Intern. J.

Neuroscience, 114:1313–1322, 2004

Copyright  Taylor & Francis Inc.
ISSN: 0020-7454 / 1543-5245 online
DOI: 10.1080/00207450490476084

QI-TRAINING ENHANCES NEUTROPHIL

FUNCTION BY INCREASING GROWTH
HORMONE LEVELS IN ELDERLY MEN

MYEONG SOO LEE

Center for Integrative Medicine
Institute of Medical Science
Wonkwang University
Iksan, Korea

HOON RYU
Department of Neurology
University of Massachusetts Medical School
Worcester, Massachusetts, USA

This article investigated the effects of Qi-training on the neuroendo-

crine response and superoxide generation by neutrophils in healthy eld-
erly men. One hour of Qi-training significantly reduced the plasma con-
centration of cortisol, and increased plasma concentrations of growth
hormone and melatonin. Generation of superoxide by neutrophils in-
creased significantly immediately after Qi-training (p < .01). The change
in neutrophil superoxide generation was significantly correlated with
the change in growth hormone concentration after Qi-training (p <
.01). These data indicate that, in elderly men, Qi-training enhances
superoxide generation by neutrophils, possibly via the changes in plasma
growth hormone concentration.
Keywords cortisol, growth hormone, neutrophil, psychoneuroimmunology,
Qigong, superoxide generation

Received 26 December 2003.

Address correspondence to Myeong Soo Lee, Ph.D., Center for Integrative Medicine, Insti-
tute of Medical Science, Wonkwang University, Iksan 579-749, Republic of Korea. E-mail:
qimed@wonkwang.ac.kr; integmed@chol.com

1313
1314 M. S. Lee and H. Ryu

The effect of exercise on immunity is an important health issue, and

has been studied over a wide range of physical activities from light
exercise to intensive training programs (Brines et al., 1996: Nieman
& Pederson, 1999: Woods et al, 1999). Moderate exercise may en-
hance resistance to infection and inflammation (Woods et al., 1999),
whereas strenuous exercise may decrease resistance to infection (Nieman
& Pederson, 1999).
Neutrophils comprise 60–70% of the total circulating white blood
cell population, and act as the first line of defense against foreign
microorganisms (Ortega Rincon, 1994). Through their phagocytic
activity, neutrophils modulate bactericidal and fungicidal function,
and ingest and destroy pathogens by releasing toxic reactive oxygen
intermediates and hydrolytic enzymes. Although the biochemistry
of neutrophil respiratory function is well known, the mechanisms
by which exercise may influence neutrophil function are not at present
fully understood.
The authors have recently reported that Qi-training alters regula-
tory functions of the neuroendocrine and immune systems (Lee
et al., 1999; Ryu et al., 1995, 2000). However, they are unaware
of any reports on the effects of acute Qi-training on innate immu-
nity and hormones in the elderly. The aim of this study was to
determine whether Qi-training modulates hormone levels or the func-
tions of neutrophils. The effect of Qi-training on the plasma con-
centrations of cortisol, growth hormone (GH), and melatonin, and
on neutrophil production of superoxide (O2–) in elderly men was
investigated.

MATERIALS AND METHODS

Subjects

The subjects were seven male volunteers who performed ChunDoSunBup

(CDSB) Qi-training of the Iksan branch in Korea. Their physical
characteristics (mean ± SD) were: age 65.3 ± 2.0 yr, height 159.4 ±
5.32 cm, and weight 60.4 ± 6.8 kg; they had trained for 1.6 ± 0.5
years. The study protocol was approved by the Ethics Committee of
the Human Subjects Review Board at Wonkwang University Hospital
 Qigong Enhances Neutrophil Function in the Elderly 1315

and School of Medicine, and all subjects signed informed consent

forms. Subjects abstained from smoking and ingesting liquids with
caffeine or alcohol for at least 6 h before testing. All subjects were
in good health without a past or recent history of chronic disease,
malnutrition, malignancy, or renal disease. None were taking any
medications, such as steroid hormones, that might affect physical
activity or the variables measured in this study.

