Chapter 2 Methods
2.4 Preparation of mononuclear cell-conditioned media (MNC-CM)
Human MNC from each subjects were isolated from the peripheral blood by density centrifugation (400 g, 30 min) in a Ficoll-Hypaque solution (1.077 g/ml) 19.
After being washed by phosphate-buffered saline three times, MNC was suspended in RPMI-1640 medium (Gibco/BRL, Grand Island, NY, USA) containing 10%
heat-inactivated autoserum, and incubated in autoserum-coated culture plates. Cells, at an initial concentration of 1 x 106 cells/ml, were cultured in 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT) with 1,000 micromole/l L-glutamine (Gibco/BRL), 100 mg/l streptomycin (Gibco/BRL), 50 mg/l penicillium (Gibco/BRL), and RPMI 1640 medium containing 5 μg/ml of phytohemagglutinin (PHA; Difco Lab., Detroit, MI, USA), respectively, at 37 oC in a fully humidified incubator with 5% CO2. The
7
collected aliquot was filtered through 0.45 μ membrane to remove MNC after cultivation for 24 hours. This collected aliquot, named as conditioned media (CM), was stored at -80 oC until use. 20 PHA-MNC-CM, prepared with 5 μg/ml PHA for 24 hours, were used to observe the relative HBsAg expression in Hep3B cells using the method described below. MNC-CM prepared with 5 μg/ml PHA was collected to assay the secretion of cytokine in CM. Hep3B cell medium, which a volume of only 30% .PBS contained with 5 μg/ml PHA replaced the MNC-CM, was named as the untreated control group. PHA, isolated from plants, is a natural mitogen of T lymphocytes. It has acted as an immuno-stimulant in many studies to simulate immune response and effect. PHA-MNC-CM from the sedentary control group was termed as SC-PHA-MNC-CM and the TCC group as TCC-PHA-MNC-CM. PBMNC stimulated with gradient concentrations of PHA was act as a model system to simulate immune reaction for evaluation of drug-induced,21,22 exercise-mediated immunomodulation in our previous studies. 19,23In this model system, various cytokines, including those relate to anti-viral and anti-tumor immunity such as γ, TNF-α and
IFN-from PBMNC by PHA stimulation.
8 2.5 Assay for cytokines
To detect the secreted cytokines in PHA-MNC-CM,commercial ELISA kits, including IL-1β, TNF-α, IFN-α, and IFN-γ (R&D Systems, Minneapolis, MN, USA), were used to determine at a wavelength of 450 nm by according to the method described by Wang et al.20 The correlation coefficients (γ) for the standard curves of three cytokines were between 0.998-0.999. Three separate experiments were each tested in duplicate. TCC-PHA-MNC-CM were pre-incubated at 37 °C for 90 min with various cytokine-neutralizing antibodies including anti-IFN-γ (30.0 μg/ml, is 10 folds more over than the concentration as near 100% of neutralization), anti-TNF-α (2.4 μg/ml), anti-IFN-α (1.0 μg/ml) and anti-IL-1β (5.1 μg/ml) in combination or alone.
Viable cells were counted after 48 hours of incubation with added antibodies of the cytokines. Three separate experiments were each tested in duplicate.
2.6 Assay for relative HBsAg expression
The HBsAg is a useful index to evaluate the viral activity24 for the present of HBsAg in serum of patients indicated a current HBV infection and the risk for developing compensated cirrhosis and hepatocellular carcinoma. 25,26,27 After been cultured in DMEM with 10% fetal bovine serum for 24 hrs, Hep3B cells were
9
transferred to serum-free DMEM with or without 30% (v/v) MNC-CM and incubated for 48 hrs. We used commercial ELISA kits (General Biological, Taipei, Taiwan, Republic of China) to determine the secreted HBsAg in the culture medium. By pretest, PHA does not interfere with the HBsAg assay by ELISA kits. The determined optical density (O.D.) values of the Enzyme-Linked Immunosorbent Assay (ELISA) kits during measurement were normalized with cell numbers. The relative HBsAg expression was determined by following formula: (HBsAg/MTT) from PHA- MNC-CM / (HBsAg/MTT) from the untreated control group culture media. The (HBsAg/MTT) from the untreated control group culture media was treated as 100%
expression.
2.7 Statistical analysis
Results are presented as mean ± standard error of the mean (SEM). Differences between the treatment groups, which consisted of matched samples, was assessed by Student’s t test. A confidence level of 5% (p < 0.05) was considered significant.
