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Published ahead of print on April 29, 2004, doi:10.1164/rccm.200308-1071OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 154-161, (2004)
© 2004 American Thoracic Society

Original Article

Differential Cytokine Gene Expression in the Diaphragm in Response to Strenuous Resistive Breathing

Theodoros Vassilakopoulos, Maziar Divangahi, George Rallis, Osama Kishta, Basil Petrof, Alain Comtois and Sabah N. A. Hussain

Critical Care and Respiratory Divisions, Department of Medicine, McGill University Hospital Center; and Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada

Correspondence and requests for reprints should be addressed to Theodoros Vassilakopoulos, M.D., Department of Critical Care and Pulmonary Services, University of Athens Medical School, 45–47 Ipsilandou Street 10675, Athens, Greece. E-mail:tvassil{at}med.uoa.gr


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Strenuous resistive breathing induces plasma cytokines that do not originate from circulating monocytes. We hypothesized that cytokine production is induced inside the diaphragm in response to resistive loading. Anesthetized, tracheostomized, spontaneously breathing Sprague-Dawley rats were subjected to 1, 3, or 6 hours of inspiratory resistive loading, corresponding to 45–50% of the maximum inspiratory pressure. Unloaded sham-operated rats breathing spontaneously served as control animals. The diaphragm and the gastrocnemius muscles were excised at the end of the loading period, and messenger ribonucleic acid expression of tumor necrosis factor-{alpha}, tumor necrosis factor-ß, interleukin (IL)-1{alpha}, IL-1ß, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-{gamma}, and two housekeeping genes was analyzed using multiprobe RNase protection assay. IL-6, IL-1ß, and, to lesser extents, tumor necrosis factor-{alpha}, IL-10, IFN-{gamma}, and IL-4 were significantly increased in a time-dependent fashion in the diaphragms but not the gastrocnemius of loaded animals or in the diaphragm of control animals. Elevation of protein levels of IL-6 and IL-1ß in the diaphragm of loaded animals was confirmed with immunoblotting. Immunostaining revealed IL-6 protein localization inside diaphragmatic muscle fibers. We conclude that increased ventilatory muscle activity during resistive loading induces differential elevation of proinflammatory and antiinflammatory cytokine gene expression in the ventilatory muscles.

Key Words: interleukin • loaded breathing • respiratory muscles • ribonuclease protection assay

Strenuous resistive breathing has been recently shown to lead to elevation of the plasma levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-{alpha} (1, 2). Resistive breathing–induced plasma cytokines might serve several functions: They stimulate the hypothalamic pituitary adrenal axis (3) leading to ß-endorphin release (1) and alterations in breathing pattern (4). They affect brain functions, including sleep (5) and sensation of fatigue (6, 7). IL-6 has a hormone-like glucoregulatory role (6), whereas TNF-{alpha} depresses muscle and especially diaphragm contractility (8) and induces insulin resistance (9). IL-6, IL-1ß, and TNF-{alpha} also enhance protein degradation and have been implicated in muscle wasting (10) of chronic diseases such as chronic obstructive pulmonary disease (11–13). Whole body exercise has also been shown to induce an increase in plasma levels of cytokines such as IL-6, IL-1ß, TNF-{alpha}, IL-1 receptor antagonist, and IL-10 (14).

The cellular origin of these cytokines remains unknown. Monocytes, a major source of immunoinflammatory mediators (15), have been excluded as sources of the resistive breathing–induced or whole-body exercise–induced elevation of plasma cytokines (2, 16–19). Myocytes have been suggested as a potential source of the exercise-induced cytokines. Indeed, muscle contraction during marathon running or knee extension increases IL-6 but not TNF-{alpha} gene expression within the exercising muscles (20–24), secondary to increased transcriptional activity (22), and leads to IL-6 protein release into the circulation (21). However, these results were not confirmed by other investigators who could not detect intramuscular cytokine upregulation secondary to treadmill running (24, 25) or electrical stimulation (24). These conflicting results suggest that activation-induced intramuscular cytokine expression might be exercise- and muscle-type specific, given that different types of exercise activate different transcription factors in a manner specific to the type of muscle (26, 27). Furthermore, the cells of origin of the exercise-induced muscle-derived cytokines are not known, and both resident and blood-derived invading cells are potential candidates.

