Leptin Receptor Isoforms mRNA
Expression in Peripheral Blood
Mononuclear Cells from Patients with
Chronic Viral Hepatitis
NIKOLAOS STEFANOU,* MARIA SATRA,* VASSILIOS PAPANIKOLAOU,* FANI KALALA,?
NIKOLAOS GATSELIS,? ANASTASIOS GERMENIS,? GEORGE N. DALEKOS,? AND
*University of Thessaly, Medical School, Department of Biology, 41222 Larissa, Greece;
?University of Thessaly, Medical School, Department of Immunology, 41222 Larissa, Greece;
?University of Thessaly, Medical School, Department of Medicine, Academic Liver Unit, 41222 Larissa,
Greece; and §University of Thessaly, Medical School, Laboratory of Cytogenetics and Medical
Genetics, 41222 Larissa, Greece
There is accumulating evidence that leptin has a pleiotropic role
in hematopoiesis, immune response, fibrogenesis, and hepato-
carcinogenesis. We investigated the expression of leptin and
leptin receptor (OB-R) at the protein level by flow cytometry and
also quantified by real-time reverse transcriptase–polymerase
chain reaction (RT-PCR) the two major leptin receptor isoforms
(OB-Rl, OB-Rs) in peripheral blood mononuclear cells (PBMCs)
of patients with hepatitis B (HBV; n ¼ 31), hepatitis C (HCV; n ¼
34), and nonviral liver disease (n ¼ 25), and healthy controls (n
¼ 36), as well as in liver tissues of HBV (n ¼ 8), HCV (n ¼ 7),
and healthy individuals (n ¼ 6). Serum leptin levels were
measured in all participants (N ¼ 126). We observed significantly
lower OB-Rl and OB-Rs mRNA levels in PBMCs of HBV and HCV
patients compared with healthy controls and nonviral liver
disease patients (P , , 0.05). Flow cytometry analysis confirmed
the real-time RT-PCR results. Expression of leptin and OB-Rl
was significantly increased in viral hepatitis liver tissues
compared with healthy tissues (P , , 0.01). OB-Rl mRNA levels
were not associated with hepatitis patients’ clinical status
(inactive, chronic hepatitis, or cirrhosis). We also found
decreased serum leptin in HBV and HCV patients compared
with healthy individuals and the nonviral liver disease group.
Leptin was expressed in 3 of 34 HCV (8.8%) and 19 of 25 (76%)
nonviral liver disease patients. Moreover, expression of OB-Rl
and OB-Rs were associated when all individuals were grouped
together (r ¼ 0.78, P , , 0.001). In conclusion, our findings may
suggest the involvement of the leptin system in the immunopa-
thology of chronic viral hepatitis. Exp Biol Med 231:1653–1663,
Key words: leptin; leptin receptors; hepatitis B virus; hepatitis C
Leptin, the 16-kDa nonglycosylated protein product of
the OB gene, is an essential hormone/cytokine that is
secreted from adipocytes and links nutritional status with
neuroendocrine and immune functions (1–4). As a hormone,
leptin regulates appetite and energy homeostasis at the
hypothalamic level by activating specific neuroendocrine
pathways (2, 5, 6). As a cytokine, leptin affects thymic
homeostasis and, similar to other proinflammatory cyto-
kines, modulates the onset of immune responses (1, 7, 8). In
humans, there are four isoforms of leptin receptor, generated
by alternative splicing, which are membrane-spanning
glycoproteins with cytoplasmic domains of varying length
(9). The longest isoform (OB-Rl) is responsible for fully
functional leptin signaling, while the shorter isoforms (OB-
Rs) have truncated cytoplasmic domains with reduced
signal transduction capabilities (10–12).
Leptin receptors are expressed in a variety of peripheral
tissues including the brain, liver, pancreas, placenta, lung,
skeletal muscle, heart, hematopoietic cells, and peripheral
blood mononuclear cells (PBMCs) (9, 13). It has been
observed that leptin-deficient (ob/ob) and leptin receptor–
deficient (db/db) mice present a complex syndrome that
consists of abnormal reproductive function, hormonal
imbalances, immune dysfunction, and reduced levels of
peripheral T and B cells, suggesting a role for leptin in
1To whom correspondence should be addressed at University of Thessaly, Faculty of
Health Sciences, Medical School, Department of Biology, 22 Papakyriazi Street,
41222 Larissa, Greece. E-mail: email@example.com
Received September 7, 2005.
Accepted April 10, 2006.
