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LeuCAM and Reactive Oxygen Species during Long-Term Exercise Combined with Sleep and Energy Deficiency


Medicine & Science in Sports & Exercise: February 2007 - Volume 39 - Issue 2 - p 275-282
doi: 10.1249/01.mss.0000241648.07382.7c
BASIC SCIENCES: Original Investigations

Introduction: During the Norwegian military ranger-training course, cadets are exposed to prolonged physical exercise combined with sleep-, energy-, and food deficiency. The open-window postexercise hypothesis indicates that after hard physical activity, there is an increased risk of contracting infectious diseases.

Purpose: The purpose of the present study was to determine leukocyte reactive oxygen species (ROS) levels, total antioxidant status (TAS), leukocyte expression of the cell adhesion molecules CD62L and CD11b, and plasma levels of soluble adhesion molecule L-selectin before, during, and in the recovery phase of a military ranger-training course.

Methods: Ten cadets from the Norwegian Military Academy were recruited to the study. Flow cytometry was used to study the intracellular levels of ROS in leukocytes (basally, as well as after in vitro stimulation with phorbol myristate acetate (PMA)), applying the probes dihydroethidium (DHE) and dihydrorhodamine 123 (DHR) and the leukocyte expression of adhesion molecules CD62L and CD11b. ELISA was used to assess the plasma levels of soluble L-selectin, and TAS in plasma was measured using the ABTS+ reduction assay kit.

Results: The basal levels of ROS as well as PMA-stimulated ROS in leukocytes declined gradually during the ranger-training course, being lowest on the last day (P < 0.05). The level of TAS increased (P < 0.01) during the course. A striking decrease (P < 0.001) was observed in leukocyte CD62L expression and was sustained even after 3 d of recovery. The leukocyte expression of CD11b remained unchanged.

Conclusion: The ranger-training course leads to a partial exhaustion of the leukocyte ROS-generating machinery and to a nearly total extinguishing of leukocyte CD62L expression. These changes may support the open-window hypothesis indicating reduced ability to combat microbial invasions before total restitution.

1Center for Clinical Research, Ullevaal University Hospital, Oslo, NORWAY; and 2Norwegian Defence Research Establishment, Kjeller, NORWAY

Address for correspondence: Hilde Grindvik Nielsen, Center for Clinical Research, Ullevaal University Hospital, N-0407 Oslo, Norway; E-mail:

Submitted for publication January 2006.

Accepted for publication August 2006.

Military personnel seem to be at particular risk ofdeveloping clinical immunosuppression (20). During the ranger-training course, the cadets are exposed to prolonged hard, continuous physical exercise combined with sleep-, energy-, and water deficiency; cold; and time pressure. Severe energy malnutrition leads to progressive deterioration in many marks of immune functions, such as impaired phagocytosis, decreased production of and response to various cytokines, and a reduced proliferative response to mitogens (20). Sleep deprivation can influence immune function both by disturbing the normal sleep/wakefulness cycle and by causing a stress-related change in the secretion of catecholamines and cortisol (20). However, the situation may be further modified by heavy exercise, which has an arousing effect on the drowsy individual and which also augments the secretion of stress hormones induced by sleep deprivation. It has been argued that prolonged physical activity in itself is a form of stress, which can interact with various psychological and environmental stressors (20). The open-window postexercise hypothesis (22) indicates that there is an increased risk of contracting infections after long-term physical exercise because of immunosuppression-viruses may invade the host, and infections can be established-but there is a demand for elucidation. However, in those who perform regular moderate exercise, the immune system will often be temporarily enhanced, providing protection from infections (22).

