Cells from virtually all organisms respond to a variety of environmental stress factors by the rapid transcription and subsequent translation of a unique, highly conserved set of polypeptides termed heat shock or stress proteins (HSP). Mammalian cells are known to synthesize HSP in vitro after brief exposures to temperatures of 3–5°C above normal (heat shock (HS)) (23). Earlier studies demonstrated that, after recovery from mild temperature increases, thermotolerance was conferred to the cells, which allowed survival during subsequent exposures to otherwise lethal temperatures and suggested a protective function for HSP (23). The mechanism of thermotolerance is still not completely understood.
HSP expression is also altered during glucose depletion and oxidative stress (27). Cells that are starved for glucose overproduce a set of proteins called glucose-regulated proteins (GRP) (23). Similarly, cells exposed to low levels of oxidative stress such as H2O2 exhibit protection against subsequent exposure to higher, normally lethal, oxidative stress levels (27). This induction of oxidative stress resistance is thought to be linked to the overexpression of genes that encode the oxidative stress proteins (OSP) (27). The functions of HSP, GRP, and OSP are incompletely understood, but evidence suggests that many stress proteins are enzymes that either provide immediate stress protection or conduct cellular repair processes (19). Furthermore, they seem to be responsible for stress tolerance after repeated stress situations (23,27).
The metabolic changes caused by exercise are similar to those known to induce stress protein synthesis (9,16). Physical exercise can elevate core temperature to 44°C and muscle temperatures up to 45°C (23). Exercise also causes oxidative stress via an increased generation of reactive oxy- gen intermediates (ROI) (5). Activation of blood neutrophils described after exercise and an increase of lipid peroxidation products suggest that oxidative stress plays a role in exercise-induced changes in the blood compartment (5,26). Furthermore, sustained physical activity results in the progressive depletion of glucose and glycogen stores, a phenomenon that is highly correlated with fatigue. Heavy exercise also induces an inflammatory reaction that includes leukocytosis and increases in host defense mediators such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNFα) (30). Given all these factors, it seems to us that exercise is an excellent tool for studying the effects of stress on gene expression and the physiological significance of stress proteins.
Recently, there has been increasing interest in a family of 70-kDa heat shock proteins (HSP70) that appear to play a role in protein translocation and assembly processes. In mammalian cells, there are three different types of HSP70-like proteins: stress-inducible HSP70, constitutively expressed cognate HSC70, and glucose regulated proteins (GRP) (19). Although structurally related, members of this gene family appear to be functionally distinct. HSC are constitutively expressed in several cell types of different organisms. The main feature of these genes is that, unlike HSP genes, their transcription is at most only moderately responsive to heat. HSC have been shown to provide essential functions needed for normal growth, and recently they have been assigned a role in translocation of proteins across membranes (13,31).
Of particular interest is that the highly stress-inducible HSP70 may be required for the transport of nuclear-encoded polypeptides that are destined for processing and assembly in mitochondria (6). In muscle, one of the major effects of regular exercise training or conditioning is an increased mitochondrial biogenesis in which the mitochondrial content can actually be doubled (4). Because precursor polypeptides from the nucleus are required for the assembly of most complete mitochondrial proteins, HSP70 could provide a vital link in the mechanism of exercise-induced mitochondrial biogenesis (23). This raises the question of whether inducible HSP70 in leukocytes is also influenced by exercise.
The regulation of expression and phosphorylation of HSP27 by the inflammatory cytokines TNFα and IL-1 (11) may be meaningful for the stress response to intensive exercise.
Mammalian HSP60 apparently functions to facilitate proper oligomeric assembly of proteins within the matrix of mitochondria (31), and HSP60 is associated with inflammation and autoimmunity (8). T cells that are reactive with HSP60, possibly triggered by high local concentration of HSP, could be involved in the initiation or perpetuation of the inflammatory reaction. An inflammatory reaction is also induced by the physiological stimulus of extensive exercise (30).
