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 .
this activity was eventually found to be due to interleukin- ( il- ) 1 and tumor necrosis factor- ( tnf- )  [ 2 , 3 ] , prompting a series of studies examining the role of these and other proinflammatory cytokines as mediators of bone resorption in periodontal disease , rheumatoid arthritis , osteolytic malignancies , and osteoporosis [ 48 ] .
in addition to il-1 and tnf- , the spectrum of cytokines with effects on bone metabolism now includes the antiosteoclastogenic cytokines il-4 and il-10 and transforming growth factor- ( tgf- )  , and two pleiotropic cytokines , interferon- ( ifn- )  and il-6 , whose effects on bone vary depending on experimental conditions [ 9 , 10 ] . 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 .
evidence indicates that the mechanical stresses associated with physical activity augment bone mineral density and improve bone quality by promoting adaptive changes in its geometry and architecture . in the microgravity of space
, however , bone loss continues at an average rate of one to two percent per month despite the institution of vigorous exercise programs designed to duplicate conditions on earth [ 13 , 14 ] , indicating that there remains much to be learned about the mechanism(s ) whereby physical exercise ( and gravity ) influences the ontogeny and functioning of hematopoietic and bone cells involved in the maintenance of bone health .
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 1 ) .
fifty - two volunteers met eligibility criteria and were enrolled in a hospital - based wellness center where they underwent 6 months of supervised training .
subjects were required to exercise for a minimum of 30 minutes twice a week . each training session consisted of a combination of aerobic ( 60% ) , resistance ( weightlifting ) ( 30% ) , and flexibility ( stretching ) ( 10% )
exercises . aerobic exercise choices included walking , running , cycling , rowing , climbing , skiing , and aerobics .
forty - three subjects ( 25 women , average age 48 years [ range 3058 ] , and 18 men , average age 49 years [ range 3559 ] ) successfully completed the study ; their risk factors for osteoporosis are listed in table 1 .
nine subjects were excluded : 8 did not meet attendance requirements and 1 moved to another location .
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 . in order to minimize seasonal influences on the results
, 2 - 3 subjects were enrolled each month over an eleven - month period .
subjects were instructed not to exercise outside of the supervised exercise program and for at least 24 hours prior to blood drawing .
all blood samples were drawn in the morning at approximately the same time of day .
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   using lysed whole blood and a facscan flow cytometer ( becton dickinson , san jose , calif . ) and fluorescein- and phycoerythrin - labeled murine monoclonal igg antibodies to measure levels of t lymphocytes ( cd3 ) ; t helper lymphocytes ( cd4 ) ; t cytotoxic lymphocytes ( cd8 ) ; t lymphocytes displaying mhc class ii antigen ( dr ) , vascular adhesion molecule-4 ( vla-4 ) ( cd49d ) , lymphocyte function - associated antigen-1 ( lfa-1 ) ( cd11a ) , fas antigen ( cd95 ) , or gamma - delta t cell receptors ( tcr ) ; b lymphocytes ( cd3cd19 ) ; b1 lymphocytes ( cd3cd19cd5 ) and natural killer cells ( nk cells ) ( cd3cd16cd56 ) .
peripheral blood mononuclear cell ( pbmc ) preparations containing 1520% monocytes and 8085% lymphocytes were isolated from venous blood using accu - prep ( accurate chemical & scientific corp . ,
westbury , ny ) , washed three times at 10c with sterile phosphate buffered saline ( ph 7.4 , 0.1  m ) , and suspended at a concentration of 2  10  cells/l in rpmi-1640 containing 5% heat inactivated human ab serum ( v / v ) , l - glutamine ( 2  mm ) , penicillin ( 50  u / ml ) , and gentamicin and streptomycin ( 50  g / ml each ) .
preparations were incubated under 5% co2 at 37c for 48 hours with and without phytohemagglutinin ( pha ) ( 5  g / ml ) , a lectin mitogen that stimulates unprimed t cells to proliferate by cross - linking their t cell receptors ( this assay provides a more definitive assessment of t cell function than assays using unstimulated ( pha ) cultures ) .
culture supernatants were rendered cell - free by centrifuging at 1,000  g for 10 minutes at 12c and stored in 1 - 2  ml aliquots at 80c for later use .
