Although it is recognized that the mechanical stresses associated with physical activity augment bone mineral density and improve bone quality, our understanding of how exercise modulates bone homeostasis at the molecular level is lacking. In a before and after trial involving 43 healthy adults, we measured the effect of six months of supervised exercise training on the spontaneous and phytohemagglutinin-induced production of osteoclastogenic cytokines (interleukin-1
Interest in cytokines as regulators of bone metabolism began with the experiments of Horton and associates who, in 1972, found that conditioned medium from PHA-stimulated peripheral blood mononuclear cells contained bone resorbing activity [
Along with fall prevention and calcium and vitamin D supplementation, the Surgeon General has recommended regular physical activity as the first line in fracture prevention in persons with low bone density [
We have investigated the possibility that long-term moderate intensity exercise improves bone health by favorably altering the production of cytokines with osteoclastogenic and antiosteoclastogenic properties by peripheral blood mononuclear cells.
This before and after clinical study was approved by the Institutional Review Board of East Tennessee State University. Each subject read and signed the informed consent in the presence of an investigator.
Subjects aged 30 to 60 were recruited from the general population by placing an outline of the study and a request for volunteers in three local newspapers. A total of 77 persons responded and all agreed to be screened for eligibility (see Figure
Flow diagram.
Forty-three subjects (25 women, average age 48 years [range 30–58], and 18 men, average age 49 years [range 35–59]) successfully completed the study; their risk factors for osteoporosis are listed in Table
Risk factors for osteoporosis
Men ( |
Women ( |
|
---|---|---|
Age, mean (SD), y | 48.1 (8.0) | 49.7 (7.2) |
Estradiol deficiency |
4 (28%) | 15 (60%) |
Inactivity |
11 (61%) | 16 (64%) |
Smoking | 1 (6%) | 4 (16%) |
Alcohol |
9 (50%) | 9 (36%) |
Eight percent of women had five risk factors, 12% had four risk factors, 24% had three risk factors, 36% had two risk factors, 16% had one risk factor, and 4% had no risk factors for osteoporosis. Fifty percent of men had one risk factor and 50% had two risk factors for osteoporosis. Twenty-four percent of women and 28% of men were obese (BMI > 30
Supervisors kept detailed records documenting attendance, the duration and type of each exercise, weights, changes in medications, changes in dietary or smoking habits, and state of health. These data, along with risk factors for osteoporosis, were analyzed for their potential effects on outcome measurements (cytokine production, C-terminal telopeptides of Type I collagen, osteocalcin levels, hormonal levels, lymphocyte phenotypes, and mitogen responses) and for group and within-group differences.
Immunologic studies were done at baseline and after completion of 6 months of training
Solid-phase enzyme-linked immunoassay kits were used to measure serum levels of osteocalcin (BRI-Diagnostics Bioresearch, Dublin, Ireland), C-terminal telopeptides of Type I collagen (CTXI) (Osteometer Biotech A/S, Herlev, Denmark), and relevant hormones (25 (OH) vitamin D, estradiol, testosterone, parathyroid hormone, and insulin-like growth factor 1) (American Laboratory Products Company, Salem, New Hampshire).
Immunophenotyping of blood lymphocytes was done as previously described [
Cytokine production was measured as previously described [
Mitogenic responses were measured by adding methyl-3H-thymidine (20
Statistical analysis was done using STATISTICA (Statsoft, Inc., Tulsa, OK). The two-sided
In this analysis, we have identified IFN-
Modulation of bone and immune cells by cytokines.