HSP90 is a very abundant protein in all cells that are grown under normal conditions, and its synthesis increases three- to fivefold after heat shock. In cells deprived of glucose or oxygen, or treated with agents that perturb calcium homeostasis, synthesis of HSP90 declines concomitantly with an increased synthesis of GRP and HSP70 (31).
Only a few studies have examined mammalian stress proteins in vivo, and the methods used primarily involved artificially raising the temperature of animal or in vitro culture models (12,16,20,21). A more natural stress that has not been adequately investigated as a possible inducer of HSP synthesis in human blood is strenuous physical exercise. Given the many similarities between the conditions observed with strenuous exercise and the factors that may cause HSP synthesis in vitro, it was of great interest to determine whether HSP synthesis is induced in immune cells of athletes undergoing strenuous endurance exercise in vivo.
The present study was designed to investigate for the first time the differential expression of HSP27, HSP60, HSP70, HSC70, and HSP90 in circulating human leukocyte subpopulations (lymphocytes, monocytes, and granulocytes) of half-marathon runners before and at different time points after the competition. Furthermore, HSP expression in the blood of trained athletes at rest compared with that of nontrained individuals was analyzed to study the influence of regular training on HSP expression in immunocompetent cells.
MATERIALS AND METHODS
Subjects
Twelve well-trained male athletes were recruited from local teams and clubs. The trained individuals were engaged in specific endurance training. The last 3 d before the race, the athletes completed only moderate endurance runs lasting up to 40 min with a running intensity below the lactate threshold. Twelve healthy and normally conditioned male adults were selected as sedentary controls. These untrained individuals did not perform any kind of sports conditioning and did not have any stress situation within 3 d before the collection of blood samples. All subjects used no drugs or mineral or vitamin supplements. Each gave written informed consent before participation in the study. The experimental protocol was approved by the Institute’s Human Ethics Committee according to the principles set forth in the Declaration of Helsinki of the World Medical Association. The anthropometric data of athletes and controls are shown in Table 1.
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Table 1: Physical characteristics and training volume of trained athletes and untrained controls (mean ± SD).
The athletes performed an official half marathon under competition conditions (21.1 km), which started at 10:00 a.m. Venous blood samples were taken in a sitting position using EDTA as an anticoagulant. The samples were collected at rest 24 h before (A, 9:00 a.m.), directly after (B, 11:30 a.m.–12:00 p.m.), 3 h (C, 2:30–3:00 p.m.), and 24 h (D, 9:00 a.m.) after competition. Additionally, blood was collected from twelve untrained controls at rest (9:00 a.m.) who did not participate in the half marathon. Sample A of the trained persons represents the corresponding resting value.
Lactate, CK, and Uric Acid
The lactate concentrations of the hemolyzed capillary blood samples were measured electrochemically using a lactate analyzer (EBIO, Eppendorf, Germany). Plasma creatine kinase activity (CK) and uric acid concentrations were determined by enzymatic analyses (Hitachi 717, Boehringer, Germany).
White Blood Cells, Hematocrit, and Hemoglobin (Hb)
Hematocrit, Hb concentration, and total and differential counts of white blood cells were determined using an automated hematology analyzer (Coulter Juniors JS, Coulter Electronics, Delkenheim, Germany). Differential analyses of lymphocytes, monocytes, and neutrophils were conducted automatically. Hematocrit and Hb were used to correct plasma concentration of uric acid, CK, myeloperoxidase (MPO), and TNFα with regard to changes in plasma volume after exercise.
Preparation of Leukocytes From Peripheral Blood
A 5-mL sample of EDTA-treated blood was carefully layered over 5 mL of Lymphoflot (Biotest, Dreieich, Germany), a solution containing Diatrizoate (9.6% w/v) and Ficoll (5.6% w/v) and allowed to settle by gravity without centrifugation for 60 min. The erythrocytes were aggregated at the interface and sedimented to the bottom of the tube. The majority of the leukocytes remained in the plasma layer and was removed. The overlay was washed two times with phosphate-buffered saline (PBS), and the cell concentration was adjusted with PBS to 1 Ă— 107 cells per mL. A sample of 100 μL of the suspension was used for analysis by flow cytometry.