Oxidative stress is of importance in many human disease processes, especially in cardiovascular disease, aging, and cancer. It has been defined as a disturbance in the balance between the production of reactive oxygen species (ROS) and antioxidant defenses, leading to tissue injuries (23). Molecules such as superoxide anion (O2 ), hydroxyl radical (OH·), nitric oxide (NO), and lipid radicals are free radicals, which possess an unpaired electron. Others such as hydrogen peroxide (H2O2), peroxynitrite (ONOO), and hypochlorous acid (HOCl) are not free radicals but have oxidizing effects that contribute to the oxidant stress. The latter chemical species are, however, also classified as ROS because of their higher reactivity relative to molecular O2. When NO and O2 react with each other, they rapidly form ONOO (17). The stability of ONOO allows it to diffuse through cells and hit a distant target. Intracellular ONOO formation will arise from elevated production of NO and its subsequent diffusion to sites of O2 formation, and it is usually minimized by the intracellular superoxide dismutase (SOD) activity (17). The most important cellular source of ROS, O2 , is produced by the NADPH oxidase-catalyzed oxidative burst reaction in macrophages and neutrophils (15) and is produced primarily to destroy invading microorganisms (15). This effect is normally advantageous, but inadvertent extracellular release of ROS may induce undue inflammatory reactions in surrounding tissues (15). The production of O2 is increased in a number of disease states (e.g., inflammation (15)), and the precise mechanisms leading to this increase have not been assessed. In athletes, oxidative stress has been associated with decreased physical performance, muscular fatigue, muscle damage, and overtraining. It has been hypothesized that the body's physiological amount of antioxidants is not sufficient to prevent exercise-induced oxidative stress and that additional antioxidants are needed to reduce oxidative stress, muscular damage, or overshooting inflammation (25).

The human body has evolved strong antioxidant defense mechanisms to protect against free radical attacks (15). Numerous natural defenses exist either to prevent ROS formation or to neutralize them after they have been generated. SOD catalyzes the superoxide anion to hydrogen peroxide, which is further degraded by catalase (7). There is also an array of antioxidant molecules capable of scavenging free radicals in the extracellular compartments (1). The most notable are vitamins C and E, various carotenoids, glutathione, ubiquinone, uric acid, and bilirubin.

Cell adhesion molecules (LeuCAM) are expressed on the surface of leukocytes and endothelial cells and have a central role in leukocyte-endothelial interactions. This interaction is an early step in inflammatory events (27) and, thus, in the recruitment of circulating leukocytes into tissues. The L-selectin (CD62L) is constitutively expressed on all leukocytes (14) and is important in the initial "tethering" process of leukocyte-endothelial cell adhesion, which is associated with leukocyte trafficking and inflammation (13). Soluble (s) L-selectin, together with sP-selectin and sE-selectin, can, when added at a high concentration, inhibit lymphocyte adhesion to the endothelium (15,16), because they are bioactive.

The integrins are found on various types of leukocytes and mediate the firm adhesion between leukocytes and vascular cells (13). The β2-integrins (i.e., the CD11/CD18 complex) consist of a β2 chain (CD18) and one of three distinct α-chains: denominated CD11a, CD11b, and CD11c (16). The CD11/CD18 belongs to the transmembrane glycoproteins and is expressed on the surface of neutrophils, monocytes, and macrophages. The expressions of cell-surface adhesion molecules are induced by inflammation, sepsis, transplant rejection, tumor metastasis, and arteriosclerosis (4).

Patients with a deficiency in the β2-integrins, leukocyte adhesion deficiency-1 (LAD-1), are predisposed to life-threatening infections because of impaired intravascular leukocyte adhesion, transendothelial emigration, phagocytosis, and target cell killing. LAD-2 patients have a deficiency in neutrophil expression of the selectin ligands such as SLex or CD15; this may reduce the ability of the cells to roll on the endothelium and to traffic to inflammatory sites (3).

The purpose of the present study was to determine leukocyte ROS levels, total plasma antioxidant status (TAS), the leukocyte expression of selected cell-bound adhesion molecules, and the plasma levels of soluble leukocyte-derived adhesion molecules during a military ranger-training course and also during the early recovery phase.

Our hypotheses, based on our earlier investigations in long-distance runners (18,19), were that leukocyte CD62L would decrease and that CD11b would increase even more during the ranger-training course because the course includes, in addition to long-term physical activity, the nearly total deprivation of sleep and energy supply. Moreover, the exercise-induced increased ROS production would partially and temporarily reduce the ROS-generating capacity of leukocytes.

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The Ranger-Training Course: Study Design

The Norwegian ranger-training course took place in the eastern part of Norway, in a forest area at 500-m altitude. The 8 d of semicontinuous physical activity were executed in good weather with temperatures between 18 and 30°C during the days and between 5 and 15°C by night. The cadets were provided no food during the course, and only 3 h·d−1 sleep was allowed (20).

