Seven days’ around the clock exhaustive physical exertion combined with energy depletion and sleep deprivation primes circulating leukocytes
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- Gundersen, Y., Opstad, P.K., Reistad, T. et al. Eur J Appl Physiol (2006) 97: 151. doi:10.1007/s00421-006-0150-8
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Both exhaustive physical exertion and starvation have been reported to induce depression of immune function. The aim of the present study was to investigate the inflammatory environment and state of activation and mediator-producing potential of circulating leukocytes during prolonged physical activity with concomitant energy and sleep deprivation. Eight well-trained males were studied during 7 days of semi-continuous physical activity. Sleep was restricted to about 1 h/24 h, energy intake to 1.5– 3.0 MJ/24 h. Blood was drawn at 07.00 a.m. on days 0, 2, 4, and 7. Plasma levels of inflammation markers were measured. The response of circulating leukocytes to lipopolysaccharide (LPS; 1 μg mL−1), and the effect of added hydrocortisone (10 and 100 nmol L−1), were measured in the supernatant after 3 h of incubation in an ex vivo whole blood model. Activation of leukocytes steadily increased as measured by plasma matrix metalloproteinase-9, tumour necrosis factor-α, interleukin-1β, and interleukin-6. Inhibitors of systemic inflammation were either unaltered (tissue inhibitor of matrix metalloproteinase-1) or elevated (plasma interleukin-1 receptor antagonist). Cortisol levels increased on days 2 and 4, but thereafter reverted to baseline values. The leukocytes responded to LPS activation with increasing release of inflammatory cytokines throughout the study period. The anti-inflammatory potency of hydrocortisone decreased. Prolonged multifactorial stress thus activated circulating immune cells and primed them for an increased response to a subsequent microbial challenge.
Strenuous and prolonged physical activity is known to affect different aspects of immune function (Pedersen and Hoffman-Goetz 2000; Nieman et al. 2001; Suzuki et al. 2000). Several investigators have reported increased concentrations of circulating inflammatory cytokines, which may also be used as a general measure to assess the impact of external stimuli on the innate immune system. The cytokine pattern provoked by physical exertion is in many ways comparable with the reaction to surgical trauma or infection, and may therefore even be used as a model for clinical pathological states of systemic inflammation (Moldoveanu et al. 2001).
The cytokines are produced locally by a variety of cells, and released into the circulation in proportion to the magnitude, duration, and mode of the activating stimulus (Pasquale et al. 1996; Halson et al. 2003; Rowbottom and Green 2000; Nielsen et al. 2004). However, the clinical significance of the accompanying deviations from normal resting values has not been definitely established (Nieman 2000). Regular and moderate physical activity is usually quoted to enhance the resistance to infection, while intense and long-lasting exertion probably has the opposite effect. Other classes of physical and psychological stressors like sleep deprivation and starvation may likewise affect immune function. Although a correlation between lack of sleep and immune deficiency is usually assumed, divergent results have been reported (Dinges et al. 1994; Irwin et al. 1996; Eversen and Toth 2000). Nutritional imbalance and overall energy deficit may further compound negative influences of accompanying stressors (Gleeson and Bishop 2000). For instance, depletion of carbohydrates may lead to increased values of circulating stress hormones, or to a competition between muscles and immune cells for key amino acids (Shephard and Shek 1995). The different classes of stressors normally act in concert to produce a response from the organism. Prolonged military field operations represent typical multifactorial and arduous strain that could conceivably jeopardize the resistance to infections.
The aim of the present study was to explore the influence of a combination of different stress factors on facets of primarily innate immune function. The measurements were undertaken during a 7-day ranger-training course with periodic round the clock physical exertion, sleep deprivation, and energy deficit.
Materials and methods
Subjects and experimental design
Physical characteristics of the study group (mean ± SEM)
25.8 ± 0.9
Body mass baseline (kg)
80.0 ± 3.7
Body mass end (kg)
72.6 ± 3.6
Loss of body mass (%)
9.3 ± 0.7
181 ± 2.5
Baseline body mass index
24.4 ± 1.0
Blood sampling and assays
With the subjects in seated position peripheral blood was collected by antecubital venipuncture on days 0, 2, 4, and at the conclusion of the course. To exclude circadian variations, sampling was started at 0700 a.m. on each day. EDTA or heparin anticoagulated vacuum tubes (Vacuette, Greiner) were employed for the collection. All specimens were put on ice immediately and processed within 1 h.
