Journal of Applied Physiology AJP: Regulatory, Integrative and Comparative Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 103: 305-314, 2007. First published April 26, 2007; doi:10.1152/japplphysiol.00226.2007
8750-7587/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
103/1/305    most recent
00226.2007v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, J.-S.
Right arrow Articles by Wong, M.-K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, J.-S.
Right arrow Articles by Wong, M.-K.

Chronic intermittent hypoxia modulates eosinophil- and neutrophil-platelet aggregation and inflammatory cytokine secretion caused by strenuous exercise in men

Jong-Shyan Wang,1 Hung-Yu Lin,1 Mei-Ling Cheng,2 and May-Kuen Wong1

1Graduate Institute of Rehabilitation Science and Center for Gerontological Research and 2Graduate Institute of Medical Biotechnology and Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Tao-Yuan, Taiwan

Submitted 23 February 2007 ; accepted in final form 23 April 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Although acclimatization to intermittent hypoxia (IH) improves exercise performance by increasing oxygen delivery and utilization, the effects of chronic IH on platelet-leukocyte interaction and inflammation-related cytokine secretion caused by strenuous exercise remain unclear. This investigation elucidates how two intensities of IH influence eosinophil- and neutrophil-platelet aggregation (EPA and NPA) as well as pro- and anti-inflammatory cytokines mediated by strenuous exercise. Twenty healthy sedentary men were randomly divided into severe (SIH) and moderate (MIH) IH groups; groups were exposed to 12% O2 (SIH) and 15% O2 (MIH) for 1 h/day, respectively, for 5 days/wk for 8 wk in a normobaric hypoxia chamber. Before IH intervention, 1) exercise up to maximal oxygen consumption promoted shear stress-, LPS-, and N-formyl-methionyl-leucyl-phenylalanine-induced EPA, increased IL-1beta and malondialdehyde levels, and decreased total antioxidant levels in plasma and 2) exposure to 12% O2, but not to 15% O2 for 1 h, enhanced LPS-induced EPA and reduced plasma total antioxidant levels. After IH for 8 wk, hypoxia- and exercise-promoted EPA, IL-1beta, or malondialdehyde levels were suppressed in both MIH and SIH groups, and plasma IL-6 and IL-10 levels in the SIH group were increased. However, the NPA induced by the shear force and chemical agonists was not changed under the two IH regimens. Therefore, both MIH and SIH regimens ameliorate eosinophil- and platelet-related thrombosis, proinflammatory IL-1beta secretion, and lipid peroxidation enhanced by strenuous exercise. Furthermore, SIH simultaneously increases circulatory anti-inflammatory IL-6 and IL-10 concentrations. These findings can help to develop effective IH regimens that improve aerobic fitness and minimize risk of thromboinflammation.

platelet-leukocyte interaction; redox status; thrombosis

THE MAIN RATIONALE FOR THE clinical use of intermittent hypoxia (IH) concerns the potential cross-protective value of adaptation to one stress, which then provides resistance to another stress (20, 21). Furthermore, as in the case of acclimatization to chronic hypoxia, the IH is characterized by a progressive increase in pulmonary ventilation (16), adaptation of the circulatory system to promote the delivery of oxygen to the tissue (17), and change in the tissue level to optimize the utilization of oxygen (9). The increases in both the stress resistance and the oxygen delivery and utilization by IH have been suggested to improve exercise performance and be of value in the prevention and treatment of various illnesses (21).

The accumulation and activation of eosinophils and neutrophil and their subsequent interaction with platelets are critical in determining the severity of inflammation and thrombosis (3, 7, 32). Some inflammation-related cytokines have been observed to enhance platelet reactivity and subsequently increase the capacity of platelets to adhere to leukocytes (5, 22, 31). Although previous studies demonstrated that severe exercise enhanced neutrophil- and eosinophil-platelet aggregation (33) and inflammatory cytokine releases (23), the effects of chronic IH on the thromboinflammatory responses to this exercise remain unclear.

The beneficial or detrimental effects of IH may vary substantially with the concentration of air oxygen exposure and the subsequent change in the circulatory redox status under the IH regimen. Animal studies established that long-term adjustment to hypoxia (10% O2) involved a decrease in the leukocyte-endothelial adherence that was caused by an increase in the production of antioxidants (40). A recent investigation from our group (36) of healthy sedentary men demonstrated that intermittent exposure to 12% O2 for 4 wk reduced antioxidative capacity and increased the amount of lipid peroxidation in circulation, resulting in the suppression of the vascular endothelial function, impairing vascular hemodynamics. When the concentration of O2 was set to 15%, the risk of vascular complications was absent (36). Earlier studies have proved that blood is subjected to oxidative stress during exercise (15) and that the changes of circulatory redox status affect leukocyte-platelet interaction (18) and inflammation-related cytokine releases (6). Hence, we hypothesize that chronic IH impacts eosinophil- and neutrophil-platelet aggregation and inflammation-related cytokine releases induced by strenuous exercise through modulation of the redox status in circulation, with reactions determined by intervening IH condition.