Flow Cytometry
Surface phenotyping.
HSP expression on the surface of different cells was discussed by Multhoff et al. (13,17). The cells were analyzed by indirect immunofluorescence using the following HSP-specific monoclonal antibodies (StessGen, Biotechnologies Corp., Victoria, Canada): SPA-800 (HSP27; IgG1, clone G3.1), SPA-806 (HSP60; IgG1, clone LK-1), SPA-810 (specific for the inducible form of human HSP70; IgG1, clone C92F3A-5), SPA-815 (HSC70; IgG2a, clone 1B5), and SPA-840 (identifies both free and complexed human HSP90; IgG2a, clone 9D2).
A total of 1 Ă— 106 cells were resuspended in a dilution of the HSP-specific monoclonal antibody shown to give a maximum of positive cells or isotype-matched monoclonal antibodies at the same concentration (1 μg/test) and incubated for 20 min on ice in the dark. After washing the cells twice and incubating in the presence of the secondary FITC-conjugated goat antimouse/rat F (ab’)2 IgG (Dianova, Hamburg, Germany), the cells were analyzed using the flow cytometer EPICS-XL-MLC (Coulter, Krefeld, Germany). Dead cells were excluded by electronic gating, and fluorescence histograms were area-corrected to 10,000 cells. The lymphocyte, monocyte, and granulocyte populations were differentiated according to granularity and size in the forward versus side scattergram and were gated. For each of the three special gates, data were presented as percentage of positive cells (%) and mean fluorescent channel (MFC), which were corrected for background fluorescence with the negative controls.
Intracellular staining.
According to the manufacturer’s instructions (Fix & Perm kit, An der Grub, Vienna, Austria), the cells were first fixed at room temperature in a solution containing formaldehyde (reagent A) and washed twice. Then the cells were permeabilized with reagent B and at the same time incubated with the primary HSP-specific antibody. After washing twice, the labeled cells were again developed with the secondary FITC-conjugated F (ab’)2IgG, washed again, and analyzed as described above. For intracellular labeling, the same set of HSP-specific antibodies as depicted above was used.
Determination of TNFα and MPO
The testing for TNFα and MPO in the plasma probes of the athletes was done by ELISA (R&D Systems, Minneapolis, MN and Calbiochem-Novabiochem GmbH, Bad Soden, Germany; detection limits 1.0 pg·mL−1 and 1.5 pg·mL−1, respectively) according to the instructions of the manufacturer.
Statistical Methods
All statistical analyses and descriptional methods were computed by the statistical software package JMP (JMP3.1-software, SAS Institute Inc., Cary, NC) for PC. Data in Tables 1 and 2 are expressed as mean ± SD. All data concerning HSP are expressed as median and minimum/maximum values. The descriptional presentation of the data was performed using a quantile box-plot visualizing also the single values (Fig. 1). Differences between preexercise and postexercise values in the trained group were tested for significance by the paired t-test. A one-factor ANOVA was used to test for significant differences between the resting values of the trained and untrained group. Data were present in a normal distribution. A value of P < 0.01 was regarded as significant.
Table 2: Counts of white blood cells and values of CK and uric acid before (A), directly after (B), 3 h (C) and 24 h (D) after the half marathon (mean ± SD).
Figure 1: Descriptional presentation of the data as a box-and-whisker plot in which the horizontal line represents the median and the edges of the box represent the quartiles. The 10th and 90th quantiles are shown as lines above and below the box. The line across the whole graph represents the total-response sample mean. The points represent the individual data.
