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Clinical and Experimental Immunology logoLink to Clinical and Experimental Immunology
. 2009 Jun;156(3):518–527. doi: 10.1111/j.1365-2249.2009.03916.x

An inhibitor of interleukin-6 trans-signalling, sgp130, contributes to impaired acute phase response in human chronic liver disease

A Lemmers *,†,1, T Gustot *,†,1, A Durnez ‡, S Evrard *, C Moreno *,†, E Quertinmont †, V Vercruysse †, P Demetter §, D Franchimont *,†, O Le Moine *,†, A Geerts ‡, J Devière *,†
PMCID: PMC2691982  PMID: 19438606

Abstract

In chronic liver disease, high circulating interleukin (IL)-6 contrasts with a poor acute phase response. We evaluated the impact of liver and circulating IL-6-receptor (IL-6R) forms on IL-6 bioactivity in chronic liver disease. IL-6, soluble IL-6-receptor and sgp130 levels were assayed in plasma from 45 patients with alcoholic liver disease, 84 with hepatitis C virus (HCV) infection undergoing transjugular liver biopsies and 15 healthy subjects. IL-6R mRNA was quantified on liver extracts from 54 patients with alcoholic liver disease with or without cirrhosis and 18 HCV-infected patients. The effect of gp130–Fc on fibrinogen secretion induced by IL-6 trans-signalling was evaluated on hepatocyte cultures. Levels of plasma IL-6 and sgp130, but not soluble IL-6R, increased with the stage of chronic liver disease, and correlated significantly with disease severity. Alcoholic liver disease patients had higher plasma IL-6 levels than hepatitis C, but lower liver IL-6R expression. In alcoholic and HCV-related liver diseases, liver IL-6R expression decreased with advanced fibrosis stage. In vitro, on hepatocytes, gp130–Fc blunted the acute phase response while soluble IL-6R enhanced IL-6 stimulation. In advanced chronic liver disease, high plasma IL-6 is associated with low liver IL-6R expression. This situation enables high plasma sgp130 to act as a major negative regulator of liver IL-6 trans-signalling, as demonstrated functionally here on hepatocytes. This might explain the poor acute phase response induced by IL-6 in chronic liver disease.

Keywords: acute phase response, cirrhosis, hepatic stellate cell, interleukin-6, sgp130

Introduction

Liver cirrhosis is associated with increased susceptibility to bacterial infections, related to the degree of liver dysfunction [1]. In the hepatocyte the induction of C-reactive protein (CRP), which has opsonic functions [2], is regulated mainly by interleukin (IL)-6 [3]. Decompensated cirrhosis is associated with a defective acute phase response, where low CRP and fibrinogen (FBG) are poor predictors of bacterial infection, and contrast with high IL-6 levels [4–6]. The mechanisms of this state of resistance to IL-6 are still unclear. Moreover, the absence of IL-6 signalling increases sepsis-related mortality in a mouse model of chronic cholestasis [7].

To induce signal transduction, IL-6 binds IL-6-receptor (IL-6R) and then interacts with the gp130 subunit. The subsequent dimerization of two gp130 proteins activates the intracellular pathways, resulting in the induction of the acute phase response [8,9]. Soluble forms of IL-6R (sIL-6R) also trigger gp130 dimerization and signalling when complexed with IL-6 [10]. This process is called trans-signalling. Conversely, the soluble form of gp130 (sgp130) binds to sIL-6R/IL-6 complexes and prevents their interactions with membrane-anchored gp130 on target cells [11]. The regulation of IL-6 bioactivity by its soluble receptors in chronic liver diseases has been poorly explored. In this study, we first showed that sgp130 is elevated in the plasma of chronic liver disease patients; we then investigated the cytokine profile of several cell types. Lastly, we tested on hepatocytes our hypothesis that sgp130 blocks the IL-6 trans-signalling-mediated acute phase response.

Patients and methods

Patients

We studied 129 consecutive patients with chronic hepatitis C virus (HCV) infection or alcoholic liver disease, undergoing transjugular liver biopsy. Their clinical characteristics are shown in Table 1. HCV (n = 84) was defined by positive HCV serology and reverse transcription–polymerase chain reaction (RT–PCR). Alcoholic liver disease patients (n = 45) had a history of excessive alcohol ingestion but no other causes of liver disease. Cirrhosis was defined by clinical and biochemical criteria and was biopsy-proven. Fifteen patients with alcoholic cirrhosis (AC) had a severe biopsy-proven alcoholic hepatitis (AH), defined by a modified discriminant function >32 [12].

Table 1.

Characteristics of patients whose plasma was assessed by enzyme-linked immunosorbent assay.

