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Published ahead of print on October 3, 2002, doi:10.1164/rccm.200203-177OC
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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 1572-1578, (2002)
© 2002 American Thoracic Society

Original Article

Strenuous Resistive Breathing Induces Plasma Cytokines

Role of Antioxidants and Monocytes

Theodoros Vassilakopoulos, Paraskevi Katsaounou, Maria-Helena Karatza, Androniki Kollintza, Spyros Zakynthinos and Charis Roussos

Department of Critical Care and Pulmonary Services, University of Athens Medical School, Evangelismos Hospital, Athens, Greece

Correspondence and requests for reprints should be addressed to Theodoros Vassilakopoulos, Critical Care Department, Evangelismos Hospital, 45-47 Ipsilandou Str., GR-10675 Athens, Greece. E-mail: tvassilakopoulos{at}yahoo.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inspiratory resistive breathing increases plasma cytokines, yet the stimulus (or stimuli) and source(s) remain unknown. We tested the role of reactive oxygen species as stimuli and of monocytes as sources of resistive breathing-induced cytokines. Six healthy subjects performed two resistive breathing sessions at 75% of maximum inspiratory pressure before and after a combination of antioxidants (vitamin E 200 mg, vitamin A 50,000 IU, and vitamin C 1,000 mg per day for 60 days, allopurinol 600 mg/day for 15 days, and N-acetylcysteine 2 g/day for 3 days before the second session). Blood was drawn before, at the end, and at 30 and 120 minutes after resistive breathing. Before antioxidants, plasma cytokine levels (determined by enzyme-linked immunosorbent assay) increased secondary to resistive breathing (tumor necrosis factor-{alpha} and interleukin [IL]-6 by twofold and IL-1ß by threefold). After antioxidants, plasma IL-1ß became undetectable. The tumor necrosis factor-{alpha} response to resistive breathing was abolished, and the IL-6 response was significantly blunted. Intracellular cytokine detection (by flow cytometry) showed no change in either the percentage of monocytes producing the cytokines or their mean fluorescence intensity both before and after antioxidants. We conclude that oxidative stress is a major stimulus for the resistive breathing–induced cytokine production and that monocytes play no role in this process.

Key Words: interleukin • flow cytometry • oxidative stress • inspiratory resistance • blood mononuclear cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Resistive breathing is encountered in many disease states, such as asthma and chronic obstructive pulmonary disease. When strenuous enough, inspiratory resistive breathing produces diaphragmatic fatigue (1) and delayed diaphragmatic structural injury (2, 3). We have reported that strenuous resistive breathing increases plasma cytokines (interleukin [IL]-1ß and IL-6) (4). Thus, strenuous resistive breathing has certain similarities with intense whole-body exercise, which also induces a plasma cytokine response (5, 6).

Resistive breathing–induced plasma cytokines might serve several functions. They stimulate the hypothalamic pituitary adrenal axis (7) and thus ß-endorphin release (4), which affects the control of breathing (8). They affect brain functions, including sleep (9) and sensations of fatigue (10). IL-6 has a hormone-like glucoregulatory role, signaling that glycogen stores are reaching critically low levels in the contracting muscles and stimulating hepatic glucose output to maintain glucose homeostasis (10, 11) and might also signal injury in muscles (12). Tumor necrosis factor-{alpha} (TNF-{alpha}) affects muscle and especially diaphragm contractility (13) and glucose metabolism (14) and enhances protein degradation (in association with IL-1 [15], and IL-6 [16]). Thus, it is implicated in muscle wasting (13, 17) of chronic diseases (18) such as chronic obstructive pulmonary disease (19–21).

The source of cytokine production during resistive breathing remains nevertheless unknown. Blood monocytes are a major source of immunoinflammatory mediators (22). When activated by either infectious or noninfectious agents, such as bacteria-derived lipopolysaccharide (endotoxin), they release a cascade of cytokines, including TNF-{alpha}, IL-1ß, and IL-6 (22, 23). Thus, it stands to hypothesize that activated blood monocytes are a likely source of cytokine production during strenuous resistive breathing, a form of intense exercise for the respiratory muscles, given that intense exercise induces (apart from cytokines) endotoxemia (24) and leukocyte mobilization and activation (25).