Cytokine | Osteoblast | Osteoclast | Osteocyte | Bone (in vitro) | Rodents (in vivo) | T cells, B cells, and macrophages | References |
---|---|---|---|---|---|---|---|
IL1- |
↑ RANKL | ↓ apoptosis | ↑ resorption | [ | |||
|
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TNF- |
↑ RANKL | ↓ apoptosis |
↑ resorption |
[ | |||
|
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IFN- |
↓ RANKL signaling pathways | ↓ collagen synthesis | ↑ bone loss |
↑ TNF- |
[ | ||
|
|||||||
IL-4 | ↓ RANKL |
↑ Th2-type |
[ | ||||
|
|||||||
Il-6 | ↑ RANKL |
↓ RANKL |
↑ production with loading | ↓ TNF- |
[ | ||
|
|||||||
Il-10 | ↑ OPG | ↓ RANKL signaling pathways | ↓ bone loss | ↓ IFN- |
[ | ||
|
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TGF- |
↓ RANKL, |
↓ osteoid |
↑ production with loading | ↑ osteoid matrix | ↓ TNF- |
[ |
RANKL, receptor activator of nuclear factor kappa B ligand, promoting osteoclastogenesis by binding to RANK on osteoclast precursors. OPG, osteoprotegerin; a decoy RANKL receptor & potent inhibitor of osteoclastogenesis. Wnt1, a protein crucial to normal bone formation. IL-1ra, interleukin-1 receptor antagonist.
Postexercise TNF-
Effect of exercise on osteoclastogenic cytokine production. Exercise attenuated the production of TNF-
Collectively, osteoclastogenic cytokine production fell by 59% in PHA+ cultures (
Exercise modality and cytokine production. Osteoclastogenic cytokine levels fell in proportion to the time subjects spent in each training session doing aerobic exercises (Pearson correlation test with regression analysis). Data points for cytokines are the sum of IL-1
Postexercise TGF-
Effect of exercise on antiosteoclastogenic cytokine production. Exercise enhanced the production of IL-6, IL-10, and TGF-
Body weight and IL-6 levels. In both PHA− and PHA+ cultures, IL-6 values correlate linearly with body weight (Pearson correlation test with regression analysis).
Collectively, antiosteoclastogenic cytokine production increased by 50% in PHA+ cultures and by 89% in PHA− cultures (
Postexercise IL-2 levels increased by 116% in PHA+ cultures (
Effect of exercise on IL-2 production. Exercise increased the production of IL-2 in both PHA− and PHA+ cultures. Preexercise values are represented by the white columns and postexercise values by the black columns. PHA− culture values are listed on the left
Levels of lymphocytes expressing LFA-1, an integrin that augments endothelial cell adhesion, increased by 21% in response to exercise (
PHA-induced proliferative responses of T cells fell from 9,
CTXI levels fell by 16% (
Scattergram showing the effects of the exercise training program on serum levels of osteocalcin and CTXI. Osteocalcin levels increased by 9.8 percent (
Postexercise CTXI levels correlated inversely with estradiol levels (Figure
Estradiol and the antiresorptive effect of exercise. Postexercise CTXI levels correlate inversely with estradiol levels (Pearson correlation test with regression analysis). The results suggest that estradiol enhanced the antiresorptive effects of the exercise training program. Note: the correlation improves with removal of the apparent outlier (425 pg/mL estrogen):
Subjects trained an average of 2.5 hours per week (range 0.3–7.4 hours). The average duration of each exercise session was 71 minutes (range 36–123 minutes), and the average number of visits per week was 2 (range 1 to 5). During each training session, subjects divided their time between aerobic (57%), resistance (weightlifting) (35%), and flexibility (stretching) (8%) exercises. Aerobic exercises included walking or running (32%), cycling (16%), aerobics (3%), rowing (3%), climbing (2%), and skiing (1%).
PBMCs taken from men prior to exercise spontaneously produced more IFN-
In both men and women, exercise attenuated the production of TNF-
Osteoclastogenic cytokines in women and men. Exercise attenuated the production of TNF-
Antiosteoclastogenic cytokines in women and men. In PHA− cultures, exercise enhanced the production of TGF-
Four subjects (9.3%) changed their diet to one that was lower in energy intake and animal fat, and 2 of the 5 tobacco users discontinued smoking during the study. There were no changes in medications or alcohol consumption.