supernatants were subsequently assayed for il-1 , il-2 , il-4 , il-6 , il-10 , il-12 , tnf- , ifn- , and tgf-1 using solid - phase enzyme - linked immunoassay kits ( quantikine , r & d systems , inc . , minneapolis , mn , for il-1 and tgf-1 ; predicta , genzyme corp . , cambridge , ma , for tnf- ; cytoscreen , biosource international , inc . ,
, westbrook , me , for il-2 , il-4 , il-6 , il-10 , and il-12 ) .
mitogenic responses were measured by adding methyl - h - thymidine ( 20  ci / ml , 50  l ) to culture samples removed 6 hours prior to supernatant harvesting .
labeled samples were incubated under 5% co2 at 37c for an additional 6 hours , and the cells were collected on glass fiber filter paper using a mash ii cell harvester .
samples were air - dried , placed in vials containing scinti - verse ii , and assayed with a beckman ls 9800 liquid scintillation counter .
proliferative responses are expressed as net counts per minute ( cpm ) , calculated as cpm in pha - stimulated cultures minus cpm in unstimulated cultures .
the two - sided t - test for dependent samples was used to determine the significance of differences between measurements .
the pearson correlation test was used to quantify the relationship between two variables and linear regression was used to determine the predictability of y from x. one - way analysis of variance ( anova ) was used to compare measurements of three or more groups . unless stated otherwise ,
in this analysis , we have identified ifn- as an osteoclastogenic cytokine and il-6 as an antiosteoclastogenic cytokine . in our opinion ,
the reader is referred to table 2 for a summary of the effects of these and the other studied cytokines on bone homeostasis and immune cell function .
postexercise tnf- levels fell by 52% and 28% in cultures with and without added pha ( p  .0031 ) .
il1- levels fell by 13% in pha cultures ( p = .0472 ) and by 3% in pha cultures ( p > .05 ) .
ifn- levels fell by 71% in pha cultures ( p < .0001 ) and by 44% in pha cultures ( p = .0065 ) ( figure 2 ) .
collectively , osteoclastogenic cytokine production fell by 59% in pha cultures ( p < .0001 ) and by 24% in pha cultures ( p > .05 ) ; in pha cultures , the percent reduction was proportionate to the average time subjects spent per training session doing aerobic exercises ( figure 3 ) .
postexercise tgf-1 levels increased by 37% and 43% in cultures with and without added pha ( p < .0001 ) .
il-10 levels increased by 9% in pha cultures ( p > .05 ) and by 940% in pha cultures ( p = .0025 ) .
il-4 levels increased by 94% in pha cultures ( p = .0012 ) and showed no change in pha cultures .
il-6 levels increased by 50% in pha cultures and by 112% in pha cultures ( p < .0001 ) ( figure 4 ) . in both pha and pha cultures , il-6 production was linearly related to body weight ( figure 5 ) .
collectively , antiosteoclastogenic cytokine production increased by 50% in pha cultures and by 89% in pha cultures ( p < .0001 ) .
postexercise il-2 levels increased by 116% in pha cultures ( p = .0470 ) and by 63% in pha cultures ( p < .0001 ) ( figure 6 ) .
il-12 levels fell by 40% in pha cultures ( p > .05 ) and showed no change in pha cultures .
levels of lymphocytes expressing lfa-1 , an integrin that augments endothelial cell adhesion , increased by 21% in response to exercise ( p = .0033 ) . b1 ( il-10 b repressor ) cell counts and percentages increased by 330% ( p = .0580 ) and 190% ( p = .0464 ) , respectively , and cd4/cd8 ratios fell by 5.6% ( p = .0129 ) .
pha - induced proliferative responses of t cells fell from 9,142  760  cpm to 3,155  584  cpm following exercise ( p < .0001 ) .
the reason for this change is not known , although it may reflect the change in cd4/cd8 ratios and an increased activity of suppressor cell populations ( e.g. , m2 monocytes , b1 cells , and t repressor cells ) .
ctxi levels fell by 16% ( p = .0128 ) and osteocalcin levels increased by 9.8% ( p > .05 ) in response to the exercise program ( figure 7 ) .
there was no significant change in serum levels of estradiol , testosterone , parathyroid hormone , or insulin - like growth factor-1 following exercise .
the average duration of each exercise session was 71 minutes ( range 36123 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- and tnf- than mononuclear cells taken from women ( p  .04 ) . following exercise
, men spontaneously produced more il1- and tnf- than women ( p  .004 ) . in both men and women , exercise
attenuated the production of tnf- and ifn- ( p  .0062 ) and enhanced the production of il-6 and tgf-1 ( p  .0028 ) in pha - stimulated cultures .
postexercise levels of tgf- also increased in pha cultures in both groups ( p  .0049 ) ( two - sided t - test for dependent samples ) ( figures 9 and 10 ) .