RESULTS
The runners completed the half-marathon race (21.1 km) in an average finishing time of 90.34 ± 12.8 min. Lactate values were 5.1 ± 2.2 mmol·L−1 directly after exercise.
Counts of White Blood Cells, CK Activity, and Uric Acid
The exercise gave rise to a marked leukocytosis that was mainly caused by a significant increase of granulocytes directly and 3 h after the half marathon (P < 0.01). The monocytes were also increased (3 h, P < 0.01), whereas the lymphocytes decreased 3 h postcompetition (P < 0.01). The leukocyte counts reached preexercise levels 24 h after exercise (Table 2).
After the race, we found an increase in plasma CK and uric acid (P < 0.01). CK values peaked 24 h after exercise. Directly and 3 h after the half marathon, uric acid showed a significant increase from baseline levels (Table 2).
Concentration of MPO and TNFα in Plasma
The content of MPO in plasma showed a significant increase immediately after the race (P < 0.01), but this decreased afterward. At a time of 24 h after the half marathon, preexercise levels were reached (Fig. 2).
Figure 2: Concentration of TNFα and myeloperoxidase (MPO) in the plasma of the athletes before (A) and immediately (B), 3 h (C), and 24 h (D) after the half marathon (N = 12). MPO is displayed as a box-and-whisker plot. For TNFα, single points are displayed and connected with lines. * Denotes significant changes compared with preexercise values (P < 0.01).
The level of TNFα in the plasma of the athletes was very low at rest and was stimulated directly after the half marathon (Fig. 2) in all athletes investigated. All values were near or below the detection limit of 1 pg·mL−1, and the changes were not statistically significant.
Flow Cytometry
Flow cytometry was used to determine two parameters: (i) the rate of positive cells (%) for a specific marker in a given population and (ii) the MFC that informed us about the fluorescence intensity of the specific marker within that population.
Intracellular HSP expression.
More monocytes and granulocytes than lymphocytes were found to be positive for HSP in their cytoplasma (Figs. 3 through 6).
Figure 3: HSP27 expression in the cytoplasma of lymphocytes, monocytes, and granulocytes of endurance athletes before (A) and immediately (B), 3 h (C), and 24 h (D) after the half marathon (N = 12) presented as percentage of positive cells and mean fluorescence intensity (MFC). Data are displayed as a box-and-whisker plot. * Denotes significant changes compared with preexercise values (P < 0.01).
Figure 4: HSP60 expression in the cytoplasma of lymphocytes, monocytes, and granulocytes of endurance athletes before (A) and immediately (B), 3 h (C), and 24 h (D) after the half marathon (N = 12) presented as percentage of positive cells and mean fluorescence intensity (MFC). Data are displayed as a box-and-whisker plot. * Denotes significant changes compared with preexercise values (P < 0.01).
Figure 5: HSP70 expression in the cytoplasma of lymphocytes, monocytes, and granulocytes of endurance athletes before (A) and immediately (B), 3 h (C), and 24 h (D) after the half marathon (N = 12) presented as percentage of positive cells and mean fluorescence intensity (MFC). * Denotes significant changes compared with preexercise values (P < 0.01).
Figure 6: Comparison of the cytoplasmic HSP27 and HSP70 expression in untrained (UT, N = 12) versus trained subjects (TR, N = 12) at rest. Presented as percentage of HSP-positive cells. Data are displayed as a box-and-whisker plot. # Denotes significant differences between TR and UT (P < 0.01).
An increase of HSP27, HSP60, and HSP70 in the cytoplasma of lymphocytes, monocytes, and granulocytes in response to the half marathon was found, although cytoplasmic HSP was nearly undetectable in the majority of lymphocytes (Figs. 3, 4, and 5). The most remarkable and statistically significant upregulation after the race was discovered in the percentage of HSP27- and HSP70-positive monocytes and granulocytes at all time points postexercise (B, C, and D, P < 0.01, Figs. 3 and 5). The fluorescence intensity for HSP70 was significantly stimulated in monocytes at all time points after the competition (B, C, and D) compared with preexercise level and 24 h postmarathon (D) in granulocytes (P < 0.01, Fig. 5). The HSP27 fluorescent signal was significantly stimulated in monocytes directly and 3 h after the run (B and C, P < 0.01, Fig. 3). Interestingly, the largest increase in percent, as well as MFC, values for HSP70 was seen in granulocytes 1 d after exercise (D, P < 0.01, Fig. 5).