The ranger-training course consisted of patrols during nighttime, and, during the day, combat training with attacks, building defense positions, and passing obstacles and narrow tubes containing water (20). The physical activities corresponded to about 35% of maximal oxygen uptake and a calorie expenditure of about 40,000 kJ·24 h−1 per cadet (20).

Blood samples were collected at the start of the ranger-training course, after 2, 4, and 8 d (end of the ranger-training course), and after 1 and 3 d of recovery (Table 1). Venous blood was sampled into EDTA-, Na-citrate-, and heparin-anticoagulated vacuum tubes (Becton Dickinson, Plymouth, UK) and was taken between 6 and 8 a.m., kept on ice, and transported to the laboratory for further preparation within 1 h.



In the present study, the use of a control group consisting of soldiers going through basic training or engaging in normal military duty would, in principle, have been possible (5). However, we have observed in healthy volunteers that the variables studied in the present study (ROS) remain stable during a 24-h inactive period (unpublished data, Nielson et al. 2004). These observations are in accordance with the general opinion that ROS-related variables mainly vary because of physical activity, diseases, etc. On the basis of this and the obvious difficulties associated with controlling a potential control group's physical activity (intake of antioxidant supplementation, sleep, etc.), we decided not to include a control group in the present study. The lack of a control group is compensated for by taking measurements for each subject a number of times during the "treatment" (the ranger-training course). Thus, we assume that the time evolution of the variable values during the training course, combined with our knowledge of the course program, will make it evident that the observed variations are actually attributable to the training course and are not attributable to natural variations.

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Ten physically well-trained and healthy male cadets between 21 and 28 yr old were recruited to this study. The cadets did not eat anything during the course, as reflected in an average weight reduction of 9.3 kg (range 7.0 kg-11.0 kg) measured after the course. The intake of water was ad libitum during the course. In the restitution period (days 9 through 11), the cadets followed regular sleeping and eating habits, as before the course. All subjects were informed about the study and gave their written consent for participation. The regional committee for medical ethics approved the test protocols.

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Hematological Analyses

White blood cell (WBC), platelet and erythrocyte counts, hemoglobin, and hematocrit were assessed in EDTA blood using the Technicon H2 System (Bayer Corporation, Tarrytown, NY), and CRP was analyzed with Cobas Integra 800 Roche (Mannheim, Germany) at the Department of Clinical Chemistry, Ullevaal University Hospital.

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Preparation of Blood Leukocytes for ROS Analysis

Basal ROS levels.

Aliquots of 50 μL of EDTA whole blood were incubated for 15 min at 37°C in a 5% CO2/humidified air atmosphere in polystyrene round-bottom tubes (2052 Falcon, Oxnard, CA) with dihydroethidium (DHE) (Sigma, St. Louis, MO) (final concentration 5 μmol·L−1), recognizing mainly the oxygen species superoxide anion (O2 ) (26), dihydrorhodamine 123 (DHR) (Sigma) (final concentration 5 μmol·L−1) (recognizing mainly the oxygen species peroxynitrate (ONOO−1) (28), and phosphate-buffered saline (PBS) (Sigma) (10 mmol·L−1 of phosphate buffer,2.7 mmol·L−1 of KCl, and 137 mmol·L−1 of NaCl, pH 7.4) as autofluorescence control. After the end of incubation, 1.5 mL of red cell lysing solution containing 156 mmol·L−1 of NH4Cl, 10 mmol·L−1 of NaHCO3, and 0.12 mmol·L−1 of NaEDTA was added to the tubes. The tubes were incubated in the dark at room temperature for 15 min and were then centrifuged at 300g for 5 min at 4°C. Subsequently, the supernatant was discarded and the leukocyte pellet was gently resuspended and washed once with 2 mL of cold PBS. The cells were finally resuspended in0.5 mL of 1% w/v paraformaldehyde (PFA) (Merck, Darmstadt, Germany) in PBS and stored in the dark at 4°C until flow cytometry could be performed.

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In vitro stimulated ROS production.

Aliquots of 1.5 mL of whole blood were incubated in polystyrene tubes with ventilation caps with 15 μL of phorbol myristate acetate (PMA) (final concentration 100 ng·mL−1) (Sigma) or PBS (control) at 37°C for 60 min in a CO2 incubator. Aliquots of 50 μL of blood were then labeled with DHE or DHR as described for basal ROS levels. The intraassay coefficient of variation (CV) was < 5% in unstimulated and < 10% in PMA-stimulated samples.

