Sleep disruption and its effect on lymphocyte redeployment following an acute bout of exercise.

Sleep disruption and deprivation are common in contemporary society and have been linked with poor health, decreased job performance and increased life-stress. The rapid redeployment of lymphocytes between the blood and tissues is an archetypal feature of the acute stress response, but it is not known if short-term perturbations in sleep architecture affect lymphocyte redeployment. We examined the effects of a disrupted night sleep on the exercise-induced redeployment of lymphocytes and their subtypes. 10 healthy male cyclists performed 1h of cycling at a fixed power output on an indoor cycle ergometer, following a night of undisrupted sleep (US) or a night of disrupted sleep (DS). Blood was collected before, immediately after and 1h after exercise completion. Lymphocytes and their subtypes were enumerated using direct immunofluorescence assays and 4-colour flow cytometry. DS was associated with elevated concentrations of total lymphocytes and CD3(-)/CD56(+) NK-cells. Although not affecting baseline levels, DS augmented the exercise-induced redeployment of CD8(+) T-cells, with the naïve/early differentiated subtypes (KLRG1(-)/CD45RA(+)) being affected most. While the mobilisation of cytotoxic lymphocyte subsets (NK cells, CD8(+) T-cells γδ T-cells), tended to be larger in response to exercise following DS, their enhanced egress at 1h post-exercise was more marked. This occurred despite similar serum cortisol and catecholamine levels between the US and DS trials. NK-cells redeployed with exercise after DS retained their expression of perforin and Granzyme-B indicating that DS did not affect NK-cell 'arming'. Our findings indicate that short-term changes in sleep architecture may 'prime' the immune system and cause minor enhancements in lymphocyte trafficking in response to acute dynamic exercise.

The impact of acute strenuous exercise on TLR2, TLR4 and HLA.DR expression on human blood monocytes induced by autologous serum.

Acute exercise alters the surface expression of toll-like receptors (TLRs) and HLA.DR on blood monocytes, which could transiently compromise immunity. As serum factors might be responsible, we examined the effects of autologous post-exercise serum exposure on TLR2, TLR4 and HLA.DR expression on resting blood monocytes and their subtypes. Eight trained cyclists completed an ergometer 60 km time trial. PBMCs and serum were obtained before, immediately after and 1 h after exercise. TLR2, TLR4 or HLA.DR expression (gMFI) was determined on blood monocyte subtypes expressing combinations of CD14 and CD16 by flow cytometry, and on resting monocytes exposed to 50% autologous serum (pre, immediately after or 1 h after exercise) for 18 h in culture. Immediately after exercise, total monocyte expression of TLR2 and TLR4 increased by 41 and 27%, respectively, while HLA.DR expression was 39% lower than baseline. TLR2 and TLR4 was 53 and 84% greater 1 h after exercise, respectively, while HLA.DR was 48% lower. Changes in TLR2 and TLR4 expression occurred on the CD14(++bright)/CD16(+dim) monocyte subtype only, while HLA.DR expression changed on the CD14(+dim)/CD16(++bright) subtype. Serum did not affect monocyte TLR2 or TLR4 expression but 1 h post serum increased expression of HLA.DR on total monocytes and the CD14(+dim)/CD16(++bright) subtype, which was in contrast to the change observed at this time after exercise. We conclude that a bout of strenuous aerobic exercise alters the surface expression of TLR2, TLR4 and HLA.DR on blood monocytes and some of their subtypes, but these changes appear to be unrelated to blood serum factors.

Total lymphocyte CD8 expression is not a reliable marker of cytotoxic T-cell populations in human peripheral blood following an acute bout of high-intensity exercise.

Cytotoxic T-lymphocytes co-express the T-cell receptor, CD3 and the MHC I restricted antigen CD8. Although total CD8 expression is often used to identify CD8(+) T-cells in blood, errors are associated with this method as some CD3 negative natural killer (NK)-cells are known to express CD8. As greater relative proportions of NK-cells are found in the blood compartment after exercise, these errors are likely to be amplified in post exercise blood samples. To test this, isolated blood lymphocytes obtained from aerobically trained male subjects before, immediately after and 1h after an exhaustive treadmill-running protocol were surface stained for CD3, CD4, CD8, CD16, and CD56 and analysed by multi-colour flow cytometry. It was found that 25.4+/-16.9% of all CD8(+) cells at rest were CD3 negative, CD8(dim+) and expressed the NK-cell markers CD16 and CD56. The magnitude of this error increased to 40.8+/-20.7% immediately after exercise due to an influx of CD8(dim+) NK-cells. Although all CD8(bright+) cells expressed CD3, gating around the CD8(bright+) cells only identified 79.2+/-8.7% of the total CD3(+)/CD8(+) T-cell population; however, the magnitude of this error did not change after exercise despite the altered proportions of CD8(bright+) and CD8(dim+) cells. In conclusion, total lymphocyte expression of CD8 should not be used as a single antigenic marker to identify CD8(+) T-cells after an acute bout of exercise. Although there are errors associated with using CD8(bright+) as a single antigenic marker to identify CD3(+) T-cells, these are not amplified in response to exercise.

