Increase in interstitial interleukin-6 of human skeletal muscle with repetitive low-force exercise

Lars Rosendal 1, 3, Karen Søgaard 1, Michael Kjaer 2, Gisela Sjøgaard 1, Henning Langberg 2,

Jesper Kristiansen 1

1: National Institute of Occupational Health, DK-2100 Copenhagen, Denmark

2: Sports Medicine Research Unit and Copenhagen Muscle Research Centre, Bispebjerg Hospital,

DK-2400 Copenhagen, Denmark 3: Pain and Occupational Medicine Centre, University Hospital, SE-581 85 Linköping, Sweden

Running title: Muscle IL-6 response to low-force exercise

Key words: Metabolism, Trauma, Inflammation, Cytokine, Microdialysis

Request for reprints and correspondence to:

Lars Rosendal, MSc, National Institute of Occupational Health

Lersø Parkallé 105, DK-2100 Copenhagen, Denmark.

Telephone: +45 3916 5454, fax number: +45 3916 5201, E-mail: LRL@ami.dk

Articles in PresS. J Appl Physiol (September 24, 2004). doi:10.1152/japplphysiol.00130.2004

Copyright © 2004 by the American Physiological Society.

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Abstract

Interleukin-6 (IL-6), which is released from muscle tissue during intense exercise, possesses

important metabolic and probably anti-inflammatory properties. To evaluate the IL-6 response to

low-intensity exercise we conducted two studies: I) a control study with insertion of microdialysis

catheters in muscle and determination of interstitial muscle IL-6 response over two hours of rest and

II) an exercise study to investigate the IL-6 response to 20 min repetitive low-force exercise. In both

studies a microdialysis catheter (cut-off 3000 kDa) was inserted into the upper trapezius muscle of 6

male subjects and the catheters were perfused with ringer-acetat at 5 µl min-1. Venous plasma

samples were taken in the exercise study.

The insertion of microdialysis catheters into muscle resulted in an increase in IL-6 from 8 ± 0 pg

ml-1 to 359 ± 171 and 484 ± 202 pg ml-1 after 65 and 110 min, respectively (P<0.001). Similarly, in

the exercise study, IL-6 increased to 289 ± 128 pg ml-1 after 55 min rest (P<0.001). During the

subsequent repetitive low-force exercise, muscle IL-6 further increased to 1246 ± 461 pg ml-1, and

reached 2132 ± 477 pg ml-1 after 30 min recovery (all P<0.001). In contrast to this, plasma IL-6 did

not significantly change in response to exercise.

We conclude that upper extremity, low-intensity exercise results in a substantial increase in IL-6 in

the interstitium of the stabilizing trapezius muscle, whereas no change is seen for plasma IL-6.

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Introduction

In recent years the interleukin-6 (IL-6) response to intense or prolonged exercise has been

characterized as well as its possible biological role. Increases in plasma IL-6 were found in response

to intense exercise (13; 20) and it was suggested that exercise-induced increase in plasma IL-6 is

due to muscle damage from intense eccentric contractions (3). However, increases were also found

during non-damaging intense exercise (2; 27). It has been demonstrated that IL-6 is produced by

muscle tissue in response to exercise and that local production in the exercising muscles can

account for the observed increase in plasma IL-6 (17; 27). A recent study applying the microdialysis

technique to connective tissue demonstrated that this tissue may also contribute to the plasma

elevations in IL-6 during intense exercise (8). There seems to be a time delay for the increase in IL-

6 in the blood during exercise under normal conditions. For example, during combined concentric

and eccentric contractions, plasma IL-6 was significantly increased after 30 min (16), and in a

concentric, one-legged exercise model, IL-6 was significantly increased after 3h (27). However, it is

unknown whether this time delay is due to a reduced initial release rate or to a delayed production.

Recently, a rapid IL-6 release was demonstrated after 10 min exercise when the subjects were

exercising in a glycogen-depleted state (11).