This work clarifies how two intensities of IH regimens (12% and 15% O2 for 1 h/day, 5 days/wk for 8 wk, respectively) affect eosinophil- and neutrophil-platelet interactions and inflammation-related cytokine secretion caused by severe exercise. Accordingly, a strategy that involved suitable IH was developed to improve aerobic capacity, while minimizing the risk of vascular inflammatory and thrombotic disorders evoked by vigorous exercise.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Subjects and interventions.   The Ethics Committee of Chang Gung Memorial Hospital approved the study protocol, which followed institutional guidelines. Twenty healthy nonsmokers, who did not use medications or vitamins, were infection free and cardiopulmonary risk-free (i.e., blood cholesterol level <200 mg/dl, blood pressure <140/90 mmHg, and force expiratory volume in 1 s/forced vital capacity >80%), and did not have diabetes mellitus, were recruited from Chang Gung University, Taiwan. No subject had engaged in regular physical activity (i.e., exercise frequency ≤1 time/wk, duration <20 min) for 1 yr. All subjects gave informed consent after the experimental procedures were explained to them. Subjects were randomly divided into moderate intermittent hypoxia (MIH) (n = 10) and severe intermittent hypoxia (SIH) (n = 10) groups. Anthropometric data for MIH and SIH groups did not differ significantly (Table 1). Subjects fasted for at least 8 h and were instructed to refrain from exercise for at least 24 h before this study. All subjects arrived at the testing center at 9:00 AM to eliminate any possible diurnal effect.


View this table:
[in this window]
[in a new window]

  		Table 1. Anthropometric parameters, basic cardiopulmonary function, and exercise performance at MIH and SIH regimens

 
The subjects in the two groups were exposed to 12% (SIH) or 15% (MIH) O2 for 1 h/day, 5 days/wk for 8 wk in an air-conditioned normobaric hypoxia chamber (Colorado Mountain Room, Boulder, CO), which was maintained at a temperature of 22 ± 0.5°C with a relative humidity of 60 ± 5%; a CO2 scrubber eliminated CO2 in air (≤3,500 ppm). All subjects recorded their daily activity via a physical activity questionnaire that was collected every week until the end of this study. During the experiment, subjects did not engage in regular physical activity (i.e., exercise frequency ≤1 time/wk, duration <20 min). Moreover, their compliance rates to the two IH interventions were 100%.

Exercise and blood collection protocol.   Each subject performed a graded exercise test 48 h before and 48 h after IH intervention (Fig. 1). The graded exercise test on a bicycle ergometer (Corvial 400; Lode, Groningen, The Netherlands) comprised 2 min of unloaded pedaling; the loading increased by 20–30 W every 3 min until exhaustion [i.e., strenuous exercise up to maximal oxygen consumption (VO2 max)]. Heart rate, minute ventilation (VE), oxygen consumption (VO2), and carbonic dioxide production (VCO2) were measured with an automated system (System 2000, Medical Graphics, Paul, MN). The VO2 max was that value at which the level of VO2 increased <2 ml·kg–1·min–1 after at least 2 min, heart rate exceeded its predicted maximum, the respiratory exchange ratio exceeded 1.2, or the venous lactate concentration exceeded 8 mM (37). Additionally, the ventilation threshold was determined when VE-to-VO2 increased without a corresponding increase in the VE-to-VCO2 ratio and end-tidal PO2 increased without a decrease in the end-tidal PCO2 or departure from linearity for VE. Blood pressure was monitored with an automatic blood pressure system (model 412; Quinton, Bothell, WA); arterial O2 saturation was measured by finger pulse oximetry (model 9500; Nonin Onyx, Plymouth, MN), and blood pH, PCO2, HCO3–, and lactate concentrations were determined with an i-STAT clinical analyzer (+CG4; i-STAT, East Windsor, NJ).


Figure 1
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 1. Design and time course of the experiment. The subjects in moderate (MIH) and severe (SIH) intermittent hypoxia groups were exposed to 12% (SIH) or 15% (MIH) O2 for 1 h/day, 5 days/wk for 8 wk in an air-conditioned normobaric hypoxia chamber, respectively. Each subject performed a grade exercise test 48 h before and 48 h after IH intervention. Before and immediately after the hypoxia test (i.e., acute exposure to various O2 concentrations) and the grade exercise test at the beginning and end of the two IH regimens, blood samples were colleted from a vein in the forearm to measure hematological parameters, leukocyte-platelet interaction, and inflammation-related cytokines.

 
Before and immediately after acute exposure to various O2 concentrations and the strenuous exercise at the beginning and the end of the two IH regimens, blood samples were collected from an anticubital vein by a clean venipuncture (20-gauge needle) under controlled venous stasis of 40 Torr. The first 2 ml were discarded, and then the remaining blood sample was used for the measurements of hematological parameters, leukocyte-platelet interaction, and inflammation-related cytokines (Fig. 1). The blood cells were counted with the use of a Sysmax SF-3000 cell counter (GMI, Ramsey, MN).

