Vol.2, No.7, 736-741 (2010)
doi:10.4236/health.2010.27112
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Health
Dynamic analysis of lymphocyte sub sets of peripheral
blood in patients with acute self-limited hepatitis B
Bo Liu, Jun Li*, Yaping Han, Yuan Liu, Lianhua Kong, Yang Cao, Zuhu Huang
Department of Infectious Diseases, the First Affiliated Hospital with Nanjing Medical University, Nanjing, China;
*Corresponding Author: dr-lijun@vip.sina.com
Received 19 March 2010; revised 12 April 2010; accepted 13 April 2010.
ABSTRACT
Purpose: To investigate dynamic changes and
significance of lymphocyte subsets (T lympho-
cytes, B lymphocytes, NK cells and T cell sub-
sets) of peripheral blood in patients with acute
self-limited hepatitis B (AHB). Methods: Immune
cells of peripheral blood were compared among
17 cases of self-limited acute hepatitis B pa-
tient s, 36 p atient s with chronic hep atitis B (CHB)
and 32 healthy controls by flow cytome try (FCM).
CD4+/CD8+ was monitored dynamically, mean-
while relations between T lymphocyte subsets
and ALT and clearance of HBV DNA were ex-
plored. Results: Dynamic changes of lympho-
cyte subsets were found in AHB, the level of
CD3+T cells was significantly higher compared
to CHB group and healthy control group. Fre-
quencies of CD3+CD4+ T cells in the third and
fourth week and CD4+/CD8+ in the second week
were higher compared to other groups. Frequ-
ency of NK cells was low and was significantly
lower compared to other groups in the third
week specially. It was showed that CD4+/CD8+
was low followed by high abnormal ALT during
early st age by dynamic mo nitoring o f CD4 +/CD8+,
and CD4+/CD8+ was increasing accompanied by
normal ALT set by set, but CD4+/CD8+ had no
significant relation to ALT and HBV DNA. Con-
clusion: Immune status of AHB, compared to
CHB and healthy controls, was significantly dif-
ferent and d y namic ch anges of lymphocyte sub-
sets may b e related to progress of disease.
Keywords: Acute Hbv; Self-Limited;
Lymphocyte Subsets; Facs
1. INTRODUCTION
Hepatitis B virus (HBV) is one of the most prevalent viral
pathogens in humans, with almost a third of the world
population having evidence of infection, and about 350
million chronically infected patients. Approximately 1
million people die annually from HBV-related disease,
such as liver failure, cirrhosis and hepatocellular carci-
noma [1]. After body was infected by Hepatitis B virus
(HBV), complex immune responses could be caused,
and cell-mediated immunity is an important factor to
determine results of HBV infection [2]. Different subsets
of lymphocyte have different responses to viral antigens
and effects on clinical course and prognosis of develop-
ment. Many precious researches focused on lymphocyte
subsets in peripheral blood of chronic hepatitis B [3,4],
which indicated that there was an imbalance in periph-
eral blood T-lymphocyte subsets and turbulence in cel-
lular immunity. Researches about stable detection of
lymphocyte subsets for adults with acute self-limited
hepatitis B (referred to as “acute hepatitis B”) [5] show-
ed that acute-phase CD4 responses were efficient and
CD8 responses were multispecific irrespective of the
outcome of infection. In this paper, by comparing dif-
ferences of lymphocyte subsets among acute hepatitis B,
chronic hepatitis B and normal controls, dynamic char-
acteristics of lymphocyte subsets in acute hepatitis B
were observed and the relationship between body’s im-
mune response to HBV infection and disease prognosis
was explored.
2. MATERIALS AND MEHTODS
2.1. Characteristics of Patients
Patients with acute hepatitis B, chronic hepatitis B were
out-patient clinics and hospitalized patients of Depart-
ment of Infectious Diseases, the First Affiliated Hospital
with Nanjing Medical University from May 2007 to May
2008, diagnosis were in line with the revised diagnostic
criteria of the Tenth National Conference on viral hepati-
tis and Liver Disease for viral hepatitis in 2000 [6] and
the “prevention and treatment of chronic hepatitis B
B. Liu et al. / HEALTH 2 (2010) 736-741
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guide” [7], and exclusion of other hepatitis viruses and
CMV, EBV, HIV co-infection. The information of pa-
tients was shown in Table 1.