IL-6 has several possible biological effects. Recent data suggest that IL-6 should be classified as an

anti-inflammatory cytokine due to its inhibitory effect on low-grade inflammation via suppressing

tumor necrosis factor (TNF)-α. TNF-α is thought to play a central role in diseases associated with

low-grade inflammation such as cardiovascular disease and type 2 diabetes (12). In a human in-vivo

model, exercise-induced increases in IL-6 as well as infusion of recombinant human IL-6 were

shown to blunt endotoxin-induced increases in TNF-α (25), and thus it has been suggested that IL-6

mediates the beneficial effects of exercise on diseases associated with low-grade inflammation (19;

25).

IL-6 is also speculated to work in hormone-like fashion exerting metabolic control by increasing

energy supply during exercise (22; 26). It has been shown that IL-6 inhibits glycogen synthase

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activity and facilitate glycogen phosphorylase activity (6), and that IL-6 gene transcription rates

increase rapidly during exercise when the muscle glycogen content is low (7). Furthermore, IL-6

induces lipolysis in humans (10) and in support of the importance of this metabolic effect, it has

been shown that IL-6 deficient mice develop mature-onset obesity (32). Thus, IL-6 has been

substantiated as an important metabolic cytokine released from muscle tissue during intense

exercise.

During low-force exercise, systemic changes in markers of metabolism are rarely detected.

However, our group recently described increased lactate and pyruvate levels locally in upper

extremity muscle tissue measured by microdialysis, without simultaneous systemic changes, in

response to a repetitive, low-force exercise task (23). Therefore we speculated that IL-6 may be

released concomitantly due to its metabolic effects. The microdialysis technique offers a unique

opportunity for in-vivo measurement of the time pattern for cytokine release locally in muscular

tissue during exercise. On this background the aim of the present study was to evaluate the local and

systemic IL-6 response to a repetitive, low-force exercise task in humans. Our hypothesis is that the

IL-6 concentration - as during intense exercise - is increased locally in muscle tissue in response to

repetitive low-force exercise.

Because the microdialysis technique is used to study the local muscle IL-6 response to exercise, a

small trauma is introduced during insertion of the microdialysis catheters. Therefore, a control study

characterizing the cytokine response to the insertion trauma alone was also conducted.

Materials and methods

Participants

Six healthy men participated in the control study, average age was 31 years (range 26 – 40), height

180 cm (176 – 184) and weight 81.5 kg (75 – 89). Six healthy men participated in the exercise study

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age 30 years (28 – 33), height 180 cm (173 – 188) and weight 81.5 kg (68 – 89). Three participants

took part in both studies. All participants were without known muscle disorders and there was no

significant difference in participant characteristics between the studies. Data from the exercise study

has been reported previously (23). The participants were instructed not to perform any shoulder or

neck-straining exercise for 48 hours prior to the study, except for ordinary daily working activities.

The participants were all students or had sedentary office jobs and did not take any medication.

None of the participants experienced discomfort or tenderness in the upper body region during the

week prior to the study. All participants gave their informed written consent and the study

conformed to The Declaration of Helsinki, and was approved by the Ethical Committee of

Copenhagen (KF 01-152/01).

Experimental protocol

In both studies, the participants reported to the laboratory at 09.00 h after an overnight fast. During

the studies, the participants were only allowed to drink water.

In the control study the participants had a microdialysis catheter inserted into the upper trapezius

muscle (see below) after which they rested while sitting upright for 120 min. The control study was

conducted to investigate the local muscle IL-6 responses to the minimal trauma caused by inserting

a microdialysis catheter. Microdialysis was sampled over 10 min intervals during rest and IL-6 was

measured at the following time points: 5 min (representing the collection interval 0-10 min), 25 min

(20-30 min), 65 min (60-70 min) and 105 min (100-110 min).