The stimulus (or stimuli) for the resistive breathing-induced cytokine production remains unknown. Resistive breathing is accompanied by increased levels of reactive oxygen species (ROS) both in the vascular and extracellular compartments and within the working respiratory muscles (26, 27). Because ROS are general mediators of cellular responses able to induce cytokine production from various cell types (28–30), it can be hypothesized that ROS might be implicated in the resistive breathing–induced cytokine production. This study was performed to test the role of monocytes (as sources) and ROS (as stimuli) in the resistive breathing–induced cytokine production in normal healthy volunteers.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Six healthy males who were 33 ± 5 years old (range 28–44, 79 ± 17 kg in weight, 176 ± 10.5 cm in height) were studied. None participated in regular physical training or had febrile illnesses during the month before testing. The local ethics committee approved the protocol, and all participants gave informed consent. Subjects refrained from exercising or strenuous activities for 24 hours before testing, which was started at 9:00 A.M. (to exclude circadian rhythm variation effects in the cytokine levels) after a carbohydrate-rich breakfast.

Protocol and Measurements
Pulmonary function tests and determination of the maximal static inspiratory pressure were performed on a separate day before the day of testing, as previously described (4). Each subject performed two resistive breathing sessions before and after antioxidant supplementation at 75% of maximal static inspiratory pressure for 45 minutes (additional details are provided in the online data supplement and in previous reports [4]).

Antioxidant Supplementation
Subjects received a combination of antioxidants, including vitamin E 200 mg, vitamin A 50,000 IU, vitamin C 1,000 mg daily for 60 days before the second resistive breathing session; allopurinol 600 mg/day for 15 days; and N-acetylcysteine 2 g/day for 3 days and 800 mg in the morning before the second resistive breathing session.

Blood Samples
Blood samples were collected at baseline, at the end of the resistive breathing session, and at 30 and 120 minutes into recovery into sterile syringes and were transferred to precooled sterile ethylenediaminetetraacetic acid tubes (for enzyme-linked immuosorbent assay) or sodium heparin tubes for flow cytometry (used within 2 hours from venipuncture). Samples in ethylenediaminetetraacetic acid tubes were immediately spun in a refrigerated centrifuge to separate plasma from cells and thus avoid ex vivo cytokine secretion and were stored in polysterene tubes at -70°C until assayed.

Enzyme-linked Immunosorbent Assays
Plasma levels of TNF-{alpha}, IL-1ß, and IL-6 were measured with commercially available enzyme-linked immunosorbent assay kits (HS Quantikine; R&D Systems, Minneapolis, MN). Plasma concentrations of cytokines were adjusted for changes in plasma volume during and after resistive breathing (31).

Intracellular Flow Cytometric Detection of Cytokines in Monocytes
Flow cytometric detection of cytokines was performed as previously described with slight modifications (32–36).

Cell stimulation and staining.
Whole blood (1 ml) was incubated with Brefeldin-A (10 µg/ml) (to block intracellular transport of proteins, thus retaining cytokines produced inside the cell) in the presence or absence of lipopolysaccharide (1 µg/ml), and was cultured at 37°C for 4 hours in 5% CO2. Cultured cells (50 µl) were incubated with anti–CD14-fluorescein isothiocyanate surface stain for 20 minutes at room temperature in the dark for staining of monocytes and were fixed with solution A (75 ml) for 15 minutes and washed with 2 ml of phosphate-buffered saline azide (containing 0.1% sodium azide and 0.1% bovine serum albumin). Afterward, permeabilization reagent B (75 µl; Sera-Lab) was added, followed by the appropriate anticytokine monoclonal antibodies, and the mixture was incubated for 20 minutes. After a final wash with 2 ml of phosphate-buffered saline azide, the cells were resuspended in 500 µL of 1% paraformaldehyde and stored at 4°C in the dark until analyzed.

