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(Journal of Leukocyte Biology. 2001;69:747-754.)
© 2001 by Society for Leukocyte Biology

Changes of HSP72-expression in leukocytes are associated with adaptation to exercise under conditions of high environmental temperature

Elvira Fehrenbach*, Andreas Michael Niess{dagger}, Roman Veith*, Hans-Herrmann Dickhuth{dagger} and Hinnak Northoff*

* Department of Transfusion Medicine, and
{dagger} Medical Clinic and Polyclinic, Department of Sports Medicine, University of Tuebingen, Tuebingen, Germany

Correspondence: Elvira Fehrenbach, Ph.D., Abteilung Transfusionsmedizin, Eberhard-Karls-Universitaet Tuebingen, Otfried-Mueller-Str. 4/1, D-72076 Tuebingen, Germany. E-mail: elvira.fehrenbach{at}med.uni-tuebingen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Overexpression of the heat shock protein HSP72 provides thermotolerance. We asked if two consecutive endurance runs 1 week apart (CR1, CR2) and additional environmental heat stress affect HSP72-expression in leukocytes of nonheat-acclimated endurance athletes. Twelve subjects were allocated randomly into two groups. Group HH completed both runs at 28°C ambient temperature, and group NH performed CR1 at 18°C and CR2 at 28°C. HSP72-expression was determined by flow cytometry and RT-PCR before and 0, 24, and 48 h after exercise. Additionally, post-exercise cells were exposed to in vitro heat shock (HS; 2 h, 42°C). The prolonged, high HSP72 protein level after CR1 in HH compared with NH may reflect thermotolerance induced by endurance exercise at high ambient temperature. Adaptation of cardiocirculatory/thermoregulatory capacity after CR2 in HH went along with a more rapid down-regulation of HSP72 compared with CR1. HSP72 mRNA demonstrated temperature-related changes after exercise. The reduced HS response in vitro after CR2 may represent exercise-related adaptation mechanisms. HSP72 concentrations in leukocytes may indicate previous exercise- and temperature-related stress conditions and adaptation in immunocompetent cells.

Key Words: heat shock protein • hyperthermia • acclimatization • thermotolerance • in vitro


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The heat-shock response represents a general homeostatic mechanism that protects cells, individual tissues, and the entire organism from the harmful effects of environmental stress factors [1 ]. Also, intensive endurance exercise was able to stimulate heat-shock proteins (HSP), especially HSP72, the inducible form of the HSP70-group, in leukocytes. Possible inducers of the exercise-related HSP72 response are oxidative, inflammatory, muscular, and, most likely, exercise-induced hyperthermia [2 3 4 5 6 7 ].

Elevated, external temperature exerts a synergistic impact on the stress response during intensive exercise. Furthermore, external heat results in detrimental effects on performance during endurance exercise [8 ]. One major factor limiting performance seems to be hyperthermia, which develops during exercise under conditions of increased external temperature and may exceed 40–41°C following strenuous exercise [9 ].

Conversely, repeated exercise bouts at high ambient temperature improve tolerance and performance, referred to as acclimatization. Maintenance of a high level of sweating, lowered heart rate, and internal body temperature constitutes the classic description of the heat-acclimated individual during exercise at high ambient temperature [10 ]. Exercising under conditions of elevated, external temperature appears to be the most effective method for developing heat acclimatization. Nevertheless, it is not clear which exact cellular and molecular mechanisms mediate adaptation to exercise in the heat.

