Detection of T lymphocyte subsets and mIL-2R on surface of PBMC in patients with hepatitis B

Abstract

AIM: To study the levels of T lymphocyte subsets and membrane interleukin-2 receptor (mIL-2R) on surface of peripheral blood mononuclear cells (PBMCs) of patients with hepatitis B and its role in the pathogenesis of hepatitis B.

METHODS: The levels of T lymphocyte subsets and mIL-2R in PBMC before and after being stimulated with PHA were detected by biotin-streptavidin (BSA) technique in 196 cases of hepatitis B.

RESULTS: In patients with hepatitis B, the levels of CD₃⁺, CD₄⁺ cells, and the ratio of CD₄⁺ cells/CD₈⁺ cells were lower, but the level of CD₈⁺ cells was higher than those in normal controls (42.20 ± 6.01 vs 65.96 ± 6.54, 38.17 ± 5.93 vs 41.73 ± 6.40, 0.91 ± 0.28 vs 1.44 ± 0.31, 39.86 ± 6.36 vs 30.02 ± 4.54, P < 0.01). The total expression level of mIL-2R in PBMC before and after being stimulated with PHA was also lower than those in normal controls (3.47 ± 1.55 vs 4.52 ± 1.49, 34.03 ± 2.94 vs 37.95 ± 3.00, P < 0.01). In all the patients with hepatitis B, the levels of T lymphocyte subsets and mIL-2R in PBMC with HBV-DNA (+) were lower than those with HBV-DNA (-), which were significantly different (39.57 ± 7.11 vs 44.36 ± 5.43, 34.36 ± 7.16 vs 40.75 ± 5.87, 37.82 ± 6.54 vs 41.72 ± 6.21, 0.88 ± 0.33 vs 0.99 ± 0.27, 2.82 ± 1.62 vs 3.85 ± 1.47, 31.56 ± 3.00 vs 35.84 ± 2.83, P < 0.01). In addition, the levels of CD₃⁺, CD₄⁺, CD₈⁺ cells, the ratio of CD₄⁺ cells/CD₈⁺ cells and mIL-2R among different courses of hepatitis B were all significantly different (F = 3723.18, P < 0.01. F = 130.43, P < 0.01. F = 54.01, P < 0.01. F = 2.99, P < 0.05. F = 7.16, P < 0.01).

CONCLUSION: Both cellular and humoral immune functions are obviously in disorder in patients with hepatitis B, which might be closely associated with the chronicity in patients.

Key Words: $[Keywords]

INTRODUCTION

Hepatitis B virus (HBV) parasitizing in hepatocytes is a pathogen of viral hepatitis B, which easily develops into hepatic fibrosis and cirrhosis, even hepatocellular carcinoma. But the pathogenesis of hepatitis B is very complex and has not been clarified until now. Generally, it is not HBV itself that damages hepatocytes directly, but the results of function disorder of cell-mediated immunity[1-18]. Peripheral blood mononuclear cells (PBMCs), which are aggregation of abundant immunologically competent cells, such as T lymphocytes, natural killer cells and lymphokine activated killer, likely play an important role in anti-HBV infection. Interleukin-2 (IL-2) has a crucial role in several immunologic functions. Its effect is dependent on the conjugation with membrane interleukin-2 receptor (mIL-2R) expressed on surface of activated T lymphocytes and other immunocompetent cells and can release from them. Biotin-streptavidin (BSA) has a high specificity and sensitivity. In order to study possible changes of T lymphocyte subsets and mIL-2R on surface of peripheral blood mononuclear cells (PBMC) of patients with hepatitis B and its role in the pathogenesis of hepatitis B, 196 patients with acute and chronic hepatitis B were detected by the BSA methods in this study. The results suggest that there is a state of depression rather than of activation of T lymphocyte subsets and mIL-2R system in viral hepatitis B, and that the pathogenesis of viral hepatitis B is related to the cellular and humoral immune function of patients.

