The effects of prednisolone and interferons on serum macrophagecolony stimulating factor concentrations in chronic hepatitis B
Yoshito Itoh, Takeshi Okanoue, Shinichi Sakamoto, Kenichi Nishioji and Kei Kashima
Third Department of internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan

Backgrounds/Aims: Serum concentrations of macrophage-colony stimulating factor (M-CSF) are
increased in parallel with hepatic inihunmation.
The aim of this study was to assess the immunologic significance of elevated M-CSF in patientswith chronic hepatitis B virus infection.
Methods: The subjects included 20 asymptomatic
HBV carriers and 45 patients with chronic hepatitis B, including 8 undergoing prednisolone treatment, 10 experiencing an acute exacerbation, and
12 undergoing daily administration of interferons.
Results: Serum concentrations of M-CSF significantly decreased during prednisolone administrabut
significantly
increasedfollowingtion,
prednisolone withdrawal, similar to the increaseduringacute exacerbation.
Changesin thelipopolysaccharide-stimulated
production of interlet&in-l-beta and tumor necrosis factor-alpha byperipheral whole blood, or of interferon-gamma
by peripheral blood mononuclear cells showed asimilar pattern. Serum concentrations of M-CSF
did not correlate with the titers of HBV-DNA or
HBV-DNA polymerase activity. However, serum
M-CSF peaked preceding seroconversion to HBeantibody in three HBe antigen positive patients.
Exogenous interferon-alpha,-beta, or -gamma
induced significant elevation in serum M-CSF concentrations, irrespective of changes in the serum
alanine aminotransferase levels.
Conchsions:
Increased serum M-CSF is closely
associated with increased serum interferons and/or proinflammatory cytokines produced by peripheral blood cells during hepatic inflammation inchronic hepatitis B. This may be a consequence of
the altered cytokine cascade resulting from thehost immune response against hepatitis B virus.

Halso stimulates liver macrophages to secrete tumor
necrosis factor (TNF), and induces chemotaxis bymonocytes (6,7). M-CSF also augments monocyteendothelium interactions through increased expression of monocyte chemotactic protein-l on endothelium (8).
The pleiotropic effects of M-CSF are mediated bya receptor on the surface of macrophages/monocytes,
which is identical to the c-fms proto-oncogene product (9). Although the main source of M-CSF in thecirculation is not well understood (lo), many types ofcells have been found to secrete M-CSF, including
endothelial cells, stroma cells, and fibroblasts (1,lO).
M-CSF mRNA is expressed in various organs, suchas liver, brain, and heart (11). Blood monocytes also
secrete M-CSF in vitro when they adhere to plastic
dishes in vitro (12) or in response to cytokines such

macrophage-colony
stimulating factor (MCSF) is a growth factor of cells in the mononuclear phagocyte lineage (1). The human M-CSF geneis located in the short arm of chromosome 1, band
p13-p21 and its 4.0 kb cytoplasmic n-RNA encodes
an 85 kDa homodimeric bioactive M-CSF protein(1,2). M-CSF acts not only as a specific growth anddifferentiation factor for progenitors in the bone marrow, but also is required for the proliferation and differentiation of mature monocytes and tissue macrophages, including Kupffer cells in the liver (3-5). It
UMAN

Received 2 April; revised II June; acc@ed

19 June 1996

Correspondence: Takeshi Okanoue, M.D., Third Department of Internal Medicine, Kyoto Prefectural University of
Medicine, Kawaramachi-Hirokoji
Kamigyou-ku,
Kyoto,
602, Japan. Tel: 81-75-251-5519. Fax: 81-75-251-0710.

244

Key words: Hepatitis B; Interferons; Macrophagecolony stimulating factor; Proinflammatory
cytokines.

