Y-Jun Zhng,Ru Wng,Pei-Jie Chen,*,Ding-Hi Yu

aDepartment of Sports Medicine,School of Kinesiology,Shanghai University of Sport,Shanghai 200438,China

bDepartment of Wu Shu,Shanghai University of Sport,Shanghai 200438,China

Original article

Effects of Tai Chi Chuan training on cellular immunity in post-surgical non-small cell lung cancer survivors:A randomized pilot trial

Ya-Jun Zhanga,Ru Wanga,Pei-Jie Chena,*,Ding-Hai Yub,*

aDepartment of Sports Medicine,School of Kinesiology,Shanghai University of Sport,Shanghai 200438,China

bDepartment of Wu Shu,Shanghai University of Sport,Shanghai 200438,China

Background:Although emerging evidence points to benefits from Tai Chi Chuan(TCC)in improving immune system function,its effects on cellular immune responses remain under-studied.The objective of this study was to evaluate the effects of TCC training on cellular immunity in non-small cell lung cancer patients.

Methods:A 2-group randomized trial design in which post-surgical,non-small cell lung cancer survivors were randomly assigned to a TCC training group(n=16)or a control group(n=16).The participants in the TCC group completed a 16-week intervention.The main immune response outcome measures assayed included the ratio of T-helper cells/T-suppressor cells(CD4+:CD8+ratio)and complement regulatory proteins status(CRPs;CD55 and CD59).Using repeated measures ANOVA,the data were analyzed for the participants who completed the study (n=27).

Results:At 16 weeks,the TCC participants showed a significantly lower increment in the expression of CD55(p＜0.05)as compared to the control group.No significant between-group differences were found in the CD4+:CD8+ratio or CD59 expression.There were also no significant correlations among the changes in CRPs or T lymphocyte subpopulations,either.

Conclusion:A 16-week TCC intervention caused no alterations in CD4+:CD8+ratio,but significantly attenuated CD55 expression among postsurgical non-small cell lung cancer survivors.

Copyright©2013,Shanghai University of Sport.Production and hosting by Elsevier B.V.All rights reserved.

Complement regulatory proteins;Immunity;Non-small cell lung cancer;Tai Chi Chuan

1.Introduction

In many countries,lung cancer is a leading cause of death withthelowestsurvivalratesandhighmortality.Inlungcancer, up to 80%of the cases are associated with non-small cell lung cancer in which malignant cells form in the tissues of the lung.1Advances in surgical technology and chemotherapy have led to asignificant increase inthe long-term survivalofnon-small cell lung cancer patients.Studies have shown that in the United States,approximately 26,000 patients per year will survive more than 5 years after their initial diagnosis.2Therefore, promoting an active lifestyle and improving the quality of life among non-small cell lung cancer survivors may offer a positive prognosis,and thereby decrease the recrudescent oncogenesis risk.

Increasing epidemiological evidence shows that physical activity is associated with a reduced risk of oncogenesis3—6and an enhanced quality of life in cancer survivors.7—10Tai Chi Chuan(TCC),a traditional Chinese exercise for health promotion,has also been shown to enhance functional capacityand health-related quality of life among breast cancer survivors.11More recent studies reported that regular TCC exercise may also help increase the ratio of T-helper/T-suppressor cells (CD4+:CD8+ratio)and mediate Th1/Th2 T-helpercellbalance and reaction toward to Th1,suggesting that TCC training may elicit healthy immune responses and/or enhance T lymphocyte anti-neoplastic activity.12,13TCC,characterized by its slow, meditative,and moderate-intensity aerobic movements,may serve as an intervention that offers potential benefit to cancer survivors by boosting their immune system.14

Based on the current literature in the areas of TCC and immunology,the purpose of this study was to conduct a pilot trial aimed at determining whether TCC would result in changes in the T-helper/T-suppressor(CD4+:CD8+)ratio and membrane expression of the complement regulatory proteins (CRPs;CD55 and CD59)in non-small cell lung cancer survivors.On the basis of previous findings,12,13it was hypothesized that TCC would increase the T-helper/T-suppressor ratio and decrease the expression of CD55 and CD59.A secondary hypothesis was that the ratio of T-helper/T-suppressor would be negatively associated with an attenuated level of expression of CD55 and CD59.