Eosinophil, neutrophil, and platelet separations.   Peripheral blood polymorphonuclear leukocytes were isolated from 40-ml whole venous blood by dextran sedimentation, followed by density separation over Ficoll-Hypaque and hypotonic lysis. Eosinophils were then prepared from the polymorphonuclear leukocytes suspensions using a MACS-negative immunomagnetic selection method; neutrophils were isolated positively by human CD16 immunomagnetic microbeads (~50 nm in size and biodegradable), as described previously (33). Both eosinophils and neutrophils were resuspended in HBSS (Sigma, St. Louis, MO) with 2 mM CaCl2 (Sigma), at pH 7.4, and adjusted to a concentration of 1 x 107 cells/ml. Platelet-rich plasma was prepared by centrifugation at 120 g for 10 min at room temperature. Four milliliters of platelet-rich plasma were then mixed with 8 ml of HBSS in a polypropylene tube with 0.8 ml of albumin (4 g/ml) acting as the "cushion" for these platelets. To prevent platelet activation during the experiment, the following inhibitors were added: 0.05 IU/ml apyrase (Sigma) to remove traces of ADP and 0.05 IU/ml hirudin (Sigma) to remove traces of thrombin. The platelet pellets were obtained after centrifugation at 700 g for 10 min and then adjusted with HBSS to 2 x 108 cells/ml (37). The analyses of eosinophil, neutrophil, and platelet functions were completed within 2 h after cell purification.

Eosinophil- and neutrophil-platelet aggregation.   Platelet suspension was incubated with a saturating concentration of monoclonal anti-human CD42b antibody conjugated with phycoerythrin (CD42b-PE) (eBioscience, San Diego, CA) in darkness for 20 min at 37°C. Then, 50 µl of platelets (2 x 108 cells/ml) were labeled with CD42b-PE, and 50 µl of neutrophils or eosinophils (1 x 107 cells/ml) were mixed on 1 mg/ml albumin (Sigma)-coated glass (32 mm diameter) and sheared at controlled shear stresses at 37°C for 5 min using a rotational viscometer (CAP2000; Brookfield, Middleboro, MA). The cell mixture was either under static condition (0 dyn/cm2, as a negative control) or under constant physiological shear stress (5 dyn/cm2, mimicking a venous circuit) for 5 min at 37°C. In some experiments, various stimuli, such as 100 µg/ml LPS (a principal component of the outer membrane of Gram-negative bacteria) (Sigma), 1 µM N-formyl-methionyl-leucyl-phenylalanine (fMLP; a chemotactic peptide) (Sigma), and 1 µM PMA (a PKC activator, as a positive control) (Sigma) were added to the cell mixture, which was then warmed to 37°C for 30 min. These cell mixtures were transferred into polypropylene tubes that contained 2% formaldehyde (Sigma) in PBS, immediately after exposure to various shear stresses and stimuli. The fluorescences from 5,000 events, representing the CD42b-PE-labeled platelets that were bound to eosinophils or neutrophils, were then calculated with the use of a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) (33). In brief, the eosinophils (or neutrophils) were gated separately from the platelets on the basis of forward or sideward scatter, and then the PE-stained events found in the eosinophil (or neutrophil) gate were expressed as the percentage of definition neutrophil- or eosinophil-platelet complexes. This result shows the percentage of neutrophils or eosinophils that bind platelets (33).

Inflammation-related cytokines, antioxidants, and malondialdehyde levels in plasma.   Before and immediately after acute exposure to various O2 concentrations and the strenuous exercise at the beginning and the end of the two IH regimens, additional 10-ml blood samples were obtained, placed in cold centrifuge polypropylene tubes containing sodium citrate (3.8 g/dl; 1–9 vol of blood), and immediately centrifuged at 10,000 g for 30 min at 4°C. These plasma samples were stored at –80°C until assay. Cytokines TNF-{alpha}, IL-1beta, IL-6, IL-8, and IL-10 were quantified by commercially available ELISA kids (R&D Systems, Minneapolis, MN). Total antioxidant was evaluated by the automated ferric-reducing ability of plasma (FRAP) assay (4). Briefly, 300 µl of freshly prepared FRAP reagent were warmed to 37°C, and a reagent blank reading was taken (M1) at 593 nm; plasma (10 µl) was then added, along with 30 µl of H2O2. Absorbance (A) readings were taken after 0.5 s and every 15 s thereafter during the monitoring period. The change in absorbance ({Delta}A593 nm) between the final reading selected and the M1 reading was calculated for each sample and related to{Delta}A593 nm of a Fe(II) standard solution tested in parallel (4). Additionally, level of plasma malondialdehyde (MDA) was determined by fluorometric liquid chromatography (1). The mobile phase consisted of 40-to-60 ratio (vol/vol) of methanol to 50 mM potassium monobasic phosphate (Sigma) at pH 6.8, pumped at a rate of 1.0 ml/min on a Hewlett-Packard Hypersil 5 µ octadecylsilica 100 x 4.6 mm (Altech Associates, Deerfield, IL) placed in a column warmer set to 37°C. Samples of plasma were treated with antioxidant and butylated hydroxytoluene (Sigma) and heat derivatized at 100°C for 1 h with thiobarbituric acid in a H3PO4 solution (Sigma). These samples were then extracted with n-butanol (Sigma), and 10 µl of the extract were injected at 1-min intervals with the use of an autosampler. The fluorescence detector was set at an excitation wavelength of 515 nm and emission wavelength of 553 nm.