Only a small number of cells expressed cytoplasmic HSP60 compared with the other HSP (note graduation of y axes). The MFC was highly variable for HSP60. Percent values were significantly stimulated directly after the run (B) in all three cell types and rapidly fell to previous levels, except in granulocytes, where it slightly rose again (P < 0.01) (Fig. 4).
HSC70 and HSP90 were highly expressed in the cytoplasma of all populations investigated (98–100%), but their expression was not influenced by the race. The untrained controls showed a correspondingly high expression (data not shown).
Surface expression of HSP.
Few cells expressed HSP on their surface compared with the intracellular staining (Fig. 7). The baseline surface expression of all HSP investigated was at nearly the same level in trained and untrained subjects and mostly below 5% (data of controls are not shown). Lymphocytes expressed no discernible HSP on their surface at all. The exercise-induced rise in monocytes and granulocytes presenting HSP on their surface was much lower than in cytoplasma and was nonsignificant (Fig. 7). Exclusively granulocytes, which were positive for HSP27, HSP60, and HSP70 on the surface, reacted 24 h after the half marathon with a significant increase in percentage (P < 0.01, Fig. 7).
Figure 7: Surface expression of HSP27, HSP60, and HSP70 on lymphocytes, monocytes, and granulocytes of endurance athletes before (A) and immediately (B), 3 h (C), and 24 h (D) after the half marathon (N = 12) presented as percentage of positive cells. Data are displayed as a box-and-whisker plot. * Denotes significant changes compared with preexercise values (P < 0.01).
HSC70 and HSP90 were not expressed on the surface of the different cells investigated (<3%, data not shown).
Comparison of Trained Versus Untrained Subjects
The percentage of HSP27- and HSP70-cytoplasma positive lymphocytes, monocytes, and granulocytes of the peripheral blood of endurance athletes at rest was significantly weaker in contrast to sedentary controls (P < 0.01, %, Fig. 6).
HSC70 and HSP90 expression in cells of trained versus untrained persons revealed no significant difference (data not shown).
DISCUSSION
The exercise-induced changes in the immune system, including leukocytosis, granulocytosis, monocytosis, and a decrease in lymphocytes, together with increases in CK, uric acid, and MPO, and the induction of TNFα corresponded with findings in other studies (2,24,30) and confirmed that the half marathon investigated here was a strenuous athletic event. In this paper, we have focused on the influence of strenuous endurance exercise on the expression of HSPs in cells of the peripheral blood. HSP may constitute a possible defense system in leukocytes against exercise-induced stress.
HSP can be induced by many methods in organisms as diverse as bacteria, animals, and humans. The effect of exhaustive exercise on HSP-expression has been studied in several animal models (9,16,23,25). Only one study to our knowledge has investigated the influence of exercise on HSP70 synthesis in human lymphocytes under conditions of external high temperature (22). The novel feature in our paper was the use of the physiological stressor of competitive endurance exercise (half marathon) to induce HSP synthesis in the human blood compartment. Leukocytes of endurance athletes before and after a half marathon were analyzed for their expression of HSP on the surface and in the cytoplasma. The HSP production of athletes at rest was compared with that of untrained subjects to further explore the phenomenon of stress tolerance and to investigate whether heat shock proteins play a role in training effects and chronic adaptation of endurance athletes.
