morales, A., Calvo, M., Enrich, C., and Fernandez-Checa, J. C. (2003) *J. Clin. Invest.* **111**, 197–208

49. Jones, B. E., Lo, C. R., Srinivasan, A., Valentino, K. L., and Czaja, M. J. (1999) *Hepatology* **30**, 215–222

50. Deaciuc, I. V., Nikolova-Karakashian, M., Fortunato, F., Lee, E. Y., Hill, D. B., and McClain, C. J. (2000) *Alcohol Clin. Exp. Res.* **24**, 1557–1565

51. Haimovitz-Friedman, A., Cordon-Cardo, C., Bayoumy, S., Garzotto, M., McLoughlin, M., Gallily, R., Edwards, C. K., Schuchman, E. H., Fuks, Z., and Kolesnick, R. (1997) *J. Exp. Med.* **186**, 1831–1841

52. Memon, R. A., Holleran, W. M., Moser, A. H., Seki, T., Uchida, Y., Fuller, J., Shigenaga, J. K., Grunfeld, C., and Feingold, K. R. (1998) *Arterioscler. Thromb. Vasc. Biol.* **18**, 1257–1265

53. Tsugane, K., Tamiya-Koizumi, K., Nagino, M., Nimura, Y., and Yoshida, S. (1999) *J. Hepatol.* **31**, 8–17

54. Wright, S. D., and Kolesnick, R. N. (1995) *Immunol. Today* **16**, 297–302

55. Delogu, G., Famularo, G., Amati, F., Signore, L., Antonucci, A., Trinchieri, V., Di Marzio, L., and Cifone, M. G. (1999) *Crit. Care Med.* **27**, 2413–2417

56. Vandevoorde, V., Haegeman, G., and Fiers, W. (1992) *FEBS Lett.* **302**, 235–238

57. Tominaga, K., Kirikae, T., and Nakano, M. (1997) *Mol. Immunol.* **34**, 1147–1156
58. Kozawa, O., Suzuki, A., Kaida, T., Tokuda, H., and Uematsu, T. (1997) *J. Biol. Chem.* **272**, 25099–25104

59. Tokuda, H., Kozawa, O., Harada, A., and Uematsu, T. (1999) *J. Cell. Biochem.* **72**, 262–268

60. Hedlund, M., Svensson, M., Nilsson, A., Duan, R. D., and Svanborg, C. (1996) *J. Exp. Med.* **183**, 1037–1044

61. Hedlund, M., Duan, R. D., Nilsson, A., and Svanborg, C. (1998) *Mol. Microbiol.* **29**, 1297–1306

62. Fiebich, B. L., Lieb, K., Berger, M., and Bauer, J. (1995) *J. Neuroimmunol.* **63**, 207–211

63. Vandevoorde, V., Haegeman, G., and Fiers, W. (1997) *J. Cell Biol.* **137**, 1627–1638

64. Beyaert, R., Cuenda, A., Vanden Berghe, W., Plaisance, S., Lee, J. C., Haegeman, G., Cohen, P., and Fiers, W. (1996) *EMBO J.* **15**, 1914–1923

65. Schulze-Osthoff, K., Beyaert, R., Vandevoorde, V., Haegeman, G., and Fiers, W. (1993) *EMBO J.* **12**, 3095–3104

66. Amtmann, E., Baader, W., and Zoller, M. (2003) *Drugs Exp. Clin. Res.* **29**, 5–13

67. Plumpe, J., Streetz, K., Manns, M. P., and Trautwein, C. (1999) *Ital. J. Gastroenterol. Hepatol.* **31**, 235–243

68. Taub, R. (2003) *J. Clin. Invest.* **112**, 978–980

69. Barton, B. E., and Jackson, J. V. (1993) *Infect. Immun.* **61**, 1496–1499

70. Barton, B. E., Shortall, J., and Jackson, J. V. (1996) *Infect. Immun.* **64**, 714–718

71. Damas, P., Ledoux, D., Nys, M., Vrindts, Y., De Groote, D., Franchimont, P., and Lamy, M. (1992) *Ann. Surg.* **215**, 356–362

72. Casey, L. C., Balk, R. A., and Bone, R. C. (1993) *Ann. Intern. Med.* **119**, 771–778

73. Chai, Z., Gatti, S., Toniatti, C., Poli, V., and Bartfai, T. (1996) *J. Exp. Med.* **183**, 311–316

74. Nakamura, A., Mori, Y., Hagiwara, K., Suzuki, T., Sakakibara, T., Kikuchi, T., Igarashi, T., Ebina, M., Abe, T., Miyazaki, J., Takai, T., and Nukiwa, T. (2003) *J. Exp. Med.* **197**, 669–674

75. Boer, U., Fennekohl, A., and Puschel, G. P. (2003) *J. Hepatol.* **38**, 728–735

76. Libert, C., Takahashi, N., Cauwels, A., Brouckaert, P., Bluethmann, H., and Fiers, W. (1994) *Eur. J. Immunol.* **24**, 2237–2242