372

HEPAT 01 I :4

Jowi~ul t# Wepa:nlo~~. 1992; 15: 372-377 i’ 1992 Elsevier Science Publishers B.V. All rights reserved. 016%827X:YZ,SO5.00

Interleukin-6 production by peripheral blood monocytes in patients with chronic liver disease and acute viral hepatitis

The in vitro production of the acute-phase mediator interleukin-6 by peripheral blood monocytes derived from patients

with various liver diseases was studied. Compared with healthy controls (n =45; 860 _t92 U/ml, mean i SEM), mono-

cytes from patients with chronic hepatitis B produced significantly lower amounts of interleukin-6 (n = 14; 424 + 126 U/

ml) after stimulation with lipopolysaccharide (~=0.02), whereas monocytes from patients with chronic hepatitis non-

A, non-B secreted normal amounts of interleukin-6 (n= 13; 672+ 151 U/ml; n.s.). In contrast, monocytes of patients

suffering from alcoholic liver cirrhosis (n = 2 2; 1310f153 U/ml) or primary biliary cirrhosis (rz=6; 1450+186 U/ml)

produced higher amounts of interleukin-6 than healthy control individuals (p = 0.03, respectively). Lipopolysaccharide-

stimulated monocytes derived from patients with acute hepatitis A, B and non-A, non-B showed an interleukin-6 production not different from that seen in healthy control individuals and did not experience a discernible change

during the course of the acute disease. These results suggest that the production of the acute-phase mediator interlewkin- 6 varies in chronic liver disease in accordance with various etiologies with a reduced lipopolysaccharide-inducible

interleukin-6 response in chronic hepatitis B and an enhanced response in alcoholic liver cirrhosis and primary biliary cirrhosis.

Key boor&: Interleukin-6 production; Monocytes; Viral hepatitis; Liver cirrhosis

-.

Several abnormalities in cell-mediated immunity have

been demonstrated in patients with chronic liver dis-

ease. Among these the diminished proliferative response

of peripheral blood mononuclear cells towards mitogens

( i,2), impaired concanavalin-A-inducible suppressor T-cell activity (3), decreased natural killer cell activity

(4) and impaired delayed-type hypersensitivity (DTH) reaction (5) are well documented. Acute viral hepatitis

has also been associated with impaired blast trans-

formation (6). a change in the CD4/CDX ratio (7) increased serum concentrations of soluble interleukin-2

receptors (8) and reduced production of lymphokines such as in?erIeukin-1 (9) Although the mechanisms

responsible for these changes are at present unclear,

lymphokines produced by macrophages which exert a regulatory function Oli 9

involved. and& T-cells could be

Interleukin-6 (IL-6). also called interferon-p, (IFN- fir), B- cell stimulatory factor (BSF-2) (IO), hybridoma

growth factor (HGF) (11) or hepatocyte-stimulating

factor (HSF) (12,13), is produced by several cell types

such as monocytes ( 13), fibroblasts ( 14) and endothelial

cells (15). Monocytes seem to be the most important in

this process. IL-6 acts as a pleiotropic cytokine and

leads to a variety of different actions including matura-

tion of B-cells for immunoglobulin synthesis (16), syner-

gistic activation of T-cells with IL-I (17) enhancement

of growth of myeloma cells in an autocrine manner (18)

and induction of the synthesis of acute-phase proteins

by hepatocytes ( 13). These studies indicate that IL-6 may have an impor-

tant role in immunoregulation and inflammation. An

altered production of this monokine may contribute to

the changes in cellular immune regulation which occur

C~~rVJ~jfUkerlcP 10: Christian Miller, M.D.. Division ol Gastroenterology and Hepatology, Department of Medicine IV, University Hospital, 18-20 W%hringer Giirtel. A-1090 Vienna. Austria.

IL-6 PRODUCTION IN LIVER DISEASE 373

in patients with acute and chronic viral hepatitis and

chronic liver diseases of other etiologies. In this study,

therefore, we investigated spontaneous and lipopolysac-

charide-(LPS)-induced IL-6 production in vitro by peri-

pheral blood monocytes of patients suffering from these diseases.