Senescent T-lymphocytes are mobilised into the peripheral blood compartment in young and older humans after exhaustive exercise.

Senescent T-lymphocytes are antigen-experienced cells that express the killer-cell lectin-like receptor G1 (KLRG1) and/or CD57; fail to clonally expand following further antigenic stimulation and prevail in the resting blood of older adults compared to the young. Physical exercise mobilises T-lymphocytes into the bloodstream and is therefore a model with which to compare age-related phenotypes of blood-resident T-cells with those T-cells entering the blood from peripheral lymphoid compartments. Eight young (Y; Age: 21+/-3 years) and 8 older (O; Age: 56+/-3 years) healthy males completed a maximal treadmill exercise protocol. Blood lymphocytes isolated before, immediately after and 1h after exercise were assessed for cell surface expression of KLRG1, CD57, CD28, CD45RA, CD45RO, CD62L and lymphocyte subset markers using three-colour flow cytometry. Lymphocyte subset numbers (CD3+, CD3+/CD4+, CD3+/CD8 and CD3-/CD56+) increased with exercise (p<0.05) but were not different between Y and O. At rest and immediately after exercise, the percentage of CD3+/CD8+ T-lymphocytes expressing KLRG1 and CD45RO was greater in O than Y, whereas Y had a greater expression of CD45RA and CD62L than O. The percentage of all CD3+/CD8+ and CD3+/CD4+ T-lymphocytes expressing KLRG1 and CD57 increased after exercise, but the magnitude of change was not age-dependent. In conclusion, there is a greater proportion of senescent CD3+/CD8+ T-lymphocytes in the blood of older adults compared to young at rest and immediately after exhaustive exercise, indicating that the greater frequency of KLRG1+/CD8+ T-lymphocytes in older humans is ubiquitous and not localised to the peripheral blood.

High-intensity exercise elicits the mobilization of senescent T lymphocytes into the peripheral blood compartment in human subjects.

Clonal expansion of T lymphocytes in response to antigenic stimulation is a fundamental process of adaptive immunity. As a consequence of clonal expansion, some T lymphocytes acquire a senescent phenotype, fail to replicate in response to further antigenic stimulation, and express the killer cell lectin-like receptor G1 (KLRG1) and/or CD57. Physical exercise elicits a mobilization of large numbers of T lymphocytes into the bloodstream from peripheral lymphoid compartments, but the frequency of senescent cells in the mobilized population is not known. Eight male runners (age: 29 +/- 9 yr; maximal O2 uptake 62 +/- 6 ml x kg(-1) x min(-1)) performed an intensive treadmill-running protocol at 80% maximal O2 uptake to volitional exhaustion. Blood lymphocytes isolated before, immediately after, and 1 h after exercise were assessed for cell surface expression of KLRG1, CD57, CD28, CD45RA, CD45RO, CD62L, and lymphocyte subset markers (CD3, CD4, CD8, CD56) by flow cytometry. The percentage of all CD3+ T lymphocytes expressing KLRG1 and CD57 increased with exercise (P < 0.01). The change in T-lymphocyte KLRG1 expression was attributed to both CD4+ and CD8 bright T cells, with the relative change being greater for the CD8 bright population (P < 0.01). Mobilized T-lymphocyte populations expressing KLRG1 and CD57 appeared to extravasate the peripheral blood compartment after 1 h of recovery. In conclusion, T lymphocytes with a senescent phenotype are mobilized and subsequently removed from the bloodstream in response to acute high-intensity exercise. This suggests that T lymphocytes contained within the peripheral lymphoid compartments that are mobilized by exercise are likely to be at a more advanced stage of biological aging and have a reduced capacity for clonal expansion than blood-resident T cells.

Blood lactate thresholds and walking/running economy are determinants of backpack-running performance in trained soldiers.

