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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 19, 2004 as doi:10.1096/fj.03-1286fje. |
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,1
,1,2
* The Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts, USA;
Saga Nutraceuticals Research Institute, Otsuka Pharmaceutical Co., Ltd, Saga, Japan; and
Tufts-New England Medical Center, Boston, Massachusetts, USA
2Correspondence: Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111, USA. E-mail: evannier{at}tufts-nemc.org
SPECIFIC AIM
Decline in muscle mass and strength with age, a protracted process termed sarcopenia, is an important cause of disability and mortality in the elderly. Whether old and young individuals retain a similar capacity to heal from injury is poorly understood. We tested whether senescence of human skeletal muscle impairs cytokine response to exercise-induced injury. We focused on a set of inflammatory (TNF-
and IL-1ß) and anti-inflammatory cytokines (IL-6 and TGF-ß1) that contribute to recruitment of leukocytes to the injured muscle and/or the remodeling of muscle tissue.
PRINCIPAL FINDINGS
1. Cytokine transcript levels in vastus lateralis from young and old subjects
Physically active young (23–35 years, n=15) and old (66–78 years, n=15) men ran downhill (16% decline) on a treadmill for three 15 min intervals at a speed set to reach 75% of each subject’s predetermined maximal oxygen consumption. Biopsies from the vastus lateralis of the nondominant leg (at the lower third portion of the thigh) were obtained 24 h before and 72 h after acute eccentric exercise. Cytokine transcripts were quantified by real-time PCR (Fig. 1
). Before exercise, there was no age-related difference in the levels of transcripts encoding TNF-, IL-1ß, IL-6, and TGF-ß1. Seventy-two hours after exercise, TNF- mRNA levels increased 2.8- and 1.8-fold (both P<0.01) in young and old subjects, respectively (Fig. 1A
). IL-1ß mRNA levels increased marginally 1.7-fold (P=0.063) in young subjects, and to a greater extent (2.8-fold, P < 0.01) in old subjects (Fig. 1B
). Acute eccentric exercise failed to increase IL-6 mRNA levels in old subjects (1.3-fold, P=0.44). In contrast, IL-6 mRNA levels increased 3.6-fold in young subjects after acute eccentric exercise (Fig. 1C
). This increase nearly reached significance (P=0.057). TGF-ß1 mRNA levels increased 3.7- and 2.0-fold in young (P=0.03) and old subjects (P<0.01), respectively (Fig. 1D
).
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2. CD18 transcript levels in vastus lateralis from young and old subjects
The extent of leukocyte infiltration was monitored by quantifying mRNA levels for integrin CD18, a pan-leukocyte surface marker. We chose CD18 because inflammatory mediators increase CD18 surface expression via mobilization of preformed intracellular stores (independent of transcription and protein synthesis). At baseline, CD18 mRNA levels did not differ between young and old subjects. Seventy-two hours after eccentric exercise, CD18 mRNA levels increased 10.1-fold (P=0.005) in young subjects but only 4.7-fold (P=0.02) in old subjects. The mean fold increase was greater (P<0.05) in young than in old subjects.
3. Age-associated differences in relationships among cytokine and CD18 mRNA levels
Changes in cytokine and CD18 transcript levels were expressed as the difference between post- and pre-exercise values divided by the pre-exercise value. There was an overall age-associated difference in correlations among cytokine and CD18 transcript levels (P<0.05; Lantz and Perlman test). Individual correlations among cytokines and CD18 were analyzed in young and old subjects. In young subjects, changes in CD18, TNF-, IL-1ß, IL-6, and TGF-ß1 mRNA levels correlated positively with each other (all but one P<0.001). In the elderly, positive correlations were noted only between changes in mRNA levels for CD18 and TNF- (P<0.01) or TGF-ß1 (P<0.01), and between changes in TNF- and TGF-ß1 mRNA levels (P=0.01). At old age, there was no correlation between IL-1ß and CD18 transcript accumulations.