Because resistive breathing is a form of exercise for the respiratory muscles associated with plasma cytokine elevation and some forms of skeletal muscle activation lead to intramuscular IL-6 production (20) and release into the circulation (21), we hypothesized that the expressions of proinflammatory and antiinflammatory cytokines are upregulated in the respiratory muscles secondary to resistive loading and that this upregulation is dependent on the duration of muscle activation. We evaluated in this study the nature and the time course of cytokine expression within the ventilatory muscles in response to increased activation secondary to inspiratory resistive loading. We have also identified the cellular sources of cytokines produced during strenuous ventilatory muscle contraction. We propose that myocytes are the main source of cytokine production in response to ventilatory muscle activation. Some of the results of these studies have been previously reported in the form of an abstract (28).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Preparation
Male Sprague-Dawley rats (300–325 g) were anesthetized with pentobarbital sodium and tracheostomized with polyethylene tubing connected to a two-way nonrebreathing valve. The inspiratory line delivered 100% O2 to prevent hypoxemia. After a short stabilization period, animals (n = 8 in each group) were randomly assigned to periods of 1, 3, or 6 hours of moderate inspiratory resistive loading (peak inspiratory tracheal pressure of approximately 50% of maximum). Other animals (n = 6 per group) were exposed to either inspiratory loading for 1 hour followed by 2 hours of unloaded breathing or intermittent loading (20 minutes of loading followed by a 30-minute recovery repeated three times). Sham-operated animals breathing against no load for 1, 3, and 6 hours served as control animals (n = 8). Animals were killed at the end of the experiment, and the diaphragm and gastrocnemius muscles were quickly excised and frozen either in liquid nitrogen or cold isopentane (20 seconds) before liquid nitrogen.

RNase Protection Assay
Total RNA was isolated with proteinase K and DNase I treatments (RNeasy kit; Qiagen Mississauga, Ontario, Canada), and mRNA expression of IL-1{alpha} , IL-1ß, TNF-{alpha}, TNF-ß, IL-3, IL-4, IL-5, IL-6, IL-10, IL-2, IFN-{gamma}, and two housekeeping genes (L32 and GADPH) was measured by Multi-Probe RNase Protection Assay System (RiboQuant; PharMingen, San Jose, CA). Briefly, the multiprobe set was hybridized in excess to target RNA in solution, after which free probe and other single-stranded RNA were digested with RNases. The remaining RNAase-protected probes were purified, resolved on a denaturing polyacrylamide gel, and detected by autoradiography. Optical densities of various mRNAs in the scanned autoradiography films were quantified with ImagePro Plus software (Media Cyberetics Inc., San Diego, CA).

Immunohistochemistry
Frozen tissue sections (5 µm in thickness) were incubated overnight at 4°C with primary goat anti-rat IL-6 or rabbit anti-rat IL-6 antibodies. After three rinses with phosphate-buffered saline, sections were incubated with biotin-conjugated anti-goat or anti-rabbit secondary antibodies followed by Cy3-labeled streptavidin. Sections were then examined under fluorescence microscopy and photographed with a digital camera.

Immunoblotting
Frozen muscle samples were homogenized in a homogenization buffer and centrifuged at 1,000 x g for 10 minutes, and supernatants (crude muscle homogenates, 80-µg total protein per sample) were separated onto tris-glycine sodium dodecyl sulfate-polyacrylamide gel. Proteins were then transferred to polyvinylidene diflouride membranes and probed overnight with rabbit anti-rat IL-6 and IL-1ß antibodies. Specific proteins were detected with horseradish peroxidase–conjugated anti-rabbit secondary antibody and an enhanced chemiluminescence kit and quantified with ImageProPlus software (Media Cybernetics Inc.).

Myeloperoxidase Activity Assay
Crude muscle homogenates (in 0.5% hexadecyltrimethylammonium bromide) were mixed with 50-mM potassium phosphate buffer (pH 6.0) containing o-dianisidine dihydrochloride and H2O2 (29). Absorbance was measured at 460 nm for 60 minutes. Myeloperoxidase activity was calculated in units: change in absorbance/minute/g protein.