10
Chapter 3 Results
3.1 The anthropometric measurement and peak oxygen uptake (V‧
O2peak) of subjects
The basically anthropometric measurement of the two subjects groups were summarized in Table 1. The SC group was measured as mean age of 54.8 ± 5.4 years (range 50 to 63 years old). The TCC group was measured as mean body weight of 58.7
± 9.6 kg (range 43 to 73 kg), mean age of 55.3 ± 5.3 years (range 48 to 66 years old), and mean body length of 161.7 ± 9.7 (range 140 to 175 cm). There was no difference in these anthropometric measurements between the SC group and TCC group (p>.05).
The V‧
O2peak observed in TCC group, as 27.9±6.1, were similar to in SC group, as 25.2±5.6. (p>.05) From Table 2, RER values exists no observable difference between before and after TCC exercise. During TCC exercise performance, the blood lactate concentration (from 1.95 to 1.76 mmol/L) seems exist no obvious change.
From the data of % HRmax (at 48.35±7.61 to 65.66±6.23 %) and of %V‧
O2peak (at 23.15±14.85 to 47.17±13.39 %), the intensity of exercise increased during TCC exercise performance slightly higher.
11
3.2 Comparison of the reduction of relative HBsAg expression in Hep3B cells stimulated by PHA-MNC-CM between the SC group and TCC group
The response in TCC-PHA-MNC-CM was much more obvious than in SC-PHA-MNC-CM. For instance, a much lower relative HBsAg expression to the extent about 68.4% of HBV in Hep3B cells, incubated with TCC-PHA-MNC-CM prepared by stimulating with PHA at the same 5 μg/ml in comparison with the SC-PHA-MNC-CM, was observed (Table 4). No apparent death of Hep3B cells was examined in both groups when the PHA administered less than 5μg/ml. No obvious inhibition of relative HBsAg expression in Hep3B was observed in the SC group (Table 4).
3.3 Effects of cytokine against HBsAg Expression in Hep3B cells
After being stimulated by PHA, the secretion of IFN-γ, IL-1β, TNF-α and IFN-α in TCC-PHA-MNC-CM all significantly raised up to (665 ± 160 pg/ml, 626 ± 167 pg/ml,1186 ± 274 pg/ml and 832 ± 215 pg/ml respectively), nevertheless, compared to those in SC-PHA-MNC-CM to (335 ± 103 pg/ml, 282 ± 107 pg/ml, 512 ± 244 pg/ml and 317 ± 132 pg/ml, respectively). (Table 3) It seemed exist no difference that the se- cretion of IL-2. Comparing to the same concentration of PHA (5 μg/ml) stimulation,
12
the secretion of TNF-α in TCC-PHA-MNC-CM was about 2 fold greater than that in the SC-PHA-MNC-CM.
When comparing the amount of PHA at 5 μg/ml in TCC-PHA-MNC-CM and SC-PHA-MNC-CM, the secretion of IFN-γ in TCC-PHA-MNC-CM was about 1 folds greater than that in SC-PHA-MNC-CM. The secretion IL-1β in TCC-PHA-MNC-CM, stimulating by the amount of PHA at 5 μg/ml, were at about 2 times greater than in SC-PHA-MNC-CM. The secretion of IFN-αin TCC-PHA-MNC-CM was also at about 2 times greater than in SC-PHA-MNC-CM (Table 3). The aliquots of CMT-PHA- MNC-CM were pre-incubated with one cytokine-neutralizing antibodies as anti-IL-1β, anti-IFN-γ, anti-IFN-α, and anti-TNF-α before cultivation of Hep3B cells to invest- igate the effects of cytokines on the reduction of relative HBsAg expression in Hep3B cells.
Results in Table 4 show that the relative HBsAg expression in Hep3B cells elevated to 81.3 ± 13.2% (from 68.4 ± 13.3%) in the presence of 500 N.U./ml anti-IFN-γ antibodies and to 77.5 ± 11.4% in the presence of 500 N.U./ml anti-TNF-α antibodies.
The relative HBsAg expression in Hep3B cells elevated to the lower level at 64.7 ± 10.6% in the presence of anti-IL-1β antibody in CMT-PHA-MNC-CM. The relative HBsAg expression in Hep3B cells elevated to 77.5 ± 11.4% in the presence of anti-
13
IFN-α antibody. Although there was a statistically significant difference when comp- ared to CMT-PHA-MNC-CM alone, there was little elevation of relative HBsAg. The most reducing effect on the relative HBsAg expression in Hep3B cells was at about 86.3± 9.7% with a combination of all three cytokine antibodies.
14
Chapter 4 Discussion
The basic anthropometric measurements of TCC group were similar to SC group, for example, the two groups were similar in gender, height weight, age, body fat and
‧V repeated sequences for 24 minutes, seems to be a moderate intensity exercise.