CONCLUSIONS AND SIGNIFICANCE
Eccentric muscle contractions induce muscle damage that results in systemic acute phase response and infiltration of leukocytes in the damaged tissue. We chose an eccentric exercise protocol of moderate intensity so that, in young and old men, serum CK and CRP levels were elevated 24 h after exercise but receded to pre-exercise levels at 72 h (data not shown). In contrast, leukocyte infiltration (assessed by CD18 transcript levels) persisted for at least 72 h after exercise. We therefore monitored cytokine transcripts 72 h after exercise, that is, during the repair phase while avoiding the early phase of muscle damage and acute systemic inflammation.
It is well established that cytokine transcripts and proteins accumulate in human skeletal muscle after acute eccentric exercise or prolonged strenuous exercise, but there is scant evidence that aging impairs the muscle-derived cytokine response to exercise. In the present study, aging altered the relationships among variables associated with inflammation (Fig. 2
). A striking feature was the markedly reduced accumulation of CD18 transcripts induced by exercise in old men. Likewise, there was a trend toward a blunted accumulation of TNF- and TGF-ß1 transcripts in the exercised muscle of old men. In young and old men, the accumulation of CD18 transcripts strongly correlated with the accumulation of TNF- and TGF-ß1 transcripts. These data indicate that leukocyte recruitment is reduced at old age, but remains strongly associated with TNF- and TGF-ß1 gene expression in both young and old muscles. This age-associated decrease in leukocyte recruitment is unlikely due to a reduced pool of circulating leukocytes since circulating neutrophil counts at baseline did not differ between young and old subjects (data not shown). Since myoblasts and myofibers remain a minor source of TNF- and TGF-ß1, we propose that resident macrophages and infiltrated monocytes are the main source of these cytokines. TGF-ß1 is a potent chemotactic factor for neutrophils and monocytes. The chemotactic activity of TGF-ß1 has been confirmed in vivo at sites of inflammation. Thus, local production of TGF-ß1 may amplify and sustain cellular recruitment to sites of exercise-induced muscle damage. The ability of TGF-ß1 to induce TNF- synthesis in monocytes may explain the strong correlation between these two cytokines. Whether TGF-ß1 antagonizes the catabolic activity of TNF- on skeletal muscle, as it inhibits other activities of TNF- on leukocytes, is unknown. The biological significance of reduced TGF-ß1 synthesis in old muscle remains uncertain since in addition to its complex pro- and anti-inflammatory properties, TGF-ß1 is an inhibitor of myogenic differentiation.
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IL-1ß and TNF- are proinflammatory cytokines produced in a coordinated fashion and acting in a synergistic manner. We observed a strong correlation between the increases in TNF- and CD18 transcripts in either young or old men. In contrast, the increases in IL-1ß and CD18 transcripts correlated weakly at young age, and did not correlate at old age. In fact, TNF- transcripts tended to accumulate to a lesser extent at old age while IL-1ß transcripts tended to accumulate to a greater extent. The lack of association between IL-1ß and CD18 transcripts at old age suggests that the IL-1ß gene is not exclusively expressed in leukocytes. Indeed, IL-1ß protein has been localized to muscle and nonmuscle cells in skeletal muscle tissue harvested from young men after eccentric exercise. By reducing the recruitment of leukocytes to the exercised muscle, aging should favor the contribution of muscle cells to total IL-1ß gene expression. This may be the case since the reduced cellularity is not compensated for by a greater IL-1ß synthesis in old mononuclear cells. Because IL-1ß transcripts tended to be higher in exercised muscle tissues at old age, our results suggest that regulation of IL-1ß gene expression in muscle cells differs with age. A recent report by Jozsi et al. lends support to this hypothesis. These authors observed that acute resistance exercise failed to induce an accumulation of IL-1ß transcripts in vastus lateralis of elderly subjects, but did so in young subjects. However, their acute resistance exercise protocol combined eccentric and concentric contractions of lower intensity than the eccentric concentrations performed by our subjects. Moreover, IL-1ß transcripts were detected in muscles of the elderly subjects when enrolled in the resistance exercise protocol for three months. Taken together, Jozsi’s and our results indicate that the propensity of skeletal muscle tissues from the elderly not to express the IL-1ß gene may be overcome by increasing the intensity or the frequency of muscle contractions. Since the repetition of low intensity exercise induces IL-1ß transcript accumulation in old muscle, muscle contraction rather than muscle damage may be sufficient to induce IL-1ß gene expression, hence a case for muscle cells as an important source of IL-1ß in skeletal muscles of the elderly.