Hepatitis/steatohepatitis (n = 83) Cirrhosis (n = 46)
Age 50 ± 12 52 ± 10
Gender (female/male) 37/46 13/33
Aetiology (HCV/EtOH) 68/15 16/30
Metavir
F0 9 0
F1 17 0
F2 32 0
F3 10 0
F4 0 16
Child–Pugh (A/B/C) _ 13/9/9
Alcoholic hepatitis 0 15
mDF _ 58·3 ± 22·8
Total bilirubin (mg/dl) 0·98 ± 0·72 7·13 ± 10·87**
Albumin (g/dl) 4·03 ± 0·88 3·15 ± 0·76**
Prothrombin time (%) 88 ± 20 56 ± 23**
HVPG (mmHg) 5·88 ± 4·75 18·7 ± 5·42**
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Hepatitis versus cirrhosis:

**

P < 0·01. mDF, modified discriminant function; HCV, hepatitis C virus; HVPG, hepatic venous pressure gradient; EtOH, ethyl alcohol.

We also studied 10 AC patients who were treated for refractory ascites [13] by a functional transjugular intrahepatic portosystemic shunt (TIPS), which reduced their portal hypertension [hepatic venous pressure gradient (HVPG) <12 mmHg], five patients treated by a TIPS that became non-functional (defined by an HVPG relapse to >12 mmHg) and 10 AC patients matched for age and gender. Their clinical characteristics are given in Table 2.

Table 2.

Characteristics of patients of the portal hypertension study.

Alcoholic cirrhosis (n = 10) Alcoholic cirrhosis functional TIPS (n = 10) Alcoholic cirrhosis non-functional TIPS (n = 5)
Age 51(39–59) 52(37–62) 48(46–57)
Gender (female/male) 4/6 4/6 2/3
Total bilirubin (mg/dl) 2·1(0·6–8·1) 3·1(0·6–5) 2·75(1·37–5·7)
Albumin (g/dl) 3·2(2·3–4·2) 3·4(2·6–4) 2·7(2·5–4)
Prothrombin time (%) 65(18–109) 47(22–99) 53(32–64)
HVPG (mmHg) 18·5(12–21) 7(1–10) 20·5(14–23)
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TIPS, transjugular intrahepatic portosystemic shunt; HVPG, hepatic venous pressure gradient.

Blood samples were drawn through a jugular catheter. Plasma was stored at −20°C until assay. A snap-frozen fragment of each liver biopsy was stored at −80°C and the recruitment of more biopsies was performed for the revision of this manuscript. The study was performed after approval by our institution's Ethics Committee, and written informed consent was obtained from each participant.

Liver histology

For HCV, liver fibrosis was evaluated semi-quantitatively (F0–F4) according to the METAVIR score [14]. Alcoholic liver disease cases were classified as cirrhosis on the basis of histological assessment and scored for fibrosis stage using the Kleiner score [15]. AH and alcoholic steatohepatitis (ASH) were defined, respectively, by the presence of satellitosis and the presence of macrovacuolar steatosis combined with portal or lobular infiltration.

Immunoassays

The enzyme-linked immunosorbent assay used to quantify IL-6, sIL-6R, sgp130 (Quantikine; R&D Systems, Abingdon, UK) and FBG (Assaypro, St Charles, MO, USA) had sensitivities of 3·12 pg/ml, 31·2 pg/ml, 0·125 ng/ml and 0·16 µg/ml respectively.

RNA extraction and RT–PCR

Snap-frozen liver biopsies were crushed with a MagNaLyser (Roche Diagnostics, Vilvoorde, Belgium). PolyA-mRNA was extracted using Magnapure (Roche Diagnostics) according to the manufacturer's instructions, including DNase treatment. RNA was quantified using a Lightcycler (Roche Diagnostics) with a one-step real-time RT–PCR (qRT–PCR). The primers and probes were designed with primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA, USA): gp130 sense 5′-AGGACCAAAGATGCCTCAACT-3′; anti-sense 5′-TTGGACAGTGAATGAAGATCG-3′; probe 5′-(6Fam)-TCCTCCTGAAGACACAGCATCCACC-(Tamra)-3′; β-actin sense 5′-GGATGCAGAAGGAGATCACTG-3′; anti-sense 5′-CGATCCACACGGAGTACTTG-3′; probe 5′-(6Fam)-CCCTGGCACCCAGCACAATG-(Tamra)-3′. β-actin was used as a housekeeping gene. For IL-6R, the probe from Applied Biosystems (IL6R Hs00169842_m1) was used. Copy numbers were calculated as described previously [16].

Cell cultures

Hepatocellular carcinoma cells (HepG2; American Type Culture Collection, Manassas, VA, USA; HuH-7 [17] were kindly provided by A. Op De Beeck, Virology, ULB, Brussels, Belgium); 5 × 105 cells/ml were seeded in Dulbecco's modified Eagle's medium (Invitrogen, Merelbeke, Belgium) with 10% fetal bovine serum (FBS) in 24-well plates. After overnight culture, cells were incubated for 24 h in fresh medium containing 5 IU/ml heparin with rhIL-6, rhsIL-6R, gp130–Fc (R&D Systems) and/or vehicle.