Flow cytometric acquisition and analysis.
An electronic acquisition gate was set on CD14-positive cells (Epics Elite Esp flow cytometer; Coulter Electronics, Miami, FL). Results were expressed as the percentage of cytokine-producing monocytes, as well as the mean fluorescence intensity in unstimulated and stimulated cultures. For negative control, intracellular isotype control antibodies were used. Additional details are provided in the online data supplement.

Statistical Analysis
Values reported are means ± SEM. Plasma cytokine levels were analyzed two different ways. First, iso–time values were compared using Friedman analysis of variance followed by Wilcoxon matched pairs tests for post hoc comparisons. Second, both the changes of cytokine values postresistive breathing over the baseline preresistive breathing values and the areas under the curve of the change in plasma cytokine levels over time were compared before and after antioxidant supplementation using Wilcoxon matched pairs tests. The percentage of cytokine-positive monocytes or mean fluorescence intensities at different time points was compared using Friedman analysis of variance followed by Wilcoxon matched pairs tests post hoc. A p value of 0.05 was initially considered as statistically significant and was accordingly adjusted using the sharper Bonferroni-type procedure for multiple comparisons described by Hochberg and Benjamini (37). Correlations were determined using the nonparametric Spearman-R test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pulmonary function tests were normal in all subjects (data not shown). The maximal static inspiratory pressure amounted to 145 ± 10 cm H2O. Before antioxidants, the duty cycle was on the average 0.33 ± 0.02 at the beginning of the resistive breathing session and 0.31 ± 0.03 at the end. After antioxidants, the duty cycle was on the average 0.34 ± 0.02 at the beginning of the resistive breathing session and 0.31 ± 0.02 at the end.

Plasma Cytokine Levels
Before antioxidants.
All three cytokines increased secondary to resistive breathing (Figures 1–3) . IL-1ß (Figure 2A) and IL-6 (Figure 3A) were increased at the time resistive breathing was terminated and reached their peak value at 30 minutes after resistive breathing. TNF-{alpha} also increased, but at a later time point (Figure 1A).



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  		Figure 1. The TNF-{alpha} response to resistive breathing before and after antioxidants. (A) The mean plasma TNF-{alpha} concentration before the resistive breathing session (0 minutes), at the end of resistive breathing (load) (45 minutes), and at 30 and 120 minutes after the end of resistive breathing (recovery) (i.e., 75 and 165 minutes after the beginning of the resistive breathing session). (Upper panel) Before antioxidant supplementation. (Lower panel) After antioxidant administration. (B) The areas under the curve of the change in plasma TNF-{alpha} concentration over time before and after antioxidant supplementation. (C) The absolute changes of plasma TNF-{alpha} concentration secondary to resistive breathing relative to the baseline concentration before resistive breathing (0 minutes) before and after antioxidants. (Closed symbols) Before antioxidant administration. (Open symbols) After antioxidant administration. *p < 0.05 compared with baseline values (0 minutes) after Bonferroni-type adjustment according to Hochberg (37); #p < 0.05 compared with values before antioxidant supplementation after Bonferroni-type adjustment according to Hochberg and Benjamini (37).

 


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  		Figure 3. The IL-6 response to resistive breathing before and after antioxidants (for legend and symbols, refer to Figure 1).

 


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  		Figure 2. The IL-1ß response to resistive breathing before and after antioxidants (A) (for legend and symbols, refer to Figure 1A). Please note that the plasma IL-1ß was below detection limit after antioxidant supplementation (B) (for legend and symbols, please refer to Figure 1C). (Hatched boxes) IL-1ß values below the detection limit.