MATERIALS AND METHODS

Subjects

According to the diagnotic criteria passed by the 10^th National Conference on Viral Hepatitis and Hepatopathy 2000 (Xi’an), 196 patients with hepatitis B (male 113 and female 83), aged 19-52 years (average 34.6 years), were chosen from our affiliated teaching hospitals. Among them, 24 patients were HBsAg-positive without symptoms, 22 with acute hepatitis B, 46 with slight chronic hepatitis, 37 with moderate chronic hepatitis, 26 with severe chronic hepatitis, 15 with severe hepatitis, 18 with posthepatitic cirrhosis and 8 with hepatocellular carcinoma. In addition, the controls were selected from HBsAb-positive volunteers (n = 10) and normal blood donors from the local central blood bank (n = 20), aged 10-45 years (average 32.6 years).

Reagents and instruments

Antibodies against T lymphocyte subsets were provided by Shanghai Jing’an Medical Institute, Ficoll-Hypaque sedimentation gradients were offered by Shanghai Second Reagent Factory, and HBV-DNA reagents were made in Shanghai Middle Asia Gene Institute. Carbon dioxide incubator (MDF-135) was made in Japan.

Samples

Five mL peripheral vein blood 5 mL from each patient with hepatitis B and the controls was collected at 8:00 a.m., and 2.5 mL was distributed in a sterile test tube and 2.5 mL into an anticoagulant test tube with heparin.

Separation of PBMC and detection of T cell subsets, mIL-2R

After the heparinized anticoagulant blood was mixed with equal volume of Hanks’ liquid without Ca²⁺ and Mg²⁺, PBMC were harvested from heparinized whole blood by centrifugation on Ficoll-Hypaque sedimentation gradient and diluted to (1-3) × 10⁶·L^-1cells suspension with RPMI 1640 culture liquid. Ten μL suspension of PBMC was smeared on sheet glass pores so that the cells with CD₃⁺, CD₄⁺, CD₈⁺ and the rest phrase of mIL-2R could be detected. Of the PBMC suspension, 0.5 mL was mixed with RPMI 1640 culture liquid, which had PHA 200 μg·L^-1. The cells were grown in continuous culture (37 °C, 50 mL·L^-1 CO₂ in atmosphere) for 72 h and its mIL-2R induced by PHA could be measured by the antibodies against membranes of T cells.

Immunocytochemical method of biotin-streptavidin system (BSA)

The different monoclonal antibodies (mAb) against CD₃, CD₄, CD₈ and Tac with biotin and SA-HRP were smeared on different sheet glasses. These smears were left dry naturally and fixed with acetone for 15-20 min. The cells were incubated in continuous culture (37 °C, 50 mL·L^-1 CO₂ in atmosphere) for 30 min. The immune sheet glass pores were measured after being stained with the color-developing agent and several washings with Tris buffer solution (TBS). The total number of 200 PBMCs was counted and its positive cells were statistically analyzed with the help of high power lens. The positive criterion was that the color of cell membrane was brown, if not being negative.

Detection of HBV-DNA

The levels of HBV-DNA in serum and PBMC were detected with negative, positive and vacuity controls being set up each time. After routine process, the samples were placed at 94 °C for 300 s for denaturation at first, then at 94 °C for 30 s, at 55 °C for 30 s and at 72 °C for 30 s. After 35 cycles, the samples were extended at 72 °C for 300 s. After electrophoresis in 2% sepharose with ethidium bromide (EB) for 20 min, products of amplification were observed with infrared-transmission meters. Samples with orange fluorescent bands as the positive controls were considered to be positive, while the others were negative.

Statistical analysis

Statistical analysis was made by t and F tests.

RESULTS

The results showed that the percentages of CD₃⁺ and CD₄⁺ cells, and the ratio of CD₄⁺ cells/CD₈⁺ cells were lower, the percentage of CD₈⁺ cells was higher, and the levels of mIL-2R before and after stimulation with PHA were lower in patients with hepatitis B than those in normal controls (P < 0.01). Among different courses of hepatitis B, T lymphocyte subsets and mIL-2R were all significantly different from each other. The detailed results are shown in Table 1 and Table 2.

Table 1 Detection of T lymphocyte subsets and mIL-2R in PBMC of patients with hepatitis B (^-x±s, %).