Patients and Methds

Patients

IL-6 production by peripheral blood monocytes

derived from patients with liver disease of different

etiologies was studied as follows:

Chronic her diseme I Table I I

(I) Chroizic hrputifis B: 14 patients (11 males, 3

females: mean age 55 + 12 years). HBsAg was present in

all patients, HBeAg in six patients. In 6 patients chronic hepatitis (3 with chronic active hepatitis, 3 with chronic

persistent hepatitis) was present without histologic signs of cirrhotic changes; in another 8 patients cirrhosis was

diagnosed either by liver biopsies (6 patients) or clinically

(2 patients). when poor coagulation did not allow liver

biopsy.

j_7/ Chronic hepatitis NANB: 13 patients (7 males, 6 females; mean age 54+ 1 P years). In 9 patients chronic

active and four patients chronic persistent hepatitis was

present; in three patients histologic signs of cirrhosis

were observed. Nine of the 13 patients were found to

have antibodies against hepatitis C virus in their serum.

13) rl/coholic cirrhosis: 22 patients (13 males. 9

females; mean age 52 &9 years). The history of each

patient was consistent with severe chronic alcoholism

(> 80 g daily alcohol intake for more than 10 years), but

all patients tested had abstained from alcohol for at

least 4 weeks prior to investigation. Seven patients had

Grade A, 6 Grade B and 9 patients Grade C cirrhosis

according to the Child-Turcotte criteria (19). Diagnosis

was established in 17 patients by liver biopsy. whereas

in the remaining 5 patients the typical combination of

clinical. laboratory and gastroscopic findings, and the

presence of ultrasound-verified ascites, strongly sug-

gested cirrhosis. These patients were not biopsied due

to poor coag:.rlation.

(4) Primary biliary cirrhosis: 6 patients (1 male, 5

females; mean age 52+ 1 I years). The diagnosis was

verified by liver biopsy in all patients. In add.ition, all

patients had antimitochondrial antibodies. Three

patients had histologic Stage II and three patients

Stage III PBC.

The patients investigated did not receive steroids or

other immunosuppressive treatment. All patients were in stable clinical condition without any recent episodes

of fever, infection, upper gastrointestinal bleeding. or renal insufficiency. There was no clinical or laboratory evidence of malnutrition or hepatocellular carcinoma. Serum levels of a-fetoprotein were below 15 ng/ml.

Acute viral hepatitis

Five patients (3 males, 2 females; mean age 34 + 12 years) suffered from acute hepatitis A, 6 patients (4

males, 2 females; mean age 37 + 12 years) from acute hepatitis B, 5 m-t’,-.“+.. )ruriblrrs (3 males, 2 females; mean age 32&g years) had acu_e hepatitis non-A, non-B and 4 patients Epstein-Barr-virus-related acute hepatitis (1

male. 3 females; mean age 20+ 5 years). Diagnosis was

based on characteristic clinical, laboratory and serologi-

cal features. The acute disease was mild to moderately severe. No supportive treatment with substitution of

electrolytes or fluid was necessary and no other therapeu- tic measures were taken. After a variable clinical course all patients recovered from acute hepatitis without com-

plications. None of the patients with hepatitis B or non-

A, non-B became chronic. Three of the patients with acute hepatitis non-A, non-B developed hepatitis C virus

antibodies within 4 months after the acute disease. Blood

for determination of lL-6 production was drawn in

weekly intervals.

Corurol indiciduals

Forty-five healthy individuals (30 males, I5 females; mean age 45 &l I years) with no known liver disease

served as controls.

Macropizage isolatiorz

Peripheral blood mononuclear cells (PBMC) were separated by a buoyant density gradient (20) on Ficoll-

Hypaque (Pharmacia, Uppsala, Sweden) from freshly

drawn heparinized (preservative free heparin, Immu- no A.G., Vienna, Austria) peripheral venous blood,

washed thrice in 0.9% saline and resuspended to l.106

PBMC/ml in RPM1 1640 media (Gibco, Paisley, U.K.) supplemented with 10% human AB serum (Flow Labs..

U.K.), IO0 1U penicillin/ml. 100 pg streptomycin/ml and

2 mM r_-glutamine (Gibco). Five milliliters of the cell

suspension were pipetted onto 100 x 20 mm tissue cul-

ture dishes (Costar, Cambridge, MA, U.S.A.) and incu- bated for I h at 37’C in a humidified atmosphere

containing 5%) C02. Thereafter, non-adherent cells wefe

removed by repeated washing of the plates, whereas adherent cells were gained bjr using a disposable cell

374 C. MijLLER and C.C. ZIELINSKI

scraper (Costar). This procedure resulted in an 85% pure macrophage population, as judged by non-specific esterase staining.