2.Methods

2.1.Study design

The study was a randomized controlled trial with the participants randomized into either a TCC or a control group. The participants in the TCC group enrolled in a structured class three times per week for 16 weeks.The study protocol was approved by the institution review board in accordancewith the Declaration of Helsinki,and all participants signed informed consent forms.The clinical study was registered in the Chinese Clinical Trial Registry(No.ChiCTR-TRC-11001404).

2.2.Participants

The subjects were recruited from the Shanghai Lung Cancer Centre in Shanghai,China.Thirty-two subjects(16 males and 16 females)were enrolled in this study,all of whom were diagnosed with non-small cell lung cancer(NSCLC)and had received surgical therapy.Of the total number of enrolled subjects,27 completed the study and five dropped out because of personal reasons(Fig.1).The inclusion criteria included:(1) diagnosed with Stage I—IIIB NSCLC as per the guidelines provided by the American Joint Committee on Cancer and had received surgical therapy,and(2)had survived the surgery for more than 2 years and had not received chemotherapy,since recurrence occurs most with physical activity.15

Participants who were receiving immune therapy were excluded from this study.

2.3.Randomization and masking

Using a randomized design with computer-generated random numbers,we assigned the eligible participants in a 1:1 ratio to one of the two groups within the strata of their gender. The randomization codes were kept by an independent member of the team.The trial was single-blinded with the assessors blind to the study conditions.

2.4.Study conditions

Participants in the TCC group exercised meditative movements involving the elements of balance,postural alignment, and mental concentration.The participants learned to perform 24 standard TCC movements,and practicing breathing exercises taught by a senior instructor with 20 years of teaching experience.Using a polar sport tester(Polar Team2Pro,Oulu, Finland),their heart rate(HR)was monitored during TCC exercise and was set at between 40%and 59%of the maximum HR.16The participants in the TCC group underwent the TCC exercise three times per week;each exercise session lasted for 60 min,with a 10-min warm-up period and a 10-min cool down.

Participants in the control group were encouraged not to modify their daily life activity routine,including diet and physical activity.

2.5.Outcome assessment

Peripheral blood samples from participants who were not infected with an infectious disease during the past 3 days were collected at 7:00 am at baseline,and again upon completion of the 16-week study.The total RBC count,hemoglobin,and total lymphocyte counts were measured using an automated hematology analyzer(BC-3000,Shenzhen Mindray Bio-Medical Electronics CO.,Ltd.,Shenzhen,China).Flow cytometry(Beckman Coulter EPICS XL;Beckman Coulter, Inc.,Brea,CA,USA)was used to detect the mean fluorescence intensity(MFI)of the CD55/CD59 cells and the percentages of CD4+/CD8+T cell subpopulations.These analyses were conducted within 24 h after the blood samples were collected.Using flow cytometry,the CD55/CD59 levels on the T lymphocytes were evaluated by direct immunofluorescence.Briefly,peripheral blood mononuclear cells(PBMC) were separated from 100 μL of whole-blood by erythrocyte lysis using Optilyze C lysing solution(Beckman Coulter),and then the freshly-isolated PBMCs were washed in strokephysiological saline solution.The cells were labeled with 10 μL each of FITC-rat anti-human CD59 or PE-rat antihuman CD59(Beckman Coulter)primary antisera,followed by incubation for 20 min at room temperature.The cells were washed again after immunostaining,and then analyzed using an EPICS XL flow cytometer(Beckman Coulter).The lymphocytes were gated on the basis of forward and side scatter.17IgG1(mouse)-PE monoclonal antibodies(mAbs) and IgG2a-FITC(mouse)mAbs were used as CD55 and CD59 controls in each assay,respectively.Cells labeled with isotypecontrols were used to set the fluorescence thresholds for positivity,and thus＜1%of the cells labeled with isotypecontrol antibodies were positive.The Mfiwere recorded for lymphocytes expressing CD55 and CD59.17

Fig.1.Consort flow chart detailing participants recruitment.