 
After antioxidants.
After antioxidant supplementation, plasma IL-1ß was below the detection limit (of the enzyme-linked immunosorbent assay that we used) both before and after the resistive breathing session (Figure 2A). The TNF-{alpha} response to resistive breathing was abolished (Figure 1A), and the IL-6 response was significantly blunted (Figure 3A). The absolute changes of the TNF-{alpha} and IL-6 values above the baseline (before resistive breathing) values secondary to resistive breathing were significantly lower after antioxidants (Figures 1C and 3C). The areas under the curve of the plasma cytokine response over time were significantly smaller from those before antioxidants (Figures 1B and 3B).

Intracellular Cytokine Production by Monocytes
The number of monocytes did not change significantly secondary to resistive breathing. Before antioxidants, monocyte counts were 235 ± 48 cells/dl before the resistive breathing, 280 ± 61 cells/dl at the end of resistive breathing, 248 ± 42 cells/dl at 30 minutes after resistive breathing, and 246 ± 53 cells/dl at 2 hours after resistive breathing. A similar response in the number of monocytes was observed after antioxidants (data not shown). The level of cytokine expression in monocytes at the various time points is presented in Tables 1– 4 . Resistive breathing did not increase the production of cytokines by monocytes. Neither the percentage nor the mean fluorescence intensity of monocytes producing the three cytokines either spontaneously or after lipopolysaccharide stimulation changed secondary to resistive breathing (Tables 1–4). The only change observed was a reduction in the mean fluorescence intensity of the TNF-{alpha}–producing monocytes (Tables 3 and 4) both before and after antioxidants, which reached statistical significance only after antioxidants (Friedman analysis of variance X2 = 10.2, p = 0.017 for spontaneous production [Table 3] and Friedman analysis of variance X2= 10.47, p = 0.015 for lipopolysaccharide-stimulated production [Table 4]).


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  		TABLE 1. Percentage of cytokine-positive cells in unstimulated monocytes

 

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  		TABLE 4. Mean fluorescence intensity of cytokine-positive cells in lipopolysaccharide-stimulated monocytes

 

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  		TABLE 2. Percentage of cytokine-positive cells in lipopolysaccharide- stimulated monocytes

 

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  		TABLE 3. Mean fluorescence intensity of cytokine-positive cells in unstimulated monocytes

 
Correlations
No correlation was observed between the plasma cytokine level and the percentage of cytokine-positive monocytes or the mean fluorescence intensity at iso–time points. Furthermore, no correlation was observed between the percentage of cytokine positive monocytes or the mean fluorescence intensity at any time point and the plasma cytokine level at all later time points (considering that an intracellular cytokine expression level might need time to be transformed into circulating cytokine level). Finally, no correlation was found between the changes in the percentage of cytokine positive monocytes or the mean fluorescence intensity between different time points and both the corresponding and the subsequent changes in the plasma cytokine concentration.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The main findings of our study are as follows: (1) Plasma TNF-{alpha}, IL-1ß, and IL-6 were induced secondary to resistive breathing. (2) Antioxidant supplementation drastically affected this induction so that plasma IL-1ß became undetectable, the TNF-{alpha} response was abolished, and the IL-6 response was significantly blunted. (3) No change was observed in either the percentage of monocytes producing the cytokines or in their mean fluorescence intensity both before and after antioxidants.

Cytokine Response to Inspiratory Resistive Breathing
We have shown that strenuous resistive breathing induces plasma IL-1ß and IL-6 (4). This study confirms the previous findings and shows for the first time that TNF-{alpha} is also induced secondary to resistive breathing, yet with a different time course. Whereas IL-1ß and IL-6 are induced by the time resistive breathing ends and exhibit their peak increases 30 minutes after resistive breathing (Figures 2A and 3A), TNF-{alpha} increases later (Figure 1A).