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Preparation of Blood Leukocytes for Adhesion Molecule Determination


Phycoerythrin-conjugated monoclonal antibodies against CD62L and fluorochrome-matchedIgG2a (isotype control) were both from Becton Dickinson (San Jose, CA). Fluorescein isothiocyanate (FITC)-conjugated anti-CD11b and the corresponding fluorochrome-matched isotype IgG1 were from Sigma.

To study the expression of leukocyte surface markers, 50μL of whole blood (EDTA) was incubated with antibodies against CD62L (20 μL) and CD11b (10 μL). Isotype-fluorochrome- and protein concentration-matched controls were run in parallel to the monoclonal antibodies. The test tubes were incubated on ice for 30 min in the dark. After the end of incubation, 1.5 mL of red cell lysing solution was added to the tubes and incubated for 15 min in the dark at room temperature before centrifugation at 300g for 5 min at 4°C. Subsequently, the supernatant was discarded and the leukocyte pellet was gently resuspended and washed once in 2 mL of cold PBS, pH 7.4. The cells were finally resuspended in 0.5 mL of 1% w/v PFA in PBS and were stored in the dark at 4°C until flow cytometry could be performed.

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Flow Cytometry Analysis

The labeled samples were analyzed within 24 h in a FACSort flow cytometer (Becton Dickinson). The flow cytometer was equipped with an argon laser and CellQuest software (BD). A common window of analysis was set with QC Windows calibration beads (Flow Cytometry Standard Corp., San Juan, Puerto Rico). To convert the fluorescence intensity into molecules of equivalent soluble fluorochrome (MESF) units, a calibration was performed weekly with Quantum 26-FITC and Quantum 27-R-phycoerythrin microbead standards (Bangs Laboratories, Fisher, IN). Both the QC Windows and Quantum beads were suspended in the same 1% PFA solution as all the test samples. The different leukocyte subpopulations (i.e., granulocytes, monocytes, and lymphocytes) were identified by means of their light scatter characteristics, enclosed in electronic gates, and analyzed separately for fluorescence intensity.

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The Randox Total Antioxidant Status kit (Crumlin, UK) was used according to the manufacturer's instructions to measure plasma TAS. This assay is based on the peroxidase-mediated conversion of a chromogen to a blue-colored radical cation, which can be detected at 600 nm. Antioxidants in the added plasma sample cause inhibition of this color production to a degree that is proportional to their concentration. Blood samples were collected in heparin tubes, centrifuged at 2300g and 4°C for 12 min, and then plasma was stored at −70°C until analysis. The inter- and intraassay CV were 2.4 and 1.2%, respectively.

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ELISA Analysis

Plasma levels of soluble CD62L (heparin plasma) were measured using the ELISA technique (R&D System Europe Ltd., Abingdon, UK) according to the manufacturer's instructions. The inter- and intraassay CV were 7.1 and 4.1%, respectively.

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Statistical Analyses

For ROS analysis, the results were calculated as the difference in mean fluorescence intensity (dMFI), which is the mean fluorescence intensity (MFI) (arbitrary units) obtained in the specific fluorescent probe sample subtracted from the MFI value obtained when the sample was incubated with PBS. Because of large interindividual variations, dMFI values before the ranger-training course were defined to be 100%, and the values during and after the course are reported relative to this. The results of adhesion molecule analyses were expressed as the difference in MESF units, which means that MESF values obtained with the specific antibodies were subtracted from the MESF values obtained with the isotype control.

Results are given as means and standard errors of the mean (SEM). Paired-sample t-tests were used to compare results from day 0 with results after 2, 4, and 8 d during the ranger-training course and after 1 and 3 d of recovery. Corrections for multiple uses of the tests were performed ad modum Bonferroni, and statistical significance was set at the 0.05 level.

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Hematological analyses.