Statistical Analysis
Values reported are means ± SEM. Comparisons were made using Friedman analysis of variance followed by Wilcoxon Matched Pairs Tests for post hoc comparisons. A p value of 0.05 was initially considered as statistically significant and was accordingly adjusted using a Bonferroni-type procedure for multiple comparisons (30).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RNase protection assay detected weak expression of IL-6, IL-1ß, IL-10, TNF-{alpha}, IFN-{gamma}, and IL-4, (highest to lowest mRNA concentration) in the diaphragm of quietly breathing (unloaded) rats. Different periods of unloaded breathing (1, 3, or 6 hours) did not change the expression of these cytokines. IL-6 mRNA was three times more abundant (p < 0.05) than the mRNAs of IL-1ß, IL-10, TNF-{alpha}, and IFN-{gamma}, which were equally abundant, whereas the expression level of IL-4 was one order of magnitude less than the other cytokines (p < 0.05). A very weak expression for these cytokines was detected in the gastrocnemius, which did not change at any time point in the unloaded animals. Expression of TNF-ß, IL-1{alpha}, IL-2, IL-3, and IL-5 mRNAs could not be detected at any time point in the diaphragm and gastrocnemius of quietly breathing rats.

Maximum peak tracheal pressure measured before resistive loading averaged 75.2 ± 11.7 cm H2O. Peak inspiratory tracheal airway pressure developed by the animals during loading averaged 35.5 ± 1.96 cm H2O (46 ± 8% of maximum peak tracheal pressure). Loaded breathing resulted in worsening hypercapnia and acidosis in a time-dependent fashion, without concomitant hypoxemia, which was prevented because of the enriched inspired oxygen used (see the online supplement).

Loaded breathing resulted in a significant differential upregulation of the expression of IL-6, IL-1ß, IL-10, TNF-{alpha}, IFN-{gamma}, and IL-4 in the diaphragm but not the gastrocnemius (Figure 1) . The increase in the cytokine mRNA expression (expressed as the fold increase above the respective value of equal duration unloaded breathing) in the diaphragms of loaded animals is presented in Figure 2 . With the exception of IL-1ß, which exhibited a nearly constant upregulation at different time points, the other cytokines were upregulated in a time-dependent manner, exhibiting the greatest increase after 6 hours of loaded breathing (Figures 2 and 3) . IL-6 exhibited the greatest fold increase both at 3 and at 6 hours of loaded breathing. At each time point of loaded breathing, IL-6 mRNA was the most abundant (expressed as a percentage of the housekeeping gene L32 or glyceraldehyde 3-phosphate dehydrogenase), whereas the mRNA for IL-4 exhibited the weakest expression (Figure 4) .



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  		Figure 1. Representative autoradiograph of RNase protection assay showing the time course of cytokine gene expression in the diaphragm and gastrocnemius muscles. Lanes 1–3: probe, the negative (–ve) and positive (+ve) control, respectively. Lane 4: diaphragm sample from control rat (quiet breathing). Lanes 5–7: diaphragm samples obtained from animals exposed to 1, 3, and 6 hours of resistive loading, respectively. Lane 8: gastrocnemius sample obtained from rats exposed to 6 hours of inspiratory resistive loading. A total of 10 µg RNA was used in each lane. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; IL = interleukin; TNF = tumor necrosis factor.

 


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  		Figure 2. Time course of differential cytokine gene expression in the diaphragm secondary to inspiratory resistive loading. Data are expressed as fold increase over equal duration of unloaded (quiet) breathing, normalized to L32 mRNA. *p < 0.05 compared with quiet breathing.

 


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  		Figure 3. Representative autoradiograph of RNase protection assay performed on diaphragm muscle samples obtained after 3 (lanes 5–9) and 6 hours (lanes 10–16) of inspiratory resistive loading. Lanes 1–3: probe, the negative (–ve) and positive (+ve) control, respectively. Lane 4: diaphragm of a quietly breathing rat. A total of 10 µg RNA was used in each lane. IRL = inspiratory resistive loading.

 


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  		Figure 4. Relative abundance of cytokine mRNAs in the diaphragm after 3 (upper panel) and 6 (lower panel) hours of inspiratory resistive loading (data normalized to L32 mRNA expression). *p < 0.05. Please note that the scale of the upper panel is triple (0–10) that of the lower panel (0–30).

 
To evaluate the time course of cytokine gene expression after termination of muscle activation, a group of animals (n = 6) completed 1 hour of loaded breathing followed by 2 hours of recovery before tissue collection (Figure 5) . With the exception of IL-10, all other cytokines were further upregulated after the termination of 1 hour of resistive loading (p < 0.05), suggesting that once initiated, contraction-induced diaphragmatic cytokine upregulation is a long-lasting process (see the online supplement). To evaluate the influence of total duration of muscle activation on cytokine gene expression, another group of animals (n = 6) underwent intermittent inspiratory resistive loading for 3 periods of 20 minutes separated by 30-minute periods of unloaded breathing for a total duration of muscle activation of 1 hour. This intermittent activation pattern resulted in marked upregulation of cytokine expression (Figure 5).