Three-color Opticlone CD4-FITC/CD8-PE/CD3-PC5 monoclonal antibodies(Beckman Coulter)were used to evaluate the levels of the CD3+/CD4+/CD8+T lymphocyte subpopulations, and Opticlone IG1(mouse)-FITC mAb/IG1(mouse)-PE mAb/ IG1(mouse)-PC5 mAbs served as the isotypic control.Briefly, 20 μL of CD4-FITC/CD8-PE/CD3-PC5 monoclonal antibodies or 20 μL of Opticlone IG1(mouse)-FITC mAb/IG1(mouse)-PE mAb/IG1(mouse)-PC5 mAbs were added to the appropriate tubes,and 100 μL of whole blood was then added into the tubes and incubated for 15 min at room temperature in the dark,followed by erythrocytes lysis.Next,the strained cells were analyzedusinganEPICSXLflowcytometer(BeckmanCoulter). Lymphocytes were identified on the basis of their forward and side scatter,and the determination of positivity/negativity of the immunostained cells were based on the isotypic control.CD3-PC5 single positive cells were analyzed,and the percentage of CD3+Tlymphocyteswaspreciselydetermined.Similarly,CD3-PC5/CD4-FITC double positive cells or CD3-PC5/CD8-PE doublepositivecells wereassayed,andthepercentagesofCD4+or CD8+T lymphocyte subpopulations were simultaneously determined.

2.6.Statistical analysis

Baseline demographic descriptors and study outcome measures were compared between experimental groups using an analysis of variance for continuous variables,a Chi-square test for categorical variables,or tests for proportions.Only data from participants(n=27)who completed both baseline and post-intervention assessments were included in the endpoint analysis.The intervention efficacy was analyzed using a two-factor(group×time)repeated measures ANOVA to compare pre-and post-intervention changes in the outcome measures.The outcome variables analyzed included changes in the percentage of CD4+and CD8+T-lymphocytes,the ratio of T-helper/T-suppressor(CD4+:CD8+)cells,and the expression of CD55 and CD59.A pairedttest was used to assess within-group differences in the outcome variables.The Pearson rank correlation test was used to evaluate the correlations between the percentage of CD4+/CD8+T lymphocyte subpopulations and the CRPs(CD55 and CD59).All data are presented as means±SD.Allpvalues are two tailed and statistical analyses were performed using SPSS,version 17.0 (SPSS Inc.,Chicago,IL,USA).Given the exploratory nature of this preliminary study,no sample size calculations or power analysis were performed.

3.Results

The baseline characteristics of the participants are presented in Table 1.There were no significant differences in the demographic details and clinical status between the control and TCC groups.Intervention group statistics(mean±SD)at baseline and post-intervention and between-group differences from pretest to posttest(difference scores)on the study outcome variables are presented in Table 2.

Although the TCC participants exhibited significant pre-topost changes in their CD4+and CD8+(p＜0.05)levels and more importantly in their CD4+:CD8+ratio(p＜0.05),there were no between-group differences in these outcome variables. Therewas a significant between-group difference in the levelof CD55 lymphocytes(p=0.040),indicating that at the end of the 16-week intervention,the TCC participants exhibited an attenuated increase from baseline(mean=1.61)as compared to the control group(mean=2.09).However,there were no significant between group differences in the lymphocyte CD59 levels.Finally,no significant correlations were observed between changes in the levels of lymphocytes CD55 or CD59and changes in the percentages of CD4+/CD8+T lymphocyte subpopulationsand CD4+:CD8+ratio ineither ofthe two study groups(Table 3).

Table 1 Anthropometric and physiologic characteristics of the participants.

4.Discussion

In this randomized pilot study,we evaluated the effects of a 16-week TCC intervention on the lymphocytes CRPs(CD55 and CD59),the ratio of helper to suppressor T lymphocytes in post-surgical non-small cell lung cancer survivors.The major finding is that a 16-week TCC intervention significantly attenuated the increase in lymphocyte CD55 levels,which has been shown to negatively impact T cell immunity.18,19However,there were no significant changes in the ratio of helper to suppressor T cells after the TCC intervention,and no significant correlations were observed amongst the changes in the lymphocyte CD55 and T lymphocyte subpopulations.

Research using animal models has shown the importance of decay accelerating factors such as CD55 in modulating T cell responses.20Thus,lymphocytes expressing decreased CD55 levels may enhance T cell immunity.However,it remains largely unclear how exercise,such as TCC,can influence lymphocytes expressing CD55 and CD59.One possible molecular mechanism might be complement activation and chronic inflammation.Research from the use of animal-basedmodels suggested that CD55 may be expressed by neurons during chronic nervous inflammation associated complement activation,21and previous studies also suggested that regular aerobic exercise could selectively down-regulate complement fragments and induce anti-inflammatory effects.22—24It is therefore possible that TCC exercise,when used as a form of complementary and alternative medicine,may share the benefits produced by aerobic exercise,thus resulting in reduced chronic inflammation and complement activation which could be linked to a down-regulation of CD55.