Statistical analysis.   Data are expressed as means ± SE. The statistical software package StatView IV was applied to analyze the data. The results were analyzed by repeated ANOVA followed by Tukey's multiple range test to compare the aerobic fitness, hematological parameters, platelet-leukocyte interaction, inflammatory-related cytokines, and plasma redox states of MIH and SIH groups at the beginning of the study and after 8 wk. The associations of measurements with TNF-{alpha}, IL-1beta, IL-6, IL-8, and IL-10 concentrations and either MDA or total antioxidants were assessed by the Spearman rank correction test. The criterion for significance was P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The two IH groups did not differ significantly in anthropometric parameters or basic cardiopulmonary functions at the beginning of the study (Table 1). Acute exposure of 12% O2 increased venous pH and was accompanied by declines in PCO2 and blood lactate concentration; these effects decayed for 8 wk of SIH (data not shown). After the IH interventions, both SIH and MIH subjects exhibited increased exercise time, work rate, VE, VO2, and VCO2 at ventilation threshold (P < 0.05), as well as VE, VO2, and VCO2 at maximal exercise performance (P < 0.05). However, the increase in the cardiopulmonary fitness of the SIH group did not differ significantly from that of the MIH group (Table 1).

Strenuous exercise increased erythrocyte, total leukocyte (neutrophil, eosinophil, basophil, lymphocyte, and monocyte), and platelet counts, whereas the enhancement of blood cell counts by severe exercise did not have any effect after either SIH or MIH intervention (Table 2). Although acute exposure to 12% O2 reduced lymphocyte and monocyte counts (P < 0.05), such exercise effects did not occur after intervening SIH for 8 wk (Table 2). However, acute exposure to 15% O2 followed by intervention of MIH did not affect erythrocyte, total leukocyte, or platelet counts in blood (Table 2).


View this table:
[in this window]
[in a new window]

  		Table 2. Effects of MIH and SIH regimens on blood cell counts

 
At the beginning of this study, 12% O2 promoted LPS- and fMLP- induced eosinophil-platelet aggregation (P < 0.05), whereas 15% O2 did not change the heterotypic cell aggregation (Fig. 2). However, the enhancement of eosinophil-platelet aggregation by exposure to 12% O2 was reduced after 8 wk of intervening SIH (Fig. 2). Although strenuous exercise increased shear stress-, LPS-, and fMLP-induced eosinophil-platelet aggregation (P < 0.05), interventions of SIH and MIH reduced the extent of exercise-induced eosinophil-platelet aggregation under physical shear force and various chemical stimuli (Fig. 3). Additionally, acute exposure to 12% or 15% O2 and strenuous exercise with the intervention of MIH or SIH did not significantly change static -, shear stress-, LPS-, fMLP-, or PMA-induced neutrophil-platelet aggregations (Figs. 2 and 3).


Figure 2
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 2. Effects of MIH and SIH regimens on hypoxia-induced leukocyte-platelet aggregation under static (SC), shear stress (SS), LPS, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and PMA conditions. N (normoxia) and H (hypoxia) indicate before (21% O2) and after acute exposure to various O2 concentrations (MIH: 15% O2; SIH: 12% O2), respectively. Neu-Plt, neutrophil-platelet aggregation; Eos-Plt, eosinophil-platelet aggregation. *P < 0.05, N vs. H; #P < 0.05, 0 wk vs. 8 wk.

 

Figure 3
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 3. Effects of MIH and SIH regimens on severe exercise-induced leukocyte-platelet aggregation under SC, SS, LPS, fMLP, and PMA conditions. R and E indicate at rest and immediately after exercise, respectively. *P < 0.05, R vs. E; #P < 0.05, 0 wk vs. 8 wk.

 
Before IH intervention, acute exposure to 12% or 15% O2 did not change TNF-{alpha}, IL-1beta, IL-6, IL-8, and IL-10 levels (Fig. 4), but the IL-1beta level (P < 0.05) did increase in response to strenuous exercise (Fig. 5). Intervention of IH for 8 wk suppressed the exercise-promoted IL-1beta production in both MIH and SIH groups (Fig. 5). Moreover, resting, postexercise, and posthypoxia IL-6 (P < 0.05) and IL-10 (P < 0.05) levels simultaneously increased in the SIH group (Figs. 4 and 5).


Figure 4
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 4. Effects of MIH and SIH regimens on hypoxia-mediated TNF-{alpha}, IL-1beta, IL-6, IL-8, and IL-10 productions. #P < 0.05, 0 wk vs. 8 wk.