The whole-blood model
The ability of circulating inflammatory cells to react to a lipopolysaccharide (LPS) challenge was studied in a whole-blood model. Two millilitres of heparinised blood was used in each sample, which was either not further stimulated, or stimulated with 1 μg mL−1 LPS (Escherichia coli, serotype 0111:B4) alone, or stimulated with LPS 1 μg mL−1 together with hydrocortisone 10 nmol L−1 or 100 nmol L−1. The tubes were incubated for 3 h at 37°C and gently rotated x6 per min during the incubation period. After centrifugation the supernatant was removed and immediately frozen at –20°C. The concentration of tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and soluble interleukin-1 receptor antagonist (sIL-1 RA) in the supernatant was determined with standard kits (R&D Systems, Minneapolis, MN, USA), that is, TNF-α with Quantikine human TNF-α, catalogue number DTA00C, detection limit 1.6 pg mL−1; IL-1β with Quantikine human IL-1β, catalogue number DLB 50, detection limit 1 pg mL−1; IL-6 with Quantikine human IL-6, catalogue number D6050, detection limit 0.7 pg mL−1; IL-10 with Quantikine human IL-10, catalogue number D1000, detection limit 3.9 pg mL−1; and sIL-1 RA with Quantikine human sIL-1 RA/IL-1F3, catalogue number DRA00, detection limit 22 pg mL−1. The sample assays were performed as single measurements; in duplicate only to assess variance.
Haematological and biochemical measurements
Haemoglobin, haematocrit, and total and differential leukocyte counts were determined in EDTA blood on an automatic blood cell counter (Advia 60, Bayer HealthCare, Tarrytown, NY, USA) within 1 h from sampling. Estimation of cortisol was performed with standard kits from R&D Systems, C-reactive protein (CRP) on Tina-quant Modular (Roche Diagnostics GmbH, Mannheim, Germany).
Cytokine and protease assays
Plasma derived from EDTA blood was used for cytokine determination with commercially available kits from R&D Systems. The same was the case for Leptin (Quantikine human, catalogue number DLP00, detection limit 7.8 pg mL−1). For MMP-9 and TIMP-1 we used Quantikine human kits with catalogue numbers DMP 900 (detection limit 0.156 ng mL−1), and DTM 100 (detection limit 0.08 ng mL−1), respectively.
Data are presented as mean ± SEM. The data were tested for normal distribution. Thereafter differences between values were estimated with one-way repeated measurements ANOVA, or one-way repeated measurements ANOVA on ranks as appropriate, followed by Student-Newman-Keul´s post hoc test. P-values less than 0.05 were considered statistically significant.
Body mass changes
The cadets lost 7.4 ± 0.5 kg in weight during the study, corresponding to 9.3 ± 0.7 per cent of baseline values (Table 1).
Selected variables (mean ± SEM)
Haemoglobin (g dL−1)
15.31 ± 0.27
14.36 ± 0.2a, b
14.21 ± 0.23a
13.48 ± 0.26a, b
44.0 ± 0.8
40.8 ± 0.7a, b
39.9 ± 0.9a
37.8 ± 0.9a, b
C-reactive protein (mg L−1)
1.38 ± 0.89
11.38 ± 3.03a
Leptin (pg mL−1)
4581 ± 886
317 ± 99a, b
245 ± 83a, b
198 ± 67a
Cortisol (nmol L−1)
599 ± 34
1072 ± 82a, b
1045 ± 78a
662 ± 40b
Plasma volume changes
Relative plasma volume (PV) changes were calculated from haemoglobin and haematocrit according to the formula: % PV change = [(Hbcontrol/Hbtest) x (100 – Hcttest)/(100 – Hctcontrol) – 1] x 100; (see Dill and Costill 1974; Lundvall and Bjerkhoel 1995). After 2 days the PV had increased to 115 ± 4% (P < 0.05 vs preceding value), after 4 days to 118 ± 4%, and after 7 days the values were 129 ± 4% (P < 0.05 vs preceding value).