The source of these cytokines remains elusive. We had speculated that blood monocytes and the respiratory muscles are the most likely candidates (4). The data presented here show that monocytes are not the sources of the resistive breathing–induced plasma cytokines. We used intracellular flow cytometric detection of cytokines, which allows for the sensitive determination of cytokine production by specific cell types, as it measures the amount of protein arrested at the Golgi complex of a specific cell population at a specific time point (32, 33). Neither the percentage nor the absolute number of cytokine-positive monocytes increased in response to resistive breathing. The mean fluorescence intensity, a rough estimate of the amount of the cytokine production by cytokine-positive cells (36), was also not altered by resistive breathing. Furthermore, no correlation was found between the level of cytokine expression in monocytes and the plasma level of cytokines. Thus, although a role for monocytes in the baseline plasma level of cytokines cannot be excluded, monocytes do not contribute to the resistive breathing–induced plasma cytokine elevation.

The source of resistive breathing–induced cytokines has not been studied before. However, the role of monocytes in the plasma cytokine induction has been investigated during whole-body exercise. Early reports using cultures of monocytes derived from sedentary and exercised subjects and stimulated with lipopolysaccharide or phytohaemagglutinin found increased levels of IL-1 and IL-6 in the culture supernatant (38), which suggested that monocytes were the sources of the exercise-induced cytokine elevation. However, the cytokine concentration in the supernatants results from the summation of cytokine synthesis over the incubation period (36), and thus does not necessarily represent the secretory output of monocytes in vivo at the specific time points tested. Ullum and colleagues (39), using a concentric bicycle exercise protocol, found (by nuclear run-off analysis) that the pre-mRNA level of TNF-{alpha}, IL-1ß, and IL-6 was not altered by exercise. Similarly, Moldoveanu and colleagues (40) reported that 3 hours of cycling and inclined walking exercise at 60–65% of maximum oxygen consumption did not change the monocyte mRNA level of TNF-{alpha}, IL-1ß, and IL-6 (determined by competitive reverse transcription-polymerase chain reaction), despite increases in plasma cytokine levels. In marathon runners, Ostrowski and colleagues (41) found no change in the monocyte mRNA level of TNF-{alpha} and IL-6. However, monocyte IL-1ß mRNA was increased in some subjects, with only modest increase in the plasma IL-1ß protein (41). Although highly suggestive, these results cannot exclude monocytes as sources of the exercise-induced cytokine production, as cytokine mRNA levels do not necessarily reflect the translation of mRNA into secreted protein (42, 43), given the well-established dissociation between transcription and translation for these cytokines (44, 45).

Our results in resistive breathing are similar to those of Starkie and colleagues during entire body exercise (35, 46), who also excluded monocytes as sources of cytokines, using the same sensitive technique of intracellular flow cytometric detection of cytokines. In contrast, Rhind and colleagues (34), using similar techniques, found an exercise-induced increase in the percentage of monocytes expressing the cytokines. We cannot completely explain with certainty the differences between the results of Rhind and associates (34) and those of Starkie and colleagues (35, 46) and ours. The exercise protocol of Rhind and colleagues (34) was very intense, incorporating both prolonged mixed aerobic–anaerobic and concentric–eccentric components, and was preceded by 1 week of exhaustive exercise. In contrast, resistive breathing is a relatively pure concentric "exercise" for the respiratory muscles, which in our study was not preceded by training. Furthermore, Rhind and colleagues (34) have not determined the mean fluorescence intensity, and thus, no firm conclusions about the contribution of monocytes in the cytokine production can be made, as increased numbers of cytokine-positive monocytes can have reduced mean fluorescence intensity and thus can actually produce less cytokine (46). Given these limitations, it might also be speculated that the response of monocytes is exercise-type specific or that a threshold of duration and intensity of exercise should be exceeded before the contribution of monocytes to the exercise-induced cytokine elevation becomes significant. Nevertheless, our results show that resistive breathing does not induce monocyte-derived cytokine production.