In the exercise study, a microdialysis catheter was inserted into the upper trapezius muscle on the

dominant side and a venous catheter into the antecubital vein of the non-dominant arm where after

the participants rested in an upright position for 60 min. The resting period was followed by a 20

min repetitive, low-force exercise period performed with the dominant arm and hand. Short wooden

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sticks (12 cm, 23 g) were moved back and forth between standardized positions 30 cm apart on a

giant pegboard at a frequency of 1 Hz indicated by an electronic metronome (Zen-on music, Tokyo,

China). The participants performed the exercise in a seated position with the pegboard placed 30 cm

in front of them, measured from the elbow with the upper arm hanging vertically and the elbow in a

90° flexion. The exercise load was characterized by electromyography (EMG) measurement and

was found to be 8 - 9 % EMGmax which has been reported elsewhere (23). Following exercise, the

participants rested for 30 min (recovery). IL-6 was measured at the following time points – during

rest: 5 min (representing the collection interval 0-10 min), 25 min (20-30 min), 55 min (50-60 min)

and during exercise: 65 min (60-70 min), 75 min (70-80 min) as well as during recovery: 85 min

(80-90 min), 105 min (100-110 min).

Microdialysis and calculations

Microdialysis was performed following the principles described by Lönnroth and co-workers (9). A

custom-made microdialysis catheter (membrane length 30 mm, molecular cut-off: 3000 kDa) was

inserted with ultrasound guidance into the upper trapezius muscle at a standard anatomical point on

the dominant side. The catheter was placed in the trapezius muscle parallel to the muscle fibers,

verified by ultra sonography. The insertion point was located on the center of the descending part of

the trapezius muscle, midway between the processus spinosus of the seventh cervical vertebra and

the lateral end of acromion. The skin and the subcutaneous tissues where the catheter entered and

exited the trapezius muscle were anaesthetized with a local injection (0.2-0.5 ml) of Xylocaine (20

mg/ml) without adrenaline, and care was taken not to anaesthetize the underlying muscle. The

distance between the entrance and exit sites of the catheters in the skin was approximately 7 cm

with at least 5 cm of the catheter in the trapezius muscle ensuring that the entire 30 mm membrane

was within the muscle. The catheters were constructed, and were sterilized by ethylene oxide before

use as previously described (8). The microdialysis catheter was perfused via a high-precision

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syringe pump (CMA 100; Carnegie Medicine, Solna, Sweden) at a rate of 5 µl min-1 with a Ringer

acetate solution (Pharmacia & Upjohn, Copenhagen, Denmark) containing 3 mM glucose and 0.5

mM lactate. 0.25 µg ml-1 [3H] human type IV collagen (130 kDa; specific activity 5.9 MBq mg-1;

NEN, Boston, USA) was added to the perfusate to mimic the in-vivo relative recovery (RR) of IL-6

as previously described (8), as no radioactive labelled IL-6 was commercially available. The distal

exteriorized tip of the microdialysis catheter was placed in a 200 µl capped microvial for dialysate

collection. Dialysate collection was timed to adjust for the transition time of the dialysate in the

non-permeable outlet part of the catheter. The sampling periods were 10 min. To ensure that the

actual flow within the catheter was 5 µl min-1, each microvial was weighed before and immediately

after each collection on a high precision electronic weight (Sartorius BP 211 D, Bie & Berntsen,

Copenhagen, Denmark). Deviation from the aimed dialysate volume by more than ± 15 % would

result in discard of the sample. No samples were discarded in the present studies. Dialysate samples

were collected and the samples were immediately frozen and stored at -80° C until the analyses

were performed.

In order to determine RR of each sample, 3 µl dialysate was pipetted into a counting vial, 3 ml of

scintillation fluid (High-flash Point LSC Cocktail UCH maGold Packard Bioscience B.V.,

Gronningen, The Netherlands) was added and the samples were counted in a beta counter. RR was

calculated for each microdialysis catheter as RR = (cpmp – cpmd)/ cpmp, where cpmp was counts per

min in the perfusate and cpmd in the dialysate. It was assumed that the RR from the interstitial fluid

to the perfusate of an unlabelled metabolite equals relative loss from the perfusate to the interstitial

fluid of a labeled metabolite. The interstitial concentrations (Ci) were calculated using the internal

reference calibration method (24) as Ci = (Cd – Cp)/RR + Cp, where Cd was dialysate concentration

and Cp perfusate concentration.