Inflammatory and anti-inflammatory markers in plasma
Plasma concentrations of the pro-inflammatory cytokines TNF-α and IL-1β were below detection at all measure points, as was the anti-inflammatory cytokine IL-10. IL-1 RA increased significantly throughout the study (from 207 ± 15 to 841 ± 437 pg mL−1). IL-6 concentrations increased significantly from below detection on day 0, to 10.6 ± 1.3 and 6.8 ± 1.6 pg mL−1 on days 2 and 4, and then below detection again on day 7. The protease matrix metalloproteinase-9 (MMP-9) steadily increased from 840 ± 167 on day 0, via 1178 ± 219 on day 4 (NS vs baseline), and ultimately 1240 ± 162 ng mL−1 on day 7 (P < 0.05 vs baseline). The anti-protease tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), on the other hand, remained unaltered. The hepatocyte-derived acute-phase protein CRP increased from 1.38 ± 0.89 to 11.38 ± 3.03 mg L−1 at the end of the study (P < 0.05). As expected, leptin values decreased rapidly from 4,581 ± 886 pg mL−1 at baseline to 245 ± 83 pg mL−1 on day 4 (P < 0.05 vs baseline) and 198 ± 67 pg mL−1 on day 7 (P < 0.05 vs baseline). Cortisol values increased from baseline levels of 599 ± 34 nmol L−1 to 1,072 ± 82 nmol L−1 on day 2 (P < 0.05). The values remained high on day 4, but almost reverted to normal at the end of the study (662 ± 40 nmol L−1; NS vs baseline) (Table 2).
Markers of leukocyte activation in the whole-blood model
The ranger-training course represents a combination of different stressors frequently found in real life, for example, during military manoeuvres, extreme sport expeditions, and as accidental isolated incidents. One week of semi-continuous physical activity, lack of sleep, and inadequate food rations, provoked a mild acute phase response. Several aspects of the innate immune system were activated significantly, but the alterations of biochemical variables were not associated with clinical signs of infections or other diseases in any of the participants.
Consistent with other studies, the total leukocyte count rapidly increased, mainly because of an enhanced number of circulating granulocytes (Bøyum et al. 1996; Pedersen and Nieman 1998). The lymphocytes, on the other hand, were reduced, and remained low, while the granulocytes reverted to normal levels on day 7. Demanding physical activity, starvation (Wing and Young 1980), and sleep deprivation (Dinges et al. 1994) are all known to be able to induce leukocytosis. Several mechanisms have been put forward, including demargination of cells that are normally adhered to the endothelium. Furthermore, enhanced concentrations of circulating catecholamines and cortisol may induce a washout of cells from different organs. The variations in total leukocytes thus closely paralleled cortisol levels. Other investigators have observed an increased number of band forms, indicating increased release from the bone marrow (Bøyum et al. 1996). The observed lymphopaenia partly may have been due to the elevated cortisol values, but persisted despite normalisation of cortisol towards the end of the study (Pedersen et al. 1997). However, the simultaneously strongly reduced leptin levels provoked by the lack of food, probably also negatively influenced lymphocytic proliferation and lymphocyte count (Goldberg et al. 2005).
Haemoglobin values fell gradually throughout the study. The average decrease of 12% within a time span of 7 days cannot be explained by the minor effect of blood sampling. Mechanical damage and oxidative injury of red cells, combined with reduced synthesis due to induction of an inflammatory response, conceivably also play only modest roles. Most important is probably the considerable plasma dilution reaching eventually 129 ± 4% of the baseline values (Fellmann 1992). This further emphasises the leukocytosis.
Our data reveal a slightly increasing upregulation of inflammation. In the circulation, this was reflected by the release of acute-phase reactants like CRP and major mediators like MMP-9 and IL-6. On the other hand, key proinflammatory mediators like IL-1β and TNF-α were below detection, which is in line with results from other studies of physical activity with submaximal work rates (Suzuki et al. 2000; Bøyum et al. 1996). When humans are exposed to higher work intensities, the literature typically reports both IL-1β and TNF-α levels to be moderately enhanced, while IL-6 may increase up to 100-fold (Pedersen 2000). As opposed to IL-6, which is produced and released also from contracting muscles during exercise (Steensberg et al. 2000), the plasma levels of basically local mediators like IL-1β and TNF-α mainly reflect overflow into the circulation. Together with the measured plasma dilution, this may explain the inability to trace these substances in the blood samples. Moreover, the cells may merely have been primed to produce increasing amounts of mediators only upon a second, and perhaps innocuous, stimulus. The very gentle handling of the samples in the whole-blood model, without additional stimulation, thus sufficed to unmask a boosted responsiveness, resulting in increasing production of IL-1β and TNF-α as the course proceeded (see Fig. 2). Although the increments on days 2 and 4 partly can be explained by an elevated number of leukocytes in the sample, the highest values were only reached on day 7 when leukocyte count had reverted to baseline levels. The response to the challenge with LPS confirmed a heightened state of responsiveness, as shown in Fig. 3.