IL-6 production IL-6 production by macrophages (3. 104/ml suspended

in RPM1 1640 medium supplemented with 10% human AB serum, 100 IU penicillin and 100 pg streptomycin) was induced by the addition of 100 ng or 0.1 ng lipopoly- saccharide (LPS) derived from E. co/i 01 I l:B4 (Sigma, St. Louis, MO, U.S.A.). Cells were incubated in 12 x 75 mm round-bottom polystyrene tubes (Falcon 2058, Becton Dickinson, Oxnard, CA, U.S.A.) for 18 h at 37 “C in a humidified atmosphere containing 5% COL. The concentration of 100 ng LPS/ml has been shown to yield maximum IL-6 levels in previous experiments. 1:1 some experiments, unstimulated (without LPS induction) IL-6 production was also studied.

IL-6 assay IL-6 secreted into culture supernatant was assessed

by a bioassay using the murine hybridoma cell line B13.29 clone B9 which is dependent on IL-6 for growth (I I). One hundred microliters of B9 cells (5.104/ml) suspended in RPM1 1640 supplemented 2-mercaptoetha- no1 (5 x IO m) were dispensed in triplicate in 96-well microtiter plates (Costar, Cambridge, MA, U.S.A.) together with IO ~1 of the appropriately diluted culture supernatant tested. Control wells with medium alone and wells containing dilutions of recombinant human IL-6 (Genzyme, MA, U.S.A.) were included to construct a calibration curve. After 18 h incubation at 37 “C, 20 pl of 3H-thymidine (Amersham Int., Amersham, U.K.) was added for 6 h. Thereafter cells were harvested with an automatic cell harvester and the amount of radioactivity incorporated into DNA was measured in a beta-counter. Results were expressed as units IL-6/ml supernatant. Stored (- 20 ‘C) serum samples of the same patients as studied for in vitro IL-6 production were also investi- gated. To remove serum inhibitors of IL-6 activity, serum samples were heat-inactivated (30 min. 56 ‘C).

Statistics Clinical characteristics of patients with chronic liver disease

Data are given as mean f standard error. Statistical analysis was performed by one-way analysis of variance and Student’s t-test.

Diagnosis AST (U’l) Bilirubin :-CT (U/l) k’dl b

Chronic hepatitis B (n = 14)

Chronic hepatitis non-A. non-B (fl= ii)

Alcoholic liver cirrhosis (n = 22)

Primary biliary cirrhosis (n = 6)

86+ IO I.1 + 0.2 41 rf I.5

81 +8 1.3+0.1 45 + 2.0

45 + 7 1.5 + 0.6 83 + 4.2

3956 1.2 & 0.3 75 + 2.1

Results

Spontaneous I L-6 production in chronic liver disease Low amounts of IL-6 were secreted by unstimulated

peripheral blood monocytes into the culture supernatant.

No difference in spontaneous IL-6 production was observed between healthy control individuals, patients with chronic hepatitis B, patients with chronic hepatitis non-A, non-B, patients with primary biliary cirrhosis (Table I). In patients with alcoholic cirrhosis a clear dichotomy in IL-6 production was seen: patients with Child C-stage cirrhosis (n=4) secreted a significantly higher amount of IL-6 into supernatant (885 +64 U/ml) than either the combined group of Child A and B patients (n=5, 120+89U/ml, pcO.001) or healthy controls (n=25, 150+32 U/ml, ~~0.01).

LPS-induced IL-6 production in chronic liver disease

Peripheral blood monocytes of patients with chronic (;-lcr.. a c _.Lir’:, B p;oduced significantly lower amounts of IL- 6 after stimulation with an optimal concentration of LPS in vitro (100 ng/ml), as compared with monocytes derived from healthy control individuals (p= 0.02; Table 2). As also shown in Table 2, monocytes derived from patients with chronic hepatitis non-A, non-B pro- duced normal amounts of IL-6 following stimulation with 100 ng LPS/ml. In contrast to what was seen in patients with chronic hepatitis B, peripheral blood monocytes of patients with alcohol-related cirrhosis and primary biliary cirrhosis secreted significantly higher amounts of IL-6 than healthy controls (p=G.O3, respec- tively; Table 2). When stimulated with a suboptimal LPS-dose (0.1 ng/ml), monocytes of patients with chronic hepatitis B secreted significantly lower amounts of IL-6 (n= 16, 346&94 Uiml) than control individuals (n=40,

730+ 106 U/ml. p< 0.01). No significant difference was found, however, in patients with alcoholic cirrhosis, primary biliary cirrhosis and chronic hepatitis non-A, non-B.

lnj?uence ofdisease stage on LPS-induced IL-6 production

No influence of disease stage on IL-6 secretion by LPS (IO0 ng/ml)-stimulated monocytes could be

TABLE !