There was a significant increase (P < 0.05) in total circulating leukocytes from day 0 to day 2 (Table 2). At day 4, the leukocyte count had declined; it increased again at day 8 and leveled off to baseline values on the third day of recovery (day 11). The increase in WBC was mainly attributable to neutrophils, and the neutrophil profile during the course closely followed the WBC profile. The monocyte counts increased (P < 0.05) during the first 2 d and remained at an increased level during the course, decreasing to baseline values during the recovery period. As opposed to this, lymphocyte counts decreased (P < 0.001) during the first 2 d, and after 3 d of recovery (day 11), the baseline level was not yet reached. Platelet counts increased significantly (P < 0.05) from day 4 and remained high during the first 3 d of recovery. Erythrocyte counts and hematocrit andhemoglobin levels fell gradually and significantly (P < 0.05) during the course, including the first phase of the recovery period. CRP rose significantly (P < 0.001) during the first 2 d (from day 0 to day 2) of the ranger-training course and then gradually approached baseline values after 3 d of recovery (day 11).



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Leukocyte ROS levels.

When monitoring with the DHR probe, basal intracellular ROS levels in granulocytes decreased gradually during the ranger-training course, with the reduction maximal between days 4 and 8 (end of ranger-training course) (P < 0.001) and remaining low during the first day of the recovery phase (Fig. 1A). The monocyte ROS levels (DHR) remained unchanged during the whole course, with a maximal reduction after 1 d of recovery (Fig. 1A). Using the DHE probe, the ROS reduction profile was displaced in time, reaching a significant reduction in granulocyte ROS at the very end of the course and maximal reduction (including also monocytes) on the first day of recovery (P < 0.001) (Fig. 1C).



Before the start of the ranger-training course, the ROS production of granulocytes increased 136-fold (± 17) in PMA-stimulated whole blood compared with PBS-incubated blood (DHE probe). The corresponding figure for the DHR probe was a 168-fold (± 13) increase. Monocytes showed considerably lower response and increased their ROS levels 31-fold (± 2) (DHE) and fivefold (± 1) (DHR) on PMA stimulation.

The ability of both monocytes and granulocytes to generate ROS on PMA stimulation was significantly reduced after 2 d of the ranger-training course, being maximally reduced at the end of the course (day 8) (Fig. 1B,D). At this stage, the residual ROS-generating capacity was 35-40% (DHE, DHR) in granulocytes (P < 0.001) and 55-60% (DHR, DHE) in monocytes (P < 0.001, P < 0.05) but was rapidly regained, reaching precourse levels or supraprecourse levels at the first day of recovery. The exception to this was DHR-monitored ROS in monocytes, which lagged behind.

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Leukocyte adhesion molecules.

In all three leukocyte subsets (i.e., granulocytes, monocytes, and lymphocytes), the expression of surface-bound CD62L decreased gradually during the ranger-training course and was close to completely extinguished from the cell surface at the end of the course (P < 0.001). It remained low during the first 3 d of recovery (Fig. 2A) (P < 0.001). In contrast, the expression of CD11b on granulocytes and monocytes did not change significantly during the course, but a significant rise in monocyte (P < 0.001) and lymphocyte (P < 0.01), as well as granulocyte CD11b (P < 0.05), was seen during the first day of recovery (Fig. 2B).



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Plasma TAS was increased (P < 0.001) during the whole ranger-training course, reaching the highest levels after 4 d (Fig. 3). At the first day of recovery, the TAS levels had returned to baseline, and after 2 d of additional recovery, TAS values reached subbaseline (78%) levels.



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Soluble adhesion molecules.

Plasma levels of soluble L-selectin (sCD62L) remained unchanged during the course but were significantly decreased (P < 0.001) at recovery, day 11 (Fig. 4).



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Moderate exercise seems to stimulate the immune system, but there is good evidence that intense exercise can cause immune deficiency (25). This may partly explain the observation that athletes are more likely to contract infectious diseases if exposed to pathogenic microorganisms in the immediate period after intensive physical activity. Earlier observations (25), which have included the actual ranger-training course (6), have shown that physical exercise leads to increased inflammatory responses that are in many respects similar to those seen in septic patients and in patients with the systemic inflammatory response syndrome. In the present study, we have followed 10 male cadets before, during, and after the ranger-training course at the Norwegian Military Academy, with the aim of analyzing leukocyte levels of ROS, changes in TAS, and the leukocyte expression of selected cellular (CD11b and CD62L (L-selectin)) and soluble (sCD62L) adhesion molecules.

During the ranger-training course, we observed a variable (maximally 1.5-fold) increase in total circulating leukocytes, and this was mainly attributable to neutrophilic granulocytes, but monocytes also increased. Our data confirm the well-documented leukocytosis after strenuous exercise (6,29,30). It is generally assumed that postexercise leukocytosis is mainly caused by demargination of WBC, induced by increased blood flow (mediated by catecholamines) (23). The lesser increase in WBC in comparison with, for instance, a marathon race, probably reflects the different type of activity (lower exercise intensity) and the lesser blood-flow increase attributed to the ranger-training situation (6).