Human primary hepatocytes were provided by Lonza (Verviers, Belgium), plated at 3·8 × 105 cells/ml in HCM™ Bulletkit culture medium on a 24-well collagen-coated plate and harvested for RNA extraction after 24 h culture.

Peripheral blood mononuclear cells (PBMC) were isolated on a Ficoll gradient and seeded in 24-well plates at 4 × 106 cells/ml in RPMI-1640 with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, either stimulated or not for 24 h by 200 ng/ml lipopolysaccharide (LPS) (Escherichia coli O55 : B5).

Human hepatic stellate cells (HSC) were isolated from normal liver far away from colon cancer metastasis and characterized as described previously [18,19]. Cells were cultured on plastic dishes (diameter: 35 mm) in standard medium [20]. Experiments were performed on cells between serial passages 3 and 8, while HSC exhibited the morphological features of the ‘myofibroblast-like’ phenotype [20].

William's E, RPMI-1640, penicillin, streptomycin and L-glutamine were purchased from Cambrex (Verviers, Belgium). Insulin, bovine transferrin, γ-aminolevulinic acid, collagen and LPS were purchased from Sigma Aldrich (Bornem, Belgium). All cell culture experiments were performed in duplicate.

Flow cytometry analysis

The HSC, HepG2 and HuH-7 cells were detached with 0·05% trypsin 0.53 mM ethylenediamine tetraacetic acid, washed twice and incubated for 20 min at 4°C with phycoerythrin-labelled anti-IL-6R, gp130 or irrelevant immunoglobulin G1 (BD Biosciences, Erembodegem, Belgium). Data were acquired with a fluorescence activated cell sorter (FACScalibur; Becton Dickinson, Mountain View, CA, USA).

Statistical analysis

Data are expressed as medians (min–max). Multiple comparisons were performed by the Kruskal–Wallis test and post hoc analysis by the Mann–Whitney U-test. Results are shown as box-plots. Correlations were explored using Spearman's rank correlation coefficient. Analyses were performed using spss 11.0 software (SPSS Inc., Chicago, IL, USA).

Results

The sgp130 Plasma levels correlate with disease progression in patients with alcoholic liver disease or chronic HCV infection Figs 1 and 2)

Fig. 1.

Fig. 1

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(a) Circulating interleukin (IL)-6, soluble IL-6 receptor (sIL-6R) and sgp130 levels in 15 healthy subjects (HS) and in 45 patients with alcoholic liver disease, comprising 15 with alcoholic steatohepatitis (ASH) including six with F0–F1 Kleiner fibrosis score and nine F2–F3 fibrosis stage, 15 with alcoholic cirrhosis and 15 with severe alcoholic hepatitis (AH). (b) In 84 HCV infected patients: Metavir fibrosis score F0: n = 9, F1: n = 17, F2: n = 32, F3: n = 10, F4: n = 16; *P < 0·05, **P < 0·01. Results are shown as box-plots. Lines represent median values, the outer limits of the box, the 25th and the 75th percentile values, and the whiskers above and below the boxes the 90th and 10th percentile values.

Fig. 2.

Fig. 2

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Correlations between sgp130 levels and the hepatic venous pressure gradient (HVPG), total bilirubin and the percentage of prothrombin time (PT) in patients with hepatitis C virus (HCV)- or alcohol-induced chronic liver disease. ρ, Spearman's correlation coefficient; P, significance.

The IL-6, as described previously [21], and sgp130 plasma levels were elevated significantly in patients with chronic liver disease compared with healthy subjects (HS; n = 15) (P < 0·05 and P < 0·001 respectively), while no difference in sIL-6R plasma levels was seen. Moreover, the increases in IL-6 and sgp130 were larger in alcoholic liver disease than in HCV patients (P < 0·001 and P < 0·01 respectively). In both liver diseases, IL-6 and sgp130 increased with the histological stage of the disease (Fig. 1). Interestingly, AC patients with severe AH (n = 15) had similar sgp130 levels to those of AC patients without AH (n = 15), but higher IL-6 levels (P < 0·05). If we stratified patients according to the fibrosis stage, in patients with cirrhosis [AC without AH, and not steatohepatitis (ASH)], the plasma IL-6 level was higher than in the HCV F4 stage [AC 16·5 (1·9–287·1) pg/ml versus HCV F4 3·9 (1·5–27·8) pg/ml; P < 0·01], but the sgp130 level did not differ between these groups [AC 613·4 (303·9–804·1) pg/ml versus HCV F4 696 (391·4–904·5) pg/ml; P = 0·093]. In ALD and HCV patients with low fibrosis stages, the IL-6 but not sgp130 plasma level was also higher in ALD than in HCV [IL-6: ALD F0–F1 5·7 (1·7–13·2) pg/ml versus HCV F0–F1 1·5 (1·5–41·3) pg/ml; P < 0·01. sgp130: ALD F0–F1 410 (206·8–756·3) pg/ml versus HCV F0–F1 306 (185·6–472·9) pg/ml; P = 0·097] (data not shown).