The induction of HSP72 may be a protective mechanism of leukocytes against heat and exercise stress. Protective functions of HSP72 include chaperoning protein assembly and degradation, translocation, and stabilization to protect cellular homeostasis [1 , 6 , 11 , 12 ]. Furthermore, the HSP72-expression level may have important implications in adaptation processes as a result of exercise- and additional temperature-related stress. In addition, it may provide information about earlier stress situations of a cell [12 13 14 15 ]. The phenomenon, acquired thermotolerance, has been associated with the accumulation of HSP induced by a short exposure to a nonlethal heat treatment mediating a transient resistance to the cytotoxic effects of a subsequent, otherwise lethal shock [16 , 17 ]. Therefore, assessing adaptation to training or to heat conditions may be monitored by the cell’s content of HSP72, which may function as a cellular thermometer and a marker of recent thermal stress [7 , 15 , 17 , 18 ]. Because of this, we investigated the expression of HSP72 in peripheral leukocytes of nonheat-acclimated endurance runners under high ambient temperature. We examined the influence of environmental heat and two consecutive, intensive endurance exercise runs (CR1, CR2) on the expression of the inducible HSP72 in leukocytes at mRNA and protein levels by comparing two groups exercising with the same intensity at different ambient temperatures. Group HH performed both runs at 28°C, whereas group NH completed CR1 at 18°C and CR2 at 28°C room temperature. We wanted to know if thermotolerance or adaptation was induced by one bout of exercise under high ambient temperature conditions. Furthermore, we investigated the effect of an additional heat shock (HS) in vitro on HSP72-expression in leukocytes, which had already been stressed by intensive exercise in vivo, to obtain more insight into the mechanisms of stress response after prolonged, heavy exercise.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Twelve male endurance athletes gave informed consent to participate in the study. They were allocated randomly to two groups of six subjects (group HH and NH). The exercise protocols were limited to six subjects because of the extreme difficulty in performing these tests at high external temperatures. The physical characteristics of the athletes are presented in Table 1 . All subjects were competitive runners with normal, dietary habits; did not take any medication; and performed regular endurance training, including more intensive workouts. The subjects were experienced in competing under elevated, ambient temperatures and were familiar with treadmill running. Exercise tests were performed during the winter months (January and February). The athletes refrained from thermal spa, sauna, or trips to warm geographic regions during the last 10 weeks before the tests. One week before, and in the week between the runs, the athletes performed only moderate endurance exercise with a running intensity below 80% of their individual anaerobic threshold (IAT). The IAT was determined to evaluate the aerobic performance by regarding the lactate-workload relationship [19 ]. The investigation was approved by the Ethics Committee of the University conformed to the 1975 Declaration of Helsinki.


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  		Table 1. Physical Data of the Subjects

 
Preliminary testing
Before participating in the main study, the athletes performed an incremental exercise test on the treadmill (Saturn, HP Cosmos, Traunstein, Germany) to determine the IAT of each subject. Initial running velocity was 6 km·h-1 with increments of 2 km·h-1 every 3 min. The incline of the treadmill was kept constant at 1%, and the ambient temperature and relative humidity during the test were 18°C and 50%, respectively. Capillary blood for lactate measurement was obtained from the earlobe after every stage (EBIO, Eppendorf, Hamburg, Germany), and heart rate was monitored continuously using a heart-rate monitor (Sporttester, Polar Electro, Finland). Running speed and heart rate at the IAT were assessed as described by Dickhuth et al. [19 ].

Maximal oxygen consumption (VO2max) was assessed using an additional ramp test until exhaustion on the treadmill. Subjects started running at 8 km·h-1 for 30 s, and thereafter, running speed was increased by 1 km·h-1 every 30 s. Ventilation (VE), VO2, and respiratory exchange ratio (R) were assessed using breath-by-breath analysis (Oxycon alpha, Jaeger, Germany) with a mouth-piece where the flowmeter was attached. Criteria for assessment of VO2max were a R >= 1.1 and a plateau in VO2 (increase<=150 ml per 30 s) paralleled by an increase in workload [20 ].

Continuous run
The main investigation consisted of two continuous runs (CR1, CR2) on the treadmill, lasting 60 min and performed 6 days apart (Fig. 1 , design of study). Running velocity during the run was adjusted to 90% of the IAT as assessed before. The exercise procedure was performed in a climatized room, where dry-bulb temperature and relative humidity were kept constant at 28°C/50% or 18°C/50%. Group NH (n=6) completed the first run (CR1) at 18°C and 1 week later, the second trial (CR2) at 28°C room temperature. Group HH (n=6) performed both exercise tests at 28°C environmental temperature. All subjects refrained from competitions or vigorous training at least 7 days before the test and in the week of rest between the runs. A dietary protocol revealed a comparable fluid intake 24 h before the test. The subjects were allowed to drink water during the run.