Materials
Hank’s balanced salt solution (HBSS) and phosphate-buffered sa-
line (PBS) were prepared by standard methods and filtered. HBSS
contained 5 mM D-glucose. A single batch of opsonized zymosan
was used for all experiments, and was prepared by incubating 10
mg of Zymosan-A particles (highly glycosylated fragments of yeast
cell walls) (Sigma Chemical Co., St. Louis, MO, USA) with 1 ml of
fresh human plasma at 37°C for 30 min. The suspension was centri-
fuged at 400 × g for 2 min, and the pellet was washed twice with
PBS before being resuspended in PBS at a final concentration of 10
mg/nil. Aliquots (1 ml) were stored frozen (4°C) and used only
once after thawing. Counting by hemocytometer revealed that this
preparation contained approximately 106 zymosan particles/µl.

Isolation of Polymorphonuclear Leukocytes (PMNs)

Human venous blood (10 ml) was collected from healthy adult
volunteers into heparinized tubes. To isolate neutrophils from pe-
ripheral blood, the heparinized blood was centrifuged for 10 min at
400 × g to remove the platelet-rich plasma. After sedimentation of
erythrocytes with 2% dextran for 30 min, neutrophils were isolated
under sterile conditions by density gradient centrifugation on Ficoll-
Paque cushions in conical tubes. The tubes were centrifuged at 400
× g for 30 min in swing-out buckets at room temperature. Contami-
nating erythrocytes were lysed with a hypotonic solution containing
NH4Cl-EDTA and then washed twice. The cells were gently resus-
pended in magnesium-free HBSS containing 1.6 mM CaCl2, and the
cell number and viability were determined. The entire procedure
was performed in sterile conditions at room temperature. The final
1316 M. S. Lee and H. Ryu

cell preparation comprised >97% neutrophils and <0.2% monocytes,

as assessed by Wright-Giemsa differential staining. The viability of
neutrophils was >98% as determined by trypan blue exclusion.

Measurement of Superoxide Anion (O2–) Production

Superoxide generation was measured by a chemiluminescence as-
say. Ten microliters of lucigenin (10,10'-dimethyl-9,9'-acridinum)
(Sigma) (0.3 mM final concentration). as a chemiluminescence source,
was added to each tube containing 1 × 106 cells in 300 µl of Veronal
buffer containing bovine serum albumin, glucose, Ca2+, and Mg2+.
The tubes were immediately placed in a lightproof and thermostatic
chamber of a six-channel Biolumant LB9505 (Berthold, Bad Wildbad,
Germany) and stabilized for 20 min. For the enhancement of O2–
production, the triggering agent, opsonized zymosan (50 particles/
cell) (Sigma) and phorbol 12-myrystate 13-acetate (150 nM, Sigma)
were added to the tubes. The number of photons from lucigenin-
dependent chemiluminescence was recorded in counts per minute
(cpm) for 60 min.

Hormone Assays
We used commercial radioimmunoassay kits were used to deter-
mine plasma cortisol (Radim, Rome, Italy), growth hormone (Radim),
and melatonin (Elias USA Inc., WI, USA). All measurements were
performed in duplicate.

Experimental Procedure and Blood Sampling

Subjects were directed by a master instructor and performed Qi-
training for 1 h, as previously reported (Ryu et al., 1995). The 1-h
session consisted of resting for 10 min before Qi-training, followed
by three types of exercise [15 min sound reciting (Chunmoon), 15
min slow movement, and 20 min meditation]. Peripheral blood was
obtained by venipuncture of the median cubital vein using heparin-
ized (10 U/ml) syringes. To determine the optimal sampling times,
three trained individuals participated in CDSB Qi-training. Prelimi-
nary data identified three sampling times to minimize the number
 Qigong Enhances Neutrophil Function in the Elderly 1317

of blood draws. Blood was sampled 10 min before Qi-training (pre),

within 10 min after the Qi-training (post I) and 2 h after Qi-training
(post II). The samples were immediately treated to separate the neu-
trophils using the methods described earlier (Ryu et al., 1997).

Statistical Analysis
Data are presented as the mean ± standard deviation. One-way re-
peated analysis of variance (ANOVA) was used to evaluate the
differences between sampling times. If the ANOVA showed a sig-
nificant effect of time (p < .05), subsequent comparisons were made
with the Scheffe’s post-hoc test. The Pearson correlation test was
used to determine the relation between variables, including changes
in hormone levels and superoxide generation by neutrophils.