2.2. Instruments and Reagents
IgG1-PE/IgG2a- FITC,CD3/CD4 /CD8/CD45,CD3 /CD4/
CD28, CD3/CD8/CD28, CD3/CD19/CD56/CD45 mono-
clonal antibodies (fluorescent-labeled), as well as hemo-
lytic agents were purchased from BD company(USA).
Flow cytometry (BD FACS Calibur, USA). Automatic
biochemical analyzer and its reagents were Beckman
Company’s products (USA). HBV DNA quantitative
detection equipment were production of Roche’s Light
Cycler 1.0(USA).
2.3. Detection of Lymphocyte Subsets
Methods were referred Ya Pinghan [8] etc. and modified
it slightly. 100 μl blood samples were collected into he-
parinized vacuum tubes and blended with mouse anti-
human monoclonal antibody CD3/CD8/CD45/CD4, CD3/
CD16 + 56/CD45/CD19 (fluorescent-labeled) 20 ul re-
spectively, reacted at room temperature protected from
light for 30 min, and was detected by FACS in 2 h. A
“gate” of the leukocyte common antigen (CD45)-posi-
tive cells was set up according to forward and side scat-
ter two-parameter point diagram and lymphocyte surface
markers were analyzed by two-parameter. Statistical
results were analyzed by use of CellQuest software.
2.4. Dynamic Detection of Lymphocyte
Subsets
The frequencies of lymphocyte subsets were measured
and dynamically analyzed for acute hepatitis B when
admitted to hospital for the first, second, third and fourth
week, compared to normal controls and chronic hepatitis
B. Dynamical CD4+/CD8+ ratios were detected and dy-
namical relationships between CD4+/CD8+ ratios and
alanine aminotransferase (ALT) were analyzed. In order
to explore relationship between CD4+/CD8+ ratios and
clearance of HBV DNA, CD4+/CD8+ ratios of acute
hepatitis B with HBV DNA positive and negative pa-
tients were analyzed and compared.
2.5. Statistical Analysis
Measurement data were expressed using
X
± s. By ap-
plication of SPSS 11.0 statistical software, means of two
groups were compared using t test or paired t test and
multiple sets of means were compared by single-factor
ANOVA analysis of variance. The correlation among
ratios of lymphocyte subsets, alanine aminotransferase
and HBV DNA level were analyzed by use of correlation
analysis. P < 0.05 was referred as statistically significant.
Table 1. Some characteristics of different groups in patients.
Group No Male Female Average age
AHB 17 11 6 32.4 ± 4.8
CHB 36 20 16 37.5 ± 6.2
Normal 32 18 14 35.5 ± 3.9
A total of 17 cases of acute hepatitis B group, 11 males and 6 females,
aged from 18 to 57 years old, and average (32.4 ± 4.8) years of age.
Chronic hepatitis B group, 36 cases were recruited, 20 males and 16
females, aged from 23 to 75 years old, and mean (37.5 ± 6.2) years of
age. Healthy controls, 32 cases without past and current HBV infection,
as a normal control group, 18 male cases and14 female, aged from 22
to 53 years old, and mean (35.5 ± 3.9 years).
3. RESULTS
3.1. Changes of Lymphocyte Subsets for
Acute Hepatitis B
For acute hepatitis B at admission and the dynamic
changes trend of lymphocyte subsets for the first, second,
third and fourth week (Figure 1(a)), and lymphocyte
subsets of normal controls and chronic hepatitis B (Fig-
ure 1(b)).Peripheral blood CD3+ T cells within 4 weeks
of acute hepatitis B hospitalization were significantly
higher, and the difference was statistically significant
(Figure 2) compared to normal controls and CHB group.