Table 2 CD4+/CD8+T lymphocyte subpopulations and CRPs(CD55 and CD59)levels on lymphocytes att=0 andt=16 weeks.

Table 3 Correlations between changes in lymphocyte CD55:CD59 levels and CD4+/ CD8+T lymphocyte subpopulations.

The results from the current study showed that TCC exercise significantly attenuated the lymphocytes CD55 increase; however,no significant correlations were observed for any changes in the lymphocyte subpopulations.It is well-known that CD55 regulates T cell immunity via its influence on lymphocytes producing INF-γ,which may cause a more polarized th1 cell response.18,19Therefore,the ratio of helper/ suppressor T cells may be not specific to changes in lymphocyte CD55 levels.In addition,a previous study showed that lymphocytes expressing low levels of CD55 and CD59 immediately increased their expression post-exercise after intensive and downhill treadmill running at 80%maximal oxygen uptake(VO2max),but showed no changes after moderate treadmill running at 60%VO2max.25Therefore,one may speculate that lymphocytes expressing low levels of CD55 and CD59 were altered due to lymphocyte redistribution during acute exercise,but do not represent lymphocyte CRPs varying at the cellular level.However,our results were congruent with those reported by Wang et al.26in that a 4-week diet and moderate exercise program induced a lymphocyte CD55 level decrease in overweight adolescents.Theseresults collectively suggest that changes in the lymphocyte CRPs(CD55 and CD59)may be related to the intensity and duration of the exercise regimen applied.Furthermore, evidence suggests that long-term,moderate exercise may be beneficial to overall health.Therefore,future studies should investigatewhetherlong-term exercise would resultin a meaningful change in lymphocytes expressing CD55,and how these changes may be related to immune health.

Inconclusion,thecurrentstudydemonstratedthata16-week TCC intervention showed no effect on changes in the CD4+:CD8+ratio,but TCC training significantly attenuated the levels of lymphocytes expressing CD55 in post-surgical, non-small cell lung cancer survivors.These results,although promising,await future corroboration at a larger scale.

Acknowledgment

This study was supported by the National Natural Science Foundation of China(No.30840046).

1.D’Addario G,Fru¨h M,Reck M,Baumann P,Klepetko W,Felip E,et al. Metastatic non-small-cell lung cancer:ESMO clinical practice guidelines for diagnosis,treatment and follow-up.Ann Oncol2010;21:116—9.

2.Jones LW,Eves ND,Kraus WE,Potti A,Crawford J,Blumenthal JA,et al. The lung cancer exercise training study:a randomized trial of aerobic training,resistance training,or both in postsurgical lung cancer patients: rationaleand design.BMC Cancer2010;10:155.http://dx.doi.org/ 10.1186/1471-2407-10-155.

3.Colbert LH,Harman TJ,Tangrea JA,Pietinen P,Virtamo J,Taylor PR, et al.Physical activity and lung cancer risk in male smokers.Int J Cancer2002;98:770—3.

4.Lee IM.Physical activity and cancer prevention—data from epidemiologic studies.Med Sci Sports Exerc2003;35:1823—7.

5.Campbell PT,Sloan M,Kreiger N.Physical activity and stomach cancer risk:the influence of intensity and timing during the lifetime.Eur J Cancer2007;43:593—600.

6.Friedenreich CM,Cust AE.Physical activity and breast cancer risk: impact of timing,type and dose of activity and population subgroup effects.Br J Sports Med2008;42:636—47.

7.Irwin ML,McTiernan A,Bernstein L,Gilliland FD,Baumgartner R, Baumgartner K,et al.Physical activity levels among breast cancer survivors.Med Sci Sports Exerc2004;36:1484—91.

8.Clark MM,Novotny PJ,Patten CA,Rausch SM,Garces YI,Jatoi A,et al. Motivational readiness for physical activity and quality of life in longterm lung cancer survivors.Lung Cancer2008;61:117—22.

9.Coups EJ,Park BJ,Feinstein MB,Steingart RM,Egleston BL,Wilson DJ, et al.Physical activity among lung cancer survivors:changes across the cancer trajectory and associations with quality of life.Cancer Epidemiol Biomarkers Prev2009;18:664—72.