 

Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 5. Effects of MIH and SIH regimens on severe exercise-mediated TNF-{alpha}, IL-1beta, IL-6, IL-8, and IL-10 productions. *P < 0.05, R vs. E; #P < 0.05, 0 wk vs. 8 wk.

 
Acute exposure to 12%, but not to 15% O2, reduced the total antioxidant content (P < 0.05), whereas strenuous exercise reduced the total antioxidant content (P < 0.05) and increased the MDA level (P < 0.05) at the beginning of the study (Table 3). However, the decrease in the total antioxidant level or the increase in the MDA level due to severe exercise and hypoxia declined after 8 wk of intervention with SIH and MIH (Table 3). In relationships between the redox status and the inflammation-related cytokines in plasma, Table 4 shows that levels of plasma IL-1beta are negatively correlated with total antioxidants (MIH: r = –0.847, P < 0.001; SIH:r = –0.721, P < 0.001) and positively correlated with MDA (MIH: r = –0.939, P < 0.001; SIH:r = –0.870, P < 0.001) in both MIH and SIH groups, whereas levels of plasma IL-6 and IL-10 are positively correlated with total antioxidants (IL-6: r = 0.428, P = 0.006; IL-10:r = 0.432, P = 0.005) and negatively correlated with MDA (IL-6: r = –0.318, P = 0.045; IL-10:r = –0.332, P = 0.037 ) in only the SIH group.


View this table:
[in this window]
[in a new window]

  		Table 3. Effects of MIH and SIH regimens on plasma total antioxidant and malondialdehyde levels

 

View this table:
[in this window]
[in a new window]

  		Table 4. Correlation between redox status and inflammation-related cytokine levels in plasma

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
By elucidating the relationship between chronic IH intervention and eosinophil and neutrophil aggregation or inflammation-related cytokines, this study attempts to provide a strategy for developing a suitable IH regimen that would improve aerobic fitness and minimize risk of thromboinflammation evoked by severe exercise. Both MIH and SIH for 8 wk increased the aerobic fitness of sedentary men by increasing their pulmonary ventilation and the tissue utilization of O2 at ventilation threshold and maximal performance, revealing that the two IH regimens improve exercise efficiency and physical endurance in daily life. Furthermore, this study is the first to demonstrate clearly that 1) acute exposure to 12% O2 but not to 15% O2 promotes the formation of eosinophil-platelet aggregates that are produced by shear force and inflammation-related stimuli; 2) chronic MIH and SIH suppress eosinophil-platelet aggregation, proinflammatory cytokine IL-1beta production, and lipid peroxidation caused by severe exercise or acute hypoxia; and 3) plasma levels of anti-inflammatory cytokine IL-6 and IL-10 increased simultaneously after SIH intervention.

Previous works have claimed that vigorous exercise increases the risk of major vascular thrombotic events and transiently increases the incidence of primary cardiac arrest (2, 26). Platelet and leukocyte colocalizations at the wall of damaged or stimulated blood vessels are essential components of a multiple cascade in thrombosis and inflammation (3, 7). Eosinophilic inflammatory is a principal factor in the pathogenesis of allergic disease (39), and neutrophils play an important role not only in bacterial infection but also in respiratory distress syndrome and asthma (28). Activated eosinophils and neutrophils produce oxygen metabolites, such as superoxide (30), which act as disinfectants and cause tissue injury at the inflamed lesion. Platelets contribute to the secondary tethering processes of the eosinophils and neutrophils to activated endothelium, promoting thrombotic formation and vascular occlusion, thereby decreasing blood flow and exacerbating tissue ischemia (3, 7, 32). A recent investigation found increases in eosinophil- and neutrophil-platelet aggregations and subsequent reactive oxygen species production of eosinophils and neutrophils after sustained strenuous exercise (80% VO2 max for 40 min); the effect of exercise on heterotypic cell aggregation was more pronounced in platelet interaction with eosinophils than with neutrophils (33). The aforementioned findings were consistent with some of the results in this study concerning progressive vigorous exercise [gradually increased in intensity until exhaustion is reached (VO2 max)]. Our results indicated that this exercise promoted eosinophil-platelet aggregation induced by shear force and inflammatory stimuli, such as LPS and fMLP. Such heterotypic adhesive interactions, strengthened by severe exercise, may promote inflammatory responses and accelerate thrombus formation in microcirculation.

Previous investigations have shown that oxidative stress increased the binding of leukocytes to surface-adherent platelets and enhanced the capacity of the heterotypic cell aggregates to withstand physiological or pathological flow shear stress (34, 35). As is well known, blood undergoes oxidative stress during hypoxia (24), and oxygen-derived free radicals generated by hypoxia facilitate a rapid microvascular inflammatory response that is characterized by enhanced leukocyte-endothelial adherence and emigration increasing vascular permeability (41). Conversely, acclimatization to chronic hypoxia weakened the leukocyte-endothelial interaction by promoting the production of antioxidants (11, 40). In addition, heavy exercise was observed to suppress antioxidative capacity of leukocytes by decreased superoxide dismutase activity and {gamma}-glutamylcysteinyl glycine content (38). In this work, a single exposure to 12% O2 and severe exercise reduced the antioxidative capacity of blood and simultaneously accelerated the formation of eosinophil-platelet aggregates under shear flow and various inflammatory conditions. However, intermittent exposures to 12% and 15% O2 for 8 wk inhibited the generation of oxidative stress during severe exercise and simultaneously suppressed the eosinophil-platelet aggregation that was promoted by this exercise. These findings imply that, although severe hypoxia (12% O2) as the period can disturb blood redox homeostasis toward pro-oxidative status, chronic IH interventions are required to adapt the body's antioxidative enzyme systems, which may, in turn, suppress the eosinophil-platelet aggregation induced by oxidative stress that is generated from severe exercise, further protecting individuals against risks of thrombosis and inflammation evoked by severe exercise.