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  		Figure 5. The influence of muscle activation pattern on diaphragmatic cytokine gene expression. Lanes 1–3: probe, the negative (–ve) and positive (+ve) control animals, respectively. Lanes 4 and 5: diaphragms of quietly breathing rats. Lane 6: diaphragm sample obtained after intermittent resistive loading (20 minutes of loading followed by 30 minutes of quiet breathing, repeated three times with a total of 1 hour of inspiratory resistive loading). Lanes 7 and 8: diaphragm samples obtained immediately after 1 hour of inspiratory resistive loading. Lanes 9 and 10: diaphragm samples obtained from rats exposed to 1 hour resistive loading followed by 2 hours of quiet breathing.

 
Figure 6 illustrates representative examples and mean values (n = 5) of the changes in IL-6 and IL-1ß protein expression in the diaphragm of rats exposed to 3 and 6 hours of severe inspiratory resistive loading. No detectable IL-6 and IL-1ß proteins were found in the diaphragms of animals breathing against no load. Inspiratory resistive loading for 3 hours elicited a significant rise in diaphragm protein expression of these cytokines (Figure 6). Six hours of inspiratory resistive loading elicited an even greater rise in protein expression of IL-6 and IL-1ß, which averaged approximately 10-fold higher than that observed after 3 hours of inspiratory resistive loading (Figure 6). No detectable protein expression of these cytokines was found in the gastrocnemius muscle samples in the three groups of animals (results not shown).



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  		Figure 6. Representative examples of immunoblotting (upper panel, A) and mean optical density values (lower panel, B) of IL-6 and IL-1ß protein expression in the diaphragm of rats exposed to 3 and 6 hours of inspiratory resistive loading. No detectable IL-6 and IL-1ß proteins were found in the diaphragms of animals breathing against no load (A, lanes 1–2). Inspiratory resistive loading for 3 hours elicited a significant rise in diaphragm protein expression of these cytokines (A, lanes 3–4). Six hours of inspiratory resistive loading elicited even greater rise in protein expression of IL-6 and IL-1ß (A, lanes 5–6), which averaged approximately 10-fold higher than that observed after 3 hours of IRL (B). OD = optical density; QB = quiet (unloaded) breathing.

 
Figure 7 illustrates localization of IL-6 protein expression in rat diaphragms. Both goat anti-rat IL-6 (Figure 7A) and rabbit anti-rat IL-6 antibody (Figure 7B) detected positive IL-6 protein staining in the diaphragms of rats exposed to 6 hours of inspiratory resistive loading. Both punctuate cytosolic and membrane-associated positive IL-6 staining (white arrows in Figures 7A and 7B) was evident inside small muscle fibers, whereas large muscle fibers showed no IL-6 staining. Blood vessels were negative for IL-6 protein (white arrow in Figure 7C). Very weak IL-6 staining was detectable in the diaphragm of quietly breathing rats (Figure 7D). The replacement of primary antibodies with nonspecific antibodies completely eliminated positive IL-6 staining (data not shown).



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  		Figure 7. Localization of IL-6 protein expression in rat diaphragms. Both goat anti-rat IL-6 (A) and rabbit anti-rat IL-6 antibody (B) detected positive IL-6 staining in the diaphragms of rats exposed to 6 hours of inspiratory resistive loading. Both membrane-associated (white arrows in A) and punctuate cytosolic positive IL-6 staining (white arrows in B) was evident inside small muscle fibers, whereas large muscle fibers showed no IL-6 staining (gray arrows). Blood vessels were negative for IL-6 protein (white arrow in C). Very weak IL-6 staining was detectable in the diaphragm of quietly breathing rats (D).

 
Inspiratory resistive loading elicited no change in the myeloperoxidase activity in the diaphragms, which averaged 72.9 ± 6.2 U in animals breathing against no load, 91.3 ± 18.0 U after 3 hours of inspiratory resistive loading, and 80.1 ± 9.7 U after 6 hours of inspiratory resistive loading (p = NS).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major finding of this study is that IL-6 and to a lesser extent IL-1ß, TNF-{alpha}, IL-10, IL-4, and IFN-{gamma} were significantly increased in a time-dependent manner in the diaphragms of animals subjected to inspiratory resistive loading. Immunohistochemical analysis and absence of any change in myeloperoxidase activity during resistive loading suggest that cytokines are produced inside muscle fibers and are not derived from infiltrating inflammatory cells up to 6 hours after inspiratory resistive loading.