Frequencies of CD4+ T cells from admission to 4th week
showed a rising trend which were higher than normal
controls and CHB group, and the differences of the third
week and fourth week were statistically significant
(Figure 3).For acute hepatitis B,CD8+ T cells showed a
upward trend first and then showed a downward trend,
and the difference was statistically significant for the
results of the second week (Figure 4).CD56+ cells in 4
weeks were lower than both normal controls and CHB
group, however, the third week compared with other two
groups and the fourth week compared with CHB group
respectively, the difference were statistically significant
(Figure 5).
3.2. Level of ALT in Acute Hepatitis B and
Changes in the Ratio of Lymphocyte
Subsets
For acute hepatitis B, the trends of CD4+/CD8+ ratio
means and ALT dynamic changes for hospitalized in the
first week, second week, third week and fourth week
were showed in Figure 6. For third week and fourth
week, difference of CD4+/CD8+ ratio was statistically
significant (P < 0.05) compared with chronic hepatitis B
B. Liu et al. / HEALTH 2 (2010) 736-741
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(a)
(b)
Figure 1. (a) Dynamic changes of acute hepatitis B lympho-
cyte subsets. Patients admitted to hospital and dynamic
changes of lymphocyte subsets CD3+,CD4+,CD8+,CD19+ and
CD56+ for the first, second, third and fourth week. (b) Fre-
quencies of lymphocyte subsets of normal controls and chronic
hepatitis B.
Figure 2. The results of dynamic changes of CD3+ T cells in
patients with AHB compared to normal controls and CHB. The
mean ratios of different weeks for AHB were 73.97 ± 5.21%,
77.67 ± 6.64%, 78.54 ± 1.78% and 78.81 ± 5.01% respectively.
And the ratios of normal controls and CHB were 64 ± 11.54%
and 62.48 ± 11.33% respectively. Compared to the other
groups, CD3+T cells of AHB were higher and the differences
were statistically significant. (*P < 0.05).
Finger 3. The results of dynamic changes of CD4+ T cells in
patients with AHB, compared to normal controls and CHB. For
AHB, the mean ratios of CD4+ T cells were 37.86 ± 9.15%,
37.93 ± 7.34%, 45.71 ± 7.42% and 46.37 ± 8.09% respectively.
The mean ratios of CD4+ T cells in normal control and CHB
were 34.41 ± 7.53% and 30.07 ± 6.67% respectively. Com-
pared to the other groups, CD4+ T cells of AHB in third and
fourth week were higher and the differences were statistically
significant. (*P < 0.05).
Figure 4. The results of CD8+ T cells in patients with AHB
compared to normal controls and CHB. The mean ratios of
CD8+ T cells in patients with AHB in four weeks were 31.58 ±
10.05%, 35.24 ± 7.79%, 26.45 ± 6.01% and 26.1 ± 5.95%,
respectively.And the mean ratios of normal control and CHB
were 27.18 ± 7.59% and 29.89 ± 8.36% respectively. The dif-
ferences between 2nd week of AHB and the other groups were
statistically significant. (*P < 0.05).
(1.12% ± 0.28%).For chronic hepatitis B, difference was
statistically significant (P < 0.05) compared with normal
control (1.41% ± 0.60%). CD4+/CD8+ ratio and ALT
changes were observed dynamically, and at early stage
high levels of abnormal ALT followed lower CD4+/CD8+
ratio. As ALT normalization, the CD4+/CD8+ ratio showed
a gradual increasing trend. The correlation between dy-
namic CD4+/CD8+ ratio and ALT was analyzed for dif-
ferent weeks, and r values were 0.700, 0.286, 0.179,
0.286, P values were 0.188, 0.535, 0.702, 0.535 (all >
0.05), respectively ,which suggested no significant cor-
relation.