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  		Figure 1. Schematic illustration of the design of the study. Two groups of athletes (HH, NH) performed two continuous runs (CR1, CR2) on a treadmill at the indicated room temperatures (RT), exercise intensity (VIAT=running velocity at the IAT), relative humidity (rH), and duration. Oxygen consumption, core temperature, and heart rate were assessed continuously during the runs, lactate at beginning and end. Blood samples were drawn pre-exercise and 0-h, 24-h, and 48-h post-exercise.

 
Sweat-loss was calculated from the differences in nude-body mass before and after exercise and corrected for fluid intake during the run. We used a telemetric-temperature analyzing system (CorTemp, Human Technologies, Greenwood, SC) to monitor body-core temperature throughout the trials. Heart rate was assessed continuously by the system described above. Blood lactate was determined before, 0 min, and 3 min after exercise. VO2 was assessed as mentioned above. Ventilatory measurements over a period of 2 min were performed every 10 min to minimize respiratory discomfort. Fluid intake was permitted as desired by the subjects. Blood samples were drawn at rest, immediately after (0 h), and 24 h and 48 h after exercise in a sitting position.

Analytical methods
Flow cytometry
The leukocytes were analyzed by intracellular, indirect immunofluorescence using the HSP72-specific monoclonal antibody (mAb) SPA-810 [specific for the inducible form of human HSP70; immunoglobulin (Ig)G1, clone C92F3A-5; StressGen Biotechnologies Corp., Victoria, BC, Canada].

In a 1 ml sample of ethylenediaminetetraacetate (EDTA)-treated blood, erythrocytes were lysed by incubating with 10 ml 1 x fluorescein-activated cell sorter (FACS) Lysing Solution (Becton Dickinson, San Jose, CA) for 15 min at room temperature. Next, cells were fixed at room temperature in a solution containing formaldehyde (reagent A) following the manufacturer’s instructions (Fix & Perm kit, An der Grub, Vienna) and washed twice. Then the cells were permeabilized with reagent B and at the same time, incubated with the primary HSP-specific mAb or isotype-matched, nonbinding antibody (negative control), and incubated for 15 min at room temperature. Previously, antibody concentrations were tested to give maximum counts of positive cells (1 µg/test). After washing the cells twice and incubating in the presence of the secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse 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, 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 then gated. Data were presented as percent-positive cells (%) and mean fluorescent channel (MFC) for each of the three special gates. They were corrected for background fluorescence by the negative controls using isotype-matched, nonbinding, primary antibody.

Isolation of RNA and reverse transcriptase-polymerase chain reaction (RT-PCR)
Low-level, constitutive synthesis of HSP72 in human peripheral blood mononuclear cells suggests that a semi-quantitative assay will be necessary to detect enhanced synthesis after heat stress [21 ].