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 2 ) .
this conclusion is supported by the finding that our exercise program caused significant reductions in serum levels of ctxi , a reliable marker of bone resorption , and modest rises in osteocalcin , a reliable marker of bone formation .
postexercise levels of ctxi correlated inversely with estradiol levels , suggesting that estradiol enhanced the antiresorptive effects of exercise , a conclusion in keeping with the report of others that the beneficial effect of exercise on bone density is blunted after menopause .
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 .
participants divided their time between aerobic exercises ( 57% ) , resistance exercises ( 35% ) , and flexibility exercises ( 8% ) and spent an average of 71 minutes in each training session . extended over a six - month period , this amount of exercise was sufficient to reduce the spontaneous and pha - induced production of osteoclastogenic cytokines by 24 and 59% , respectively , and to increase the spontaneous and pha - induced production of antiosteoclastogenic cytokines by 89% and 50% , respectively .
in pha - stimulated cultures , osteoclastogenic cytokine production fell in proportion to the time subjects spent in each session doing aerobic exercises .
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 [ 56 , 57 ] .
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 a study involving 56 previously inactive young women , lester and associates found that 8 weeks of resistance or combined aerobic - resistance training increased serum levels of osteocalcin but had no significant effect on markers of bone resorption . in a study more closely resembling ours , alghadir and associates found that 12 weeks of moderate intensity aerobic training done for 45 to 60 minutes three times weekly by 65 healthy subjects ( 36 males , 29 females ) aged 3060 increased serum osteocalcin levels and decreased serum levels of deoxypyridinoline , a marker of bone resorption . 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 / sec 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 [ 5 , 30 , 31 ] .
although it can stimulate osteoclastogenesis and bone resorption by upregulating rankl expression in osteoblasts , its effects on bone metabolism are predominantly osteogenic and antiresorptive .
il-6 enhances bone formation by promoting the differentiation of osteoblast precursors [ 33 , 34 ] and by protecting osteoblasts against apoptosis [ 5 , 35 ] ; it can inhibit bone resorption directly by downregulating rankl signaling pathways in osteoclasts   and indirectly by suppressing the production of tnf- and il-1 and stimulating the production of il-4 , il-10 , and il-1 receptor antagonist by immune cells [ 37 , 38 ] .
it is also an essential growth factor for b cells , the primary source of opg in bone marrow stroma , and can induce il-2 production in t cells [ 38 , 40 , 41 ] .
il-6 is produced in osteoblasts and osteocytes in response to bone loading signals and , like tgf- , plays an important role in bone remodeling .
thus , studies similar to that described in this report would be of particular interest if done on persons exercising in the microgravity of space .
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 ( vo2max ) done before and after the exercise program was completed would have added potentially useful information .
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 .
 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 , tumor necrosis factor- ) , antiosteoclastogenic cytokines ( transforming growth factor-1 and interleukins 4 and 10 ) , pleiotropic cytokines with variable effects on osteoclastogenesis ( interferon- , interleukin-6 ) , and t cell growth and differentiation factors ( interleukins 2 and 12 ) by peripheral blood mononuclear cells . 
 we also measured lymphocyte phenotypes and serum markers of bone formation ( osteocalcin ) , bone resorption ( c - terminal telopeptides of type i collagen ) , and bone homeostasis ( 25 ( oh ) vitamin d , estradiol , testosterone , parathyroid hormone , and insulin - like growth factor 1 ) . a combination of aerobic , resistance , and flexibility exercises done on average of 2.5 hours a week attenuated the production of osteoclastogenic cytokines and enhanced the production of antiosteoclastogenic cytokines . 
 these changes were accompanied by a 16% reduction in collagen degradation products and a 9.8% increase in osteocalcin levels . 
 we conclude that long - term moderate intensity exercise exerts a favorable effect on bone resorption by changing the balance between blood mononuclear cells producing osteoclastogenic cytokines and those producing antiosteoclastogenic cytokines . 
 this trial is registered with clinical trials.gov identifier : nct02765945 . 
1. Introduction 
2. Methods
3. Results
4. Discussion
5. Conclusions