The similar results were also observed middle aged by Chen and in elderly by Lan.28 The factor that% V‧
O2peak after TCC exercise at about 47.17 % can also support the other strong evidence to define TCC as moderate intensity exercise. The MET after TCC practicing was about 3.9 METs in 24 min. It has shown that the exercise intensity is neither high nor low.
From the little to no change of blood lactate between exercise duration, the TCC practicing can be defined as aerobic exercise and low to moderate intensity exercise.
The data present in our study have shown that greater immunomodulatory response of
15
PBMNC isolated from middle-aged people with Tai Chi Chuan exercise can significantly inhibit HBsAg expression of HBV in an ex vivo experimental model.
The greater response by secretion of cytokines, mainly the TNF-α, IFN-α and IFN-γ, may cause the inhibitory effects. The basic anthropometric characteristics of TCC group exhibited no difference between SC group. Moreover, theV‧
O2peak were also similar. The TCC exercise with 108 postures and some repeated sequences, which performed by TCC group, seems to be exercise with moderate intensity. The TCC, a moderate intensity exercise, can avoid some risks of exercise damage. In our other data, the TCC as moderate exercise intensity was assumed by the measurement of MET after TCC practicing at about 3.5 METs in 24 min. There are little to no change of blood lactate during TCC exercise. It indicated that TCC practicing can be defined as aerobic exercise.
To enhance anti-viral immunity, moderate intensity exercise might be a suitable way. In vivo study by Lowder29, moderate exercise training inhibited activity of influenza virus. Due to the removal of MNC after stimulation by various concentrations of PHA, the soluble mediators produced by MNC in CM may be the factors about inhibiting HBsAg expression in Hep3B cells.
The levels of IFN-α, IFN-γ and TNF-α existed in the TCC-PHA-MNC-CM were
16
much greater than those in the SC-PHA-MNC-CM. It partially lost the inhibitory activity against relative HBsAg expression when the TCC-PHA-MNC-CM is neutralized by anti-IFN-γ, anti-TNF-α and/or anti-IFN-α antibodies (except anti-IL-1β).
The results of our experiments of antibody neutralization showed that IFN-γ, IFN-α and TNF-α ,in soluble factors CM, might contribute to the greater inhibitory activity of TCC-MNC-CM. It must be pointed out that no cytotoxicity to MNC and Hep3B cells was observed (data not shown). The greater inhibitory activity of TCC-MNC-CM did not come from the cytotoxicity of Hep3B cells.
TNF-α was well documented to be and activator of antiviral effects in the immune system. For instance, noncytopathic suppression of hepatitis B virus DNA replication in hepatocytes was caused by the administration of TNF-α.30 A marked diminish of HBV replication occurred after administration of TNF-α to Hep3B cells.31
The prominent role played by TNF-α were assumed by our both data about the greater secretion of TNF-α and about the lower relative HBsAg expression with anti-TNF-α antibody neutralization in TCC-PHA-MNC-CM. The HBsAg inhibitory effects of IFN-γ has been presented in some clinical studies.31,32,33 Other study about active habitual older runners with moderate exercise showed that the greater blood IFN-γ concentration stimulated with PHA than that of sedentary older people was
17 observed.34
The much lower relative HBsAg expression with anti-IFN-γ antibody neutralization and higher IFN-γ in TCC-PHA-MNC-CM both provide evidences to assumed that the crucial inhibitory role played by IFN-γ. the similar phenomena have been presented in other study. The negative correlation of cell cytoplasmic HBV DNA and secreted level of IFN-γ in the supernatants of PBMNC medium was observed.35.36 Other evidence in similar experimental model about the anti-virus activity of immuno-cells activated by increased secretion of IFN-α has been demonstrated. For example, the neutralization by antibody of IFN-α in MNCs diminished the viral suppressing effects against Herpes simplex viruses.37
In addition, IFN-α has been used to inhibit the virus replication in clinical therapy. The administered IFN-α to HBeAg-positive patients led to loss of HBeAg from 25 to 40%.38,39 With both experimental data about much higher production of IFN-α and about lower relative HBsAg expression after the neutralization of anti-IFN-α antibody in TCC-PHA-MNC-CM, we can postulated that IFN-α exerts critical effects on suppression of hepatitis B virus.
In spite of the greater secretion of cytokines related closely to the reduction of HBsAg expression, the host HBV-harboring Hep3B cells did not killed by TCC-PHA-
18
MNC-CM in our study. It is logical to assume that the inhibitory activity of TCC- MNC-CM against HBsAg expression may not attribute to the cytotoxicity to Hep3B cells. It has been illustrated that both of the TNF-α and IFN-γ secreted fromHBV- specific cytotoxic T lymphocytes can inhibit HBV gene expression and replication without cytopathy.
