Chao-Pin Li, Ke-Xia Wang, Jian Wang, Department of Aetiology and Immunology, School of Medicine, Huainan University of Technology, Huainan 232001, Anhui Province, China
Bo-Rong Pan, the Fourth Military Medical University
Supported by the Youth Scientific Foundation of the Ministry of Coal Industry of China, No. 96-072
Correspondence to: Dr. Chao-Pin Li, School of Medicine, Huainan Institute of Technology, Huainan 232001,
Anhui Province, China. yxfy@hnit.edu.cn
Telephone: +86-554-6658770 Fax: +86-554-6662469
Received 2001-09-14 Accepted 2001-10-11

Abstract
AIM: To study the levels of membrane interleukin-2 receptor (mIL-2R) and T cell subsets in peripheral blood mononuclear cells (PBMC) from patients with hepatitis C and their role in the pathogenesis of hepatitis C.

METHODS: The levels of mIL-2R and T cells subsets in PBMC were detected by biotin- streptatividin (BSA) technique before and after stimulation with PHA in 203 patients with hepatitis C with HCV-RNA(+), anti-HCV(+), anti-HCV(-).

RESULTS: The total expressive levels of mIL-2R before and after stimulation with PHA(0.03±0.01, 0.03±0.02, 0.04±0.02, 0.36±0.03), and T cell subsets in PBMC (0.62±0.06, 0.37±0.05, 0.35±0.07) were all lower in patients with hepatitis C than those in normal controls (0.66±0.07, 0.41±0.06, 0.31±0.05, P<0.01). Among the patients, the levels of mIL-2R were lower in silence than those in situation of PHA inducting (P<0.01). However, the levels of mIL-2R were similar in acute hepatitis C to that in chronic hepatitis C (P>0.05). The levels of CD₃⁺, CD₄⁺, CD₄⁺/CD₈⁺ were lower and CD₈⁺ was higher in patients with acute and chronic hepatitis C with anti-HCV(+) than those in normal controls (0.62±0.06, 0.37±0.05, 0.35±0.07, 1.18±0.30, 0.61±0.07, 0.37±0.05, 1.39±0.33, 0.31±0.05, P<0.05-P<0.01).

CONCLUSION: The cellular immunity is obviously changed in patients with hepatitis C. The levels of mIL-2R and activation of T cells are closely associated with chronicity of hepatitis C.

Li CP, Wang KX, Wang J, Pan BR. mIL-2R, T cell subsets & hepatitis C.World J Gastroenterol 2002;8(2):298-300

INTRODUCTION
In acute and chronic hepatitis C virus (HCV) infection liver damage is thought to be the results of T-lymphocyte-mediated destruction of virus infected liver cells^[1-51]. The factors that affected the immune response to viral antigens, particularly those that regulate the function of virus-specific cytotoxic T-lymphocyte-mediated lymphocytes, most likely play an important role in determining the states of liver injury. Interleukin-2 (IL-2) has a crucial role in several immunologic functions and its effect is dependent on the interaction with IL-2 receptors (IL-2R) expressed on surface of activated T lymphocytes and other immunocompetent cells^[52-54]. Decreased IL-2 activity in supernatants of mitogen-activated peripheral blood mononuclear cells (PBMC) has been measured in patients with chronic HCV infection who had active liver disease, while normal levels have been detected in chronic HCV carriers with milder or no liver damage. The impaired IL-2 activity of PBMC, which has been claimed to be associated with high levels of virus duplication, has been interpreted as the reflection of an immune regulatory disorder of chronic active hepatitis C, which may be responsible for reduced differentiation of cytotoxic, virus-specific T cells and could explain the failure to eliminate all infected cells. Recently, methods for detecting membrane interleukin-2 receptor (mIL-2R), which exists on surface of T cells and can be released from activated lymphocytes, have been available, and high levels of these serum factors have been detected in a variety of pathologic conditions. To analyze IL-2R dependent immunoregulatory function in viral hepatitis, the levels of mIL-2R in the course of acute and chronic HCV infection in relation to the activity of liver disease and of virus replication have been investigated. The results suggest that in chronic active hepatitis C there is a state of activation rather than of depression of the IL-2 system, and the pathogenesis is related to the cellular immune function of the patients. PBMC are aggregation of immune cells,which have a lot of T lymphocytes, and play an important role in the anti-HCV infection. The cellular immune function of the patients may be the reflection of the level of T cell subsets and mIL-2R. The method of biotin-streptavidin (BSA) has higher specificity and sensitivity. In order to study the influence on the cellular immune function after being infected by HCV in PBMC and the effect on the mechanism of hepatitis C, the T cell subsets and mIL-2R of 203 patients with acute and chronic hepatitis C were detected by the above methods.

MATERIALS AND METHODS
Subjects
The 203 patients with confirmed diagnosis of hepatitis C (male 116 and female 87), aged 19-52 (average: 36.4 years), from our affiliated and teaching hospitals were chosen whose HCV-RNA in serum and PBMC were detected. Among them, the positive number of HCV-RNA in acute and chronic patients was 58 and 145 respectively. All diagnoses were made according to the diagnosis criterion of Chinese Hepatitis Conference 1995 (Beijing). The controls (n=20) were normal blood donors from the local Central Blood Bank.

Reagents and instruments
The antibodies against T cell subsets, Ficoll-Hypaque sedimentation gradient were produced by Shanghai Jingan Medical Institute, the Second Reagent Factory of Shanghai, and Huamei Bioengineering Company of Shanghai (No. 981010, 980215, 980422). Carbon dioxide gas incubator (MDF-135) was made in Japan.