Hematocrit, hemoglobin, and red blood cell counts declined gradually during the whole course and remained low after 3 d of recovery. Bøyum et al. (6) reported that the observed decrease of hematocrit, hemoglobin, and red blood cells could be a result of the physical strain the cadets are exposed to, rather than the energy- and sleep deprivation (6). These changes are generally believed to be a result of foot-strike hemolysis, but, as recently pointed out by Robinson et al. (24), intravascular hemolysis could also be caused by intramuscular destruction, osmotic stress, and membrane lipid peroxidation caused by free radicals released by activated leukocytes. The cadets lost, on average, 9.3 kg of weight during the ranger-training course because of the high physical activity combined with starvation (no food intake was allowed). Platelet counts increased significantly (1.2-fold) during the course and remained high during the recovery phase. Similar increases in platelet counts have been described in other studies by measuring platelet counts after prolonged endurance activity (18,19) and have been associated with a prothrombotic state. Platelet responses to exercise are dependent on several factors, such as exercise intensity, the exercise protocols used, and the physical fitness of the individual (25).

The mean plasma levels of CRP increased approximately 10-fold during the first phase of the ranger-training course and remained elevated throughout the course but dropped toward baseline levels in the recovery period. CRP is, among other acute-phase reactants, produced in the liver on cytokine stimulation. CRP enhances host survival by elimination of inflammatory agents and further promotes repair processes (10). It has previously been shown that IL-6 is produced by, and released from, contracting skeletal muscle during exercise (9). In the present 10 cadets, we documented a mean twofold increase in plasma IL-6 levels (unpublished observations), and this might explain the CRP increase.

During the ranger-training course, we observed equal or reduced basal leukocyte ROS levels measured with both the DHE and the DHR probes compared with baseline levels. At first glance, this seems to be variant from the findings of Wiik et al. (29,30). They have demonstrated, during the same type of ranger-training course, a priming of the granulocytes for accentuated chemiluminescence response to serum-opsonized zymosan particles, with maximal increases on days 1-3. The reason for this discrepancy probably is of methodological origin in that they measured the overflow of ROS to the extracellular medium, whereas we measured the intracellular ROS levels. The main reasons that basal intracellular ROS levels may be kept low despite accelerated ROS production include sequestration of the most activated cells in the microcirculation, continuous equilibration of produced ROS with the extracellular milieu, and recruitment of virginal demarginated cells to the circulation.

After the ranger-training course, the capacity of leukocytes (both monocytes and granulocytes) to respond with ROS synthesis on a standardized in vitro PMA stimulus was clearly suppressed (60-65% in granulocytes and 40-45% in monocytes). It thus seems that the leukocyte potential for ROS production was partly exhausted, but not to the same extent as we have earlier observed after a marathon race (5% residual capacity in granulocytes) (19). Therefore, this finding strongly suggests that an ongoing, more or less continuous stimulation of leukocyte ROS production took place to temporarily wear the ROS-generating machinery. The effect of this was probably dichotomous; continuous extracellular leakage (overflow) of ROS may have mediated untoward negative effects such as muscular dysfunction, and intracellular partial exhaustion of the ROS-generating systems may have challenged the microbicidal capacity of the leukocytes. In the recovery phase, after 3 d with food and sleep, the ROS-generation capacity had almost returned to baseline values or even higher.

The available data to support the role of ROS in relation to physical exercise are highly inconsistent and partly controversial. ROS generation and release have been assessed using a variety of methods. The most commonly used methods detecting extracellular release of O2 or H2O2 are spin trapping (26), luminol chemiluminescence (30), and cytochrome c reduction spectrophotometry (12). Flow cytometry, as used in this study, has the unique ability to supply quantitative measurements of individual cells in large numbers within seconds (2). The cellular content of constituents that have been labeled using specific fluorescent probes can be determined. In this study, we used the ROS-sensitive fluorescent probes DHE and DHR for instant determination of intracellular ROS levels in leukocytes harboring their natural habitat (whole blood). In our opinion, the flow cytometry method constitutes a useful supplement to (and, in some respects, an improvement on) other methods used for detecting ROS where time-consuming and potentially cell-activating isolation procedures followed by a period of in vitro cell culture are necessary before ROS production can be studied. Knowing that ROS responses are "bursty" in nature, maximal efforts should be executed to minimize the flaws of excessive in vitro handling (19).