Commonly, IL-6 levels increased with the progression of chronic liver disease and correlated with bilirubin levels (ρ = 0·609, P < 0·001), and correlated inversely with albumin levels (ρ = −0·645, P < 0·001) and with the percentage of prothrombin time (ρ = −0·547, P < 0·001). Moreover, plasma IL-6 levels also correlated strongly with portal hypertension measured by HVPG (ρ = 0·6, P < 0·001).

Interestingly, sgp130 levels also increased with the alteration of liver function (Fig. 2) and correlated positively with bilirubin (ρ = 0·616, P < 0·001) and inversely with albumin levels (ρ = −0·388, P < 0·001), and with the percentage of prothrombin time (ρ = −0·553, P < 0·001). Plasma sgp130 levels also correlated strongly with chronic liver disease-induced portal hypertension (ρ = 0·682, P < 0·001).

Increased sgp130 and plasma IL-6 levels are not because of portal hypertension

Portal hypertension itself is associated with an inflammatory process, which may affect the expression of sgp130 [22]. To assess this possible effect, we measured plasma sgp130 and IL-6 levels in AC patients with a TIPS (n = 10) in AC patients matched for age and gender (n = 10) and in AC patients with a TIPS which became non-functional (n = 5). Plasma IL-6 levels did not differ in these groups [21·05 (<3·12–221·23), 8·41 (<3·12–58·84) and 37·41 (<3·12–69·67) pg/ml respectively]. Similarly, plasma sgp130 levels remained high in all three groups (Fig. 3).

Fig. 3.

Fig. 3

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(a) Hepatic venous pressure gradient (HVPG) in 10 alcoholic cirrhosis patients with a functional transjugular intrahepatic portosystemic shunt (TIPS), 10 alcoholic cirrhosis patients matched for age and gender and five alcoholic cirrhosis patients with a non-functional TIPS. Cirrhosis versus functional TIPS: **P < 0·01; functional versus non-functional TIPS: ##P < 0·01. (b) Circulating sgp130 in the same groups of patients. Results are shown as box-plots. Lines represent the median values, the outer limits of the box, the 25th and the 75th percentile values, and the whiskers above and below the boxes, the 90th and 10th percentile values.

Potential cellular origin of IL-6 and sgp130 in chronic liver disease

We investigated two liver-derived cell types, activated HSC (aHSC) and HepG2, as well as circulating PBMCs, to assess their possible contribution to the plasmatic pattern of soluble forms of the IL-6R complex found in chronic liver disease.

Activated HSC produce sgp130 but no sIL-6R in vitro

At confluence, the 24-h culture supernatant of aHSC contained 788 (640–3670) pg/ml sgp130, 3451 (2780–4906) pg/ml IL-6 and <3·12 pg/ml sIL-6R. Similarly, qRT–PCR for the gp130 gene in aHSC disclosed a much higher expression rate than the rate for IL-6R [2·5 × 104 (2·3 × 104–2·9 × 104) versus 39 (23–51) mRNA copies/106β-actin mRNA copies]. Using flow cytometry, we confirmed the absence of IL-6R expression in aHSC, and found low membrane gp130 expression (Fig. 4a).

Fig. 4.

Fig. 4

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Flow cytometry analysis of (a) human hepatic stellate cells (HSC), (b) HepG2 cells and (c) HuH-7 cells interleukin-6 receptor (IL-6R) (left) and gp130 (right) membranous expression. Labelling is compared with control isotype immunoglobulin (Ig)G1 (in shadow). Figures show representative results of two (HSC, HuH-7) or three (HepG2) independent experiments.

HepG2 cells produce sgp130 and sIL-6R spontaneously

The 24-h culture supernatant of HepG2 cells contained 2260 (2140–2300) pg/ml sgp130 and 70·04 (64·56–75·75) pg/ml sIL-6R. Accordingly, qRT–PCR on HepG2 cell extracts revealed 2·0 × 104 (1·9 × 104–2·1 × 104) gp130 mRNA copies/106β-actin copies and many more IL-6R mRNA copies [1·3 × 102 (1·2 × 102–1·3 × 102) copies/106β-actin copies] than in aHSC.

We confirmed the results of qRT–PCR by flow cytometry. On the surface of HepG2 cells, IL-6R expression was weak and gp130 expression strong (Fig. 4b). These data illustrate the gp130high IL-6Rlow phenotype of HepG2 cells. HuH-7 cells had also a gp130high IL-6Rlow phenotype (Fig. 4c). Human primary hepatocytes had similar IL-6R and gp130 expression to HepG2 [3·9 × 102 (3·7 × 102–4·0 × 102) IL-6R mRNA copies/106β-actin copies and 1·3 × 104 (1·2 × 104–1·3 × 104) gp130 mRNA copies/106β-actin copies].