Samples
The peripheral vein blood 5mL from each of the patients with hepatitis C was collected at 08:00, which was distributed 2.5mL in a sterile test tube and an anticoagulant test tube with heparin respectively.

Separation of PBMC and detection of T cell subsets, mIL-2R
After the anticoagulant heparinized blood was mixed with equal volume of Hanks' liquid without Ca²⁺ and Mg²⁺, the PBMC were harvested from heparinized whole blood by centrifugation on Ficoll-Hypaque sedimentation gradient and diluted to (1-3)×10⁹·L^-1 cells suspension with RPMI 1640 culture liquid. The 10μL suspension of PBMC was smeared on sheet glass pores so that the cells with CD₃⁺, CD₄⁺, CD₈⁺ could be detected. Of the PBMC suspension, 0.5mL was mixed with RPMI 1640 culture liquid, which had PHA 200mg·L^-1 . The cells were grown in continuous culture (37℃, 50mL·L^-1 CO₂ in atmosphere) for 72h and its mIL-2R induced by PHA could be measured by the antibodies against membrane of T cells.

Immunocytochemical method of Biotin- streptavidin system (BSA)
The different 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 15-20min. The cells were incubated in continuous culture (37℃, 50mL·L^-1 CO₂ in atmosphere) for 30min. 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 PBMC was counted and its positive cells were statistically analyzed with the help of high power lens. The positive criterion was that the color of endoplasm or cell membrane was brown, if not being negative.
      Statistical analysis was made by using Student's t test.

RESULTS
The results showed that the absolute positive rates of CD₃⁺, CD₄⁺ and CD₄⁺/CD₈⁺ were lower and CD₈⁺ was higher in patients with anti-HCV(+) than those in normal controls (P<0.01, Table 1). However, there was no significant difference between the patients with acute hepatitis C and normal controls (P>0.05). But there was a higher significant difference between the patients with chronic hepatitis C and normal controls (P<0.05). The reactivity of T cells in peripheral blood before and after being induced by PHA was all lower in acute, chronic hepatitis C than those in normal controls (P<0.01).

Table 1 The detective results of T cell subsets and mIL-2R in peripheral blood of patients with hepatitis C (mean±SD)

^aP<0.05, ^bP<0.01, vs normal control. AH: acute hepatitis; CH: chronic hepatitis

DISCUSSION
The chronicity of hepatitis is easier in patients with hepatitis C than that with hepatitis B^{[6,12,20,26,36]}. The T cell subsets of the 203 patients with hepatitis C were detected by the method of BSA, and the results showed that the positive rate of T cell subsets and their mutual proportion were significantly different (P<0.01), and there were significant disorders of cellular immune function and obvious pathologic injury.
      It seems probable that mIL-2R is an important symbol of active T cells and plays a key role in biologic effect and its expression level can reflect the course of T cells activity and immune situation. The expressive level of T cells in silence was lower in hepatitis C patients than in normal controls (P<0.01), which showed that the T cell activity was interfered. The possible reasons seemed to be as follows: (1)The mIL-2R on surface of T cells of the patients was inhibited by HCV; (2)The infection, duplication, proliferation of HCV in PBMC promoted the soluble interleukin-2 receptor to (sIL-2R) produced and released from T cells and inhibited the expression of mIL-2R ^[55-59]. This manifestation was caused by the low inducing cellular immune ability. Some active Tc cells could be combined tightly with the complex of HCV antigen-MHC-Ⅰmolecule on the surface of liver cells by T cells receptor (TCR) and released perforin. This irrevocable and progressive injury was one of the important reasons for chronicity of hepatitis C; (3)sIL-2R and mIL-2R could competitively combine with IL-2, and sIL-2R, being an immunosupperssive factor (similar to blocking factor), which could activate the IL-2 around the T cells so that the self-secretion effect of T cells decreased and the disorder of cellular immune function was enforced and the infection of HCV would be persisted^[60-62]. These conclusions were in agreement with the concept that both acute and chronic active hepatitis C liver cell damages were caused mainly by cytotoxic T lymphocytes directed against viral antigens expressed on the surface of infected cells and that CD₈⁺ lymphocytes were abundant in the infiltrates of the liver.
      The present study demonstrates that the receptor of PHA exists on the surface of T cells. The level of mIL-2R obviously increased after stimulation with PHA, which showed that mIL-2R could be induced by PHA and was significantly different between acute hepatitis C patients and chronic hepatitis C patients (P<0.05). The more serious the patient's condition, the longer the course of disease, and the lower the level of mIL-2R. The low level of mIL-2R was not beneficial for clearing away HCV and easy to reduce chronicity of hepatitis C.
      CD₃⁺, CD₄⁺ and CD₈⁺ are major function subsets of T cells and play a key role in response to HCV. Its counts and mutual relationship could be used to identify the cellular immune level and immunoregulation and is one of the valuable immunologic targets to forecast the change of patients' condition. The results showed that the lower positive rate of CD₃⁺ in chronic hepatitis C and the immunity to HCV were partly deficient. The lower positive rate and poor activation of CD₄⁺ are important reasons for the disorder of the immune function and can not clean HCV in time. Another key reason is the high level of CD₈⁺ in chronic active hepatitis C, which can cause injury of liver cells and increase of ALT persistently. The proportion of CD₄⁺/CD₈⁺ could play an important role in the network of immunoregulation. The high level of CD₈⁺ identified the injury of liver cells caused by CTL. These findings indicated that the immune response of the body to HCV was limited, and the infection of HCV existed persistently so that the patient's condition would change from acute to chronic.

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