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Blood samples

In the exercise study, a polyethylene catheter was inserted into the antecubital vein of the non-

dominant control arm and blood was sampled in glass tubes containing EDTA before insertion of

the microdialysis catheters as well as before, during, and after the repetitive low-force exercise

period. The blood samples were immediately iced, centrifuged at 3000 rpm for 15 min at 4 °C and

plasma was stored at –80 °C until analysis.

Chemical analysis

Interleukin-6 was measured by a high-sensitivity Quantikine® assay (R&D systems, Minneapolis,

MN, USA). In order to meet the minimum sample volume requirements of the IL-6 assay, dialysate

samples were diluted 100-fold with calibrator diluent, while plasma samples were measured

undiluted. The lowest calibration standard was used as the detection limit. Hence, the detection

limit for cytokine in the dialysate was 15.6 pg/ml for IL-6 when adjusting for the dilution factor. If

the measured concentration was less than the level of detection, half of that value (8 pg ml–1) was

used as the result. Accuracy of the assay was checked by spiking buffer and a low sample in

duplicate with interleukin-6 international standard (NIBSC 89/548). The measured recovery of the

spike did not differ substantially between the two sample types. The average relative recovery

(mean ± SD) was 93 ± 9% (n=4).

Statistics

Data are presented as mean ± standard error of the mean (SEM). The IL-6 data were log-

transformed to serve the criteria of a normal distribution of the residuals in the statistical model.

One-way analysis of variance (ANOVA) for repeated measures approached by general linear

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modeling was used to test for time effect during the two studies. If the ANOVA test revealed

significant changes, post hoc analyses by Tukey’s multiple comparison were used to compare

specific pairs of means. To examine if IL-6 concentrations and patient characteristics differed

between the two studies the independent sample t-test was used (SPSS Standard Version 11.0). A

probability of <0.05 (two-tailed) was accepted as criteria for significance.

Results

The interstitial muscle IL-6 concentration was below the detection level (< 15.6 pg ml-1) in 11 of 12

participants (control study + exercise study) in the first microdialysis sample (5 min). In the control

study, interstitial muscle IL-6 increased in response to the trauma caused by inserting a

microdialysis catheter into the trapezius muscle. The initial value (mean ± SEM) was 8 ± 0 pg ml-1

which increased after 65 min to 359 ± 171 pg ml-1 (P<0.001, Fig 1) but the subsequent increase to

484 ± 202 pg ml-1 after 110 min was not significant compared to the 65 min value (P=0.694). In the

exercise study, interstitial muscle IL-6 also increased over time and reached a value of 289 ± 128 pg

ml-1 at 55 min of rest (Fig. 1). In response to the repetitive low-force exercise, IL-6 further

increased, reaching a mean of 1246 ± 461 pg ml-1 during the last 10 min of the 20 min exercise

period. Of note is that IL-6 levels continued to increase after the cessation of exercise and reached

the highest level of 2132 ± 477 pg ml-1 during the last 10 min of the 30 min recovery (Fig. 1). The

increase in IL-6 during and after exercise (75, 85, 105 min) was highly significant compared to the

pre-exercise level at 55 min (P<0.0001, Fig. 1) and also significantly higher than the IL-6

concentrations reached in the control study at corresponding times relative to insertion of the

microdialysis catheter (all P<0.05). In contrast, although the average plasma IL-6 numerically

increased by 45 % over time in the exercise study, this was not statistically significant (Fig. 1).

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The average relative recovery of the microdialysis catheters (labeled type IV collagen) was found to

be 61 ± 3 % (control study) and 62 ± 2 % (exercise study) without significant difference between

neither studies nor periods within each study.