Cellular priming is usually defined as an enhanced response to an agonist, induced by an antecedent stimulus so that subsequent stimulation results in an exaggerated cell-by-cell response (Friese et al. 1994). The nature of the secondary response is heavily dependent on magnitude, character, and temporal relationship of the priming event or agent, and in the case of LPS challenge may be turned into a state of “endotoxin tolerance”. It is worth noting that while heavy trauma and resuscitation result in an almost immediate and profound downregulation of responsiveness to LPS (Gundersen et al. 2005), the present study shows that exposure to semi-continuous and enduring multifactorial stress induces quite the opposite reaction. Reduced post-traumatic sensitivity to endotoxin has been related to poorer clinical outcome in post-traumatic ICU patients, presumably due to impaired ability to respond to bacterial invasion (Heagy et al. 2003). Our results imply that cellular priming may serve to counteract a supposedly increased disease susceptibility after severe strain.
The ranger-training course represents a model of moderate systemic inflammatory response syndrome (SIRS). The results present a putative normal response to powerful and long-lasting external immune-modulating stimuli. Obviously, a healthy individual rapidly reacts to changes in the surroundings, for example, as reflected in the initial leukocytosis. Despite enduring exposition to more or less the same level of strain, the nearly normal values at the conclusion of the course may be interpreted as an adaptation to the novel and more demanding conditions. However, one cannot exclude that reduced ability to maintain the planned activity throughout may have played a role. Initially the inflammatory response was opposed by a simultaneous release of cytokine inhibitors, anti-inflammatory cytokines like sIL-1 RA, and cortisol. While the selective sIL-1 RA steadily increased during the study, cortisol values reverted to baseline after 7 days of exercise. The resistance of circulating leukocytes to hydrocortisone was also enhanced. This is in line with previous studies, and may be important to avoid increased susceptibility to infections in circumstances of extreme multifactorial stress (Smits et al. 1998).
While it is unclear whether lack of sleep has any negative impact on immune function, these adaptations are probably more endangered when starvation is added to demanding and enduring physical exertion. The food allowed during the study represents a drastic nutritional insult, and it is well established that nutritional deficiency often is associated with impaired immune responses, particularly cell-mediated immunity. But also aspects of innate immunity are commonly attenuated, including phagocyte function and cytokine production. Although the changes are heavily dependent on magnitude and length of starvation, studies in rodents have shown that even short periods of low-protein diets can compromise several aspects of cellular immune function (Chan et al. 1996). When appearing together with energy and carbohydrate depletion, low protein ingestion is further aggravated by increased protein losses (Marable et al. 1979). The enduring food deficit manifested itself in a conspicuous reduction of leptin levels, which eventually reached a mere 4.3% of baseline values. In our case, the consequences of inadequate food rations completely overshadowed the anticipated stimulus from inflammation (Sarraf et al. 1997). In inflammatory states the synthesis of leptin seems to be modulated in a manner similar to the cytokine response to infection and injury, but its exact role in the regulation of cytokine production has not yet been defined. Although not revealed in this study, it may be surmised that starvation can inhibit aspects of the exertion-induced inflammatory response via the downregulating effect on leptin synthesis, especially cell-mediated immunity. In line with this, exogenous leptin has been shown to reverse starvation-induced immunosuppression (Lord et al. 1998).
So far the clinical significance of the immune alterations of exhaustive physical stress has not been firmly established, partly because different parts of the immune system are variably influenced. If solely based on the results obtained in the present study, exhaustive and long-lasting exposure to stressors should be supposed to raise the resistance to infection, as suggested by the heightened state of alert of the innate immune system as the course proceeded. However, the response is compound, and a coincident inhibition of the adaptive immune system may also take place (Nielsen and Pedersen 1997). The net influence is a mixed-up picture of effects on the different subpopulations of cells and their mediators (Nielsen et al. 2004; Bøyum et al. 1996; Smith et al. 1996). Infection rates after prolonged physical stress are thus often reported to be increased (Heath et al. 1992), while others find them to be unaffected or even reduced (Pedersen and Hoffman-Goetz 2000). The mixed results may be attributed to a host of factors, for example, the intensity and duration of strain, whether the stressors are imposed before infection or during the incubation period, or nature of the offending pathogen.
In conclusion, we have shown that 7 days with round the clock physical activity, severe energy deficit, and lack of sleep induced a moderate SIRS. Within the limited span of time covered by this study, circulating leukocytes were primed to release increasing amounts of acute phase reactants and mediators of inflammation. In isolation, this may be expected to heighten the resistance to invading micro-organisms. Clinical signs of infection were not found.
We thank the cadets at the Norwegian Military Academy who volunteered to take part in this study and thus were exposed to additional strain.