Systemic inflammation is involved in the pathogenesis and progression of atherosclerosis and other cardiovascular diseases (10). In the inflammation-related cytokine cascade (13), TNF-{alpha} and IL-1beta are initially released from the site of inflammation and are then referred to as proinflammatory cytokines in the early stage; then, IL-6 (27) and IL-10 (25) are released, having an anti-inflammatory effect by inhibiting the production of TNF-{alpha}, IL-1beta, and chemokine IL-8. Numerous studies have shown that some cytokines such as IL-1beta promote platelet reactivity and the capacity of activated platelets to adhere to leukocytes by modulating the expression of adhesion molecules on platelets and leukocytes (5, 22, 31). According to previous investigations, vigorous, resistive, or eccentric exercises increased the amount of IL-1beta released from exercising muscles, but this cytokine remained unchanged in response to aerobic and submaximal exercise (8, 14, 23, 29). The findings of this work were similar, as strenuous, acute exercise increased IL-1beta concentration in blood. However, chronic SIH and MIH interventions ameliorated such effects of exercise on IL-1beta. The mechanisms by which the two IH regimens decrease the amount of circulatory IL-1beta induced by severe exercise are unclear. A possible reason is that chronic IH increases circulatory anti-inflammatory cytokine levels, such as IL-6 and IL-10, to inhibit the production of IL-1beta during severe exercise. Although recent evidence has demonstrated an increase in plasma IL-6 level after chronic high-altitude exposure (12, 19), the IL-10 response to chronic IH has been not examined. This study demonstrated increases in circulatory IL-6 and IL-10 levels after SIH for 8 wk, and higher levels of either IL-6 (r = –0.316, P = 0.046) or IL-10 (r = –0.320, P = 0.044) were associated with lower IL-1beta levels in plasma (data not shown), which is a change that seems plausibly to explain why chronic SIH inhibits the secretion of IL-1beta by severe exercise. Although the decline in exercise-induced IL-1beta secretion by chronic MIH was not associated with changes in circulatory IL-6 and IL-10 levels, circulatory IL-1beta levels were negatively correlated with antioxidants (r = –0.847, P < 0.001) and positively correlated with MDA (r = –0.939, P < 0.001) in the MIH group. Because circulatory redox status affects the generation of inflammation-related cytokines (6), we speculate that the suppression of exercise-induced oxidative stress by chronic MIH may help to prevent exercising muscles from oxidative damage and further reduce the release or production of IL-1beta during exercise.

As in numerous other investigations, one limitation of the present work is that the subjects used tended to be young and healthy, and thus further clinical evidence is required to extrapolate the present results to patients with abnormal or diseased cardiovascular systems.