Data are given as mean + standard error.

IL-6 PRODUCTION IN LIVER DISEASE 375

TABLE 2

Production of IL-6 (U,‘ml) by 3.10 peripheral blood monocytes after stimulation with 100 ng LPS for 18 h

Diagnosis

Healthy controls Chronic liver disease

lL-6 production (mean f SE) - Spontaneous

n=25 150*32

LPS-induced

II = 45 860 + 92 -

Chronic hepatitis Chronic hepatitis non-A, non-B Alcoholic liver cirrhosis Primary biliary cirrhosis

Acute viral hepatitis (1st week) Hepatitis A Hepatitis B Hepatitis non-A, non-B EBV-related hepatitis

n= II t39* 18 n= 14 424 + 126” n= 7 89k22 n= I3 672 + I51 n= 9 442kl30 n=22 l3lO& i53**

n= 6 96*20 n= 6 1450+186

II= 4 104k21 n= 4 ll60+250 n= 3 s9+ 17 t*= 4 820 * 404 n= 3 95+ 15 n= 4 1020+368 not done n= 4 700 _+ 370

* p = 0.02: ** p = 0.03.

observed in patients with either alcoholic cirrhosis

(100 ng LPS/ml, Child A, n=7, 1368 +232 U/ml;

Child B, n = 6, 1302 + 365: Child C, n = 9, 1260 & 248 U/

ml) or chronic hepatitis non-A, non-B (with cirrhosis,

n=3, 430+ 105 U/ml vs. without cirrhosis, n= 10,

642 + 181 U/ml, n.s.). No significant differences were

observed between patients with chronic hepatitis B

without cirrhosis (n = 6,262 f 34 U/ml) and patients with

cirrhosis (M = 8, 550 f 207 U/ml), although the latter

tended to produce more IL-6 after LPS stimulation. No

significant correlation could be found between paramet- ers of acute liver injury (AST, bilirubin) and LPS-induced

IL-6 production by peripheral blood monocytes in

patients with alcoholic cirrhosis (I= 0.49, p=O.O7 and

r = 0.42, p = 0.15, respectively ).

IL-6 serwn levels

IL-6 activity was undetectable (< 30 U/ml) in sera of

healthy controls (n = 20), patients with chronic hepatitis

B (n = 10) and patients with chronic hepatitis non-A,

non-B (n = 9). Of the I2 patients suffering from alcoholic

cirrhosis whose serum samples were tested, 7 patients had detectable IL-6 activity between 30 and 80 U/ml.

IL-6 prod,uction in acute viral hepatitis LPS (MO ng/ml)-stimulated monocytes derived from

patients with acute hepatitis A, B, or non-A, non-B

produced amounts of IL-6 during the first week after

onset of jaundice that were not different from those seen

in healthy control individuals (Table 2). No change in

LPS-induced IL-6 production cou!d be found during the

first 3 weeks after onset of jaunctice in all 3 forms of acute hepatitis. Patients with Epstein-Barr-virus-related

acute hepatitis (first week, n = 4,700 f 370 U/ml), studied

as disease controls, also had IL-6 production which was

well within normal ranges. Also, spontaneous IL-6 pro-

duction by monocytes from patients with acute hepati- tis A, B and non-A, non-B was not diilIerent from those

in healthy controls (Table 2).

Discussion

In the present study we found impaired in vitro

production of IL-6 by LPS-stimulated peripheral blood

monocytes from patients with chronic hepatitis B, and increased monocyte production in patients with alco-

holic cirrhosis and primary biliary cirrhosis. Ftirther-

more, unstimulated production of IL-6 was enhanced in Child C, but not in other stages of alcoholic cirrhosis

or other chronic liver diseases. LPS-induced IL-6 pro-

duction by monocytes in acute hepatitis A, B, and non-

A, non-B was unchanged during the course of the disease.