RESULTS

Table 1 presents the mean and SD of plasma concentrations of cor-

tisol, GH, and melatonin. ANOVA showed significant effects of Qi-
training on the levels of cortisol [F(2, 12) = 4.22, p < .05], GH
[F(2, 12) = 4.49, p < .05], and melatonin [F(2, 12) = 3.76, p = .05].
Cortisol concentration decreased from pre to post I (p < .05), GH
concentration increased from pre to post I (p < .05), and melatonin
concentration increased nonsignificantly from pre to post I.
Figure 1 shows the mean values of neutrophil superoxide genera-
tion. ANOVA showed a significant effect of time on neutrophil
function [F(2, 12) = 8.88, p < .005]. The response to opsonized
zymosan increased significantly, by 32%, from pre to post I (p <
.01), and returned to the baseline value by post II.

TABLE 1. Changes in hormone concentrations by Qi-training in elderly men

Variables Pre Post I Post II F p

Cortisol (ng/ml) 74.83 ± 23.51 56.76 ± 17.22* 64.09 ± 7.59 4.22 0.04
GH (ng/ml) 0.27 ± 0.20 0.68 ± 0.51* 0.36 ± 0.23 4.49 0.04
Melatonin (pg/ml) 27.05 ± 1.80 30.11 ± 4.22 26.09 ± 2.88 3.76 0.05

Note. Values are mean ± SD (n = 7). *, significantly different from pre-training, p < .05, Scheffe’s post-
hoc test. F(2, 12) by one-way repeated ANOVA. Pre = before Qi-training; Post I = immediately after Qi-
training; Post II = 2 h after Qi-training.
1318 M. S. Lee and H. Ryu

FIGURE 1. Effect of Qi-training on mean superoxide (O2–) generation in isolated neu-

trophils stimulated in vitro with opsonized zymosan in elderly men. The figure shows a
typical response measured before (Pre), immediately after (Post I) and 2 h after (Post II)
Qi-training. Values are mean ± SD. **, significantly different from pre, p < .01, Scheffe’s
Post-hoc test.

Figure 2 shows that modest changes in the plasma concentration

of GH were significantly correlated with larger increases in the pro-
duction of superoxide (p < .01); changes in the plasma concentra-
tions of cortisol and melatonin were not significantly correlated with
changes in superoxide production.

DISCUSSION

Epidemiological evidence suggests that a single bout of Qi-training

(acute effects) and prolonged training over several months (chronic
effects) can produce positive and significant changes in the psycho-
logical, neuroendocrine, and immune systems (Lee et al., 1999; Ryu
et al., 1995, 2000). It has been proposed that Qi-training stimulates
psychological health and the immune system via secretion of neuro-
endocrine factors such as hormones. The present results demon-
strate that a single session of Qi-training enhances neutrophil oxida-
tive burst activity and modulates hormone levels in elderly men.
This study also found that Qi-training reduced blood cortisol con-
 Qigong Enhances Neutrophil Function in the Elderly 1319

FIGURE 2. Correlation between changes in superoxide production (Post I – Pre) and

changes in plasma GH concentration after Qi-training in elderly men.

centration. Other studies have documented decreased cortisol levels

in response to other techniques designed to induce a calm, positive
state of mind, such as transcendental meditation, and Tai Chi (Jin,
1989, 1992; MacLean et al., 1997). GH is a classical anterior pitu-
itary hormone that regulates and promotes cell growth and metabo-
lism (Kappel et al., 1993; Thelen et al., 1993). GH also influences
the development and function of the immune system (Spadoni et
al., 1991; Wiedermann et al., 1991). The level of GH rose signifi-
cantly after Qi-training; the authors propose that this increase may
influence neutrophil function.
Melatonin concentration also changes in response to Qi-training.
Regular practitioners of transcendental meditation have higher day-
time levels of the serotonin metabolite, 6-hydroxyindole-3-acetic
acid (5-HIAA), than controls, and the levels increase following medi-
tation (Bujatti & Riederer, 1976; Walton et al.. 1995). Similarly,
Massion et al. (1995) reported that melatonin levels increased in
subjects who practiced mindfulness meditation. Their study sug-
gested that the pineal gland might be sensitive to the practice of
meditation, which may cause increased levels of melatonin. The
1320 M. S. Lee and H. Ryu