To our knowledge, this study is the first to investigate the expression of the heat shock proteins HSP27, HSP60, inducible HSP70, constitutive HSC70, and HSP90 in the cytoplasma of lymphocytes, monocytes, and granulocytes of half-marathon runners before and after the competition. HSP27, HSP60, and HSP70 were increased, whereas HSC70 and HSP90 were not influenced by the race. These results reveal that the alterations caused by a half marathon were able to stimulate stress protein synthesis also in the blood compartment of endurance athletes. Strenuous endurance exercise such as competing in a half marathon causes metabolic reactions that include changes in intracellular pH and calcium as well as a decrease in glycogen storage. Furthermore, an activation of immunocompetent cells, which is caused in part by an increase in stress hormone levels, seems to stimulate the release of cytokines and the generation of ROI in terms of an inflammatory response. All of these factors, as well as an exercise-induced increase of body core temperature, are known to be potential stimulators of HSP expression (5,9,16,23,26,30). Although it is possible that exercise causes the synthesis of HSP through some unique pathway, it is more probable that a common mechanism is shared between exercise and several other metabolic stressors.
The most prominent and significant increase was seen in HSP70 expression in monocytes and granulocytes, which still increased somewhat on the day after the half marathon. A stimulation of the rate of HSP70-positive cells and their fluorescence intensity was detected. Because of increased core temperatures to 44°C after the marathon, we assume that there were denatured proteins inside the cell, which possibly induced HSP70 synthesis via a positive-feedback loop (13,23). Ryan et al. (22) evaluated an altered HSP70 response of human leukocytes after exercise depending on different rectal temperatures. In rat muscles it was shown that either heat exposure alone or exercise could induce HSP expression (25). Currently, it is not known whether humans synthesize HSP in response to exercise per se or to hyperthermia associated with exercise.
HSP70-positive monocytes and granulocytes responded to the half marathon with a significant rise (P < 0.01), whereas a very small number of lymphocytes expressed HSP70 in their cytoplasma. Exercise-induced stress mainly seems to affect monocytes and granulocytes. These two kinds of cells have also been activated through strenuous exercise as already described (26). To deal with phagocytic material and microbes, both use ROIs. Phagocytosis as well as physiological activators of the oxidative burst that are both associated with ROI production induce HSP synthesis in macrophages (13,31). Therefore, monocytes and granulocytes, in particular, need to protect themselves from the noxious molecules they produce. At least in part, this may be achieved by HSP synthesis. Interestingly, HSP70 increase is associated with intracellular but not extracellular ROI generation (29). This may help to explain why those cells in particular that are able to produce ROI also showed an exercise-induced HSP70 increase. It could be an effective physiological antioxidative mechanism for monocytes and granulocytes to protect themselves against intracellular oxidative stress.
HSP70 was described as interfering with the TNFα-induced signal transduction pathway. There may be a common mechanism of regulating transcription of HSP70 and TNFα because of the direct location of their genes in the neighborhood of the MHC-class II region (7). This relationship is compatible with our studies, which revealed a parallel increase of TNFα in the plasma of the athletes.
Especially concerning HSP27, it has been demonstrated that its expression and phosphorylation is regulated by the inflammatory cytokines TNFα and IL-1 (11,28). Thus, the increased expression of HSP27 after the half marathon observed in our study may be due to cytokines released by leukocytes and may mediate a protective function for the cells against the cytotoxic effects of TNFα. This hypothesis is supported by the slight increase of TNFα in the plasma postcompetition. The rise was nonsignificant, but the trend was seen in all athletes investigated.
Our experiments have shown that monocytes and granulocytes, stained positive for HSP60 in the cytoplasma, rose directly after the race. However, all cell populations investigated lacked a high constitutive expression of HSP60. In particular, very low amounts of lymphocytes, monocytes, and granulocytes expressing HSP60 were observed in athletes at rest. The significantly increased percentage of HSP60 positive cells after the competition may be associated with the activation of macrophages and granulocytes due to exercise and the need to protect themselves from the harmful molecules they produce. The great interindividual variations in HSP60 expression may be an indicator of individual inflammatory reactions of the single subjects at this special time or a lasting effect of some previous incident (14).