In vitro, PBMCs do not seem responsible for the elevated profile of sgp130 in alcoholic liver disease

As shown previously [23], stimulation of PBMCs from AC patients by LPS produced more IL-6 than the stimulation of PBMCs from HS [AC 185 (73·1–308·5) ng/ml versus HS 96·6 (11·8–179) ng/ml; P < 0·001]. However, the amount of sgp130 produced by such stimulation did not differ in the two groups [AC+LPS 0·7 (0·1–2·2) pg/ml versus HS+LPS 0·5 (0·1–2·2) pg/ml; P = 0·53], and nor did spontaneous sgp130 production by PBMCs [HS 0·1 (0–1) pg/ml versus AC 0·3 (0–1·8) pg/ml; P = 0·19] (Fig. 5).

Fig. 5.

Fig. 5

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(a) Interleukin (IL)-6 and (b) sgp130 levels in 24-h cultures of peripheral blood mononuclear cells (PBMCs) from 11 healthy subjects (HS) and nine patients with alcoholic cirrhosis (AC), with or without stimulation by 200 ng/ml lipopolysaccharide (LPS). HS+LPS versus AC+LPS: **P < 0·01; no stimulation versus LPS: §P < 0·05, §§P < 0·01. Results are shown as box-plots. Lines represent the median values, the outer limits of the box, the 25th and the 75th percentile values; and whiskers above and below the boxes, the 90th and 10th percentile values.

The IL-6 trans-signalling-mediated acute phase response by hepatocytes is blocked in vitro by gp130–Fc fusion protein

The IL-6-induced acute phase response in HepG2 cells was assessed by measuring the FBG in culture supernatants. When sIL-6R was added to culture wells, FBG synthesis was higher with than without sIL-6R for each stimulatory IL-6 concentration. This result showed that HepG2 cells, which express the gp130high IL-6Rlow phenotype, respond to IL-6 trans-signalling (Fig. 6a).

Fig. 6.

Fig. 6

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Acute phase response mediated by interleukin (IL)-6, assessed by fibrinogen (FBG) measurement in supernatants of 24-h cell cultures: (a) HepG2 cells stimulated with increasing doses of recombinant human IL-6 with or without soluble IL-6 receptor (sIL-6R). HepG2 cells (b) or HuH-7cells (c) stimulated with 50 ng/ml recombinant human IL-6, and increasing doses of gp130–Fc fusion protein, with 100 ng/ml sIL-6R (grey bars) or without sIL-6R (black bars). Values are means ± standard error of the mean of three to five experiments. §P = 0·05; *P < 0·05 no sIL-6R versus sIL-6R.

As shown previously in several experimental models, gp130–Fc fusion protein can block IL-6 trans-signalling.[24–26] Here, when gp130–Fc was added to HepG2 culture wells, IL-6-induced FBG synthesis was down-regulated in a dose-dependent manner, but only when sIL-6R was present. This showed that gp130–Fc block IL-6 trans-signalling in gp130high IL-6Rlow phenotype hepatocytes (Fig. 6b). These results were reproduced on other hepatocyte types, namely HuH-7 cells (Fig. 6c)

In advanced chronic liver disease liver IL-6R mRNA expression decreases, but gp130 mRNA expression does not

To explore whether the chronic high IL-6 level in cirrhosis is associated with IL-6R down-regulation in the liver in vivo, we performed qRT–PCR for IL-6R on mRNA extracted from transjugular liver biopsies (alcoholic liver disease n = 54; including 10 steatofibrosis, five steatofibrosis and AH, 19 AC only and 20 with AH, HCV F0–F2 n = 9, HCV F3–F4 n = 9). Liver IL-6R mRNA levels were lower in alcoholic liver disease than in HCV patients [347 (10–1485) versus 751 (221–2490) IL-6R mRNA copies/106 copies β-actin] (P < 0·01), whereas plasma IL-6 levels were higher in alcoholic liver disease than in HCV patients (Fig. 7a). The presence of AH foci among AC liver extracts did not modify IL-6R expression (data not shown). Furthermore, in alcoholic and HCV liver samples, IL-6R expression decreased with fibrosis progression [alcoholic steatofibrosis versus cirrhosis 963 (191–1485) versus 265 (10–1343) P < 0·001; HCV F0–F2 891 (438–2490) versus F3–F4 614 (221–1354) IL-6R mRNA copies/106 copies β-actin; P = 0·11, Fig. 7b]. When comparing high fibrosis stages, alcoholic liver samples IL-6R mRNA expression was lower than in HCV liver disease (AC versus HCV F3–F4; P < 0·05). Consequently, alcoholic liver disease progression is associated with higher plasma IL-6 levels and lower liver IL-6R mRNA levels, compared with early-stage or HCV liver disease, but the same plasma sIL-6R level. Liver gp130 mRNA levels did not differ in alcoholic liver disease with or without cirrhosis and early or advanced HCV disease (Fig. 7c and d).

Fig. 7.

