Dynamic analysis of lymphocyte subsets of peripheral blood in patients with acute self-limited hepatitis B

doi:10.4236/health.2010.27112

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Vol.2, No.7, 736-741 (2010)

doi:10.4236/health.2010.27112

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

737

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

± 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

738

(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.

B. Liu et al. / HEALTH 2 (2010) 736-741

739

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

740

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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