10.Smith AW,Alfano CM,ReeveBB,Irwin ML,Bernstein L, Baumgartner K,et al.Race/ethnicity,physical activity,and quality of life in breastcancersurvivors.CancerEpidemiolBiomarkersPrev2009;18:656—63.

11.Mustian KM,Palesh OG,Flecksteiner SA.Tai Chi Chuan for breast cancer survivors.Med Sport Sci2008;52:209—17.

12.Yeh SH,Chuang H,Lin LW,Hsiao CY,Eng HL.Regular Tai Chi Chuan exercise enhances functional mobility and CD4CD25 regulatory T cells.Br J Sports Med2006;40:239—43.

13.Yeh SH,Chuang H,Lin LW,Hsiao CY,Wang PW,Liu RT,et al.Regular Tai Chi Chuan exercise improves T cell helper function of patients with type 2 diabetes mellitus with an increase in T-bet transcription factor and IL-12 production.Br J Sports Med2009;43:845—50.

14.Mansky P,Sannes T,Wallerstedt D,Ge A,Ryan M,Johnson LL,et al.Tai Chi Chuan:mind-body practice or exercise intervention?Studying the benefit for cancer survivors.Integr Cancer Ther2006;5:192—201.

15.Sugimura H,Nichols FC,Yang P,Allen MS,Cassivi SD,Deschamps C, et al.Survival after recurrent nonsmall-cell lung cancer after complete pulmonary resection.Ann Thorac Surg2007;83:409—17.

16.Heyward VH.Advanced fitness assessment and exercise prescription. Champaign,IL:Human Kinetics;2006.

17.Christmas SE,de la Mata Espinosa CT,Halliday D,Buxton CA, Cummerson JA,Johnson PM.Levels of expression of complement regulatory proteins CD46,CD55 and CD59 on resting and activated human peripheral blood leucocytes.Immunology2006;119:522—8.

18.Heeger PS,Lalli PN,Lin F,Valujskikh A,Liu J,Muqim N,et al.Decayaccelerating factor modulates induction of T cell immunity.J Exp Med2005;201:1523—30.

19.Liu J,Miwa T,Hilliard B,Chen Y,Lambris JD,Wells AD,et al.The complement inhibitory protein DAF(CD55)suppresses T cell immunityin vivo.J Exp Med2005;201:567—77.

20.Li Q,Huang D,Nacion K,Bu H,Lin F.Augmenting DAF levelsin vivoameliorates experimental autoimmune encephalomyelitis.Mol Immunol2009;46:2885—91.

21.vanBeekJ,vanMeursM,’tHartBA,BrokHP,NealJW,ChatagnerA,etal. Decay-accelerating factor(CD55)is expressed by neurons in response to chronic but not acute autoimmune central nervous system inflammation associated with complement activation.J Immunol2005;174:2353—65.

22.Smith JK,Chi DS,Krish G,Reynolds S,Cambron G.Effect of exercise on complement activity.Ann Allergy1990;65:304—10.

23.Wolach B,Eliakim A,Gavrieli R,Kodesh E,Yarom Y,Schlesinger M, et al.Aspects of leukocyte function and the complement system following aerobic exercise in young female gymnasts.Scand J Med Sci Sports1998;8:91—7.

24.Gleeson M,Bishop NC,Stensel DJ,Lindley MR,Mastana SS, Nimmo MA.The anti-inflammatory effects of exercise:mechanisms and implications for the prevention and treatment of disease.Nat Rev Immunol2011;11:607—15.

25.Simpson RJ,Florida-James GD,Whyte GP,Black JR,Ross JA,Guy K. Apoptosis does not contribute to the blood lymphocytopenia observed after intensive and downhill treadmill running in humans.Res Sports Med2007;15:157—74.

26.Wang R,Chen P,Chen W.Effect of diet and exercise-induced weight reduction on complement regulatory proteins CD55 and CD59 levels in overweight Chinese adolescents.J Exerc Sci Fit2011;9:46—51.

26 September 2012;revised 21 November 2012;accepted 18 January 2013

*Corresponding authors.

E-mail addresses:chenpeijie@sus.edu.cn(P.-J.Chen),yudinghai0730@ yahoo.com.cn(D.-H.Yu)

Peer review under responsibility of Shanghai University of Sport

2095-2546/$-see front matter Copyright©2013,Shanghai University of Sport.Production and hosting by Elsevier B.V.All rights reserved. http://dx.doi.org/10.1016/j.jshs.2013.02.001