Fig. 7

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Expression of interleukin-6 receptor (IL-6R) (a,b) and gp130 (c,d) genes in mRNA extracted from transjugular liver biopsy fragments from 54 patients with alcoholic liver disease (ALD), including 10 with alcoholic steatofibrosis (SF), five with alcoholic steatofibrosis and alcoholic hepatitis (SF+AH), 19 with alcoholic cirrhosis (AC) only, 20 with AC and alcoholic hepatitis (AC+AH) and 18 hepatitis C virus (HCV)-infected patients (F0–F2: n = 9; F3–F4: n = 9). Data are expressed as the number of gene copies normalized by 106 of β-actin copies. ALD versus HCV (F0–F4): §§P < 0·01; SF±AH versus AC±AH: **P < 0·01; AC±AH versus HCV F3–F4: #P < 0·05. Results are shown as box-plots. Lines represent median values, the outer limits of the box, the 25th and the 75th percentile values; and whiskers above and below the boxes, the 90th and 10th percentile values.

Discussion

This human study revealed that chronic liver disease are associated with certain complex dysregulations in cytokine production and their membrane or soluble receptors expression, which became more obvious with increased severity of the disease. Two major findings emerged from our investigation.

First, like plasma IL-6, the plasma sgp130 level rises in alcoholic liver disease, with higher levels in the cirrhotic stage. For HCV-infected patients, plasma sgp130 increases with the fibrosis score.

Second, gp130–Fc fusion protein can block the IL-6 trans-signalling-dependent acute phase response on human hepatocytes expressing low membrane IL-6R levels. This is of particular interest, as qRT–PCR analysis of liver biopsies of AC has revealed an IL-6Rlow phenotype, arguing for a situation where IL-6 trans-signalling is essential for IL-6 to trigger an acute phase response. Taken together, our results show that sgp130 is probably of critical importance in regulating IL-6 trans-signalling in chronic liver disease.

The role of high plasma IL-6 in chronic liver disease is still unclear. In mice, IL-6/gp130 pathways are protective in terms of acute phase response-mediated bacterial defence [7] and in terms of fibrosis severity in non-parenchymal liver cells [27]. IL-6 induction of the acute phase response has been investigated extensively [28]. We found that ALD patients had higher plasma IL-6 levels than HCV-infected patients, even after stratification for fibrosis score. Two elements might explain these differences. First, monocytes from AC but not HCV-infected patients were shown to be primed to a LPS response with an exacerbation in IL-6 release [23,29]. Second, ethanol itself was incriminated in majoring gut permeability, leading to bacterial translocation [30]. Therefore, the higher plasma IL-6 level of ALD might reflect a higher incidence of bacterial translocation in ALD, which would be clinically relevant in contributing to haemodynamic perturbations associated with liver diseases and with the incidence of spontaneous bacterial peritonitis [31,32].

The gp130 subunit is expressed ubiquitously. We found soluble forms of gp130 in culture supernatants of aHSC, HepG2 and PBMCs. However, unlike IL-6, LPS did not revealed priming of PBMC from AC patients to produce more sgp130 than HS. Although other immune cells such as intrahepatic lymphocytes might release sgp130 in chronic liver disease, our data argue against PBMC as a source of the high plasma sgp130 levels found in vivo in AC. Conversely, in HCV-infected patients, the tendency of the sgp130 level to increase with the fibrosis score suggests a link between the fibrosis process and sgp130 release. Furthermore, we showed that AC patients had similar sgp130 plasma levels even though they underwent portal decompression by TIPS. This is in agreement with the notion of a potential fibrosis-related origin for sgp130 release in chronic liver disease, and we showed in vitro that aHSC are able to secrete it.

Soluble gp130 inhibits the binding of the IL-6/sIL-6R complex to membrane gp130, thus preventing the activation of the subsequent intracellular cascade. Plasma sIL-6R levels did not differ in HS and chronic liver disease patients in our study. This probably means that sIL-6R is not limiting for IL-6 to exert its action by trans-signalling, and that the regulation of this pathway is dependent upon both IL-6 and sgp130. The selective blocking of the IL-6 trans-signalling pathway makes sgp130 a specific negative regulator of IL-6 signalling for cells with low membrane-bound IL-6R levels. On liver biopsies, we found that the level of IL-6R mRNA is lower in chronic liver disease situations, where plasma IL-6 is higher. These findings suggest that in such situations, particularly in AC, when IL-6 increases the liver is more sensitive to trans-signalling. Therefore, circulating sgp130 should be the major regulatory determinant of IL-6 signalling in the liver. Accordingly, the high plasma sgp130 profile in cirrhosis might explain the state of resistance to high IL-6 plasma levels, particularly regarding the most-studied IL-6 effect on liver: acute phase response.

In vitro, IL-6-induced acute phase responses on HepG2 cells rose dramatically when sIL-6R was added to the culture medium, confirming that HepG2 cells respond to IL-6 trans-signalling [33] even though they express low membrane-bound IL-6R. The addition of gp130–Fc blocked the acute phase response only when trans-signalling was involved, thus confirming the results from Jostock et al.[32] by a more physiological experiment protocol.