Group	n	CD3+	CD4+	CD8+	CD4+/CD8+	mIL-2R
						Silent	Induced
Control	30	65.96 ± 6.54a	41.73 ± 6.40b	30.02 ± 4.54c	1.44 ± 0.31d	4.52 ± 1.49e	37.95 ± 3.00f
Anti-HBs (+)	10	66.34 ± 5.16	42.82 ± 6.52	29.03 ± 4.50	1.51 ± 0.27	5.06 ± 1.45	40.26 ± 3.10
NBD	20	65.80 ± 6.92	41.20 ± 6.36	30.45 ± 4.62	1.39 ± 0.33	4.24 ± 1.52	36.30 ± 2.95
Hepatitis B	196	42.20 ± 6.01a	38.17 ± 5.93b	39.86 ± 6.36c	0.91 ± 0.28d	3.47 ± 1.55e	34.03 ± 2.94f
A HBsAg (+)	24	58.83 ± 7.44g	41.34 ± 5.16h	35.34 ± 7.15i	1.20 ± 0.33j	3.94 ± 1.75k	35.05 ± 3.05l
AH	22	57.38 ± 7.73g	40.21 ± 6.12h	39.47 ± 6.25i	1.01 ± 0.30j	3.67 ± 1.68k	34.22 ± 2.25l
SCH	46	38.54 ± 7.56g	39.56 ± 6.44h	41.10 ± 7.64i	0.98 ± 0.31j	3.44 ± 1.40k	31.96 ± 3.80l
MCH	37	40.14 ± 5.85g	37.22 ± 5.38h	41.45 ± 6.23i	0.88 ± 0.25j	3.25 ± 1.50k	32.81 ± 2.76l
SCH	20	40.01 ± 6.23g	35.51 ± 6.33h	42.86 ± 5.58i	0.81 ± 0.22j	3.06 ± 1.56k	33.83 ± 3.52l
SH	15	37.85 ± 6.54g	34.57 ± 6.20h	42.92 ± 5.65i	0.80 ± 0.21j	3.95 ± 1.54k	33.85 ± 2.65l
PC	18	38.72 ± 6.22g	36.11 ± 4.23h	41.89 ± 8.98i	0.90 ± 0.19j	3.31 ± 1.60k	31.55 ± 2.34l
HC	8	39.44 ± 6.78g	34.15 ± 5.50h	43.46 ± 7.88i	0.83 ± 0.24j	3.36 ± 1.68k	30.38 ± 2.15l

^at = 19.9295, ^aP < 0.001.

^bt = 3.0300, ^bP < 0.01.

^ct = 8.1551, ^cP < 0.001.

^dt = 9.5153, ^dP < 0.001.

^et = 3.4722, ^eP < 0.001.

^ft = 6.7832, ^fP < 0.001.

^gF = 3723.18, ^gP < 0.01.

^hF = 130.43, ^hP < 0.01.

ⁱF = 54.01, ⁱP < 0.01.

^jF = 2.99, ^jP < 0.05.

^kF = 7.16, ^kP < 0.01.

^lF = 1.60, ^lP > 0.05. NBD: Normal blood donors, A HBsAg (+): Asymptomatic HBsAg (+), AH: Acute hepatitis, SCH: Slight chronic hepatitis, MCH: Moderate chronic hepatitis, SCH: Severe chronic hepatitis, SH: Severe hepatitis, PC: Posthepatitic cirrhosis, HC: Hepatocellular carcinoma.

Table 2 Detection of T lymphocyte subsets and mIL-2R in PBMC with HBV-DNA (+) before and after induced with PHA (^-x±s, %).

Group	n	CD3+	CD4+	CD8+	CD4+/CD8+	mIL-2R
						Silent	Induced
Control	30	65.96 ± 6.54	41.73 ± 6.40	30.02 ± 4.54	1.44 ± 0.31	4.52 ± 1.49	37.95 ± 3.00
Anti-HBs (+)	10	66.34 ± 5.16	42.82 ± 6.52	29.03 ± 4.50	1.51 ± 0.27	5.06 ± 1.45	40.26 ± 3.10
NBD	20	65.80 ± 6.92	41.20 ± 6.36	30.45 ± 4.62	1.39 ± 0.33	4.24 ± 1.52	36.30 ± 2.95
Hepatitis B	196	42.20 ± 6.01	38.17 ± 5.93	39.86 ± 6.36	0.91 ± 0.28	3.47 ± 1.55	34.03 ± 2.94
HBV-DNA (+) of PBMC	118	39.57 ± 7.11a	34.36 ± 7.16b	37.82 ± 6.54c	0.88 ± 0.33d	2.82 ± 1.62e	31.56 ± 3.00f
HBV-DNA (-) of PBMC	78	44.36 ± 5.43a	40.75 ± 5.87b	41.72 ± 6.21c	0.99 ± 0.27d	3.85 ± 1.47e	35.84 ± 2.83f

^at = 5.3466, ^aP < 0.001.