Copyright ? 2006 by the Society for Experimental Biology and Medicine
lymphopoiesis (14). Human leptin deficiency caused by a
missense mutation in the OB gene can also lead to immune
system dysfunction as evidenced by decreased T helper cell
1 (Th1) and increased Th2 responses (15). Leptin can also
induce proliferation, differentiation, and functional activa-
tion of hematopoietic cells and increase the secretion of
proinflammatory mediators. More specifically, leptin pro-
motes the Th1 immune responses by increasing c-interferon
(IFN-c), tumor necrosis factor-a (TNF-a), and interleukin-2
(IL-2) secretion by Th1 or naive T cells. Leptin promotes
activation and secretion of proinflammatory factors such as
leukotriene B4, cyclooxygenase-2 (Cox-2), and nitric oxide
(1, 14, 16–21) in monocytes and macrophages.
Leptin has also been associated with chronic inflam-
matory diseases, such as pelvic endometriosis, chronic
pulmonary inflammation, inflammatory nephritis, Behcet’s
syndrome, Graves’ disease, rheumatoid arthritis, and non-
alcoholic or viral hepatitis (1). It has been suggested that
activation of T cells and macrophages is one of the initial
events during viral hepatitis infection (22). Activated T cells
are either directly cytotoxic to hepatocytes or release
proinflammatory cytokines, which mediate hepatocyte
damage in various animal models (23, 24). The T cell–
modulating activity of leptin, the observation that OB-Rl is
expressed on CD4 and CD8 positive cells, and the presence
of OB-Rl and OB-Rs in hepatic sinusoidal cells suggest that
leptin may be directly involved in the pathophysiological
mechanisms that take place during viral hepatitis (25, 26).
To further assess the role of leptin and its receptors in
chronic viral hepatitis infection, we measured serum leptin
levels and investigated, for the first time to our knowledge,
expression of leptin receptor isoforms in PBMCs of
chronically-infected hepatitis B virus (HBV) and hepatitis
C virus (HCV) patients and in patients with nonviral liver
disease. We measured mRNA levels of leptin receptor
isoforms using quantitative real-time reverse transcriptase–
polymerase chain reaction (RT-PCR) and protein expression
using flow cytometry. We found that leptin receptor mRNA
and protein expression is significantly decreased in PBMCs
of HBV and HCV patients compared with healthy controls
and patients with nonviral liver disease. We also observed a
notable increase of leptin receptor mRNA in the livers of
HBV and HCV patients compared to healthy controls,
suggesting a possible involvement of the leptin system in
the immunopathogenesis of HBV and HCV.
Materials and Methods
A total of 126 human subjects were included in the
study. This consisted of 65 consecutive patients with
chronic HBV and HCV infections, 25 patients with nonviral
liver disease, constituting the disease control group, and 36
healthy individuals. All patients were followed-up at the
Outpatient Clinic of the Academic Liver Unit, University
Hospital of Larissa, Larissa, Greece. Thirty-one patients had
chronic HBV infection (17 male, 14 female; mean age: 51.1
years; age range: 25–72 years; mean body mass index
[BMI]: 26.6 6 4.562) and 34 patients had chronic HCV
infection (13 male, 21 female; mean age: 45.7 years; age
range: 18–76 years; mean BMI: 25.05 6 3.207). Control
groups consisted of 36 healthy individuals (15 male, 21
female; mean age: 47.5 years; age range: 23–65 years; mean
BMI: 24.52 6 3.343) and 25 patients with non-viral liver
disease (15 with alcoholic liver disease and 10 with
autoimmune liver disease; a total of 14 male, 11 female;
mean age: 46.1 years; age range: 20–73 years; mean BMI:
27.1 6 3.794). There were no age differences between the
four study groups. The mean 6 SD of disease duration in
HBV and HCV patients was 95 6 73.9 and 125 6 141.57
months, respectively. The duration of disease was calculated
from the age at diagnosis, as stated in other reports from our
Patients with chronic viral liver disease included in the
study were classified into three clinical groups: (i) patients
with normal levels of aminotransferases determined at 3-
month intervals for at least one year before entry into the
study (inactive state group; n ¼ 25: 14 with HBV and 11
with HCV), (ii) patients with histologically, virologically,
and biochemically proven chronic HBV and HCV (chronic
hepatitis group; n ¼ 31: 13 with HBV and 18 with HCV),
(iii) patients with HBV or HCV–related cirrhosis (cirrhosis
group; n ¼ 9; four with HBV and five with HCV). None of
the patients had during the follow-up and at the time of
investigation any clinical, laboratory, or radiological
evidence of hepatocellular carcinoma (HCC).