In conclusion, alcohol- and HCV-induced chronic liver diseases are associated with dysregulations of IL-6 and its soluble receptor production. Chronically increased plasma IL-6 is associated with a down-regulation of liver IL-6R and high plasma sgp130. These dysregulations are concordant with a state in which liver IL-6 signalling is more dependent upon trans-signalling, thus making sgp130 the major determinant of liver IL-6 signalling. Therefore, the rise in plasma sgp130 might largely explain the resistance to IL-6 observed in cirrhotic patients, particularly in terms of defective acute phase response. Accordingly, when developing gp130–Fc therapies for inflammatory disorders, the possible consequences of a blunted acute phase response should be kept in mind, especially in situations where liver IL-6R is down-regulated.

Acknowledgments

A. Lemmers, T. Gustot and D. Franchimont are researchers for the Belgian National Fund for Scientific Research (FNRS), which supported this work. We thank N. Nagy MD for assistance in treating pathological tissues, and I. Henry MD for helping with patient recruitment.

Disclosure

None of the authors has any potential financial conflict of interest related to this manuscript.

References

  • 1.Borzio M, Salerno F, Piantoni L, et al. Bacterial infection in patients with advanced cirrhosis: a multicenter prospective study. Dig Liver Dis. 2001;33:41–8. doi: 10.1016/s1590-8658(01)80134-1. [DOI] [PubMed] [Google Scholar]
  • 2.Black S, Kushner I, Samols D. C-reactive protein. J Biol Chem. 2004;279:48487–90. doi: 10.1074/jbc.R400025200. [DOI] [PubMed] [Google Scholar]
  • 3.Kushner I, Jiang SL, Zhang D, et al. Do post-transcriptional mechanisms participate in induction of C-reactive protein and serum amyloid A by IL-6 and IL-1? Ann NY Acad Sci. 1995;762:102–7. doi: 10.1111/j.1749-6632.1995.tb32318.x. [DOI] [PubMed] [Google Scholar]
  • 4.Le Moine O, Deviere J, Devaster JM, et al. Interleukin-6: an early marker of bacterial infection in decompensated cirrhosis. J Hepatol. 1994;20:819–24. doi: 10.1016/s0168-8278(05)80155-2. [DOI] [PubMed] [Google Scholar]
  • 5.Mackenzie I, Woodhouse J. C-reactive protein concentrations during bacteraemia: a comparison between patients with and without liver dysfunction. Intensive Care Med. 2006;32:1344–51. doi: 10.1007/s00134-006-0251-1. [DOI] [PubMed] [Google Scholar]
  • 6.Park WB, Lee KD, Lee CS, et al. Production of C-reactive protein in Escherichia coli-infected patients with liver dysfunction due to liver cirrhosis. Diagn Microbiol Infect Dis. 2005;51:227–30. doi: 10.1016/j.diagmicrobio.2004.11.014. [DOI] [PubMed] [Google Scholar]
  • 7.Wuestefeld T, Klein C, Streetz KL, et al. Lack of gp130 expression results in more bacterial infection and higher mortality during chronic cholestasis in mice. Hepatology. 2005;42:1082–90. doi: 10.1002/hep.20912. [DOI] [PubMed] [Google Scholar]
  • 8.Heinrich PC, Behrmann I, Müller-Newen G, et al. Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem J. 1998;334:297–314. doi: 10.1042/bj3340297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Luchtefeld M, Schunkert H, Stoll M, et al. Signal transducer of inflammation gp130 modulates atherosclerosis in mice and man. J Exp Med. 2007;204:1935–44. doi: 10.1084/jem.20070120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Rose-John S, Heinrich PC. Soluble receptors for cytokines and growth factors: generation and biological function. Biochem J. 1994;300:281–90. doi: 10.1042/bj3000281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Narazaki M, Yasukawa K, Saito T, et al. Soluble forms of the interleukin-6 signal-transducing receptor component gp130 in human serum possessing a potential to inhibit signals through membrane-anchored gp130. Blood. 1993;82:1120–6. [PubMed] [Google Scholar]
  • 12.Carithers RL, Jr, Herlong HF, Dielh AM, et al. Methylprednisolone therapy in patients with severe alcoholic hepatitis. A randomized multicenter trial. Ann Intern Med. 1989;110:685–90. doi: 10.7326/0003-4819-110-9-685. [DOI] [PubMed] [Google Scholar]
  • 13.Rössle M, Ochs A, Gülberg V, et al. A comparison of paracentesis and transjugular intrahepatic portosystemic shunting in patients with ascitis. N Engl J Med. 2000;342:1701–7. doi: 10.1056/NEJM200006083422303. [DOI] [PubMed] [Google Scholar]
  • 14.The French METAVIR Cooperative Study Group. Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. Hepatology. 1994;20:15–20. [PubMed] [Google Scholar]