Cytoplasmic RNA for RT-PCR analyses was isolated from whole blood with the RNeasy-blood kit (Qiagen, Hilden, Germany). RNA (200 ng) was reverse-transcribed (10' 20°C, 15' 42°C, 5' 99°C, 5' 5°C) and amplified (3' 95°C, 1' 95°C, 1' 55°C, 1' 72°C) in the thermal cycler (PTC200, M.J. Research, Watertown, MA) using specific primers for HSP70B (StressGen Biotechnologies Corp.) [22 ] and ß-actin [23 ]. RT and subsequent amplification by the PCR were performed using a GeneAmp RNA PCR kit (Perkin Elmer, Foster City, CA). The RT master mix contained 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 5 mM MgCl2, 1 mM each deoxyribonucleoside triphosphates (dNTPs), 1 U/µL RNase inhibitor, 2.5 U/µL RT, and 2.5 µL oligo d(T). The final RT-reaction volume was 20 µL. The PCR master mix contained 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.25 mM MgCl2, 200 µM each dNTPs, 2.5 U/100 µL AmpliTaq DNA polymerase, and 0.15 µM of each primer. A final 25 µL PCR reaction solution contained 5 µL RT product (cDNA) and 20 µL PCR master mix. Control experiments were performed for each primer pair to determine the range of cycles in which a given amount of cDNA would be amplified in a linear fashion: HSP72, 30 cycles; actin, 27 cycles. Furthermore, a dilution assay was performed to determine the proper input-RNA concentration. The resulting amplified products for HSP70B (234 bp) were confirmed by sequence analysis (SEQLAB, Goettingen, Germany). Photographs of ethidium bromide-stained DNA gels (2%) were scanned by the Lumi-Imager-System (Boehringer Mannheim, Mannheim, Germany), which allowed semi-quantitative analyses of the specific HSP72-mRNA expression. The data generated were normalized to transcript levels for the constitutively expressed ß-actin gene.

In vitro stimulation with HS
Immediately after exercise, EDTA blood of the athletes was incubated for 2 h in a waterbath heated to 42°C (HS). Control blood samples of the same individuals were incubated at 37°C for 2 h instead of the HS exposure. The samples were prepared for further analyses as described above.

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 means and SD. Comparisons of repeated measurements were tested for significance by the Wilcoxon signed ranks test. The nonparametric test of Mann-Whitney was used to evaluate significant differences between the groups HH and NH. A p value of <0.05 was regarded as significant.


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  		Table 2. Lactate Values, Core Temperature, Relative Oxygen Consumption, and Heart Rate Immediately after Two Continuous Runs (CR1, CR2) of Two Athlete Groups HH (28°C/28°C) and NH (18°C/28°C)

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lactate, oxygen consumption, core temperature, heart rate
Maximal blood-lactate values directly after exercise were significantly higher after CR2 in group NH than after CR1 (Table 2) . Group HH revealed comparable lactate data between CR1 and CR2. Comparison of lactate between the groups revealed no significant differences but showed tendencies of higher levels in HH after CR1 and lower levels after CR2 in HH compared with NH.

Body-core temperature showed significant differences between the groups and CR1/CR2 (Table 2) . Core temperature was diminished in group HH after the second run in the heat compared with CR1. In contrast, we found an increased body-core temperature in group NH after CR2. Core temperature was lower after the first exercise (CR1) at 18°C (NH) than at 28°C (HH) but lower in HH compared with NH after CR2.

Mean relative VO2max (%VOi2max) revealed no significant changes (Table 2) .

The maximal rise in heart rate at the end of exercise, presented here as deviation of maximal heart rate from heart rate at IAT, was significantly higher in HH than NH after CR1 and vice versa after CR2 (Table 2) . Deviation of heart rate in group HH at the end of CR2 was lower than after CR1, although not significant.

HSP72-expression
Flow cytometry revealed a significant increase in expression of HSP72 protein after both exercise sessions (CR1, CR2) in group HH. This was apparent in percent-positive cells (%) and mean fluorescence channel (mfc; p<0.05; Fig. 2 ). Monocytes and granulocytes showed comparable increases with a maximum at 24 h after CR1 and only slightly decreased after 48 h. The 48-h values were significantly higher than the pre-exercise expression levels (p<0.05). The second round of exercise (CR2) induced a further increase in HSP72 protein, mainly causing an increase in antigen density (mfc; Fig. 2 ). The maximal expression after 24 h was followed by a steep decrease resulting in nearly basal expression levels 48 h after the second trial.



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  		Figure 2. Cytoplasmic expression of HSP72 in monocytes and granulocytes of group HH determined by flow cytometry. The values before, and 0 h, 24 h, and 48 h, after the two exercise sessions (CR1, CR2) at 28°C (group HH) are presented as mean fluorescence intensity (mfc), a marker of antigen density and percent-positive cells. Data are displayed as means ± SD. *, Significant changes during exercise. §, Significant differences between CR1 and CR2.