The concentration of ROS depends on the balance between the rate of production and the rate of clearance by various antioxidant compounds and enzymes (intracellular redox state). Cells are in a stable state if rates of oxidant formation and antioxidant scavenging capacity are constant and in balance. Increased production of ROS during exercise may be quenched by antioxidant vitamins such as ascorbic acid and enzymes (e.g., SOD (8)). We observed during the ranger-training course an increase (~30%) in plasma TAS, with a decline (~22%) below baseline in the recovery period. The temporary increase in antioxidant status may indicate that the body's endogenous antioxidant system was activated and exploited as a response to the exercise-induced burst of ROS production. The demonstrated deficit in antioxidant capacity in the recovery period is probably a delayed effect of the constant wearing of endogenous antioxidants during the ranger-training course, combined with the lack of supplementation of dietary antioxidant principles.

One of the most prominent findings of the present study was that leukocyte CD62L (L-selectin) expression gradually decreased during the ranger-training course to be nearly extinguished from the cell surfaces at the end of the course, and also during the initial recovery period. This phenomenon also has been recorded after marathon/half-marathon races (18), but the deexpression of CD62L was not nearly as complete in these situations as it seemed to be at the later phase of the ranger-training course. The long-lasting duration of the physical exercise, added to the neuroendocrine aberrations attributable to energy-, sleep-, and nutrient deprivation, most likely accounts for this effect (i.e.,the lack of rebuilding of the leukocyte L-selectin stores).

L-selectin may be shed from the surface of leukocytes and can be recovered as a bioactive molecule in plasma (18). Taking into consideration the observed significant cellular peeling of CD62L, one could imagine that at least part of the bioactive molecules would have been recovered in soluble form in plasma. However, this was not the case. For the most part, sCD62L seemed bound to vascular receptors and was not reflected as shedded molecules in plasma. In essence, the low cellular levels of CD62L, in combination with occupancy of endothelial receptors for CD62L by sCD62L ligands, probably influence the ability of leukocytes to roll on endothelium and to eventually traverse the endothelial layer to reach inflammatory sites. In this respect, the ability to combat microbial invasions is, in all probability, temporarily insufficient (6). Thus, these results support the open-window hypothesis (22) indicating an increased susceptibility of contracting infections during the period immediately after long-term strenuous physical activity. However, no obvious signs of infections were noted in the involved cadets during or after the training course. This may have been because the ranger-training course is organized at a time of the year when the incidence of, for instance, contagious viral upper-respiratory tract infections, is at a minimum.

The influence of exercise on the cell-surface receptor CD11b/CD18 is still unclear (27). We found that the expression of CD11b on leukocytes was minimally influenced during the ranger-training course, but a slightly increased expression was observed on all leukocyte subsets on the first recovery day. We have previously shown a similar result in marathon runners (average running time 3h 40 min), whereas in a half-marathon race, a moderate increase in leukocyte (all subsets) CD11b expression was seen (18). Incremental exercise to exhaustion (e.g., test of maximal oxygen consumption (V˙O2max)) has also been shown to increase the CD11b expression of neutrophils (21). The pulmonary vascular bed is an important reservoir for the marginated pool of leukocytes, and it is believed that this pool of leukocytes can be mobilized by short-term, high-intensity exercise (e.g., a V˙O2max test). The finding of the present study that leukocyte (all subsets) CD11b was increased on the first recovery day suggests that even long-term moderate exercise, in combination with neuroendocrine disturbances (e.g., catecholamine increase (21)), can affect the mobilization of marginated leukocytes.

Long-term physical activities combined with sleep- and energy deprivation during an 8-d ranger-training course suppressed the intracellular leukocyte (monocyte and granulocyte) ROS-generating capacity and nearly extinguished the surface expression of CD62L (L-selectin) on all subsets of leukocytes. These changes support the open-window hypothesis and indicate that the military cadets may be more prone to infections in the days after such physical and psychological strain.

The authors would like to thank MLT Lisbeth Saetre for excellent technical and practical assistance.

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