Diagnosis of chronic HBV infection was based on
clinical, laboratory, and histological evaluations according
to our previous reports (28, 29) and the recent European
Association for the Study of the Liver (EASL) International
Consensus Conference on Hepatitis B (30). Briefly, patients
in the chronic inactive HBV state (n ¼ 14) had reactivity
against hepatitis B surface antigen (HBsAg; Abbott
Laboratories, Wiesbaden, Germany), normal aminotrans-
ferases, and no viral replication as attested by the absence or
the presence of low levels of serum HBV DNA (below 105
copies/ml) by PCR (cut-off: 200 copies/ml; Cobas Amplicor
HBV Monitor; Roche, Branchburg, NJ). HBV patients with
chronic hepatitis B (n ¼ 31) met the following criteria: (i)
serological evidence, using commercially available enzyme
immunoassays (Abbott Laboratories), of chronic infection
with HBV for at least 6 months before entry into the study,
(ii) active virus replication as defined by the detection of
HBV DNA (.105copies/ml) using a commercially
available PCR kit (Cobas Amplicor HBV Monitor; Roche),
(iii) elevated levels of alanine aminotransferase (ALT; at
least 2-fold that of the upper limit) for at least 6 months
before entry into the study, (iv) histologically proven
chronic HBV without any sign of cirrhosis. The diagnosis
of cirrhosis due to HBV was also based on the above criteria
(i), (ii), and (iii) and liver histology compatible with
cirrhosis. None of the HBV patients tested positive for
antibodies to HCV (anti-HCV; Murex Diagnostics, Temple
1654STEFANOU ET AL
Hill, Dartford, UK) or antibodies to HIV (anti-HIV; Abbott
Diagnosis of chronic HCV infection was based on
clinical, laboratory, and histological evaluations (27–29).
Briefly, all HCV patients included in the study met the
following criteria: (i) serological evidence of chronic
infection with HCV as determined by the detection of
anti-HCV antibodies using a third-generation enzyme
immunoassay at least twice within 6 months before
enrollment into the study, (ii) active virus replication as
defined by detection of HCV RNA using a commercially
available qualitative PCR kit (HCV Monitoring Cobas
Amplicor; Roche; cut-off: 50 U/ml). None of the HCV
patients were positive for HBsAg or anti-HIV.
Diagnosis of alcoholic liver disease was based on a
history of alcohol consumption (more than 50 g/day
ethanol) along with compatible clinical, laboratory, and
liver histology. Diagnosis of autoimmune liver disease (five
patients with autoimmune hepatitis and five with primary
biliary cirrhosis) was based on the revised descriptive
criteria for diagnosis of autoimmune hepatitis reported by
the International Autoimmune Hepatitis Group (31) and, for
patients with primary biliary cirrhosis, the following
previously-described criteria (32): positivity for antimito-
chondrial antibodies (positive titer ?1/40) by indirect
immunofluorescence using in-house rodent tissue substrates,
elevated cholestatic enzymes, and histological lesions
compatible with the disease. All disease control patients
tested negative for HBsAg, anti-HCV and anti-HIV.
Liver tissue specimens were collected from six healthy
individuals of the control group, for whom an operation was
performed for cholecystectomy (three male, three female;
mean age: 43 6 16.09 years; age range: 28–64 years), seven
patients of the HCV group (three male, four female; mean
age: 44.14 6 16.94 years; age range: 28–69 years), and
eight patients of the HBV group (four male, four female;
mean age: 47 6 14.89 years; age range 25–61 years). None
of the patients with chronic viral hepatitis had any clinical,
laboratory, or radiological evidence of HCC during follow-
up at the time of investigation.
None of the patients received antiviral treatment (a-
interferon alone or in combination with either ribavirin or
lamivudine) for at least 24 months before entry into the
study. All healthy individuals had normal ALT values (26.5
6 5.3 U/liter), tested negative for HBsAg, anti-HCV, and
anti-HIV antibodies, and denied ever having used hepato-
toxic drugs or herbals, or having abused alcohol or injected
drugs. All subjects consented at the time of interview to
participation in the study. The study protocol conformed to
the ethical guidelines of the 1975 Declaration of Helsinki as
reflected in a priori approval by the Local Ethical
Committee of the University Hospital of Larissa (Larissa,
Total RNA Isolation. PBMCs were isolated from
whole blood according to standard procedures and total
cellular RNA was extracted using Trizol (Life Technologies,
Paisley, UK; Ref. 29). Tissue samples from patients with
chronic hepatitis were obtained by needle biopsy. Samples
were immediately frozen and stored at ?808C until use.
RNA was further purified using the RNase-free DNase kit
(Qiagen, Manheim, Germany). The presence of 28S and
18S rRNA species was used to assess RNA integrity. Only
samples with prominent 28S and 18S rRNA components
were included in the study.