M-CSF in hepatitis B

as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or granulocyte macrophage-colony stimulating factor (GM-CSF) (13-15).
In contrast to other growth factors for monocytes,
such as interleukin-3 or GM-CSF, circulating concentrations of M-CSF are constantly detectable in theserum (16-19). We have shown previously that theserum concentration of M-CSF rises in parallel withthe serum alanine aminotransferase level in viral hepatitis (19).
In the present study, to assess the immunologic
significance of the increase in serum M-CSF concentrations in hepatic inflammation, we examined theeffects of prednisolone (PSL), interferon-alpha, (IFNalpha), interferon-beta(IFN-beta), and IFN-gammaon the serum M-CSF concentration in patients withchronic hepatitis B. We also investigated the production of proinflammatory
cytokines, such as interlet&in- l-beta (IL-l-beta) and TNF-alpha by peripheral whole blood, or the production of IFN-gamma by
peripheral blood mononuclear cells (PBMCs). To
clarify the correlation between the serum concentration of M-CSF and the status of hepatitis B virus(HBV) infection, the titers of HBV-DNA and HBVDNA polymerase activity and the presence of hepatitis B e antigen (HBeAg)/hepatitis
B e antibody(HBeAb) in the serum were also examined.

Materials and Methods
Patients and sera
Sixty-five symptomatic or asymptomatic hepatitis B
surface antigen (I-IBsAg) carriers (37 male and 28
female), ranging from 9 to 63 years old, were enteredinto this study. Informed consent was obtained fromeach patient and this study was approved by thehuman research committee of the hospital. The clinical and laboratory characteristics of the patients are
summarized in Table 1. The presence of HBsAg andthe predominance of HBeAg/HBeAb were assessedby commerciallyavailable radioimmunoassay
kits(Abbott Laboratories, N Chicago, IL, USA). The titerof serum HBV-DNA was quantitated using HBVDNA probe assay (20) and HBV-DNA polymerase
activity was measured by the method of Kaplan et al.
(21). All the patients were negative by second-generation assay for anti-HCV antibody (Abbott). Patientswith a history of significant disease except forchronic hepatitis B were excluded from this study.
None of the patients received any significant treatment for at least 6 months prior to entering this study,
except for water, electrolytes,
and vitamins. The
asymptomatic
HBV carriers (HBV ASC) were
defined as patients with HBsAg showing no abnor-

mal blood chemistries for at least a year. Liver biopsy
was performed in patients showing abnormal liver
tests except for five patients with severe disease prior
to entering this study. Histologic diagnoses weremade according to the previously published criteriaof Desmet et al. (22). Although liver biopsy was not
performed in five patients with severe chronic hepatitis B, their high ALT levels (more than 500 IU/l) confirmed the diagnosis. In this study, we used the term
“acute exacerbation” to mean the state of hepatitis
showing serum ALT levels more than 400 IU/l (morethan IO-fold as compared with the upper limit of normal range).
Whole blood was drawn with a sterile syringe,transferred to a centrifuge tube, allowed to clot for 30
min at room temperature and centrifuged at 1500 g
for 15 min at 4°C. One portion of the serum was used
for serologic, virologic, and biochemical examinations, and 1 ml ahquots were stored in microtubes at-20°C to be used in radioimmunoassay for M-CSF.
Treatment with PSL and IFN
Eight patients with HBeAg positive mild or moderatechronic hepatitis B received PSL. Oral administrationof PSL was given at a daily dose of 40 mg for 3
weeks and then withdrawn, or 40 mg for the first
week, 30 mg for the second week, 20 mg for the third
week, then withdrawn. Phlebotomy was performed
just prior to, 2 weeks after, and at the end of PSL
administration, then 2 and 4 weeks following withdrawal of PSL. Twelve patients with HBeAg positive
mild or moderate chronic hepatitis B received IFNs.
Three million units of IFN-alpha (Sumitomo Pharm.
Co., Osaka, Japan) were administered daily for 4
weeks intramuscularly in six patients. Three million
units of IFN-beta (Mochida Pharm. Co., Tokyo,
Japan) were administered daily for 4 weeks intravascularly in three patients. One million units of IFNgamma (Otsuka Pharm. Co., Tokyo, Japan) was
administered daily for 7 days intramuscularly in threepatients prior to initiating IFN-alpha therapy for 4
weeks. Phlebotomy was performed just prior to, and1, 3, 5, and 7 days after administration of IFNs.
Production of proinjlammatory cytokines by
peripheral blood
Peripheral blood was drawn into sterile syringes(Term0 Inc., Tokyo, Japan) containing 20 U heparin/
ml (Green Cross Inc., Tokyo, Japan) and used for thefollowing experiments. The production of IL- 1-beta
and TNF-alpha was examined using the whole blood
induction system as we have reported previously(23). In brief, 100 ~1 of whole blood was plated into245