We developed a standardized laboratory treadmill protocol for assessing physiological responses to a simulated backpack load-carriage task in trained soldiers, and assessed the efficacy of blood lactate thresholds (LTs) and economy in predicting future backpack running success over an 8-mile course in field conditions. LTs and corresponding physiological responses were determined in 17 elite British soldiers who completed an incremental treadmill walk/run protocol to exhaustion carrying 20 kg backpack load. Treadmill velocity at the breakpoint (r = -0.85) and Δ 1 mmol l(-1) (r = -0.80) LTs, and relative V˙O2 at 4 mmol l(-1) (r = 0.76) and treadmill walk/run velocities of 6.4 (r = 0.76), 7.4 (r = 0.80), 11.4 (r = 0.66) and 12.4 (r = 0.65) km h(-1) were significantly associated with field test completion time. We report for the first time that LTs and backpack walk/run economy are major determinants of backpack load-carriage performance in trained soldiers.

Perceived exertion and heart rate models for estimating metabolic workload in elite British soldiers performing a backpack load-carriage task.

Identifying field measures to estimate backpack load-carriage work intensity in elite soldiers is of interest to the military. This study developed rating of perceived exertion (RPE) and heart rate models to define metabolic workload for a backpack load-carriage task valid for a population of elite soldiers using serial data. Male soldiers (n = 18) from the British Parachute or Special Air Service Regiment completed an incremental treadmill walking and (or) running protocol while carrying a 20-kg backpack. Heart rate, RPE, and oxygen uptake were recorded at each incremental stage of the protocol. Linear mixed models were used to model the RPE and heart rate data in the metric of measured peak oxygen uptake. Workload was accurately estimated using RPE alone (SE = 6.03), percentage of estimated maximum heart rate (%E-MHR) (SE = 6.9), and percentage of measured maximum heart rate (%M-MHR) (SE = 4.9). Combining RPE and %E-MHR resulted in a field measure with an accuracy (SE = 4.9) equivalent to the %M-MHR model. We conclude that RPE, %E-MHR, and %M-MHR provide accurate field-based proxy measures of metabolic workload in elite British soldiers performing a backpack load-carriage task. The model is accurate for the metabolic range measured by these serial data for the backpack load-carriage task.

Apoptosis does not contribute to the blood lymphocytopenia observed after intensive and downhill treadmill running in humans.

The lymphocytopenia that occurs during the recovery stage of exercise may be a result of apoptosis through an increased expression of CD95, a loss of the complement regulatory proteins CD55 and CD59, or both. Trained subjects completed intensive, moderate, and downhill treadmill-running protocols. Blood lymphocytes isolated before, immediately after, 1h after, and 24h after each exercise test were assessed for markers of apoptosis (Annexin-V(+), HSP60(+)), and CD55, CD59, and CD95 expression by flow cytometry. Lymphocytopenia occurred 1h after intensive and downhill running exercise, but no changes in the percentage of Annexin-V + or HSP60 + lymphocytes were found. Numbers of CD95(+), CD55(dim), and CD59(dim) lymphocytes increased immediately after intensive and downhill exercise, which were attributed to the selective mobilization and subsequent efflux of CD8(+) and CD56(+) lymphocyte subsets. No differences were found between the intensive and downhill protocols. In conclusion, apoptosis of circulating lymphocytes does not appear to contribute to exercise-induced lymphocytopenia.

The effects of marathon running on expression of the complement regulatory proteins CD55 (DAF) and CD59 (MACIF) on red blood cells.

Exercise is known to result in the haemolysis of red blood cells (RBCs). Although mechanical stressors such as footstrike and an increased velocity of blood flow may be involved, the biological mechanisms that underpin RBC haemolysis remain elusive. RBCs are potentially susceptible to lysis by autologous complement activation. RBCs are protected from the lytic effects of complement by regulatory proteins (CRPs) bound to the cell membrane via glycosylphosphatidylinositol (GPI) anchors. This study aimed to determine if marathon running would result in RBC haemolysis through a loss of membrane expression of the CRPs CD55 (decay accelerating factor) and CD59 (membrane attack complex inhibitory factor). Blood samples were obtained from 14 male runners before, within 30 min after, and 24 h after completion of the 2004 London Marathon. RBCs were assessed for cell surface CD55 and CD59 expression using indirect immunofluorescence assays and flow cytometry. No significant changes in the total RBC count, haematocrit or haemoglobin concentrations were found in response to running the marathon (P > 0.05). Blood bilirubin concentrations after the marathon were significantly greater than the pre-race values (P < 0.01). The relative fluorescent intensity (arbitrary units) of CD55 and CD59 expression on RBC membranes did not change in response to the marathon race (P > 0.05). In conclusion, marathon running did not alter the expression of CD55 or CD59 on RBCs, despite concomitant elevations in blood bilirubin concentrations. Consequently, any haemolysis of RBCs that occurred in response to the marathon was not likely due to a loss of membrane bound CRPs and subsequent cell lysis by autologous complement.