After eccentric exercise, circulating IL-6 levels rise to a lesser extent in the elderly. Although skeletal muscles are now recognized as the major source of circulating IL-6 after exercise, the effect of aging on IL-6 gene expression in skeletal muscle has not been explored. In young subjects, we observed a 3.6-fold increase in IL-6 transcripts 72 h after cessation of eccentric exercise. The increase in IL-6 transcripts correlated strongly with the increase in CD18 transcripts, but failed to correlate with the increase in serum CK measured 24 h post-exercise. These results corroborate previous observations that IL-6 gene expression after eccentric exercise is not induced by muscle damage, but rather associated with leukocyte activation during muscle repair/regeneration at young age. At old age, eccentric exercise did not induce an accumulation of IL-6 transcripts despite a robust (4.8-fold) increase in CD18 transcripts. The inability of eccentric exercise to induce IL-6 gene expression was not due to a lower "metabolic workload" in the elderly since blood lactate levels rose to a greater extent in the elderly. Unlike moderate endurance exercise or prolonged strenuous running, our acute eccentric exercise protocol increased the constitutive IL-6 synthesis in mononuclear cells from young and old subjects. Since IL-6 synthesis in blood mononuclear cells also increases with aging, our findings suggest that exercised skeletal muscle in the elderly produces inhibitors of IL-6 gene expression by the newly recruited mononuclear cells. Because exercise would be a requirement for the production of inhibitors, leukocytes residing in elderly muscles would retain the ability to produce IL-6, hence the strong positive correlation between CD18 and IL-6 transcripts in both young (r=0.59, P=0.02) and old muscles (r=0.54, P=0.04) before exercise. An alternative (or possibly complementary) hypothesis is that the newly recruited leukocytes not only produce IL-6, but also induce IL-6 gene expression in myofibers and/or myoblasts. If so, aging may render exercised muscles either unable to synthesize IL-6 or able to produce inhibitors that act in an autocrine fashion to suppress the leukocyte-mediated induction of IL-6 synthesis.
Our study establishes that human skeletal muscle senescence disrupts the coordinated expression of inflammatory and anti-inflammatory cytokines that otherwise follows an acute bout of eccentric exercise. With aging, the reduction in the pan-leukocyte integrin CD18 is closely associated with a reduction in TNF- and TGF-ß1, suggesting that TGF-ß1 is a major factor in the recruitment of leukocytes to injured muscles, and that these leukocytes are a major source of TNF- in injured muscles. In contrast to this triad, the age-associated increase in IL-1ß points to muscle cells themselves as an alternative source of cytokines with aging, and may represent an attempt of muscle cells to compensate for the reduction in TNF- and IL-1ß of leukocyte origin. The lack of IL-6 in exercised muscles of the elderly further speaks to the dissociation between leukocyte infiltration and cytokine production in these elderly men. Because muscle-derived IL-6 is released in the circulation and promotes liver glycogenolysis, the lack of muscle-derived IL-6 may affect the maintenance of glucose homeostasis during exercise of the elderly. Our findings suggest that senescence of human skeletal muscle may impair its adaptive response to acute eccentric exercise by preventing a coordinated cytokine gene expression necessary for adequate muscle contractile function and adequate remodeling of injured skeletal muscle.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1286fje;
1 These authors contributed equally to the work. 
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0.06; *P < 0.05; **P < 0.01 vs. pre-exercise.