Y. ltoh et al.

24-well dishes (Corning Glass Works, Corning, N.Y.,
USA) containing 1 ml of complete RPM1 1640
medium (Nissui Pharm. Co. Ltd., Tokyo, Japan) buffered with NaHCO,, and supplemented
with Lglutamine (Nissui Pharm.Co., Japan), 100 U/ml penicillin, 100 mu/ml streptomycin(Sigma Co., St.
Louis. MO, USA) and 10 yglml of lipopolysaccharide (LPS from E. coli 026: B6, Difco Co., Detroit,
MI, USA), then incubated for 24 h. The supernatants
were aspirated and stored at -20°C until assay.
Because the production of IFN-gamma by peripheral whole blood was undetectable in our assay system (23), we measured the production of IFN-gamma
by peripheral blood mononuclear cells (PBMCs) inthe presenceof the patients’sera. In brief,
heparinized whole blood was diluted with phosphatebuffered saline. The PBMCs then were prepared bygradient centrifugation using “Lymphoprep”(Nyegaard, Oslo, Norway). PBMCs thus obtained were
resuspended in complete RPM1 1640 medium at adensity of 1x1 O6 cells/ml containing 10 p,g/ml of
LPS. An aliquot of 1 ml supplemented with 100 ~1 ofsera from the patients was incubated in 24-well culture dishes for 24 h. The supernatants then were aspirated and stored.
Radioimmunoassay and enzyme-linked immunosorbent assay
Radioimmunoassayof M-CSF was performed asreported previously (19). In brief, duplicate samplesof the patients’ sera were mixed with 1251-labeled
recombinant human (rh) M-CSF and rabbit antiserum
against rhM-CSF, then incubated for 48 h at 37°C.
The bound product was separated from free 1251labeled rhM-CSF by the addition of goat anti-rabbit
IgG and 6% polyethylene
glycol. The tubes wereshaken and then centrifuged at 1000 g for 15 min at
4°C. The supernatants were aspirated and the precipitates were counted for 1 min in an automated gamma
spectrometer. The detection limit for M-CSF was 0.1
ng/ml in this study. Irma- and inter-assay variations
were less than 10%.
The concentrations of IL-l-beta, TNF-alpha, andIFN-gamma were measured by essentially the same
method as previously reported (24). In brief, using a
96-well microplate (Nunc, Napervillem, IL, USA)duplicate samples were incubated with anti-rhIL- lbeta, -IFN-gamma rabbit polyclonal antibodies, or
anti-rhTNF-alpha monoclonal antibody overnight at
4°C. After blocking with 0.1% bovine serum albumin, either the serum or serial dilutions of rhIL-lbeta, rhTNF-alpha, and rhIFN-gamma standards wereadded and incubated at 4°C for 24 h. After washing 3
246

times, peroxidase-conjugatedpolyclonal rabbit antirhIL- 1-beta,
andanti-rhIFN-gamma
antibodies(diluted to l/1000), or monoclonal anti-rhTNF-alphaantibody (diluted to l/4000) were added to the wells
and incubated for 24 h at 37°C. After washing 3
times, 100 l.tl of enzyme substrate (1 pg/ml O-phenylene- diamine on 0.1 M sodium citrate buffer) wereadded. Five minutes later, the reaction was stopped
by adding 100 l_tlof H,SO, to each well of the microplate. The absorbance at 492 nm was determined byan automatic microtiter reader (Multiscan; Labosystern, Finland). The sensitivity of detection was 20 pg/
ml for each cytokine.
The concentrationof N-terminal propeptide oftype III procollagen (PIIIP) was determined by acommercially available radioimmunoassay
kit (RIA
gnostic PIIIP, Behringwerk AG, Mat-burg, Germany)and that of type IV collagen 7s (IVcol-7s) also wasdetermined by a commercially available radioimmunoassay kit (Nippon DPC Corp., Tokyo, Japan)
according to the manufacturer’s instructions. These
two parameters were used to ascertain the hepatic
necro-inflammatory
process by the degree of hepaticconnective tissue turnover.
Statistical analysis
Data are expressed as medians and/or ranges. The
nonparametric Mann-Whitney U test and two-sample
t-test were used for the comparison of data between
the two groups. Wilcoxon’s matched pairs test wasused to compare the two variable parameters. The
correlation between two parameters were calculatedby the linear regression test. A p-value x0.05 wasconsidered to be significant.