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Figure 5. The results of CD56+ cells in patients with AHB
compared to normal controls and CHB. The mean ratios of
CD56+ cells in patients with AHB were 12.28 ± 6.32, 11.15 ±
7.12, 7.26 ± 5.04% and 10.89 ± 3.33 within four weeks, re-
spectively. The mean ratios of normal control and CHB were
14.38 ± 4.23% and 18.93 ± 11.22% respectively. The differ-
ence between the 3rd of AHB and the other groups was statis-
tically significant (*P < 0.05) and the difference between the
4th of AHB and the CHB was statically significant. (#P < 0.05).
Figure 6. Trends of CD4+/CD8+ ratio means and ALT dynamic
changes. It showed that ALT became gradual normalization
followed CD4+/CD8+ gradually increased over time.
3.3. The Ratio of Lymphocyte Subsets and
HBV DNA
HBV DNA of acute hepatitis B on admission were de-
tected, of which 5 cases were negative (HBV DNA < 103
copies/ml) and 12 cases were positive (for the 103 cop -
ies/ml < HBV DNA < 107 copies/ml), and positive pa-
tients happened to be HBV DNA negative conversion
within two weeks. The CD4+/CD8+ ratio of 12 patients
with positive HBV DNA (mean 1.18% ± 0.93%) was
lower than that of negative patients (mean 1.59% ±
0.47%), but no statistically significant difference was
found between them (t = 0.701, P = 0.501).Twelve
cases were positive in patients with CD4+/CD8+ ratio
and the presence or absence of HBV DNA and no sig-
nificant correlation (r = 0.433, P = 0.466). The
CD4+/CD8+ of and HBV DNA of 12 cases of patients
with positive HBV DNA were no significant correlation
(r = 0.433, P = 0.466).
4. DISCUSSION
When adults are infected with HBV, the vast majority of
people can clear virus and only a little infected persons
continued to be chronic infection. Most studies have
confirmed that the prognosis of HBV infection is closely
related to functional status of the immune system which
is an important factor to determine the prognosis. Acute
hepatitis B triggers the body’s immune response, par-
ticularly cell-mediated immunity. In our study, dynamic
changes of peripheral blood lymphocyte subsets in acute
hepatitis B and characteristics of peripheral blood lym-
phocyte subsets with normal controls and chronic hepa-
titis B were observed and analyzed, in order to initially
explore the relationship between lymphocytes change
and disease progression and prognosis.
CD4+ T cells play a important role in viral clearance
[9], and play a central role in controlling immune re-
sponse. Subsets of CD8+ T cells include cytotoxic T cells
(CTL) and suppressive T cells (Ts), however, inactivated
CTL are differentiated to special killing effective T cells
dependent on identifying processing antigen and by IL-2
secreted by CD4+ T cells. Past studies about lymphocyte
subsets of peripheral blood such as Urbani S, D. Vassi-
lopoulos and others [10,11] used more static detection,
but we detected lymphocyte subsets dynamically. It
showed that lymphocyte subsets showed a certain degree
of dynamic change for acute hepatitis B: CD3+ T cells
increased significantly within four weeks of hospitaliza-
tion and CD4+ T cells showed increasing trend compared
to normal controls and chronic hepatitis B. However, for
chronic hepatitis B, CD3+ T cells and CD4+ T cells ratios
were lower than normal controls. These figures showed
that the weaker CD4+ T cells immune response may be
related to chronic infection. Differences of CD3+ T and
CD4+ T cells ratios between acute and chronic hepatitis
B suggested that when adults happened to be acute in-
fection, T lymphocytes of peripheral blood, especially a
higher proportion of CD4+ T cells, may be related to a
strong Th1 cell response. As disease progression, more
obvious advantage of CD4+ T cell ratio was essential to
be ridding of virus. CD8+ T cells ratio of acute hepatitis
B had a peak, and was higher than other two groups, but
the ratio has fallen and were lower than other two groups
in third and fourth week. As for chronic hepatitis B,
CD8+ T cells slightly increased compared with normal
controls. As we know, CD8+ T cells have different sub-
B. Liu et al. / HEALTH 2 (2010) 736-741
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sets and most studies confirmed that for acute hepatitis B
there was a strong polyclonal, multi-specific CTL re-
sponse. In our study CD8+ T cells had advantage of ratio
during early stage, however, specific CTL proportion
might explain the phenomenon, which injured body's
own cells while clearing the virus. But for chronic hepa-
titis B, CTL response was not likely to dominate and Ts
cell ratio mainly increased which inhibited cellular im-
mune function, leading to sustained and persistent infec-
tion.