Because monocytes are not the sources of the resistive breathing–induced cytokine induction, the question remains as to which cells–tissues are the actual culprits. Although our study does not address this issue, accumulated evidence from whole-body exercise studies could provide some answers. IL-6 originates from the exercising muscles themselves (5, 41, 47, 48). Although the cells of origin have not been determined, in vitro results suggest that myocytes are the IL-6–producing cells. Accordingly, we have shown (in a model of C2C12 myocytes transformed in culture into myotubes) that muscle cells can produce IL-6 in a ROS-dependent pathway (49). Thus, the respiratory muscles are likely sources of the IL-6 induction during resistive breathing. The origin(s) of the IL-1ß and the TNF-{alpha} is less clear. They might originate from the respiratory muscles, as both cytokines are expressed in muscles at the mRNA and protein level (14, 50–53).

It is also likely that the increased transpulmonary pressure gradient during resistive breathing causes cytokine induction from cells resident in the lung, such as epithelial cells (28, 30) and fibroblasts, secondary to the augmented extraluminal stress imposed. This possibility is supported by studies of isolated, perfused, mouse lung preparations, where increased transpulmonary pressure (yet generated by profound overdistention which is unlikely in our subjects) led to IL-6 and TNF-{alpha} release (54, 55). The adipose tissue is another potential source of the exercise-induced TNF-{alpha} production (56), and could also contribute to the elevated postexercise IL-6 levels (57). Finally, the liver, a major source of cytokines during endotoxemia, could also contribute to the resistive breathing–induced cytokine elevation. More studies are needed to elucidate the tissues–cells of cytokine origin during resistive breathing.

Antioxidant Supplementation and the Cytokine Response to Resistive Breathing
Antioxidants significantly blunted the strenuous resistive breathing–induced cytokine response. This indicates that oxidative stress was a significant stimulus for this cytokine induction. In fact, resistive breathing increases the production of ROS both within the working respiratory muscles and in the extracellular and vascular compartments (26). ROS are general mediators of cellular responses able to induce cytokine production from various cell types (28–30, 49). Our finding that antioxidants significantly blunted the strenuous resistive breathing–induced cytokine response is in concert with the role of ROS as stimuli for the cytokine induction. These cytokines are being rapidly upregulated both at the translational (48) and the posttranslational level (43, 44), and the kinetics of ROS production are suitable for signaling purposes in such a quick response.

To our knowledge, the role of antioxidants in the strenuous resistive breathing–induced cytokine response has not been studied before. However, most studies addressing the role of antioxidants in whole-body exercise-induced cytokine responses have not shown significant effects (58–60). There are many potential explanations for the different findings between these works and ours. Inspiratory resistive breathing is a form of "exercise" restricted to the respiratory muscles, which in various responses are different from limb skeletal muscles. Thus, the results obtained during whole-body exercise experiments are not directly comparable to resistive breathing. Previous studies have used exercise protocols that included an eccentric component (58, 60), whereas resistive breathing is a concentric type "exercise" for the respiratory muscles. Eccentric contractions produce direct muscle injury and leukocyte infiltration, as contrasted to concentric contractions, which are less injurious for the muscles (5, 61). Furthermore, some of these negative results were obtained in trained athletes (58), and regular training may increase the natural antioxidant defense system so that the effect of antioxidants might be accordingly attenuated (61). In contrast, we studied healthy volunteers who were not athletes to eliminate the possible confounding factor of training on antioxidant defense systems.