Physiological variables and performance markers of serving soldiers from two "elite" units of the British Army.

The aim of this study was to compare selected physiological variables and performance markers of soldiers from two "elite" units of the British Army. Ten soldiers from each of the two units were recruited for this study (n = 20). All participants completed three tests while carrying a 20 kg backpack load: (1) a maximal treadmill test using the Bruce protocol; (2) a 2 mile backpack run test specific to Unit A on a consistently flat tarmac road; and (3) a 29 km time-trial over hilly terrain typical of a mountainous area used by Unit B for performance assessment. Heart rate, maximal blood lactate concentration and performance (run time) were assessed during all three tests, with peak oxygen uptake also being measured during the maximal treadmill test. Measurements of anthropometry, isokinetic strength and mental toughness (MT48) were also recorded. There were no significant differences in terms of performance markers between the units (P > 0.05). Performance on the maximal treadmill test correlated with performance on the 2 mile backpack run test (r = -0.57) and 29 km time-trial (r = -0.66). Performance on the 2 mile backpack run test in turn correlated with 29 km time-trial performance (r = -0.77), accounting for 59% of the variance. In conclusion, the maximal treadmill test and the 2 mile backpack run test are useful indicators of performance on the arduous hill march and could be employed in the screening and selection of potential recruits.

The effects of intensive, moderate and downhill treadmill running on human blood lymphocytes expressing the adhesion/activation molecules CD54 (ICAM-1), CD18 (beta2 integrin) and CD53.

This study examined the effects of intensive, moderate and downhill treadmill running on blood lymphocyte expression of adhesion/activation (AA) molecules. Trained subjects completed three treadmill-running protocols of identical duration: (1) an intensive protocol at 80% VO2max to volitional exhaustion, (2) a moderate protocol at 60% VO2max and (3) a -10% downhill (eccentric) protocol at 80% VO2max. Blood samples were taken before, immediately after, 1 and 24 h after exercise. Isolated lymphocytes were assessed for expression of the AA molecules CD54, CD18 and CD53 by flow cytometry. Lymphocyte counts increased immediately after all running protocols. Lymphocytopenia was observed 1 h after the intensive and eccentric protocols only. Plasma creatine kinase increased 24 h after the downhill protocol only. Increases in the number and percentage of CD54+, CD18bright and CD53bright lymphocytes were observed immediately after the intensive and eccentric protocols, with the numbers falling below pre-exercise values at 1 h post-exercise for all protocols. No differences were found between the intensive protocol and the eccentric protocol at the same relative intensity. Analysis of lymphocyte subsets showed that the total number of CD3+, CD4+, CD8+ and CD56+ lymphocytes increased after the intensive protocol before falling below pre-exercise values at 1 h post-exercise. A relatively greater mobilisation of CD56+ and CD8+ cells accounts for the changes in CD54+, CD18bright and CD53bright cell populations. Lymphocytes that enter and exit the circulation following exercise express high levels of AA molecules, which may mediate extravasation and post-exercise lymphocytopenia. This effect appears to be influenced by exercise intensity and not muscle damage.

Immune alterations, lipid peroxidation, and muscle damage following a hill race.

Hill races usually include large downhill running sections, which can induce significant degrees of muscle damage in a field setting. This study examined the link between muscle damage, oxidative stress, and immune perturbations following a 7-km mountainous hill race with 457 m of ascent and 457 m of descent. Venous blood samples were taken from 7 club level runners before, immediately after, and 48 hrs postrace. Samples were analysed for total and differential leukocyte counts, markers of muscle damage (CK), lipid peroxidation (MDA), and acute phase proteins (CRP; fibrinogen; alpha-1-ACT). The total antioxidant status (TEAC) and plasma levels of the proinflammatory cytokines IL-6, IL-8, and TNF-alpha were also determined. Subjective pain reports, and plasma activities of CK, MDA, and circulatory monocytes reached peak values at 48 hrs postrace (p < 0.05). TEAC and the cytokine IL-8 increased immediately after the race (p < 0.05). Plasma TNF-alpha remained unchanged (p > 0.05). Despite the reports of muscle damage and soreness, no evidence of an acute phase response was observed (p > 0.05), which may be explained by the failure of the race to induce a plasma TNF-alpha response. Future studies should examine the link between muscle damage, oxidative stress, and the acute phase response following hill races of longer duration with larger eccentric components.