present authors propose that the meditative effects of Qi-training

might be responsible for the increased melatonin level after Qi-
training.
These results showed that a single session of Qi-training had
significant effects on the neuroendocrinc axis. Qi-training may provide
a beneficial method for coping with stress by affecting emotional
resources and reducing the responses of stress-sensitive physiological
systems.
Qi-training increased neutrophil function; these data on the ef-
fects of Qi-training on immunological responses are of special in-
terest. Many studies have focused on the role of anxiety and stress
on cellular immune functions (Easterling & Babin, 1987; Peavey et
al., 1985). A study by Peavey et al. (1985) on health volunteers
showed a significant correlation between high stress levels and low
phagocytic capacity, and that the phagocytic potential can be aug-
mented following relaxation training.
GH, melatonin, and cortisol can each affect immune function
(Maestroni, 1995; Pieri et al., 1998; Smith et al., 1996; Zhou et al.,
1997). The present study demonstrated a significant correlation be-
tween an increase in plasma GH concentration and increased super-
oxide production by neutrophils after Qi-training in elderly men. In
contrast, neutrophil function was not significantly correlated with
changes in cortisol or melatonin levels. Because this study included
only a small sample of healthy older men, these results should be
replicated in a carefully controlled larger study. Future studies might
also include patients with psychological, hormonal, or immunological
impairments to determine optimal interventions to influence immune
function.
In summary, this article shows that Qi-training contributes to
enhanced neutrophil respiratory function and modulation of circu-
lating hormone levels. Given that neutrophils function as a first line
of defense against foreign microorganisms, the authors propose that
Qi-training may increase the resistance of trained individuals to
common infections and inflammation. Further research is needed to
determine which aspects of Qi-training contribute to these changes
in neutrophil functions, and to ascertain whether other types of
exercise would cause similar changes, or if they result from the
meditative aspects of Qi-training.
 Qigong Enhances Neutrophil Function in the Elderly 1321