In this study, we used a cocktail of ROS scavengers (vitamins A, E, and C and N-acetylcysteine) and inhibitors of ROS-producing enzymes (allopurinol) (61–65), which prevents us from being able to ascertain the exact mechanism of effect. Vitamin E is the major lipid-soluble antioxidant in cell membranes, and is particularly efficient at quenching free radicals originating from the mitochondria and biomembranes. It protects against lipid peroxidation by reacting with a variety of oxygen radicals, including singlet oxygen, lipid peroxide products, and the superoxide radical, to form a relatively innocuous tocopherol radical. Vitamin C is a water-soluble antioxidant in the cytosol, and the extracellular fluid that can directly scavenge superoxide, hydroxyl radicals, and singlet oxygen and can also interact with the tocopherol radical to regenerate reduced tocopherol. Vitamin A is the most efficient "quencher" of singlet oxygen, whereas allopurinol inhibits the ROS-producing enzyme xanthine oxidase. N-acetyl-cysteine improves cysteine availability for the biosynthesis of reduced glutathione, which serves multiple functions in protecting tissues from oxidative damage by scavenging hydroxyl radicals, and singlet oxygen, reducing tocopherol radicals, and hydrogen and organic peroxides via a reaction catalyzed by glutathione peroxidase. Because multiple sources of ROS exist in the cell (such as the mitochondrial electron transport chain, the cytosolic NADH oxidase, the xanthine oxidase, and probably membrane bound oxidases) (26, 66–68), it is to be expected that a combination of scavengers and inhibitors of ROS-producing enzymes would be more effective in lowering ROS levels than any single agent. In fact, the defense against oxidative stress depends on an orchestrated synergism between several antioxidants (69). We used doses of antioxidants known to lower the exercise-induced ROS levels in humans. Accordingly, oral N-acetylcysteine (even in lower doses than the ones we used) increased the ROS scavenging capacity of human plasma secondary to bicycle exercise (62). Vitamin E supplementation (300 mg/day for 4 weeks) lessened the exercise-induced ROS-mediated lipid peroxidation (63). Vitamin C supplementation (1,000 mg, 2 hours before exercise) prevented the exercise-induced oxidative stress in healthy humans (64), and allopurinol (300 mg/day for 2 days) prevented the exercise-induced blood glutathione oxidation and lipid peroxidation in patients with chronic obstructive pulmonary disease (65). Thus, although we have not determined any surrogate marker of the level of oxidative stress in our subjects, which is a limitation of our study, we are confident that the antioxidant cocktail that we administered was effective in reducing the oxidative stress response to resistive breathing. Consequently, the observed attenuation of the cytokine response to resistive breathing was most likely due to reduced ROS production, though pharmacologic effects not related to ROS attenuation cannot be excluded.

The antioxidant supplementation blunted but did not completely abolish the IL-6 response to resistive breathing. This suggests that the stimuli for the IL-6 production during resistive breathing might be multiple. Although these stimuli during resistive breathing have not been studied, results from whole-body exercise studies suggest that carbohydrates (5, 35) attenuate the IL-6 response to exercise, and glycogen depletion greatly augments it (48). Furthermore, adrenergic stimulation could also have a contributing role (70, 71). The stimuli for the IL-1ß and TNF-{alpha} induction during exercise are less well studied. Our results suggest that oxidative stress is a strong stimulus for the resistive breathing–induced IL-1ß and TNF-{alpha} response. Antioxidants abolished the resistive breathing–induced elevation of TNF-{alpha} (Figure 1A), whereas IL-1ß became undetectable during the second resistive breathing session, which suggests that antioxidants affected both the constitutive (baseline) and the resistive breathing–induced production of IL-1ß (Figure 2A). Thus, it is possible that the plasma cytokine induction during resistive breathing is differentially regulated by various stimuli, some of them being common (e.g., ROS), whose relative importance varies with each respective cytokine. Although attenuation of the resistive breathing–induced cytokine production is the most likely cause of the effect of antioxidants in our study, increased cytokine clearance cannot be excluded, as a complex balance between production and clearance determines the concentration of cytokines, and the effect of oxidative stress on the latter is largely unknown.

In conclusion, we have shown that oxidative stress is a strong stimulus for the strenuous inspiratory resistive breathing–induced increase in plasma cytokines and that the blood monocytes are not responsible for this response.


   Acknowledgments
 
The authors thank Drs. S. N. Hussain, K. Pantopoulos, and A. Comtois (McGill University, Montreal, Canada) for their helpful suggestions and for carefully reviewing the manuscript.


   FOOTNOTES
 
Supported by the THORAX Foundation, Athens, Greece (T.V. was a Fellow of Alexander S. Onassis Public Benefit Foundation).

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

Received in original form March 5, 2002; accepted in final form September 30, 2002


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TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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