Real-time, Quantitative RT-PCR of OB-Rl and
OB-Rs mRNA. Transcription to cDNA was performed
using the AMV Kit (Roche, Indianapolis, IN). Retinoic
acid receptor-a (RAR-a) cDNA sequences were amplified
in separate reactions as positive cDNA controls. Quanti-
fication was performed by real-time RT-PCR (LightCycler
Instrument; Roche Molecular Systems, Alameda, CA) with
0.2 ll cDNA per sample using the LightCycler FastStart
DNA Master HybProbe kit (Roche, Penzberg, Germany)
according to the manufacturers’ instructions. The oligo-
nucleotide primers used for OB-Rl were 59-GCTA-
TTTTGGGAAGATGT-39 (forward, bases 288–306 in
exon 19) and 59-TGCCTGGGCCTCTATCTC-39 (reverse,
bases 553–570 in exon 20), for OB-Rs were 59-TGT-
TGTGAATGTCTTGTGCC-39 (forward, bases 402–421 in
exon 6) and 59-TGCTCCAGTCACTCCAGATTCC-39
(reverse, bases 649–668 in exon 8), and for leptin were
59-TTCTTGTGGCTTTGGCCCTA-39 (forward, bases 81–
100 in exon 2) and 59-GGAGACTGACTGCGTGTGTGT-
GAA-39 (reverse, bases 191–212 in exon 2). The thermal
cycling conditions for OB-Rl were 588C annealing for 47
cycles, and for OB-Rs and leptin were 578C and 558C
annealing, respectively, for 45 cycles. PCR products were
separated on a 2% agarose gel and visualized with
ethidium bromide staining. Specific PCR products were
identified by direct sequencing (Lark Technologies, Ltd.,
Essex, UK). A 100–base pair DNA ladder (Gibco BRL,
Paisley, UK) was used as a molecular weight standard.
The expected PCR products were 132 base pairs (bp) for
leptin, 501 bp for OB-Rl, and 394 bp for OB-Rs. Five
different dilutions of cDNA from adipose tissue were used
to obtain a standard curve. All samples were analyzed in
duplicate and the average value was used for quantifica-
tion. Variation between the two measurements for each
sample was ranged from 0.1% to 10%. If the variation
exceeded 10%, measurements were carried out in triplicate
for that sample. Data were expressed as a ratio between the
target gene mRNA level and the mRNA level of the
housekeeping gene porphobilinogen deaminase (PBGD)
(OB-Rl or OB-Rs mRNA copies/PBGD copies), which was
used as an internal control.
Serum Leptin Determination. Venous blood sam-
ples were taken in the morning after a 12-hr fasting period
and serum samples were stored at ?808C until assayed;
assays were performed in duplicate. As we described
previously (33), serum leptin concentration was measured
using a commercial sandwich enzyme-linked immunosor-
bent assay (ELISA; DRG Leptin EIA-2395; DRG Interna-
LEPTIN RECEPTOR ISOFORM EXPRESSION IN HEPATITIS PATIENTS1655
tional Inc., Marburg, Germany) with a limit of detection of
1.0 ng/ml. The intra- and inter-assay coefficients of variation
were 6.91% and 8.66%, respectively.
OB-R Detection on PBMCs by Flow Cytome-
try. PBMCs isolated from whole blood were incubated
with 10 ll monoclonal anti-human leptin receptor (OB-R)
antibody (R&D Systems, Minneapolis, MN) or mouse
IgG2a Isotype Control (R&D Systems) and 10 ll goat anti-
mouse IgG2a conjugated to fluorescein isothiocyanate
(IgG2a-FITC; R&D Systems). Flow cytometry was per-
formed using the EPICS XL-MCL Counter (Beckman
Coulter, Miami, FL) and data were acquired and analyzed
with CellQuest software. Data were acquired from 10,000
cells (events) and the proportion of PBMCs with cell-
surface OB-R was determined. Results are displayed as
percentage of events positive for OB-R staining.
Statistical Analysis. All calculations were per-
formed using SPSS Software (Version 10.0; Chicago, IL).
Data were analyzed, were applicable, by unpaired Student’s
t test, Mann-Whitney U test (MWU), Kruskal–Wallis,
ANOVA, or the Fisher protected least significant difference
(PLSD) as the post-hoc test corrected for multiple
comparisons. Correlation coefficients were calculated by
simple regression analysis (r) and nonparametric Spearman
rank correlation (rs) when appropriate. Two-sided P values
of ,0.05 were considered statistically significant.
OB-Rl and OB-Rs mRNA in PBMCs of HBV,
HCV, and Nonviral Liver Disease Patients. In order
to test the immunomodulatory role of leptin receptor
isoforms in chronic HBV and HCV infections, we
quantified using real-time RT-PCR the mRNA levels of
leptin receptors in PBMCs from HBV, HCV, and nonviral
liver disease patients as well as from healthy individuals.