Discussion

The novel findings from the present study are a) the fast and marked increase in interstitial IL-6

locally in the trapezius muscle during repetitive low-force exercise that is not reflected systemically,

and b) the high absolute IL-6 concentrations achieved in the muscle interstitium.

It has previously been shown that IL-6 is produced by skeletal muscle during intense exercise and

that overflow from muscle to plasma can account for the exercise-induced increase in plasma IL-6

(17). However, it has recently been speculated that release from connective tissue may also

contribute to the increase in plasma IL-6 during intense exercise (8). The increasing number of

studies on the IL-6 response to exercise has focused on intense or prolonged exercise. The only

exception to our knowledge is a study investigating the local and systemic production of cytokines

in response to a 10 min moderate-intensity, unilateral wrist flexion exercise (14). The method used

was venous effluent blood from the exercising arm (referred to as local) and venous blood from the

contralateral arm (systemic). The study showed no change in IL-6 during exercise but a small

increase of approximately 2 pg ml-1 during the post exercise period in venous blood from both arms.

The authors suggested that the increase in IL-6 was due to a systemic response rather than a local

response (14). The increase in systemic IL-6 during the moderate-intensity exercise protocol is

comparable to the numerical systemic increase found in the present study (4 pg ml-1) although ours

did not reach a statistical significance. It is noteworthy that in the above-mentioned study (14),

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actual muscle IL-6 levels were not determined, and one limitation of the used method is that venous

effluent blood only reflect local IL-6 levels if the IL-6 is released from the local tissue to the blood.

Biological effects of IL-6

The biological role of IL-6 is not fully elucidated but it is speculated that IL-6 released from muscle

to blood during exercise is acting in a hormone-like fashion exerting metabolic control (22).

Supporting this idea, it has been shown that IL-6 has the capability to increase hepatic glucose

release and to induce lipolysis in adipose tissue and thereby increasing the total substrate

availability (6; 21; 28; 29). In the present study, circulating IL-6 was not statistically changed which

could suggest that the exercise performed did not necessitate a systemic recruitment of substrates

for metabolism. However, since effluent venous blood was not measured, it is not possible to

determine whether IL-6 was not released from muscle to blood and/or whether hepatic clearance (4)

was sufficiently high to maintain systemic plasma IL-6 at a fairly constant level.

To what extent IL-6 exerts local effects on the exercising muscle itself cannot be proved in this

study. However, it has been shown that IL-6 stimulates myoblast proliferation and enhances

myoblast differentiation (15), and it has been suggested that IL-6 is a hypertrophying factor released

during resistance exercise (31). In contrast to this idea is the notion that IL-6 primarily is released

during endurance type exercise (18), and that transgenic mice overexpressing IL-6 is in a muscle

atrophic state due to increased protein catabolism (30). Interestingly, it has also been suggested that

IL-6 may potentially function as a local metabolic regulator during exercise having glucose

releasing and lipolytic properties (22), thus increasing substrate availability during exercise. It is an

intriguing idea that IL-6 may exert local metabolic effects during exercise and we have found some

support for this in the previously reported data from the low-force exercise protocol (23). The

metabolic activity locally in the trapezius muscle was shown to be relatively high during exercise,

despite the low-force demands. We showed that the interstitial muscle lactate concentration increase

by 50 % during and after exercise followed by an increase in pyruvate indicating an exercise-

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induced rise in anaerobic metabolism (23). Furthermore, the local changes in metabolism were

devoid of a systemic response, perhaps due to the small absolute muscle mass activated. Although it

is speculative, the local increase in IL-6 found in the present study may contribute to an acceleration

of local muscle glycogenolysis, releasing glucose in order to meet the increased metabolic demands

during exercise. The effect of metabolic demand and metabolic status during exercise on local

muscle IL-6 levels has been analyzed in a study by Pedersen and co-workers in which an increase in

IL-6 mRNA was found after 30 min exercise together with a more pronounced increase in the gene

transcription rate when the muscle glycogen content was reduced (7). Recently it has also been

shown that IL-6 is only released from exercising muscle when the muscle is in a glycogen-depleted

state (11).