The impaired ability of monocytes from patients with chronic hepatitis B to secrete IL-6 after stimulation with

LPS suggests a direct effect of the hepatitis B virus on

IL-6 production. Recently, hepatitis B virus has been

shown to infect not only hepatocytes but also bone marrow cells (21-23) possibly causing a decreased pro-

duction of lymphokines in the infected cells. In fact, an impaired production of IL-l has been demonstrated in

both monocytes which have been naturally infected in

vivo during acute viral hepatitis (9) and monocytes infected with hepatitis B virus in vitro (24). In addition,

a reduced production of other cytokines, such as

interferon-/? (25), interferon-7 (26-283 or the tumour necrosis factor (36) has been found in HBV-infected

blood cells. An HBV-related trans-acting inhibitory

factor, similar to that recently described for the suppres- sion of human interferon-p gene in HBV-infected cells (29) is one mechanism which might lead to impaired IL-

6 production.

376 C. MiiLLER and C.C. ZlELlNSK.1

Normal spontaneous LPS-induced produc.,ion Of IL- 6 in the monocytes of patients with acute viral hepatitis

contrasts with the reduced production of other mono- kines involved in the regulation of acute-phase phen- omena such as IL-l in acute hepatitis A and B.(9) and

the tumour necrosis factor in acute hepatitis B (36). Although we might expect reduced IL-6 production in acute HBV infection similar to that seen in chronic

hepatitis B, this was not observed in our patients.

In contrast to the decreased production of IL-6 in patients with chronic hepatitis B, monocytes derived

from patients with alcoholic cirrhosis and PBC, pro- duced more IL-6 after LPS stimulation than healthy

controls. Our results corroborate the findings of Deviere

et al. (30) and Khoruts et al. (37) who recently described increased IL-6 production in monocytes of patients with

alcoholic cirrhosis. Enhanced production of IL-6 in two disease entities of completely different etiologies indicates

a common mechanism associated with liver disease

per se. In these conditions, monocytes might be in a

preactivated state induced by endotoxin in the circula- tion (31) from the reduction in reticuloendothelial cell

function associated with chronic liver disease. This assumption is further supported by the fact that in

alcoholic cirrhosis spontaneous IL-6 secretion by mono-

cytes .is stage-dependent according to the Child classifi- cation with the highest production occurring in Child

C stage cirrhosis. Dependence on the stage of liver

disease is also seen in chronic hepatitis B, associated with impaired IL-6 production. In this case, monocytes

derived from patients who developed cirrhosis tended to secrete more IL-6 after LPS stimulation than those

derived from patients without cirrhotic changes. Simi-

larly, spontaneous IL-6 production in Child C alcoholic cirrhosis was significantly increased, compared with

Child A and Child B patients. Stage dependency of

spontaneous IL-6 production could not be observed in chronic liver disease of other etiologies, however.

Due to its pleiotropic effects, the altered production

of IL-6 could have several consequences. Since this

cytokine is known to induce the final maturation of B-cells into antibody-producing cells (16), an increase in

production could be one possible co-factor for the

Presence of hypergammaglobulinemia (32) and enhanced immunoglobulin production in vitro (33) in patients with cfrhosis of alcoholic origin or primary biliary cirrhosis.

However, increased IL-6 production is clearly not the

only cause of hypergammaglobulinemia, since chronic hepatitis B is associated with impaired production of

IL-6 and shows this characteristic feature.

IL-6 seems to be the major mediator of the acute-

phase response in infectious diseases and inflammatory processes inducing fever, leukocytosis, depressed zinc

and iron serum levels, and a dramatic increase in the

hepatic synthesis of acute-phase proteins. TNF and IL- I are both potent activators of the acute-phase reaction

and are thought to mediate this via IL-6 (34); a reduced induction of IL-6 production in chronic hepatitis B

might be one of the reasons for the modest acute-phase

response manifested by the absence of fever, leukocytosis,

normal serum iron levels, and normal or only slightly increased acute-phase proteins seen in that condition

(35). In summary, we found a reduced LPS-inducible IL-6

production by peripheral blood monocytes derived from

patients with chronic hepatitis B, whereas IL-6 pro-

duction in alcoholic cirrhosis and primary biliary cirrho-

sis was increased as compared with control monocytes.

These findings are further examples of changes in immu-

noregulation occurring in chronic liver diseases, and also

show that various liver diseases might be associated with

very distinct shifts in certain aspects of the immune

system.

Acknowledgements

The authors are grateful to Ms. W. Kalinowska and Ms. D. Petermann for expert technical assistance.

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