In part CD4+/CD8 + reflects state of immune system
within a certain range [12]. The up-regulation of ratio
indicates that immune response is strong and the reduc-
tion of ratio or even less than one indicates that low im-
mune function. As for chronic hepatitis B, Yin Ying [13],
You Jing [14] and so on confirmed existence of CD4+/
CD8+ down. Our study showed that CD4+/CD8+ of acute
hepatitis B in the first, third and fourth week were higher
than that of normal controls and chronic hepatitis B
which because activation and reproduction of specific
CTL depending on CD4+ T cells activated, and a higher
proportion of CD4+ T cells was behalf of inducing a
strong anti-viral response. As CD4+ T cells of acute
hepatitis B increased significantly, compared to chronic
hepatitis B, it indicated that arise of CD4+/CD8+ in acute
hepatitis B showed advantage of positive regulation and
a good prognosis disease.
For acute hepatitis B, ALT became gradual normaliza-
tion followed CD4+/CD8+ gradually increased over time.
Although a significant correlation between them was not
found, a moderate cellular immune response happened
during acute infection, which was conducive to com-
pletely remove virus, meanwhile which played a role in
immune regulation function to restore tissue damage.
HBV DNA was likely to have been largely cleared be-
fore liver damage for majority of acute hepatitis B, and
for twelve cases of positive patients, HBV DNA contin-
ued to decline rapidly and serology turned to be negative.
Virus was cleared mainly through hepatic cell disruption
or non-dissolving mechanism [15]. For acute hepatitis B
with positive and negative HBV DNA, CD4+/CD8+ of
them had no statistical difference and HBV DNA level
and CD4+/CD8+ also had no significant correlation.
However as for chronic hepatitis B, You Jing [9] sug-
gested that there was significant negative correlation
between CD4+/CD8+ and HBV DNA level, which sug-
gested that immune status of chronic hepatitis B was at a
low level and viral replication was relatively stable, and
immune response could not completely inhibit virus rep-
lication, leading to persistent infection. Nevertheless, for
acute hepatitis B, whether positive or negative HBV
DNA, immune response was at a relatively positive ad-
justment advantage, and a strong particularly cellular
immunity, which could rapidly clear HBV DNA and play
a strong and appropriate regulation function in later re-
action, ultimately got to be clinical recovery.
NK cells are component of innate immune. They ex-
press molecular CD56 and play the role of first line of
defense by leading to infectious cell disrupted non-spe-
cifically. Moretta L. et al. [16] confirmed that in early
stage of acute hepatitis B, NK cells could play a role
compared to chronic hepatitis B and normal controls.
Our study showed that NK cells of acute hepatitis B had
a high proportion in early stage, then dropped to a trough
in the third week. NK cells of chronic hepatitis B was
higher than normal controls. Chronic hepatitis B had a
protracted course and persistent inflammation, leading to
NK cells always maintained a relatively high proportion.
For acute infection NK cells of peripheral blood showed
dynamic changes, and it may be related to balance and
accumulation of NK cells between blood circulation and
liver tissue, so that it was more conducive for NK cells
to produce IFN, and thus inhibited viral replication and
cleared infected cells.
In summary, by dynamically analyzing lymphocyte
subsets of peripheral blood in acute self-limited hepatitis
B in a certain period of time, the relationship between
clearing virus and immune system changes was explored.
It showed that when acute hepatitis B occurred, there
was a more comprehensive and effective immune re-
sponse, which can be completely rid of virus and access
to clinical recovery. However, for different individuals
infected with HBV, how body activates a strong immune
response in order to completely remove virus, as well as
limitations of this study to a certain extent, additional
studies are required further study in the future.
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