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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 4, 2003 as doi:10.1096/fj.03-0311fje. |
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Department of Medical Anatomy, The Panum Institute, and
* Department of Infectious Diseases and The Copenhagen Muscle Research Centre, Rigshospitalet, University of Copenhagen, DK-2100 Copenhagen, Denmark
2Correspondence: Department of Infectious Diseases 7641, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark.
SPECIFIC AIMS
It is well known that the cytokine interleukin-6 (IL-6) is markedly increased during exercise. Recently, it has been demonstrated that contracting skeletal muscle produces and releases IL-6 into the circulation in high amounts. However, the cell source within the muscle has not been identified. The aim of the present study was to address the question of whether muscle fibers are the major producers of IL-6 in response to exercise.
PRINCIPAL FINDINGS
1. Marked IL-6 protein expression within skeletal muscle fibers following exercise
IL-6 staining of muscle biopsies was performed using a monoclonal mouse anti-human IL-6 at 0, 3 h, 4.5 h, 6 h, 9 h, and 24 h for 12 subjects who performed exercise for 3 h and at the same times for six subjects at rest. In the muscle tissue from resting volunteers, IL-6 staining was absent. Representative data are shown for 0 and 6 h (one resting subject) (Fig. 1
A, B) and for one subject before exercise (Fig. 1C
). Immediately after the end of exercise (3 h), expression of IL-6 had increased significantly (Fig. 1C, D
). IL-6 levels remained significantly increased by 4.5, 6, and 9 h; by 24 h, expression of IL-6 had clearly decreased again (Fig. 1E-H
). However, IL-6 levels by 24 h were still higher than those of resting muscles. The IL-6 staining appeared exclusively as a homogeneous staining of the cytoplasm of skeletal muscle fibers in all subjects who had performed exercise. There was no IL-6 staining present between muscle fibers.
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2. IL-6 is expressed by muscle fibers of all types
When myofibrillar staining was compared with IL-6 staining, it appeared there was no difference between muscle fiber types (type I, type IIa, or type 2x) with regard to IL-6 expression (Fig. 2
).
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3. IL-6 mRNA and plasma-IL-6
The IL-6 mRNA level increased (P<0.05) with exercise. Thus, compared with the prevalue, fold changes of IL-6 mRNA compared with prevalue were 11.0 + 2.6, 8.0 + 5.7 fold, 1.4 + 0.4, 0.4 + 0.2, and 1.2 + 0.3 at 3, 4.5, 6, 9, and 24 h, respectively, peaking at the end of exercise. Plasma IL-6 concentrations increased significantly with time, being 2.2 + 0.2, 18.6 + 2.8, 16.9 + 3.9, 9.5 + 1.5, 9.1 + 1.4, and 3.1 + 1.7 pg/mL at 0, 3, 4.5, 6, 9, and 24 h.
There was no sign of muscle damage as indicated by measurement of creatine kinase and myoglobin.
CONCLUSIONS
The present study demonstrated that physical exercise induces expression of the IL-6 protein in the cytoplasm of muscle fibers. The finding of a marked IL-6 protein expression within skeletal muscle fibers strongly indicates that exercise-induced release of IL-6 from working muscle, the so-called muscle-derived IL-6, has its origin from muscle cells per se (Fig. 3
).
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The fact that IL-6 is exclusively located in the cytoplasm of muscle fibers and there is no IL-6 staining present between muscle fibers supports IL-6 as a genuinely muscle fiber-derived factor. Although brain and peritendon tissue have been demonstrated to release IL-6 in response to exercise, it seems plausible that muscle fibers are the major source of IL-6 in response to exercise, given the massive protein expression in the muscle fibers in addition to muscle cells being the dominant cell type within skeletal muscle. Furthermore, it has been established that blood mononuclear cells do not contribute to the production of IL-6 during exercise.
Several studies have demonstrated a large increase in IL-6 gene expression in skeletal muscle biopsies. A number of studies have demonstrated that the IL-6 mRNA in muscle biopsies peak at the end of exercise or shortly afterward in accordance with the findings from this study. Transcription rates of IL-6 are increased in isolated myonuclei in response to exercise. The IL-6 mRNA kinetic fits nicely with the finding that the IL-6 protein expression within muscle fibers peaked at 6 h (3 h postexercise). Numerous studies have also demonstrated that plasma-IL-6 levels increase in an exponential fashion during exercise, peak at the end of exercise or shortly thereafter, then gradually decline. However, it has also been demonstrated that release of IL-6 from an exercising limb is 
17-fold higher than the amount accumulated in the plasma, thus indicating a very high turnover of IL-6. The finding of IL-6 protein accumulation within skeletal muscle fibers 3 h after the exercise further supports the idea of a high IL-6 turnover.