In conclusion, both SIH and MIH for 8 wk improved the aerobic fitness of subjects by enhancing pulmonary ventilation and tissue O2 utilization. Moreover, the two IH regimens can also simultaneously suppress eosinophil- and platelet-related thrombosis, proinflammatory cytokine IL-1beta production, and lipid peroxidation caused by vigorous exercise. Additionally, chronic SIH is associated with increased contents of anti-inflammatory cytokines IL-6 and IL-10 in circulation. These experimental findings can help to determine effective IH regimens to increase aerobic capacity and minimize the risk of inflammatory and thrombotic disorders associated with exercise.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
The authors thank the National Science Council of the Republic of China, Taiwan, for financially supporting this research under contract no. NSC 95-2314-B-182-035-MY3.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank the volunteers for enthusiastic participation in this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J.-S. Wang, Graduate Institute of Rehabilitation Science and Center for Gerontological Research, Chang Gung Univ., 259 Wen-Hwa 1st Rd., Kwei-Shan, Tao-Yuan 333, Taiwan (e-mail: s5492{at}mail.cgu.edu.tw)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Agarwal R, Chase SD. Rapid, fluorimetric-liquid chromatographic determination of malondialdehyde in biological samples. J Chromatogr A 775: 121–126, 2002.[CrossRef][ISI]
  2. Albert CM, Mittleman MA, Chae CU, Lee IM, Hennekens CH, Manson JE. Triggering of sudden death from cardiac causes by vigorous exertion. N Engl J Med 343: 1355–1361, 2000.[Abstract/Free Full Text]
  3. Bazzoni G, Dejana E, Del Maschio A. Platelet-neutrophil interactions possible relevance in the pathogenesis of thrombosis and inflammation. Haematologica 76: 491–499, 1991.[ISI][Medline]
  4. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay. Anal Biochem 239: 70–76, 1996.[CrossRef][ISI][Medline]
  5. Beretz A, Klein-Soyer C, Archipoff G, Camilla C, Brisson C, Freyssinet JM, Cazenave JP. Modulation by cytokines of leukocyte-endothelial cell interactions. Implications for thrombosis. Biorheology 27: 455–460, 1990.[ISI][Medline]
  6. Crapo JD. Oxidative stress as an initiator of cytokine release and cell damage. Eur Respir J 44: 4s–6s-, 2003.
  7. De Gaetano G, Cerletti C, Evangelista V. Recent advances in platelet-polymorphonuclear leukocyte interaction. Haemostasis 29: 41–49, 1999.[CrossRef][ISI][Medline]
  8. Dennis RA, Trappe TA, Simpson P, Carroll C, Huang BE, Nagarajan R, Bearden E, Gurley C, Duff GW, Evans WJ, Kornman K, Peterson CA. Interleukin-1 polymorphisms are associated with the inflammatory response in human muscle to acute resistance exercise. J Physiol 560: 617–626, 2004.[Abstract/Free Full Text]
  9. Elander A, Idstrom JP, Holm S, Schersten T, Bylund-Fellenius AC. Metabolic adaptation in response to intermittent hypoxia in rat skeletal muscles. Adv Exp Med Biol 169: 507–513, 1984.[ISI][Medline]
  10. Fiotti N, Giansante C, Ponte E, Delbello C, Calabrese S, Zacchi T, Dobrina A, Guarnieri G. Atherosclerosis and inflammation. Patterns of cytokine regulation in patients with peripheral arterial disease. Atherosclerosis 145: 51–60, 1999.[CrossRef][ISI][Medline]
  11. Gonzalez NC, Wood JG. Leukocyte-endothelial interactions in environmental hypoxia. Adv Exp Med Biol 502: 39–60, 2001.[ISI][Medline]
  12. Hagobian TA, Jacobs KA, Subudhi AW, Fattor JA, Rock PB, Muza SR, Fulco CS, Braun B, Grediagin A, Mazzeo RS, Cymerman A, Friedlander AL. Cytokine responses at high altitude: effects of exercise and antioxidants at 4300 m. Med Sci Sports Exerc 38: 276–285, 2006.
  13. Hanada T, Yoshimura A. Regulation of cytokine signaling and inflammation. Cytokine Growth Factor Rev 13: 413–421, 2002.[CrossRef][ISI][Medline]
  14. Hirose L, Nosaka K, Newton M, Laveder A, Kano M, Peake J, Suzuki K. Changes in inflammatory mediators following eccentric exercise of the elbow flexors. Exerc Immunol Rev 10: 75–90, 2004.[ISI][Medline]
  15. Jenkins RR. Free radical chemistry. Relationship to exercise. Sports Med 5: 156–170, 1988.[ISI][Medline]
  16. Katayama K, Sato Y, Morotome Y, Shima N, Ishida K, Mori S, Miyamura M. Intermittent hypoxia increases ventilation and SaO2 during hypoxic exercise and hypoxic chemosensitivity. J Appl Physiol 90: 1431–1440, 2001.[Abstract/Free Full Text]
  17. Katayama K, Shima N, Sato Y, Qiu JC, Ishida K, Mori S, Miyamura M. Effect of intermittent hypoxia on cardiovascular adaptations and response to progressive hypoxia in humans. High Alt Med Biol 2: 501–508, 2001.[CrossRef][Medline]
  18. Li N, Hu H, Lindqvist M, Wikstrom-Jonsson E, Goodall AH, Hjemdahl P. Platelet-leukocyte cross talk in whole blood. Arterioscler Thromb Vasc Biol 20: 2702–2708, 2000.[Abstract/Free Full Text]
  19. Mazzeo RS. Altitude, exercise and immune function. Exerc Immunol Rev 11: 6–16, 2005.[ISI][Medline]