  • 15.Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41:1313–21. doi: 10.1002/hep.20701. [DOI] [PubMed] [Google Scholar]
  • 16.Stordeur P, Poulin LF, Cracium L, et al. Cytokine mRNA quantification by real-time PCR. J Immunol Methods. 2002;259:55–64. doi: 10.1016/s0022-1759(01)00489-6. [DOI] [PubMed] [Google Scholar]
  • 17.Nakabayashi H, Taketa K, Miyano K, et al. Growth of human hepatoma cell lines with differentiated functions in chemically defined medium. Cancer Res. 1982;42:3858–63. [PubMed] [Google Scholar]
  • 18.Friedman SL. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies 2. N Engl J Med. 1993;328:1828–35. doi: 10.1056/NEJM199306243282508. [DOI] [PubMed] [Google Scholar]
  • 19.Pinzani M. Novel insights into the biology and physiology of the Ito cell. Pharmacol Ther. 1995;66:387–412. doi: 10.1016/0163-7258(94)00072-b. [DOI] [PubMed] [Google Scholar]
  • 20.Caligiuri A, De Franco RS, Romanelli RG, et al. Antifibrogenic effects of canrenone, an aldosteronic drug, on human hepatic stellate cells. Gastroenterology. 2003;124:504–20. doi: 10.1053/gast.2003.50058. [DOI] [PubMed] [Google Scholar]
  • 21.Deviere J, Content J, Denys C, et al. High interleukin-6 serum levels and increased production by leukocytes in alcoholic liver cirrhosis. Correlation with IgA serum levels and lymphokines production. Clin Exp Immunol. 1989;77:221–5. [PMC free article] [PubMed] [Google Scholar]
  • 22.Lopez-Talavera JC, Merrill WW, Groszmann RJ. Tumor necrosis factor α: a major contributor to the hyper dynamic circulation in prehepatic portal hypertensive rats. Gastroenterology. 1995;108:761–7. doi: 10.1016/0016-5085(95)90449-2. [DOI] [PubMed] [Google Scholar]
  • 23.Devière J, Content J, Denys C, et al. Excessive in vitro bacterial lipopolysaccharide-induced production of monokines in cirrhosis. Hepatology. 1990;11:628–34. doi: 10.1002/hep.1840110416. [DOI] [PubMed] [Google Scholar]
  • 24.Atreya R, Mudter J, Finotto S, et al. Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in Crohn disease and experimental colitis in vivo. Nat Med. 2000;6:583–8. doi: 10.1038/75068. [DOI] [PubMed] [Google Scholar]
  • 25.Nowell MA, Richards PJ, Horiuchi S, et al. Soluble IL-6 receptor governs IL-6 activity in experimental arthritis: blockade of arthritis severity by soluble glycoprotein 130. J Immunol. 2003;171:3202–9. doi: 10.4049/jimmunol.171.6.3202. [DOI] [PubMed] [Google Scholar]
  • 26.Rabe B, Chalaris A, May U, et al. Transgenic blockade of interleukin 6 transsignaling abrogates inflammation. Blood. 2008;111:1021–8. doi: 10.1182/blood-2007-07-102137. [DOI] [PubMed] [Google Scholar]
  • 27.Streetz K, Tacke F, Leifeld L, et al. Interleukin 6/gp130-dependent pathways are protective during chronic liver diseases. Hepatology. 2003;38:218–29. doi: 10.1053/jhep.2003.50268. [DOI] [PubMed] [Google Scholar]
  • 28.Ramadori G, Christ B. Cytokines and the hepatic acute-phase response. Semin Liver Dis. 1999;19:141–55. doi: 10.1055/s-2007-1007106. [DOI] [PubMed] [Google Scholar]
  • 29.Perrin-Cocon L, Agaugué S, Diaz O, et al. Th1 disabled function in response to TLR4 stimulation of monocyte-derived DC from patients chronically infected by hepatitis C virus. PLOS One. 2008;3:e2260. doi: 10.1371/journal.pone.0002260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Rao RK, Seth A, Sheth P. Recent advances in alcoholic liver disease. Role of intestinal permeability and endotoxemia in alcoholic liver disease. Am J Physiol Gastrointest Liver Physiol. 2004;286:G881–4. doi: 10.1152/ajpgi.00006.2004. [DOI] [PubMed] [Google Scholar]
  • 31.Albillos A, de la Hera A, Gonzalez M, et al. Increased lipopolysaccharide binding protein in cirrhotic patients with marked immune and hemodynamic derangement. Hepatology. 2003;37:208–17. doi: 10.1053/jhep.2003.50038. [DOI] [PubMed] [Google Scholar]
  • 32.Guarner C, Soriano G. Bacterial translocation and its consequences in patients with cirrhosis. Eur J Gastroenterol Hepatol. 2005;17:27–31. doi: 10.1097/00042737-200501000-00006. [DOI] [PubMed] [Google Scholar]
  • 33.Jostock T, Müllberg J, Özbek S, et al. Soluble gp130 is the natural inhibitor of soluble interleukin-6 receptor transsignaling responses. Eur J Biochem. 2001;268:160–7. doi: 10.1046/j.1432-1327.2001.01867.x. [DOI] [PubMed] [Google Scholar]

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