The mean OB-Rl and OB-Rs levels differed significantly
among the four study groups (HBV, HCV, nonviral liver
disease, and healthy). In more detail, after post-hoc analysis
with the Fisher PLSD test, we observed significantly lower
mean OB-Rl and OB-Rs mRNA levels in patients with
chronic HBV (1.133 6 0.361 3 10?2and 2.086 6 0.55 3
10?2, respectively) and HCV (1.167 6 0.469 3 10?2and
2.267 6 0.648 3 10?2, respectively) infections compared
with healthy individuals (1.804 6 0.629 3 10?2and 2.953
6 0.562 3 10?2, respectively) and in the nonviral liver
disease group (3.05 6 1.292 3 10?2and 7.02 6 2.092 3
10?2, respectively) (Fig. 1). A representative gel that shows
leptin and leptin receptor (OB-Rl and OB-Rs) expression in
all four study groups is presented in Figure 2. OB-Rl and
OB-Rs values did not differ between HBV and HCV
patients (3.11 6 1.12 3 10?2and 7.12 6 1.954 3 10?2,
respectively) or between alcoholic and autoimmune liver
disease patients (2.96 6 1.576 3 10?2and 6.87 6 2.386 3
10?2, respectively). Moreover, the average level of OB-Rs
expression was approximately 2-fold higher than that of
OB-Rl in all samples (HBV, HCV, healthy, and nonviral
liver disease, P , 0.001). Mean OB-Rl mRNA levels in
PBMCs were not associated with the clinical status of the
patients (inactive state, chronic HBV and HCV, and HBV
and HCV–related cirrhosis) (ANOVA, P . 0.05), even if
they were divided according to the type of viral infection
(Student’s t test, P . 0.05). Interestingly, we found a
significant difference in the expression of OB-Rs between
inactive HBV patients (n ¼ 14) and patients with chronic
HBV or HBV-related cirrhosis (n ¼ 17) (2.228 6 0.764 3
10?2and 1.944 6 0.516 3 10?2respectively, P , 0.05).
The mean OB-Rl and OB-Rs mRNA levels did not differ
between male and female HBV patients (1.159 6 0.86 3
10?2vs. 2.06 6 0.618310?2and 1.107 6 0.491310?2vs.
2.114 6 0.731 3 10?2, respectively), HCV patients (1.194
6 0.393 3 10?2vs. 2.311 6 0.521 3 10?2and 1.149 6
0.713 3 10?2vs. 2.226 6 1.027 3 10?2, respectively),
Figure 1. Comparison of PBMCs from healthy individuals, and HCV, HBV and nonviral liver disease patients with respect to mean OB-Rs and
OB-Rl expression levels (OB-Rs or OB-Rl mRNA copies/PBGD copies) after real-time RT PCR analysis. Bars, means 6 standard deviation.
1656STEFANOU ET AL
healthy individuals (1.787 6 0.711 3 10?2vs. 3.026 6
0.6193 10?2and 1.823 6 0.557 310?2vs. 2.881 6 0.441
3 10?2, respectively), and nonviral liver disease patients
(3.17 6 1.11 3 10?2vs. 2.90 6 1.536 3 10?2and 7.16 6
2.013 3 10?2vs. 6.85 6 2.275 3 10?2, respectively).
Leptin Expression in PBMCs of HBV, HCV, and
Nonviral Liver Disease Patients. Leptin was not
expressed in PBMCs from any healthy individuals (n ¼
36) or HBV patients (n ¼ 31), but was expressed in 3 of 34
samples from the HCV group (8.8%) and in 19 of 25
samples from the nonviral liver disease group (76%). No
significant correlation was observed between either type of
viral infection and leptin expression (v2¼ 2.868, P . 0.05),
while a significant association was observed between leptin
expression and the presence of nonviral liver disease (v2¼
39.7, P , 0.001).
OB-Rl, OB-Rs, and Leptin Expression in Liver
Tissue from HBV and HCV Patients. There were
differences among the mean levels of OB-Rl and OB-Rs
mRNA in livers from patients with HBV (0.289 6 0.12 and
4.04 6 1.844 3 10?2, respectively), HCV (0.542 6 0.207
and 4.97 6 2.507 3 10?2, respectively), and the healthy
control group (1.62 6 0.398 3 10?2and 2.93 6 0.234 3
10?2, respectively) (ANOVA, P , 0.01 for all OB-Rl
comparisons; Fig. 3). Leptin was not expressed in any
healthy liver tissues (n ¼ 6), but was expressed in five out
Figure 2. Representative gel showing OB-Rl, OB-Rs, and leptin mRNA levels in PBMCs from HBV, HCV, and nonviral liver disease groups and
healthy groups. (a) (lane 1) OB-Rs, healthy; (lane 2) OB-RI, healthy; (lane 3) leptin, healthy; (lane 4) OB-Rs, HBV; (lane 5) OB-RI, HBV; (lane 6)
leptin, HBV; (lane 7) OB-Rs, HCV; (lane 8) OB-RI, HCV; (lane 9) leptin, HCV; (lane 10) OB-Rs, nonviral liver disease; (lane 11) OB-RI nonviral
liver disease; (lane 12) leptin, nonviral liver disease; (lane 13) negative control; (lane 14) OB-Rs, positive control; (lane 15) OB-RI, positive
control; (lane 16) leptin, positive control; (lane 17) marker, 100 bp ladder. (b) mRNA levels of PBGD.