Results
The clinical backgrounds of 65 patients with chronic
hepatitis B who entered into this study are summarized in Table 1. The serum level of M-CSF in eachgroup of patients is presented in Fig. 1. The medianconcentration of M-CSF in healthy volunteers was
1.90 ng/ml, with a range of 1.65 to 2.32 rig/ml, which
was not significantly different from the M-CSF concentrations of HBeAg positive HBV ASC or HBeAb
positive HBV ASC. The M-CSF concentrations in
HBeAg positive HBV ASC (median 2.00 ng/ml,
range 1.72-2.29 ng/ml) were not significantly different from the M-CSF concentrations in HBeAb positive HBV ASC patients [1.95 (1.75-2.56) ng/ml],
although the titers of HBV-DNA and HBV-DNA
polymerase
activity were significantly
higher in
HBeAg positive HBV ASC [HBV-DNA;
6815

M-CSF in hepatitis B
TABLE 1
Clinical profiles of patients with chronic hepatitis B
Controls(20)

HBV asymptomatic carrier

Chronic hepatitis B
SevereeAg(+)(7)

eAb(+)(13)

Mild

ModerateeAg(+)(12)

eAb(+)(5)

eAg(+)(13)

eAb(+)(5)

eAg(+)
(10)

Sex
(F/M)

8/12

215

518

616

312

6il

213

416

Age
(years)
ALT
(W/l)

34
(254)
12
(8-14)

26(13-39)34(20-54)36(28-60)42(27-63)

38(26-50)32(25-41)

21(12-32)15(5-35);;5-73);:&92)

185(55-342)38(9-45)
172(71-267)

DNA

N.D.

6815
(2130-9980)

0(Pg/ml)
DNA-p
(cpm)

784
(O-10000)215(O-6538)

25(O-178)622(ck1886)

N.D.

3227
(8-6670)01574(O-3884)

5(O-13)
4(o-12)

2253(14-6890)

88(U-680)866(33-4480)

412(224-866)

eAg=hepatitis B e antigen, eAb=hepatitis B e antibody, ALT=alanine aminotransferase, DNA=hepatitis B virus DNA, DNA-p=hepatitis B virus
DNA polymerase activity.
Eight eAg(+) patients (3 mild and 5 moderate hepatitis B) were treated with prednisolone, and 12 eAg(+) patients (5 mild and 7 moderate hepatitis) were treated with interferons (6 alpha, 3 beta, 3 gamma).

(2130-9980) pg/ml versus 0(0-O) pg/ml, HBV-DNA
polymerase activity 3227 (86670) cpm versus 0 (O0) cpm, p<O.OOl]. Moreover, concentrations of M-

c

b

b

1

i

I,),

:::
..:::::,_i,c1IImlvvvIw
Diagnosis

Fig. 1. Serum concentrations of M-CSF in different groupsof patients and controls.
C; healthy controls.
I; HBeAg +ve asymptomatic carriers.
II; HBeAb+ve asymptomatic carriers.
III; HBeAg+ve mild chronic hepatitis.
rV; HBeAb+ve mild chronic hepatitis.
V; HBeAg+ve moderate chronic hepatitis.
VI; HBeAb+ve moderate chronic hepatitis.
VII: HBeAg+ve severe chronic hepatitis.
Data are expressed as ranges.
(a) is not signt$cantly different from controls, (b) is significantly (p<O.OS), and (C) is signi$cantly (~~0.01) higher
than controls.