By completion of the study, 32% of the women and 27% of the men had lost weight, and 4% of the women and the 11% of men had gained weight; there was no significant change in the mean weight of either group.
No group or within-group differences could be demonstrated as a result of weight change, menopause, use of medications, alcohol consumption, or smoking (one-way ANOVA).
The primary effect of our exercise program was to change the balance between PBMCs producing osteoclastogenic cytokines and those producing antiosteoclastogenic cytokines. It is likely that similar changes occurred in hematopoietic cells occupying the microenvironment of bone where they are ideally situated to influence the ontogeny and functioning of cells responsible for bone formation (osteoblasts), bone resorption (osteoclasts), and the transduction of bone loading signals (osteocytes) (see Table
Our study participants averaged two and one-half hours of moderate intensity exercise per week: the same amount of exercise recommended for adult men and women by the World Health Organization for maintenance of health [
Cross-sectional studies involving adult subjects and using bone mineral density measurements have shown that exercises involving high impact (e.g., jumping) and high resistance (e.g., weightlifting) appear to be particularly effective in improving bone mass and content, especially when the intensity of the exercise is high and the speed of movement is elevated. Loaded (weight-bearing) aerobic exercises such as walking or running also have the potential to improve bone mass in adults and have the added benefit of improving cardiovascular conditioning [
Studies on the impact of different exercise modalities on biomarkers of bone formation and resorption support the importance of both resistance and aerobic training in maintaining bone health. Fujimura and associates found that high intensity resistance training (weightlifting) done three times weekly for four months by 17 young male subjects caused a sustained increase in serum calcitonin levels, although plasma procollagen Type I C-terminal levels, a marker of bone resorption, did not change [
In our study, it is of interest that postexercise culture levels of IL-6 were proportionate to weight, which is a measure of one’s mass times the intensity of the gravity field (9.8 m/sec2 on Earth). It is possible, therefore, that the failure of exercise programs to attenuate bone loss in the microgravity of space is related, at least in part, to suboptimal production of this pleiotropic cytokine. IL-6 exerts context-dependent effects on bone metabolism [
This study would have benefitted from the inclusion of a nonexercising age- and sex-matched control group. Phenotypic analysis of Th17 and T regulatory cells and an assay for the mononuclear cell production of IL-17, a cytokine with osteoclastogenic activity, would also have been beneficial. Cardiorespiratory fitness measurements (
Long-term moderate intensity exercise exerts a favorable effect on bone resorption by changing the balance between peripheral blood mononuclear cells producing osteoclastogenic cytokines and those producing antiosteoclastogenic cytokines. This beneficial effect may be enhanced by estradiol, emphasizing the importance of this hormone as a regulator of bone metabolism. The results provide a new insight as to how physical exercise contributes to the maintenance of bone health and suggest a possible molecular mechanism to explain the difference in the antiresorptive effects of exercise done on Earth as compared to exercise done in the microgravity of space.
The funders/supporters had no role in the design and conduct of the study; collection, management, analysis, or interpretation of the data; review or approval of the paper; or decision to submit the paper for publication.
The authors declare that there is no conflict of interests regarding the publication of this publication.
Dr. J. Kelly Smith has full access to all of the data in the study and takes responsibility for the integrity of the data and accuracy of the data analysis. Study concept and design, paper preparation, and funding were done by J. Kelly Smith. Acquisition of data was carried out by J. Kelly Smith, Rhesa Dykes, and David S. Chi. Paper review was carried out by Rhesa Dykes and David S. Chi.
This study was supported by the Tennessee Chair of Excellence Grant no. 20233 (J. Kelly Smith). The authors gratefully acknowledge Scott Reynolds, B.S., and Karen Cantor, B.S. (James H. Quillen College of Medicine, East Tennessee State University), for their assistance in lymphocyte phenotyping.