REFERENCES

Brines, R., Hoffman-Goetz, L., & Pedersen, B. K. (1996). Can you exercise to make your
immune system fitter? Immunology Today, 17, 252–254.
Bujatti, M., & Riederer, P. (1976). Serotonin, noradrenaline, dopamine metabolites in trans-
cendental meditation-technique. Journal of Neural Transmission, 39, 257–267.
Easterling, B., & Babin, S. (1987) Stress-induced alternations of T-lymphocyte subset and
humoral immunity in mice. Behavioral Neuroscience, 101, 115–119.
Jin, P. (1989). Changes in heart rate, noradrenaline, cortisol and mood during Tai Chi.
Journal of Psychosomatic Research, 33, 197–206.
Jin, P. (1992). Efficacy of Tai Chi, brisk walking, meditation, and reading in reducing
mental and emotional stress. Journal of Psychosomatic Research, 36, 361–370.
Kappel, M., Hansen, M. B., Diamant, M., Jorgensen, J. O., Gyhrs, A., & Pedersen, B. K.
(1993). Effects of an acute bouts growth hormone infusion on the human immune sys-
tem. Hormone and Metabolic Research, 25, 579–585.
Lee, M. S., Kang, C. W., Ryu, H., Kim, J. D., & Chung, H. T. (1999). Effects of ChunDoSunBup
Qi-training on growth hormone, insulin-like growth factor-I, and testosterone in young
and elderly subjects. American Journal of Chinese Medicine, 27, 167–175.
MacLean, C. R. K., Walton, K. G., Wenneberg, S. R., Levitsky, D. K., Mandarino, J. P.,
Waziri, R., Hillis, L., & Schneider, R. H. (1997). Effects of the transcendental medita-
tion program on adaptive mechanism: Changes in hormone levels and responses to
stress after 4 months of practice. Psychoneuroendocrinology, 22, 277–295.
Maestroni, G. J. (1995). T-helper-2 lymphocytes as a peripheral target of melatonin. Jour-
nal of Pineal Research, 18, 84–89.
Massion, A. O., Teas. J., Herbert, J. R. , Wertheimer, M. D., & Kabat-Zinn, J. (1995).
Meditation, melatonin and breast/prostate cancer: Hypothesis and preliminary data. Medical
Hypotheses, 44, 39–46.
Nieman, D. C., & Pedersen, B. K. (1999). Exercise and immune function. Recent develop-
ments. Sports Medicine, 27, 73–80.
Ortega Rincon, E. (1994). Physiology and biochemistry: Influence of exercise on phago-
cytosis. International Journal of Sports Medicine, 15, S172–S178.
Peavey, B. S., Lawlis, G. F., & Goven, A. (1985). Biofeedback-assisted relaxation: Effects
on phagocytic capacity. Biofeedback and Self-Regulation, 10, 33–47.
Pieri, C., Recchioni, R., Moroni, F., Marcheselli, F., Marra, M., Marinoni, S., & Di Primio,
R. (1998). Melatonin regulates the respiratory burst of human neutrophils and their
depolarization. Journal of Pineal Research, 24, 43–49.
Ryu, H., Jeong, S. M., Jun, C. D., Lee, J. H., Kim, J. D., Lee, B. S., & Chung, H. T.
(1997). Involvement of intracellular Ca2+ during growth hormone induced priming of
human neutrophils. Brain Behavior and Immunity, 11, 39–46.
Ryu, H., Jun, C. D., Lee, B. S., Choi, B. M., Kim, H. M., & Chung, H. T. (1995). Effect
of qigong training on proportions of T lymphocyte subsets in human peripheral blood.
American Journal of Chinese Medicine, 23, 27–36.
Ryu, H., Lee, M. S., Jeong, S. M., Lee, J. H., Kang, C. W., Lee D. Y., & Chung, H. T.
(2000). Modulation of neuroendocrinological function by psychosomatic training: Acute
effect of ChunDoSunBup Qi-training on growth hormone, insulin-like growth
factor (IGF)-I, and insulin-like growth factor binding protein (IGFT3P)-3 in men.
Psychoneuroendocrinology, 25, 439–451.
Smith, J. A., Gray, A. B., Pyne, D. B., Baker, M. S., Telford, R. D., & Weidemann, M. J.
(1996). Moderate exercise triggers both priming and activation of neutrophil subpopu-
lation. American Journal of Physiology, 270, R838–R845.
Spadoni, G. L., Spagnoli, A., Cianfarani, S., Del Principe, D., Menichelli, A., Di Giulio,
S., & Boscherini, B. (1991). Enhancement by growth hormone of phorbol diester-stimulated
respiratory burst in human polymorphonuclear leukocytes. Acta Endocrinologica, 124,
589–594.
1322 M. S. Lee and H. Ryu

Thelen, M., Dewald, B., & Baggiolini, M. (1993). Neutrophil signal transduction and ac-
tivation of the respiratory burst. Physiological Reviews, 73, 797–821.
Walton, K. G., Pugh, N. D., Gelderloos, P., & Macrae, P. (1995). Stress reduction and
preventing hypertension: Preliminary support for a psychoneuroendocrine mechanism.
Journal of Alternative and Complementary Medicine, 1, 263–283.
Wiedermann, C. J., Niedermuhlbichler, M., Beimpold, H., & Braunsteiner, H. (1991). In
vitro activation of neutrophils of the aged by recombinant human growth hormone.
Journal of Infectious Diseases, 164, 1017–1020.
Woods, J. A., Davis, J. M., Smith, J. A., & Nieman, D. C. (1999). Exercise and cellular
innate immune function. Medicine and Science in Sport and Exercise, 31, 57–66.
Zhou, J., Olsen, S., Moldovan, J., Fu, X., Sarkar, F. H., Moudgil, V. K., & Callewaert,
D. M. (1997). Glucocorticoid regulation of natural cytotoxicity: Effects of cortisol on
the phenotype and function of a cloned human natural killer cell line. Cellular Immu-
nology, 178, 108–116.