After administration combined all of anti-TNF-α, anti-IFN-α and anti-IFN-γ antibodies together, the inhibitory effect against HBsAg expression in TCC-PHA- MNC-CM blocked the greatest. In other words, above three cytokines induced by TCC exercise played the mainly immunomodulatory anti-viral role. In summary, greater immunomodulatory response against HBsAg expression of HBV cells line exhibit in TCC group was illustrated by using an ex vivo antiviral immunity model. The much more secretion of cytokines, mainly IFN-γ, TNF-α and IFN-α from human peripheral blood MNC contributed to the greater immunomodulatory response against HBsAg expression of HBV cells line.
19
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24 List of Figure and Table
Table 1. The anthropometric measurement of subjects
TCC (n=12) SC (n=12) t p
Age (years) 55.3 ± 5.3 54.8 ± 5.4 0.438 >.05 Height (cm) 161.7 ± 9.7 163.6 ± 5.4 0.391 >.05
Weight (kg) 58.7 ± 9.6 63.8± 8.4 0.133 >.05
Body Fat (%) 26.4 ± 6.1 29.2 ± 4.9 0.124 >.05
‧V
O2peak(ml/[kg x min] ) 27.9±6.1 25.2±5.6 0.136 >.05
The middle-aged people with Tai Chi Chuan were designated as (TCC) and the sedentary control group as (SC). V‧
O2peak : peak oxygen uptake. Results are expressed as mean ± SEM.
25
Table 2. The blood lactate concentration and energy expenditure before and after Tai Chi Chuan exercise
Before TCC exercise After TCC exercise Blood lactate (mmol/L) 1.95 ± 0.36 1.76 ± 0.42
%HRmax 48.35±7.61 65.66±6.23
%V‧
O2peak 23.15±14.85 47.17±13.39
METS 1.4±0.9 3.9±1.2
RER 0.75±0.07 0.78±0.09
The middle-aged people with Tai Chi Chuan were designated as (TCC). % HRmax:
percentage of exercise intensity of maximal heart rate achieve during incremental exhaustive exercise;%V‧
O2peak: percentage of exercise intensity of peak oxygen uptake;
26
Table 3. The cytokines secreted in PHA-MNC-CMs.
SC-PHA-MNC-CM TCC-PHA-MNC-CM
IL-1β 282 ± 107 626 ± 167
IFN-α 317 ± 132 832 ± 215
TNF-α 512 ± 244 1186 ± 274
IFN-γ 335 ± 103 665 ± 160
The middle-aged people with Tai Chi Chuan were designated as (TCC).
TNF-α: tumor necrosis factor α; IFN-α: interferon-α, IL-1β: interleukin-1β and
anti-IFN-γ: interferon-γ. Triplicated data from separate experiments are expressed as mean ± SEM.
27
Table 4. Effects of PHA-MNC-CMs, cytokine antibody neutralization on the relative HBsAg expression.
Relative HBsAg expression (%) MTT (%)
Untreated-control 100 100
SC-PHA-MNC-CM 84.3 ± 11.7 99.3± 13.4
TCC-PHA-MNC-CM 68.4 ± 13.3 96.1 ± 11.2
+anti-IL-1β 71.3 ± 11.4 98.9 ± 12.1
+anti-IFN-γ 81.3 ± 13.2 96.4 ± 13.1
+anti-IFN-α 77.5 ± 11.4 96.7 ± 14.1
+anti-TNF-α 78.7 ± 12.7 97.4± 12.4
+anti-TNF-α + anti-IFN-α + anti-IFN-γ 86.3 ± 9.7 96.7 ± 12.8
Aliquots of TCC-PHA-MNC-CM were pre-incubated with or without two cytokine neutralizing antibodies anti-TNF-α (anti-tumor necrosis factor α, 2.4 μ g/ml), anti-IFN-α (anti-interferon-α, 1.0 μg/ml), anti-IL-1β (anti-interleukin-1β, 5.1 μg/ml) and anti-IFN-γ(anti-interferon-γ, 30.0 μg/ml) at 37 oC for 90 min before addition to Hep3B cell culture. Triplicated data from separate experiments are expressed as mean
Aliquots of TCC-PHA-MNC-CM were pre-incubated with or without two cytokine neutralizing antibodies anti-TNF-α (anti-tumor necrosis factor α, 2.4 μ g/ml), anti-IFN-α (anti-interferon-α, 1.0 μg/ml), anti-IL-1β (anti-interleukin-1β, 5.1 μg/ml) and anti-IFN-γ(anti-interferon-γ, 30.0 μg/ml) at 37 oC for 90 min before addition to Hep3B cell culture. Triplicated data from separate experiments are expressed as mean