 
We found less-pronounced increases in group NH as a result of both exercise sessions compared with HH (Fig. 3 ). The HSP72-protein expression was significantly lower in group NH compared with HH at 24 h and 48 h after CR1 and at 24 h after CR2. The changes of monocyte-mfc compared with pre-exercise levels are presented as representative examples to compare the groups (Fig. 3) .



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  		Figure 3. Changes of the HSP72-protein expression (differences of mfc to pre-exercise values) after two continuous runs (CR1, CR2) at indicated ambient temperatures in monocytes comparing the two groups of athletes (HH and NH). Data are displayed as means ± SD. *, Significant changes during exercise. #, Significant differences between groups.

 
The HSP72-mRNA level in the leukocytes of all athletes, analyzed by semi-quantitative RT-PCR, showed an immediate increase after both exercise sessions, which was mostly down-regulated after 24 h (Figs. 4 and 5 ). The data of the individual athletes are calculated as mRNA amounts in relation to the actin-PCR. The individual data of group HH connected with matching lines are presented in Figure 4 . Original PCR gels of two individual athletes of group HH demonstrating the HSP72-mRNA increase were given as representative examples in Figure 4 . Comparing the exercise-induced increases of the mRNA expression in group HH presented as mean values, the tendency of a more reduced up-regulation as a result of CR2 (0.13±0.07) rather than CR1 (0.24±0.09) can be detected. This may be the result of the higher basal mRNA expression before CR2 (0.29±0.07) compared with the pre-exercise level before CR1 (0.16±0.06). Comparing the trials at different ambient temperature in group NH, we found a more pronounced increase after exercise at 28°C (CR2) compared with 18°C (CR1; Fig. 5 ). Expression of HSP72 mRNA after CR1 revealed no differences between the groups/temperatures. In contrast, the exercise-induced increase after CR2 tended to be higher in group NH than in HH, although the difference was nonsignificant (p=0.05).



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  		Figure 4. HSP72-mRNA expression pre-exercise, 0 h, and 24 h after two exercise trials (CR1, CR2) under high external temperature (28°C, group HH) analyzed by semi-quantitative RT-PCR. In the upper panel, the specific mRNA values are described in relative units normalized to transcript levels of ß-actin. The single points of the individual athletes were presented and connected with lines. In the lower panel, original ethidium bromide-stained PCR-gels of two athletes are shown as representative examples. HSP72 and actin were amplified under conditions to allow relative comparisons for a given mRNA. M = size marker; bp = base pairs.

 


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  		Figure 5. Relative expression of HSP72 mRNA comparing the two athlete groups HH and NH before, 0 h, and 24 h after two continuous runs (CR1, CR2) at the indicated ambient temperatures detected by RT-PCR. HSP72 and actin were amplified under conditions to allow relative comparisons for a given mRNA. Data are displayed as means ± SD.

 
HS in vitro
Additional HS in vitro applied to the blood (native), which was already exercise-stressed in vivo, resulted in a further, significant increase of HSP72-antigen density (mfc) in monocytes and granulocytes detected by flow cytometry. The obviously reduced HS reaction after the second rounds of exercise (CR2) in the heat compared with the first trials (CR1) in both groups is remarkable, in particular, with regard to the significant difference of the core temperatures between the groups and the different environmental temperatures during the preceding runs (Fig. 6 ). The changes are not significant.



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  		Figure 6. Influence of HS in vitro on HSP72-expression in already exercise-stressed monocytes. Analysis of antigen density (mfc) by flow cytometry. Blood samples, drawn immediately after exercise, were exposed to additional, experimental HS (2 h, 42°C). Data are presented as a HS-induced increase compared with the native, exercise-stressed values. Results are presented as means ± SD.