Regular exercise has been shown to be protective against insulin resistance, type 2 diabetes and

cardiovascular disease (1), and it has been speculated that the anti-inflammatory effect of IL-6 by

suppressing low-grade inflammation via TNF-α, may be one mechanism by which physical

exercise has a positive effect on these diseases (25). The substantial increase in muscle IL-6

demonstrated in the present study could indicate that low to moderate intensity physical exercise,

utilizing many muscle groups, may have an effect on low-grade inflammation and insulin sensitivity

and thereby yield positive health effects.

Control vs. exercise

The muscle concentrations of IL-6 during and after exercise were high compared to plasma levels

and up to 15-20 fold higher than peak plasma levels previously reported during intense exercise (8;

13; 17) and this is the first study to demonstrate an increase in interstitial IL-6 of an upper extremity

muscle group in response to low-force exercise.

Because we use the microdialysis technique to study local events, a local trauma is unavoidable

when the microdialysis catheter is inserted into the muscle and thus one of the aims of this

investigation was to determine to what extent the insertion trauma affects local IL-6 concentrations.

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The data demonstrate that the insertion trauma caused a local increase in IL-6 in the muscle, which

was significant 65 min after the insertion and which remained at approximately the same level 40

min later. In rats, it has previously been shown that the concentration of IL-6 mRNA increases after

a blunt trauma (5). In early exercise studies, it was hypothesized that the increase in IL-6 in

response to intense exercise was due to muscle damage (3) and the trauma data in the present

control and exercise studies support that muscle damage per se results in an IL-6 increase. As we

have used the microdialysis technique to determine the intramuscular IL-6 response to exercise, it

can not be excluded that friction between catheter and the surrounding tissue could create a small

additional trauma during exercise, however, the friction is most likely limited because the trapezius

muscle is primarily functioning as a stabilizer of the scapula during the hand and arm movement as

well as a stabilizer of the neck, and is thus exerting minimal length changes. This is also

demonstrated by the lack of fluctuations and oscillations in trapezius electromyography in the

present low-force exercise protocol (23). Furthermore, the increase in the interstitial muscle IL-6

concentration in response to the insertion trauma did only account for 20 - 30 % of the increase seen

at comparable time points during and after exercise, and therefore the major IL-6 contribution was

most likely due to the exercise per se.

In conclusion, a substantial increase in interstitial muscle IL-6 was demonstrated in response to

repetitive, low-force exercise of the upper extremity that was not reflected systemically. An

increase in muscle IL-6 was also demonstrated in response to the insertion trauma, but it only

accounted for 20-30 % of the exercise-induced increase. Thus, muscle IL-6 seems to be highly

responsive to exercise, even at low-force levels, which may be attributable to local changes in

metabolic demands during exercise.

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Acknowledgement

We thank Anne Abildtrup for excellent technical assistance. This study was supported by the

Danish Research Agency (641-01-0035).

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Legends

Figure 1. Response of plasma and interstitial muscle IL-6 to a combination of a minimal

trauma and 20 min repetitive low-force exercise and the interstitial muscle IL-6 response to a

minimal trauma alone.

The muscle interstitial concentration of IL-6 increased in response to trauma alone (insertion of a

microdialysis catheter, Control study), and further increased dramatically during and after exercise.

No significant changes were found for plasma IL-6. Note that only a minor part of the muscle IL-6

increase could be attributed to the insertion trauma.

The gray bar indicates measurements performed during exercise. Interstitial muscle IL-6 is the

concentration over a 10 min sampling period. Data are given as mean ± SEM.

* Significantly different from corresponding baseline level (5 min) (P<0.05), # Significantly

different from baseline level (5 min, P<0.05) and from pre-exercise level (55 min, P<0.05).

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