Figure 3. Comparison of liver tissues of healthy individuals, and HCV and HBV patients with respect to mean OB-Rs and OB-Rl expression
levels (OB-Rs or OB-Rl mRNA copies/PBGD copies) obtained after real-time RT PCR analysis. Bars, means 6 standard deviation.
LEPTIN RECEPTOR ISOFORM EXPRESSION IN HEPATITIS PATIENTS 1657
of eight tissues of the HBV group (62.5%) and four out of
seven samples of the HCV group (57.14%). OB-Rl and OB-
Rs mRNA levels were not associated with age, BMI, sex,
disease duration, or level of aminotransferases. Liver leptin
expression was significantly associated with HBV and HCV
disease (v2¼ 6.3, P , 0.05).
Correlation of PBMC OB-Rl and OB-Rs mRNA
with Clinical Characteristics.
mRNA levels were not associated with age, BMI (Pearson
rank correlation, P¼NS), sex (Student’s t test, P . 0.05),
disease duration, or level of aminotransferases (Spearman
rank correlation, P¼NS) when all patients were considered
together. However, a significant negative correlation was
observed between OB-Rs level and age (Pearson rank
correlation, r ¼ ?0.269, P , 0.05). Additionally, when
patients were considered separately in groups (HBV, HCV,
nonviral liver disease), there was a correlation of OB-Rl and
OB-Rs levels with age, BMI, sex, disease duration, and
aminotransferase level. A notable negative correlation was
observed between OB-Rs levels and AST levels in the HBV
group (r ¼?0.341, P , 0.05) and a positive correlation was
observed between OB-Rl and AST levels in the autoimmune
liver disease group (r ¼ 0.752, P , 0.05).
OB-Rl and OB-Rs
Concerning the association between OB-Rl and OB-Rs
mRNA levels and age, BMI, sex, disease duration, and
levels of aminotransferases in the chronic viral disease
group, we found no significant associations for the inactive
state group, the chronic hepatitis group, and the cirrhotic
group. Also, no correlation was observed between OB-Rl
and OB-Rs mRNA levels and either HBV or HCV viral
loads, measured by HBV DNA and HCV RNA levels,
respectively, or HCV virus genotype.
The levels of OB-Rl and OB-Rs mRNA were associated
when all samples were considered together as a single group
(N ¼ 126; Pearson rank correlation, r ¼ 0.78, P , 0.001;
Serum Leptin Measurements. Serum leptin levels
were decreased, although not significantly, in HBV (6.522
6 5.659 ng/ml) and HCV (13.175 6 17.856 ng/ml) patients
compared with healthy individuals (16.822 6 15.45 ng/ml)
and the nonviral liver disease group (17.632 6 19.629 ng/
ml) (Table 1 and Fig. 5). Moreover, females had higher
serum leptin levels than males in the HBV patient group
(8.412 6 6.89 ng/ml vs. 5.077 6 4.154 ng/ml; Student’s t
test, P . 0.05), HCV patient group (19.582 6 21.06 ng/ml
vs. 4.024 6 2.769 ng/ml; Student’s t test, P , 0.02), among
Figure 4. Correlation between OB-Rs and OB-RI expression levels (OB-Rs or OB-RI mRNA copies/PBGD copies) obtained after real-time RT-
PCR analysis in all individuals studied (N ¼ 126).
Table 1. Oligonucleotide Primers for Real-Time RT-PCR and Their Respective Product Sizes
Transcript Primer sequence (59–39)Location (bases) Product size (bp)
288–306 in exon 19
553–570 in exon 20
402–421 in exon 6
649–668 in exon 8
81–100 in exon 2
191–212 in exon 2
1658 STEFANOU ET AL
healthy individuals (17.122 6 14.67 ng/ml vs. 16.403 6
17.019 ng/ml; Student’s t test, P . 0.05), and in the
nonviral liver disease group (30.12 6 21.031 ng/ml vs. 7.82
6 11.641 ng/ml; Student’s t test, P , 0.01).