To our knowledge, this is the first study showing that proinflammatory and antiinflammatory cytokines exhibit a low level of constitutive expression within the respiratory muscles under conditions of quiet-unloaded breathing, similar to what is observed in peripheral skeletal muscles (9, 21, 31, 32). More importantly, strenuous contraction of the respiratory muscles resulted in significant upregulation of IL-6 expression and to a lesser extent expressions of IL-1ß, TNF-{alpha}, IL-10, IL-4, and IFN-{gamma}. The upregulation of intradiaphragmatic cytokine expression was not due a generalized increase in transcription because no upregulation was observed in the noncontracting gastrocnemius. Furthermore, it was not due to surgical manipulation (as previously demonstrated for the soleus) (24), because no increase was observed in the diaphragms of the animals that were subjected to the same surgical procedures without inspiratory loading. Thus, the intradiaphragmatic cytokine upregulation was a specific response to increased activation of the diaphragm secondary to resistive loading.

It should be emphasized that we detected that the messenger RNA expression of cytokines using a multiprobe RNase protection assay, which does not amplify the RNA signal, is less prone to variability and errors and is significantly less sensitive from the usually used reverse transcription-polymerase chain reaction. The RNase protection assay requires 104 to 105 larger quantities of RNA to be present in the tissues for positive signal detection (33) compared with the reverse transcription-polymerase chain reaction that has been used for RNA detection in peripheral skeletal muscles (20, 21, 25). Because RNase protection assay is less sensitive than reverse transcription-polymerase chain reaction, some cytokine expression that was below the detection limit of the method might have been missed. On the other hand, this secures that the upregulation of cytokine expression within the diaphragm secondary to resistive loading that we observed represents relatively abundant tissue messenger RNA levels.

The mRNA upregulation was accompanied by commensurate increases in the cytokine protein levels, at least for the IL-6 and IL-1ß. Although we have not detected the rest of the cytokines at the protein level (which is a limitation of our study), there is no reason to expect a different response for these cytokines, because whenever cytokine messenger RNA levels change within muscles, similar changes of protein levels occur (34–39).

Cellular origins of muscle activation-induced cytokine expression are not yet established. Our results show that IL-6, the most abundantly expressed and upregulated cytokine secondary to increased muscle activation, originates from the myocytes themselves. In fact, IL-6 exhibited both a cytoplasmic and a perisarcolemmal staining pattern, which is characteristic of a secreted protein. This finding is in keeping with in vitro results showing that myocytes are capable of producing IL-6 (38, 40, 41) secondary to stimuli relevant for exercise, such as exposure to reactive oxygen species (40) and increased intracellular Ca++ (41). Similar to what we found in the diaphragm, cytokines are upregulated within cardiac myocytes secondary to loading (35, 42), which suggest that IL-6 upregulation is a general response of myocytes to increased muscle activation. We have not evaluated the cellular origin of the rest of the cytokines; however, because myocytes are capable of producing a variety of cytokines in vitro (38), it is likely that myocytes are the sources of the augmented cytokine expression within the diaphragm, although other cells could not be excluded.

The stimulus for the upregulation of cytokine expression during diaphragmatic activation is not known. We speculate that reactive oxygen species are important modulators of muscle cytokine production, as indicated by the blunting by antioxidants of the elevation in plasma IL-6, IL-1ß, and TNF-{alpha} (2, 19) induced by either resistive loading (2) or whole-body exercise (19) and by the induction of IL-6 production from cultured myocytes exposed to reactive oxygen species (40). Depletion of glycogen muscle stores during muscle activation could also regulate cytokine production as indicated by augmentation of muscle IL-6 expression after glycogen depletion (22, 23). Finally, preliminary data suggest that the rise in intracellular Ca2+ can also lead to IL-6 secretion by myocytes (41).