 
The mRNA expression was also increased further by HS, but the extent of stimulation could not be differentiated because the HS-induced expression level reached the plateau phase of amplification (unpublished results).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Endurance exercise under elevated, ambient temperature stimulated HSP72-expression at protein and mRNA level. The combination of external heat and exercise induced a prolonged, high HSP72-expression persisting at protein level until 48 h after the first exercise trial (group HH, CR1) compared with a run with the same intensity but at lower ambient temperature (18°C, group NH). Significantly higher heart-rate values, core temperature at similar VO2max, and slightly higher lactate values show that CR1 at 28°C was more exerting than at 18°C, as expected. These effects were paralleled by a more pronounced HSP72 reaction in group HH compared with NH, primarily at protein level. We could not detect significant differences at the mRNA-expression level, comparing the two groups after CR1, which let us assume mainly post-transcriptional regulatory mechanisms. Up-regulation in one instance and increased stability in the other may be interpreted as thermotolerance on the HSP72 level in leukocytes. Generally, thermotolerance is defined as the accumulation of HSP72 while recovering from the first heat-stress, which grants protection against a subsequent stress [13 , 16 , 17 , 24 ]. Exercise-induced HSP production has already been shown to protect skeletal and heart-muscle cells against future injury [25 26 27 28 29 ]. This protective function may also be assumed in our study.

The more pronounced down-regulation of HSP72 protein and the minor increase at mRNA level after the second trial (CR2) in group HH may indicate parameters of adaptive mechanisms in leukocytes induced by intensive, prolonged exercise under conditions of increased temperature. Group NH, which performed the first run under cooler conditions, revealed no such effects after the second run. The content of HSP72 might provide information about earlier stress situations of a cell, including heat and/or exercise [12 , 14 , 15 , 30 ]. Therefore, HSP72-expression could be a useful marker for assessing adaptation to training or to heat conditions [26 , 31 , 32 ].

Parallel to the effects on HSP level, one bout of endurance exercise in the heat reduced the rise in body-core temperature and cardiac drift during a subsequent, identical trial of endurance exercise (HH, CR2) in nonheat-acclimated runners. This reflects the simultaneous adaptation of the thermoregulatory and cardiocirculatory capacity [33 ]. In contrast, these adaptive effects were not induced by a previous exercise session at a lower ambient temperature (group NH after CR2). Core temperature and heart rate were even higher after CR2 in NH compared with HH. The changes of HSP72 expression in group HH as a result of the second exercise trial may result in part from the more favorable thermo- and cardiocirculatory capacity, leading to less stress in general. Comparable %VO2max, lactate values, and leukocyte counts revealed that the intensity of exercise was equal in both heat sessions. It is suggested that the high ambient temperature is one factor responsible for thermotolerance and adaptive effects on HSP72 level in leukocytes of these nonheat-acclimated athletes. Additional, secondary mechanisms, such as reactive oxygen intermediates (ROI) or cytokines, may be involved. Cytokines, ROI, and nitric oxide have been shown to increase HSP synthesis [2 3 4 5 6 , 34 ]. Thus, the simultaneous regulation of cardiocirculatory/thermoregulatory and HS responses may be interpreted as partially decompensated hemodynamics, which reduce heat dissipation, stimulate thermal stress, and up-regulate HSP synthesis. However, core temperature seems not to be the only factor responsible for HSP induction when looking at the significantly higher core temperature in group NH after CR2 compared with HH. It cannot be excluded completely that the differences in expression between the two heat sessions were related to the exercise rather than to the heat input. This, however, seems less probable because such regularly and well-trained athletes are used to similar exercise protocols. It has already been documented that physically fit men acclimated to heat more rapidly than less-fit men [10 ]. Heat-clamping experiments revealed that exercise and a rise of core temperature contribute to changes in white-cell counts immediately after exercise, potentially mediated by cortisol and growth-hormone changes [35 ]. A relationship between heat exposure and its impact on hormonal and immune responses to exercise has been suggested [36 , 37 ]. In our study, we also found reduced increases in granulocyte and monocyte counts and growth hormone concentrations in the second trial [38 ].