Serum leptin levels and BMI showed a significant
correlation (Spearman rank correlation, r ¼ 0.443, P ,
0.001) when all samples were considered together (N ¼
126) (Fig. 6). However, no correlation was observed
between serum leptin levels and age, disease duration, or
the levels of aminotransferases. Also, no correlation was
observed between serum leptin levels and OB-Rl or OB-Rs
mRNA levels in groups with HBV (r ¼ ?0.052, P . 0.05
and r ¼ 0.002, P . 0.05, respectively), HCV (r ¼ ?0.172,
P . 0.05 and r ¼ ?0.399, P , 0.05, respectively), healthy
individuals (r ¼ ?0.168, P . 0.05 and r ¼ ?0.228, P .
0.05, respectively), and nonviral liver diseases (r ¼?0.195,
P . 0.05 and r ¼ 0.071, P . 0.05, respectively). Similarly,
when all subgroups where taken together, no correlation
was observed between levels of serum leptin and OB-Rl
expression (r ¼ 0.026, P . 0.05) or OB-Rs expression (r ¼
0.135, P . 0.05) (Fig. 7).
Leptin Receptor Expression on PBMCs. Flow
cytometric analysis revealed the presence of OB-R on 2.32
6 0.8% of PBMCs from 10 of 65 (15.3%) patients of the
combined HBV and HCV group, 3.1 6 0.9% of PBMCs
from the control group, and 4.57 6 0.9% of PBMCs from
the nonviral liver disease group (Fig. 8). Flow cytometry
analysis of the HBV and HCV groups demonstrated an
increased monocyte population, consistent with the phe-
nomenon of monocytosis present in chronic viral diseases
The leptin system is likely to have an important role in
inflammatory states such as multiple sclerosis, rheumatoid
arthritis, nonalcoholic steatohepatitis, and other diseases,
probably by the induction of proinflammatory factors such
as TNF-a, IFN-c, IL-2, Cox-2, and nitric oxide (1, 3, 34,
35). In the present study, we investigated, using quantitative
real-time RT-PCR, the mRNA levels of OB-Rl and OB-Rs in
PBMCs from patients with HBV, HCV, and nonviral liver
disease in order to investigate the possible immunomodu-
latory role of leptin in chronic viral liver disease. We
observed significantly lower expression levels of OB-Rl and
OB-Rs in HBV and HCV patients compared with healthy
individuals and nonviral liver disease patients. Flow
cytometric analysis revealed that OB-R protein levels
exhibited a pattern of expression similar to that of their
mRNAs, with the nonviral liver disease group showing
increased levels compared with healthy control, HBV, and
HCV groups. The reduction in observed OB-R protein
expression in a low number of HBV and HCV samples
could have resulted from virus-induced molecular dysregu-
lation and increased sensitivity to apoptosis of PBMCs,
Figure 5. Serum leptin levels in healthy individuals, and HCV, HBV,
and nonviral liver disease patients. Bars, means 6 standard
Figure 6. Correlation between serum leptin levels (ng/ml) and BMI in all individuals studied.
LEPTIN RECEPTOR ISOFORM EXPRESSION IN HEPATITIS PATIENTS1659
which would result reduction or absence of translation (36–
It has been suggested that leptin, acting through OB-R,
activates the Janus kinase–signal transducers and activators
of transcription (JAK-STAT), phosphatidylinositol 3-kinase
(PI3K), and mitogen-activated protein kinase (MAPK)
signaling pathways in PBMC, which in turn stimulate
cytokine induction by monocytes and macrophages, pro-
liferation of naive T cells, Th1 immune responses, Th2
inhibition, antiapoptotic effects on T cells, and increased
cytotoxicity of natural killer (NK) cells (1, 20). Our findings
suggest an involvement of OB-Rl and OB-Rs in immune
dysfunction in patients with chronic HBV and HCV
infection, as decreased OB-R expression and subsequent
reduction of protein on the cell surface diminishes the ability
of leptin to stimulate PBMCs, decreases the number of
activated immune cells, and possibly increases the number
of apoptotic immune cells. Our results provide a model for
hepatitis virus–induced downregulation of the immune
system that is in accordance with previous studies, which
have reported that HBV and HCV antigens directly
influence activation-induced cell death in peripheral T cells
and generally in PBMCs in both HBV patients and an HCV
transgenic murine model (36, 37). A recent study from our
group (29) has suggested that immunodeficiency in HBV
and HCV patients correlates with the observed reduction in
Figure 7. Correlation between serum leptin (ng/ml) and (a) OB-Rl mRNA levels and (b) OB-Rs mRNA levels (OB-Rl or OB-Rs mRNA copies/
PBGD copies) in all individuals.
Figure 8. Flow cytometry analysis of OB-R protein on the surface of PBMCs incubated with either IgG2a isotype control (a, c, e, and g) or
antibody against OB-R (b, d, f, and h). OB-R–positive and OB-R–negative cells are represented in the lower right and lower left quadrants,
respectively. (a and b) Healthy individuals. (c and d) Nonviral liver disease group. (e and f) HBV and HCV OB-R–positive group. (g and h) HBV
and HCV OB-R–negative group. Circles in g, the discrete lymphocyte (1) and monocyte (2) populations.