Implications
Resistive breathing–induced intradiaphragmatic cytokine production may serve several local and systemic functions, which could be both adaptive and maladaptive. For instance, cytokines may play an important role at the local level by promoting muscle fiber injury. Resistive loading achieved in our study was of such magnitude that likely produces diaphragmatic injury (43–47). Our results raise the interesting possibility that intradiaphragmatic cytokine induction could be involved in mediating the injurious process by upregulating the expression of adhesion molecules on the surface of endothelial cells (48) and by enhancing transendothelial migration of blood-derived inflammatory cells (49), responses that would augment infiltration of neutrophils and promotion of muscle fiber injury. Although myeloperoxidase activity—an index of tissue infiltration by neutrophils—was not increased in the diaphragms of animals up to 6 hours of resistive loading, this might be due to inadequate time (neutrophilic influx taking place later) or to inadequate power of our study to document a statistically significant response (a 25% increase in myeloperoxidase activity observed would require 70 animals per group). Proinflammatory cytokines such as TNF-{alpha} may also promote fiber injury by augmenting muscle reactive oxygen species production (10). These species are well known players in ventilatory muscle injury (50). The majority of evidence suggests that TNF-{alpha} also suppresses diaphragmatic contractility (8, 51, 52), although earlier studies had suggested that TNF-{alpha} has either no effect (53) or affects diaphragmatic contractility only at high doses (54), which might explain the observation that force decline after resistive loading is proportionally greater than the observed muscle injury (44).

We should emphasize that not only proinflammatory cytokines such as IL-1ß, TNF-{alpha}, and IFN-{gamma} were induced inside the diaphragm during resistive loading but antiinflammatory cytokines such as IL-4, IL-10, and IL-6 (which has some proinflammatory but mainly antiinflammatory properties) (55) were also upregulated, suggesting that few of these cytokines may serve to oppose local muscle inflammation (55). Cytokines are also essential in orchestrating muscle recovery after injury. Cytokines such as TNF-{alpha}, IL-6, leukemia inhibitory factor, and IL-1ß (31, 56–58) and their cognate receptors (59) are upregulated in skeletal muscle after injury. These cytokines enhance proteolytic removal of damaged proteins (60, 61) and damaged cells (through recruitment and activation of phagocytes). TNF-{alpha} and leukemia inhibitory factor are important signaling molecules for the regeneration of muscle fibers after injury (57, 62). TNF-{alpha} receptor double knockout mice or mice receiving TNF-{alpha}–neutralizing antibodies exhibit a reduced muscle strength recovery after injury compared with wild-type mice, associated with a reduced expression of the myogenic transcription factor MyoD (57). This is in concert with data suggesting that TNF-{alpha} promotes differentiation of myoblasts by increasing nuclear factor-{kappa}B activity (63) and both activates satellite cells to enter the cell cycle from the normally quiescent state and enhances their proliferation once it has been initiated (64). Nevertheless, it has to be acknowledged that the differentiation promoting effect of TNF-{alpha} has been debated (65–67). More studies are needed to elucidate the exact role of cytokines in skeletal muscle injury and recovery.

The significantly greater induction of IL-6 within the diaphragm compared with other cytokines suggests that IL-6 might be involved in physiologic muscle signaling (68). Diaphragmatic contraction leads to glycogen depletion, which greatly augments IL-6 production from skeletal muscles (22, 23). IL-6 has an hormone-like role, signaling that glycogen stores are reaching critically low levels in the contracting muscles and stimulating hepatic glucose output to maintain glucose homeostasis and muscle glucose supply (6, 69). IL-6 also mobilizes free fatty acids from triglycerides stored in fat tissue, thus increasing the energy that is available to the muscle.

We also speculate that diaphragm-derived cytokines might spill into the circulation leading to elevation of plasma cytokine levels. Ventilatory muscle production of cytokines could have been the source of elevated plasma cytokines observed after resistive loading in normal humans (1, 2) or in diseases of increased respiratory load, such as chronic obstructive pulmonary disease (70, 71) and sleep apnea (72). Elevation of circulating cytokines derived from the ventilatory muscles might have systemic effects, including changes in breathing pattern (1) and sensation of fatigue (6, 7). Muscle-derived cytokines may also contribute to the cachexia observed in some chronic obstructive pulmonary disease patients (11–13). Further studies are needed to elucidate these interesting possibilities.

In conclusion, we have shown that inspiratory resistive loading results in differential cytokine expression in the diaphragm. Both proinflammatory and antiinflammatory cytokines are expressed in a time-dependent manner, which might have both local and systemic effects.


   FOOTNOTES
 
Supported by a grant from the Canadian Institute of Health Research and the Alexander Onassis Public Benefit Foundation (T.V.). S.N.A.H. is National Scholar of the Fonds de la recherche en santé du Québec; T.V. was a postdoctoral fellow of the Meakins-Christie Laboratories.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: T.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; G.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; O.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; B.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.N.A.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 1, 2003; accepted in final form April 23, 2004


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