CSF did not correlate with the titers of HBV-DNA(eO.215, N=65, NS) or the HBV-DNA polymerase
activity (-0.189, N=65, NS). Levels of M-CSF
increased in proportion to the degree of hepatic
inflammation as judged by the grade of liver injury
and the serum ALT level (~0.754, N=65, p~O.001).
In patients with HBV ASC, mild chronic hepatitis B,
and moderate chronic hepatitis B, serum concentrations of M-CSF were not significantly different
between the patients with HBeAg and those with
HBeAb (Fig. 1).
The serum concentration of M-CSF was examinedin ten patients during acute exacerbation. As shownin Fig. 2, serum concentrations of M-CSF significantly increased during acute exacerbation of hepatitis (serum levels of ALT more than 400 IU/ml), as
compared with the concentrations in the relatively
stable phase (levels of ALT less than 100 II-J/ml) prior
to and following the exacerbation [4.92 (3.96-6.93)
ng/ml versus 2.46 (1.94-3.08) ng/ml, and 2.39 (1.993.02) @ml, p<O.OOl, respectively]. During PSL
administration, serum concentrations of M-CSF significantly decreased in comparison with the concentrations prior to PSL administration [2.19 (1.79-2.72)
ngfml and 2.00 (1.65-2.20) ng/ml versus 2.74 (2.253.29) ng/ml, p<O.OOl] (Fig. 3A). Four weeks after
PSL withdrawal, the peak serum concentrations of
M-CSF coincided with the increase in the serum ALT
levels. Serum concentrations of M-CSF in this phase,
4.13 (2.20-5.44) ng/ml, were comparable to the concentrations during acute exacerbation [4.92 (3.96
6.93) ng/ml], but significantly higher than the concentrations prior to [2.74 (2.25-3.19) ng/ml] and dur247

Y. ltoh et al.

M-CSF
(w/m0

M-CSF

6

Wml)

5

7.51

I

43-

5.0-

21.

2.5-

I

A

beforeduringexacerbation

after

BeforeCytokine production[% Of Control] IL-1D

PSLl
TNFo

PSL2

w 1

W2

ylFN
-

Fig. 2. Time course analysis of the increase in serum MCSF concentrations in lopatients with acute exacerbationof chronic hepatitis B. Serum concentrations of M-CSF
signi@antly (p-cO.001) increased during the acute exacerbation (ALT levels more than 400 X.X!) as compared withthe concentrations before and after the exacerbation (ALT100%.

levels less than 100 IV/l).

,I3
ing PSL administration [2.19 (1.79-2.72) ng/ml and2.00 (1.65-2.20) @ml] @<O.OOl, respectively).
The LPS-stimulated production of proinflammatory cytokines, such as IL-l-beta and TNF-alpha byperipheral whole blood and that of IFN-gamma by
PBMCs, was examined serially in eight patients who
received PSL (Fig. 3B). The concentrations of thesecytokines in the culture supematants were expressedas the percent (%) increase/decreasein comparison
with the values prior to PSL administration in eachpatient. During prednisolone administration, medianvalues significantly decreased [IL-l-beta;
54 (3372)% and 47 (24-63)%, TNF-alpha; 60 (22~81)%
and 57 (36-75)%, IFN-gamma; 73 (35-llO)% and 82(56-105)%, ~~0.01, respectively].
However, theysignificantly increased during the PSL withdrawal
phase [IL-l-beta; 228 (158-28 l)% and 182 (117266)%, TNF-alpha; 208 (122-274)% and 220 (120316)%, IFN-gamma; 164 (89-192)% and 135 (92184)%, p~O.01, respectively]. In a similar fashion,
the serum levels of ALT, PIIIP, and 1~~01-7s
decreased after starting PSL administration,butincreased following PSL withdrawal (Fig. 3C).
In three patients, an exacerbation of hepatitis wasfollowed by seroconversion
to HBeAb. The timecourse of a representative patient who received PSL
is shown in Fig. 4. Following an increase in theserum M-CSF concentration, or coinciding with apeak, the titer of HBV-DNA
and HBV-DNA
polymerase activity rapidly decreased to undetectablelevels, followed by the appearance of HBeAb. Following seroconversion,concentrationsof M-CSF
248

ALT(U;ml)

Before

PSLl

PSU

Wl

w2

PUYP lvcol-7s(“‘,“‘/(U/d”l)

600

600

400

200

I

C

BeforeFig. 3 Time course

PSLlchangetions and other parameters
PSL.