In this context, it could be discussed that thermotolerance on HSP72 level may contribute to acclimatization of athletes to heat on a cellular level and be related to adaptation processes, such as increases in plasma volume and sweating rate, efficient nutrient metabolism, lower core temperature, and heart rate [33 ].

Conversely, adaptation to heat after a single exposure seems questionable because acclimatization processes are described as resulting from several daily, repeated exercise protocols at respective elevated, external temperatures [39 ]. However, these studies also revealed the beginning of heat acclimatization on the first day of exposure and that 4–5 days are sufficient to reach about 75% of the maximal, individual adaptive capacity. Furthermore, even endurance-exercise training in temperate conditions is shown to contribute, in part, to adaptation processes to high ambient temperature, and to offer some protection [40 ]. Moreover, the relatively high intensity of stress input (60-min exercise at 90% IAT plus heat of 28°C) performed during our exercise protocol has to be considered, resulting in maximal core temperatures of up to 40.5°C. The classic physiological adjustments during exercise-heat acclimatization, heart rate, and internal temperatures were reduced as a result of the second exercise trial at high environmental temperature (HH). This was not the case if the run was preceded by a run at lower ambient temperature (NH).

Influence of in vitro HS
Additional HS in vitro applied to blood samples directly after exercise resulted in a further increase in HSP72-expression at protein and mRNA levels when compared with the exercise-induced HSP level. This enhanced HS response may be interpreted as residual, protective resources, which immune cells have after exercise to protect against denaturing heat and additional, extraordinary stress. The experimental HS temperature of 42°C was 2°C above the mean-core temperature achieved by the exercise protocol. Such a high temperature in vivo will be reached by exercise solely in the skeletal musculature [28 ]. Exposure of cells to high temperatures causes their membranes to undergo a rapid decrease in molecular order, which is an important factor in determining HS gene expression [41 ]. Accumulation of specific HSP at or in the membrane during the transient HS response causes a rigidification of the heat-fluidized membrane. Thus, the membrane returns to its pre-exercise state [42 ]. It has been shown in epithelial cells that sufficient heat applied to induce HSP70 synthesis attenuated the heat-induced alterations. This suggests that HSP may be involved in stabilizing cytoskeleton and/or tight-junction proteins [43 , 44 ].

The reduced heat responses, which occur immediately after the second exercise sessions compared with the responses after the first trials in both groups, independent of ambient temperatures, are noteworthy. Ryan et al. [15 ] found that leukocytes obtained after exercise and incubated at 41°C demonstrated a decreased HSP72 synthesis compared with pre-exercise incubations. They suggested that the thermal history of a cell might be evaluated by the cell’s content of HSP72. We questioned if HSP72-expression in leukocytes might be used as an indicator of earlier sets of exercise. The exercise-induced attenuation of the HS response in vitro in both groups after CR2 compared with CR1 may be interpreted as short-time and early adaptation in terms of tolerance to repeated exercise. The differences of exercise-related core temperatures and preceding environmental heat conditions between the groups seem to be of minor importance for this in vitro response. The response of HSP72-expression to additional HS in vitro might be used as an indicator of earlier, severe stress conditions.

In summary, we conclude that even one bout of intensive, endurance exercise on a treadmill under elevated, ambient temperature results in adaptation of cardiocirculatory/ thermoregulatory capacity and of HSP72-expression in leukocytes of nonheat-acclimatized, endurance runners. Furthermore, HSP72 thermotolerance is induced and may afford some protection against subsequent stress. The content of HSP72 in leukocytes may represent an indicator of earlier exercise and temperature-related stress conditions. This marker possibly allows conclusions about adaptation to exercise under high ambient temperature.


   ACKNOWLEDGEMENTS
 
This investigation was supported by a grant from the Bundesinstitut für Sportwissenschaft (Koeln, Germany; VF 0407/01/21/97). We would like to thank the athletes who volunteered to participate in the study.

Received March 30, 2000; revised October 11, 2000; accepted January 5, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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