^bt = 6.8525, ^bP < 0.01.

^ct = 4.2414, ^cP < 0.001.

^dt = 2.5641, ^dP < 0.05.

^et = 4.6355, ^eP < 0.001.

^ft = 10.0352, ^fP < 0.001. NBD: Normal blood donors.

DISCUSSION

Recently, studies have shown that patients with hepatitis B are usually accompanied by disorder of immune function, and hepatocytic damage is mainly caused by immunological injury[15-30]. Immunologically competent cells and cytokines are the key points for body to eliminate hepatitis B virus[12]. Alterations of T lymphocyte subsets are an important reason for the disorder of immune function due to HBV infection. A lots of cytokines, especially IL-2, can facilitate proliferation of immunologically competent cells[14,31-37], such as T lymphocytes, natural killer cells and lymphokine activated killer cells. While IL-2 is conjugated with mIL-2R on surface of the proper target cells, it will be efficient in immunoregulation. In order to further explore the pathogenesis of hepatitis B, we designed a series of correlation experiments to detect the levels of T lymphocyte subsets and mIL-2R on surface of PBMC in patients with hepatitis B.

CD₃⁺, CD₄⁺ and CD₈⁺ cells are major function subgroups of T cells and play an important role in response to HBV infection, which can reflect the situations of cellular immune function and immunoregulation and are usually regarded as a valuable index to forecast the changes of patients’ immunity[11,38-47]. In this study, the percentages of CD₃⁺, CD₄⁺ cells and the ratio of CD₄⁺ cells/CD₈⁺ cells decreased, and CD₈⁺ cells increased, suggesting that disorders of cellular immune function and pathologic damages occurred in the 196 patients with hepatitis B detected by the method of BSA.

PBMCs are easily infected by HBV[48,49]. When entering into PBMCs, HBV can integrate with host cells and interfere with metabolism of cells, and can depress the expression of CD₃⁺ and CD₄⁺. As seen in this study, the expression levels of T lymphocyte subsets between positive and negative HBV-DNA and HBV-DNA in PBMC were significantly different (P < 0.01).

mIL-2R plays a key role in biologic effect of IL-2 and its expression levels can reflect the course of T cell activity and the immune situation of body[50]. From this study, we can see the expression levels of mIL-2R in PBMCs in silence and induction were lower in hepatitis B patients than in normal controls (P < 0.01), and the expression levels of mIL-2R in PBMCs were lower in HBV-DNA positive patients than in HBV-DNA negative patients (P < 0.01). After stimulation with PHA, the levels of mIL-2R obviously increased, which showed that mIL-2R could be induced by PHA, but its expression levels were still significantly lower than those in normal controls (P < 0.001). In addition, due to deterioration and chronicity of hepatitis B, the expression levels of mIL-2R had a tendency of descent in the patients. These also showed that T cell activity was interfered and humoral immune function was decreased in patients with hepatitis B.

The levels of CD₃⁺, CD₄⁺, CD₈⁺ cells (P < 0.01), the ratio of CD₄⁺ cells/CD₈⁺ cells (P < 0.01) and mIL-2R (P < 0.05) among different courses of hepatitis B were all significantly different, which suggested that there is a close correlation between levels of T lymphocyte subsets and different courses of hepatitis B. According to the above findings, it is concluded that in patients with hepatitis B, there is an obvious disorder of cellular and humoral immune functions, and a close relationship between the body’s immune function and the course of illness. While infecting PBMC, HBV can interfere with the normal metabolism of these cells, and prevent lymphocytic membranes from accepting signals from antigen presenting cells (APC), and depress the expression of mIL-2R. All of these do no good to eliminating HBV and contribute to chronicity of hepatitis B.