  20. Meerson F, Pozharov V, Minyailenko T. Superresistance against hypoxia after preliminary adaptation to repeated stress. J Appl Physiol 76: 1856–1861, 1994.[Abstract/Free Full Text]
  21. Neubauer JA. Physiological and pathophysiological responses to intermittent hypoxia. J Appl Physiol 90: 1593–1599, 2001.[Abstract/Free Full Text]
  22. Neumann FJ, Marx N, Gawaz M, Brand K, Ott I, Rokitta C, Sticherling C, Meinl C, May A, Schomig A. Induction of cytokine expression in leukocytes by binding of thrombin-stimulated platelets. Circulation 95: 2387–2394, 1997.[Abstract/Free Full Text]
  23. Petersen AM, Pedersen BK. The anti-inflammatory effect of exercise. J Appl Physiol 98: 1154–1162, 2005.[Abstract/Free Full Text]
  24. Prabhakar NR, Kumar GK. Oxidative stress in the systemic and cellular responses to intermittent hypoxia. Biol Chem 385: 217–221, 2004.[CrossRef][ISI][Medline]
  25. Pretolani M. Interleukin-10: an anti-inflammatory cytokine with therapeutic potential. Clin Exp Allergy 29: 1164–1171, 1999.[CrossRef][ISI][Medline]
  26. Siscovick DS, Weiss NS, Fletcher RH, Lasky T. The incidence of primary cardiac arrest during vigorous exercise. N Engl J Med 311: 984–987, 1984.
  27. Steensberg A, Fischer CP, Keller C, Moller K, Pedersen BK. IL-6 enhances plasma IL-1ra, IL-10, and cortisol in humans. Am J Physiol Endocrinol Metab 285: E433–E437, 2003.[Abstract/Free Full Text]
  28. Sustiel AM, Joseph B, Rocklin RE, Borish L. Asthmatic patients have neutrophils that exhibit diminished responsiveness to adenosine. Am Rev Respir Dis 140:1556–1561, 1989.[ISI][Medline]
  29. Suzuki K, Nakaji S, Yamada M, Totsuka M, Sato K, Sugawara K. Systemic inflammatory response to exhaustive exercise. Cytokine kinetics. Exerc Immunol Rev 8: 6–48, 2002.[ISI][Medline]
  30. Takashi S, Okubo Y, Horie S. Contribution of CD54 to human eosinophil and neutrophil superoxide production. J Appl Physiol 91: 613–622, 2001.[Abstract/Free Full Text]
  31. Todoroki N, Watanabe Y, Akaike T, Katagiri Y, Tanoue K, Yamazaki H, Tsuji T, Toyoshima S, Osawa T. Enhancement by IL-1beta and IFN-{gamma} of platelet activation: adhesion to leukocytes via GMP-140/PADGEM protein (CD62). Biochem Biophys Res Commun 179: 756–761, 1991.[CrossRef][ISI][Medline]
  32. Ulfman LH, Joosten DP, van Aalst CW, Lammers JW, van de Graaf EA, Koenderman L, Zwagina JJ. Platelets promote eosinophil adhesion of patients with asthma to endothelium under flow conditions. Am J Respir Cell Mol Biol 28: 512–519, 2003.[Abstract/Free Full Text]
  33. Wang JS, Yen HL, Yang CM. Warm-up exercise suppresses platelet-eosinophil/neutrophil aggregation and platelet-promoted release of eosinophil/neutrophil oxidant products enhanced by severe exercise in men. Thromb Haemost 95: 490–498, 2006.[ISI][Medline]
  34. Wang JS, Liao CH. Moderate-intensity exercise suppresses platelet activation and polymorphonuclear leukocyte interaction with surface-adherent platelets under shear flow in men. Thromb Haemost 91: 587–594, 2004.[ISI][Medline]
  35. Wang JS, Chow SE, Chen JK. Strenuous, acute exercise affects reciprocal modulation of platelet and polymorphonuclear leukocyte activities under shear flow in men. J Thromb Haemost 1: 2031–2037, 2003.[CrossRef][ISI][Medline]
  36. Wang JS, Chen LY, Fu LL, Chen ML, Wong MK. Effects of moderate and severe intermittent hypoxia on vascular endothelial function and haemodyamic control in sedentary men. Eur J Appl Physiol 100: 127–135, 2007.[CrossRef][ISI][Medline]
  37. Wang JS, Cheng LJ. The effect of strenuous acute exercise on {alpha}2-adrenergic agonist-potentiated platelet activation. Arterioscler Thromb Vasc Biol 19: 1559–1565, 1999.[Abstract/Free Full Text]
  38. Wang JS, Lee T, Chow SE. Role of exercise intensities on oxidized low-density lipoprotein-mediated redox status of monocyte in men. J Appl Physiol 101: 740–744, 2006.[Abstract/Free Full Text]
  39. Wardlaw AJ, Moqbel R, Kay AB. Eosinophils: biology and role in disease. Adv Immunol 60: 151–266, 1995.[ISI][Medline]
  40. Wood JG, Mattioli LF, Gonzalez NC. Hypoxia causes leukocyte adherence to mesenteric venules in nonacclimatized, but not in acclimatized, rats. J Appl Physiol 87: 873–881, 1999.[Abstract/Free Full Text]
  41. Wood JG, Johnson JS, Mattioli LF, Gonzalez NC. Systemic hypoxia promotes leukocyte-endothelial adherence via reactive oxidant generation. J Appl Physiol 87: 1734–1740, 1999.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Exp. Biol. Med.Home page
T. V. Serebrovskaya, E. B. Manukhina, M. L. Smith, H. F. Downey, and R. T. Mallet
Intermittent Hypoxia: Cause of or Therapy for Systemic Hypertension?
Experimental Biology and Medicine, June 1, 2008; 233(6): 627 - 650.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
103/1/305    most recent
00226.2007v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, J.-S.
Right arrow Articles by Wong, M.-K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, J.-S.
Right arrow Articles by Wong, M.-K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.