As discussed, we previously demonstrated that the nuclear transcriptional rate of the IL-6 gene is remarkably rapid after the onset of exercise, with a 10- to 20-fold increase when comparing 30 min of exercise with rest. We hypothesized that this rapid increase in nuclear transcriptional rate was related to a glycogen-independent mechanism, possibly the cytosolic Ca2+ levels, since mechanical load is a potent stimulus for liberating Ca2+ from the lateral sacs of the sarcoplasmic reticulum Therefore, muscle cells isolated from human biopsies were harvested and grown in a culture medium until they fused into myotubes, then stimulated with the Ca2+ ionophore ionomycin. IL-6 mRNA increased progressively over 48 h compared with preincubation levels. It is clear from an examination of the literature there is a signaling cascade in other cell types that indeed implicates intracellular Ca2+ ion concentration ([Ca2+]i) as a potent signaling factor for IL-6 transcription. [Ca2+]i control a diverse range of cellular functions. In lymphocytes, the amplitude and duration of the [Ca2+]i control the differential activation of the proinflammatory transcriptional regulators nuclear factor 
B (NF-B), c-Jun amino-terminal kinase (JNK), and nuclear factor of activated T cells (NFAT). NF-B and JNK are selectively activated by a large [Ca2+]i rise whereas activation of NFAT was induced by a low sustained [Ca2+]i. Therefore, during prolonged contractile activity that results in an increase in IL-6 mRNA in skeletal muscle, initial IL-6 transcription occurs via a Ca2+/NFAT-dependent pathway. Although NFAT in itself can lead to cytokine gene transcription, it can bind to the transcription factor AP-1, which can lead to cytokine gene transcription. Although this pathway is likely to lead to IL-6 gene transcription during sustained muscular contractions, it is possible that large Ca2+ transients as seen with maximal contraction can activate IL-6 via NF-B and JNK. It is known that skeletal muscle expresses JNK and that muscle contraction markedly increases JNK activation. Although the degree to which IL-6 is activated in skeletal muscle by these signaling pathways is not known, it is possible that during more intense muscular activity serial activation of these various pathways gives rise to the more pronounced IL-6 response.
The biological roles of IL-6 appear to be several. The IL-6 production is modulated by the glycogen content in muscles and thus appears to work as an energy sensor. IL-6 exerts its effect on adipose tissue, inducing lipolysis, and increases whole body lipid oxidation. The findings from the present study have further supported our hypothesis of IL-6 as a muscle-fiber derived hormone being released from the site of energy turnover.
We found that IL-6 was produced equally by type 1 and type 2 muscle fibers. Because IL-6 works as an energy sensor, it could be expected that the slow, more metabolically active type 1 muscle fibers would express more IL-6 than type 2 fibers. It could be argued that IL-6 is produced by type 1 fibers but taken up by type 2 fibers. However, the immunohistochemical appearance does not support such a notion, as there appears to be no gradient between the fiber types. It has been argued above that Ca plays a key role in inducing the signaling of IL-6. This brings up the question of whether the signaling pathway is the same in the two fiber types as the free Ca-concentration in the cytosol various when the type 1 vs. type 2 fibers are activated. The phasic response of the type 2 fibers, when activated, may, however, be modified when they are recruited in more prolonged dynamic exercise and have a pattern that mimics the activation of the type 1 fibers. But it makes sense that both fiber types express IL-6, as it is well documented that human type 2 fibers are recruited in prolonged dynamic exercise and are able to oxidize fat for its energy needs. However, single fiber studies are required to finally decide whether IL-6 is differentially expressed by fiber types.
IL-6 is not only related to energy mobilization in response to exercise. Recent findings from our group have demonstrated an anti-inflammatory effect of exercise in humans in response to endotoxin-induced TNF-
plasma levels, an effect that can fully be mimicked by IL-6 administration alone. The fact that IL-6 can inhibit endotoxin-induced TNF- demonstrates another potential benefit of muscle-derived IL-6. TNF- has been linked to insulin resistance by inhibiting glucose uptake in skeletal muscle. As muscle-derived IL-6 can inhibit a TNF- response, IL-6 may directly bring glucose uptake back to normal levels, reversing the detrimental effects of TNF-. For this effect to occur, it seems reasonable that the muscle cells themselves would secrete IL-6 in an attempt to reverse the effects of TNF- on muscle cell glucose uptake. These speculations are in accordance with the findings from the present study. In conclusion, this study demonstrates that skeletal muscle cells are the dominant cell source of exercise-induced, muscle-derived IL-6.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0311fje; doi: 10.1096/fj.03-0311fje 
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