1660 STEFANOU ET AL
LEPTIN RECEPTOR ISOFORM EXPRESSION IN HEPATITIS PATIENTS1661
human telomerase reverse transcriptase (hTERT) mRNA in
PBMCs from infected patients.
observed between OB-Rl and OB-Rs expression (expression
of OB-Rs was approximately twice that of OB-Rl) when all
patients and healthy controls were taken together. The above
correlation indicates a stable and parallel expression pattern
of OB-Rl and OB-Rs, suggesting that OB-Rs could serve
either as an auxiliary to leptin signaling through the OB-Rl
isoform in PBMCs or as a regulator of serum leptin clearance
from the circulation through the soluble leptin receptor (10,
11). Based on the latter possibility, the observed reduction of
serum leptin in HBV and HCV patients could lead to the
downregulation of OB-Rs expression in PBMCs. Regarding
serum leptin levels in chronic hepatitis patients, there are
conflicting data in the literature (33, 39). Our results are in
agreement with a report that indicates that serum leptin levels
are lower in chronic hepatitis patients compared with healthy
individuals (39). It is possible that a reduction of leptin
the negative energy balance, caused by either reduced energy
intake or increased energy expenditure, that occurs in the
course of chronic viral liver disease.
We detected leptin mRNA in 3 of 34 samples from the
HCV group (8.8%) and in 19 of 25 samples from the
nonviral liver disease group (76%). The observed elevated
leptin expression in PBMCs from patients with nonviral
liver disease, accompanied with the increase of OB-Rl and
OB-Rs mRNA and protein levels, suggests that leptin could
be part of an autocrine signaling loop in PBMCs. This may
reflect the immunoactivity that might be responsible for
hepatocyte cytotoxicity in autoimmune liver diseases and
possibly in alcoholic liver disease (40). In addition, elevated
leptin secretion from PBMCs may contribute to the elevated
serum leptin levels observed in the nonviral liver disease
group. Similarly, leptin expression was observed in
activated inflammatory Th1 lymphocytes during experimen-
tal autoimmune encephalomyelitis, in an animal model of
multiple sclerosis (41). The relationship between HBV and
HCV viral diseases and the autocrine role of leptin will need
to be evaluated in future studies.
In the present study, a significant increase in OB-Rl
mRNA and a high percentage (60%) of leptin expression
was observed in liver tissue from patients with chronic viral
hepatitis compared with healthy livers. The functional OB-
Rl isoform exhibited an approximately 10-fold higher
expression in HBV and HCV liver samples compared with
healthy tissues. Recent reports have also shown that leptin
induces leptin receptor expression in the liver (42), acts as a
profibrogenic factor (26), increases susceptibility to hep-
atotoxicity by regulating cytokine production and T cell
activation (24), and upregulates proinflammatory and
proangiogenic cytokines in human liver (43). In leptin-
deficient ob/ob mice, protection from concanavalin A (Con
A)–induced hepatitis has been associated with reduced
production of proinflammatory cytokines, increased levels
of the protective cytokine IL-10, diminished T cell
activation, and a decrease in the number of hepatic NK T
cells (44, 45). There is therefore increasing evidence for an
extended inflammatory role of leptin in liver tissue.
When we grouped patients according to their clinical
status, there was a significant difference between patients
with inactive HBV infection and those with chronic HBV or
HBV-related cirrhosis with respect to OB-Rs mRNA levels.
Moreover, expression of the OB-Rs isoform was negatively
correlated with AST levels in the HBV group. These results
may indicate that OB-Rs functions differently in HBV
disease, with a role that extends beyond that of leptin
transport and degradation. We also observed a negative
correlation between OB-Rs mRNA level and age in the
patients group, which might reflect an age-dependent
influence of chronic HBV and HCV infection on the immune
system.Apositivecorrelation betweenserum leptinandBMI
role for adipose tissue in regulating leptin production.
In conclusion, for the first time to our knowledge, we
have used quantitative real-time RT-PCR and flow cytom-
etry to observe decreased OB-Rl and OB-Rs expression in
PBMCs, and increased expression of OB-Rl and leptin in the
liver, of HBV and HCV infected patients. This suggests a
possible involvement of the leptin system in immuno-
pathogenesis of these viral liver diseases. There is increas-
ing evidence that chronic HBV and HCV diseases are
characterized by a complicated immunopathology and
promote, through a multilateral mechanism, immunodefi-
ciency, which increases chronicity, accelerates liver disease,
and induces mortality. Further studies are needed to confirm
the involvement of the leptin system in the immune
response to HBV and HCV disease pathogenesis.
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