PSL2

Wl

in serum M-CSF

w2
concentra-

in eight patients who received(A) serum M-CSF concentrations.
(B) production of IL-I-beta and

TNF-alphaby whole

blood, and IFN-gamma by PBMCs (median values prior to
PSL administration are expressed as lOO%.)(C) serum ALT, PIIIP, and IVcol-7s levels (median values).
Before; just prior to PSL administration.
PI; 2 weeks following PSL administration.
P2; at the end of PSL administration (3 weeks).
WI; 2 weeks afrer PSL withdrawal.
W2; 4 weeks after PSL withdrawal.

declined in parallel with the normalization of ALTlevels.
We examined the effects of IFNs (IFN-alpha, IFNbeta, and IFN-gamma) on the serum M-CSF concentration in viva (Fig. 5). The serum concentration of
M-CSF was significantly increased at the 3rd day of

M-CSF in hepatitis B

HBV
DNA
Ipep)

4

400

Fig. 4. Time course study of serum M-CSF (a), ALT (O),HBV-DNA (A), HBV-DNA polymerase activity (A), in a
representative patient who experienced a seroconversion to
HBeAb.
M-CSFconcentration
f % Ot COntrOt

1 , , FNo1, FN8

250%

200%

I FNv

Max

IJ i

MU

Mi”

Min

before

3rd

Max
Min

la,

.

5th

7th

Fig. 5. The effects of in vivo administration of IFN-alpha,-beta, -gamma on the serum concentrations of M-CSE
IFN-alpha (3 MU), IFN-beta (3 MU), or IFN-gamma (1MU) were administered daily for 7 days. Phlebotomy was
performed on the lst, 3rd, 5th, and 7th day following administration of IFNs. Values are expressed as ranges. Median values prior to IFN administration are expressed as
100%.
(Cr) ~~0.05, (k) ~~0.01, versus the values before IFN administration.

IFN administration with all forms of IFNs (‘~~0.01).
Serum concentrationsof M-CSF continued to be
increased until the end of the 7th day. Although thedoses and kinds of IFNs differed, no significant difference was noted in the concentration or the patternof increase of serum M-CSF in our study. The
increase in the concentration of serum M-CSF was
independent of changes in serum ALT, HBV-DNA,and HBV-DNA
polymerase
activity(data notshown).

Discussion
In the present study, we have extended our previous
finding that the serum concentration of M-CSF rises

in parallel with hepatic inflammation (19) by classifying the patients with chronic HBV infection
according to the grading of chronic hepatitis activity,and by serially examining patients with chronic hepatitis B during acute exacerbation and during PSL
administration
followed by withdrawal, prospectively. The administrationof PSL suppressed theserum concentration of M-CSF with a decrease in thelevel of ALT, PIIIP, and IVcol-7s. The production of
EN-gamma, IL- l-beta, and TNF-alpha by peripheral
blood cells was also suppressed by administration of
PSL. In contrast, the serum concentration of M-CSF
significantly increased during the PSL withdrawal
phase, coinciding with the increase in the levels of
ALT, PIIIP, and IVcol-7s, and with the enhanced production of IL-l-beta, TNF-alpha, and EN-gamma by
peripheral blood cells. In that phase, an increase in
the serum concentration of M-CSF also coincidedwith a decrease in the level of HBV-DNA and HBVDNA polymerase activity.
These results suggest that the serum concentrationof M-CSF is associated with the production of proinflammatory cytokines by peripheral blood cells andwith the hepatic necroinflammatory
process. A direct
anti-viral effect of M-CSF on HBV is unlikely,
because the concentrations of M-CSF were unrelated
to the titers of HBV-DNA and HBV-DNA polymerase activity. Moreover, serum concentrations of MCSF were similar between HbeAg positive ASC
showing high titers of HBV-DNA or high HBV-DNA
polymerase activity and HBeAb positive ASC having
undetectable levels of HBV-DNA and HBV-DNA
polymerase activity.
We speculate that the increased serum concentrations of M-CSF in hepatic inflammation may be
induced by proinflammatory
cytokines and/or interferons produced in viva. Our finding that exogenous
administration of ENS enhanced the serum concentration of M-CSF in viva, irrespective of changes in
the levels of ALT, HBV-DNA,or HBV-DNA
polymerase activity supports this hypothesis. In fact,
it has been reported that the production of M-CSF isregulated by several proinflammatory
cytokines inmany kinds of cells. In monocytes, the production of
M-CSF is enhanced by IL-l-beta, TNF-alpha, andIFN-gamma (25,26). Similarly, it is enhanced by IL1-beta, TNF-alpha, IL-6, or EN-gamma in bone marrow stroma (27) or fibroblasts (28), mesangial cells(29,30), or in astrocytes (31). In vivo, IL-l-alpha and
TNF-alpha elevate the serum concentration of MCSF in mice (32,33), and EN-alpha also enhances
the serum concentration of M-CSF in hepatitis C
patients (34).
249