The other compartment of a cell where HSP expression was monitored in this study is the cell surface. HSP on the surface was nearly undetectable on the majority of cells of athletes at rest and of untrained persons. However, after the run, an increase of monocytes and granulocytes expressing HSP27, HSP60, or HSP70 on their surface was detected in some athletes. The rise of HSP-positive granulocytes was even significant 24 h after the competition.
Besides the expression of HSP in the cytoplasma, its localization on the surface of several cells in free form and/or in context of MHC class I molecules is described (13). HSPs are expressed selectively on tumor surface of virally or bacterial-infected cells or on tumor cells, but not on the surface, of vital normal cells. It is hypothesized that alterations in calcium and pH levels, hypoxia, and nutrient depletion in the cells are responsible for conformational changes of HSP that result in cell-surface localization. The HSP expression in stressed cells is stimulated in the cytoplasma and may subsequently be presented on the cell surface, thus enabling recognition by other immune competent cells that can eliminate them. The idea that T cells directed against autologous HSP can detect and subsequently eliminate host cells maximally stressed by a variety of incidents is attractive (13,17). It may be hypothesized that maximally stressed cells due to the half marathon also react with HSP expression on their surface to be recognized by other immune cells.
Another major finding of the present investigation was the significantly lower percentage of HSP27- and HSP70-expressing monocytes and granulocytes in trained half-marathon runners at rest versus untrained subjects. Regular endurance training appears to downregulate HSP-positive leukocyte counts in blood. This may be a mechanism of tolerance to exercise, which is described in this study for the first time. Marathon runners represent a selected population subset because they are able to tolerate core temperatures between 40 and 42°C without signs of heat illness (22). The relevance of the here-detected lower constitutive HSP expression in the blood of well-trained half-marathon runners at rest for the development of this heat tolerance is unclear. On the other hand, several other functions seem to be diminished due to regular training. Neutrophil-killing capacity is reduced in elite athletes compared with untrained controls (10,18). Smith at al. (26) described a depressed ability of blood neutrophils to produce ROI in trained versus untrained individuals, and a lower superoxide anion generation by trained athletes was reported (1). This led us to conclude that the training-induced lower HSP production in blood cells of trained individuals was possibly caused by their reduced RIS generation. It means that the training-induced downregulation of HSP was not directly a mechanism of chronic adaptation. The reactions that lead to chronic adaptation are likely to be regulated before the HSP level. We suggest that other systems such as superoxide dismutase, catalase, or glutathione peroxidase, which are stimulated during training (3,15), are so efficient in downregulating the oxidative potential that the leukocytes of trained athletes can afford to reduce their stress protein production. The depressed HSP expression can, therefore, in our view be interpreted as a consequence of modulations of earlier systems.
In summary, heavy endurance exercise significantly stimulated HSP expression in peripheral blood leukocytes, whereas it was downregulated by regular endurance training. The biological significance of this finding is not completely understood, but a role of HSP as defense mechanism of leukocytes against exercise-induced stress enhancing exercise tolerance must be discussed. Temperature increase, ROI-generation, and inflammatory reactions should be considered as possible causal mechanisms. The lower percentage of HSP-positive leukocytes in trained subjects at rest may reflect a reduced need of HSP-expressing immune cells in blood as a result of adaptation to regular endurance training. The HSP system could represent an important molecular response mechanism to other antistress systems. Further research is needed to gain more insight into the meaning and regulatory pathways of HSP expression during acute and chronic endurance exercise.
We wish to express our appreciation to Mrs. M. Faigle for her excellent technical assistance, especially with the MPO- and TNFα-ELISA. We would also like to thank the volunteers who participated in the study.
This investigation was supported by a grant from the Bundesinstitut fĂ¼r Sportwissenschaften (Köln, Germany, VF 0407/01/21/97).
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