Y. ltnh et al.

Although the mechanisms regulating M-CSF production in viva are not well understood (lo), we postulate that the serum concentration of M-CSF depends
on the production of M-CSF in compartments such as
the liver or bone marrow, and on circulating peripheral
blood monocytes. In chronic hepatitis B patients withacute exacerbation, activated monocytes recruited tothe inflamed liver may be one of the sources ofincreased M-CSF concentrations in the serum, considering our previous result that the number of liver infiltrating M-CSF positive mononuclear cells is increasedin patients with acute hepatitis (19). Moreover, serum
concentrationsof M-CSF are correlated with thenumber of liver macrophages in patients with chronic
hepatitis (unpublished data). However, because fat
storing cells or hepatocytes also secrete M-CSF in
vitro (35,36), production of M-CSF by these types ofcells in viva cannot be neglected. Further study of theexpression of M-CSF mRNA in the liver tissue is necessary to solve this issue.
In studies using HBV transgenic mice, antigenindependent recruitment of macrophages or lymphocytes amplifies an inflammatory process initiatedby antigen-specific cytotoxic T cells in the liver by
inducing
theproductionof proinflammatory
cytokines (37). In acute exacerbation of chronic hepatitis B, increases have been reported in the mitogenstimulated production of EN-gamma by PBMCs andin the hepatic mRNA expression of IFN-gamma(38,39). The production of TNF-alpha increases bothin PBMCs and in liver infiltrating mononuclear cells
in parallel with the hepatic inflammation of chronic
hepatitis (40-42). We speculate that both in the
peripheral blood cells and in the inflamed liver,increased production of proinflammatory
cytokines
such as TNF-alpha or IFN-gamma produced by lymphocytes and/or monocytes might induce the production of M-CSF by monocytes in a paracrine or an
autocrine manner. We showed in this study that LPSstimulated production of IFN-gamma by PBMCs, orof IL- l-beta and TNF-alpha by peripheral whole
blood increased during the PSL withdrawal phase inparallel with the increased serum concentration of MCSF. This result thus supports our hypothesis.
There are conflicting reports concerning the effectsof M-CSF on the production of proinflammatory
cytokines by monocytes/macrophages(43-45); however, there seems to be a general consensus that production of M-CSF is enhanced by several proinflammatory cytokines and IFNs in many kinds of cells in
vitro or in vivo (25-36). In our study, three types of
IFNs increased the serum concentration of M-CSF in
vivo. The enhanced serum concentration of M-CSF in
250

hepatitis B patients may be a partial consequence ofenhanced production of IFNs in vivo. A recent report(46) showing that the serum level of IFN-gamma iscorrelated with active hepatic inflammation in hepatitis B patients supports our hypothesis. IFN-alpha,which is produced during HBV infection in the
peripheral blood cells and within the liver (47,48),
also enhances the production of EN-gamma
by
PBMCs in hepatitis B patients (49).
In the present study, we have shown that theincreased serum M-CSF concentrations in patientswith chronic HBV infection is associated with theincreased
levels of IFNs and proinflammatory
cytokines produced by peripheral blood cells during
hepatic inflammation, which may be a consequenceof the altered cytokine cascade resulting from hostimmune response against HBV. We are not surewhether or not the increased M-CSF has any pathophysiological significance; however, it is reasonable
to think that a cytokine network supports proliferation of macrophages/monocytes
in the liver in parallel with the severity of the hepatic necroinflammatory
process in patients with chronic hepatitis B.

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