Journal of Applied Physiology AJP: Gastrointestinal and Liver Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 103: 1196-1204, 2007. First published July 5, 2007; doi:10.1152/japplphysiol.00242.2007
8750-7587/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
103/4/1196    most recent
00242.2007v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamada, P. M.
Right arrow Articles by Schneider, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamada, P. M.
Right arrow Articles by Schneider, S. M.

Effect of heat acclimation on heat shock protein 72 and interleukin-10 in humans

Paulette M. Yamada,1 Fabiano T. Amorim,1 Pope Moseley,2 Robert Robergs,1 and Suzanne M. Schneider1

1Department of Physical Performance and Development, University of New Mexico, and 2Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, New Mexico

Submitted 28 February 2007 ; accepted in final form 3 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Heat acclimation (HA) results in whole body adaptations that increase heat tolerance, and in addition, HA may also result in protective cellular adaptations. We hypothesized that, after HA, basal intracellular heat shock protein (HSP) 72 and extracellular IL-10 levels would increase, while extracellular HSP72 levels decrease. Ten male and two female subjects completed a 10-day exercise/HA protocol (100-min exercise bout at 56% of maximum O2 uptake in a 42.5°C DB, 27.9% RH environment); subjects exhibited classic adaptations that accompany HA. Peripheral blood mononuclear cells (PBMCs) were isolated before and after each acclimation session on days 1, 6, and 10; plasma and serum were collected before and after exercise on the 1st and 10th day of HA. SDS-PAGE was used to determine PBMC HSP72 levels during HA, and ELISA was used to measure plasma IL-10 and serum HSP72 concentrations. The increase in PBMC HSP72 from pre- to postexercise on the 1st day of HA was not significant (mean ± SD, 1.0 ± 0 vs. 1.6 ± 0.6 density units). Preexercise HSP72 levels on day 1 were significantly lower compared with the pre- and postexercise samples on days 6 and 10 (mean ± SD, day 6: 2.1 ± 1.0 and 2.2 ± 1.0, day 10: 2.0 ± 1.3 and 2.2 ± 1.0 density units, respectively, P < 0.05). There were no differences in plasma IL-10 and serum HSP72 postexercise or after 10 days of HA. The sustained elevation of HSP72 from days 6 to 10 may be evidence of a cellular adaptation to HA that contributes to improved heat tolerance and reduced heat illness risk.

exercise; peripheral blood mononuclear cell; serum; plasma; in vivo

INTRACELLULAR (IC) HEAT SHOCK protein (HSP) 72 are highly conserved proteins, which are involved in maintaining cellular protein conformation and homeostasis during stress (33). Examples of such stressors are hyperthermia, hypoxia, hyperoxia, cytokines, inflammation, and injury, increases in metabolism and IC calcium, and a decrease in glycogen and ATP (13), acidosis, stress hormones, and ischemia-reperfusion (32). Extracellular (EC) HSP72 is thought to stimulate innate immunity (31), act as a danger signal resulting in increased immune responses, and facilitate host defense to pathogenic challenges (15). If a pathogenic challenge does not occur, then EC HSP72 will have little impact on innate immune cell production of proinflammatory cytokines. However, if a pathogenic challenge ensues, then EC HSP72 could help to eliminate bacterial pathogens (15).

Studies have shown that marathon runners can tolerate core body temperatures (Tc) as high as 41.5°C without exhibiting clinical symptoms of heat illness (3, 20, 29). These observations challenge current guidelines, which state that heatstroke will typically occur as Tc approaches 40.6–41.3°C (39). Furthermore, runners who have collapsed during a triathlon were not more hyperthermic than other noncollapsed runners (34), so it appears that some athletes are more "protected" from heat illness. Some athletes may acquire heat acclimatization and cellular preconditioning during training, enabling them to withstand a severe, subsequent stress. IC HSP72 may be involved in improving cellular and animal thermotolerance. Dokladny et al. (7) showed that HSP72 expression plays a role in protecting the intestinal epithelial tight junction barrier in Caco-2 cells when exposed to modest increases in temperature (37–41°C). The preservation of tight junctions prevents large amounts of endotoxin release into the portal vein, activation of macrophages, and the subsequent proinflammatory cytokine cascade (24, 27). In extreme cases, this inflammation can lead to sepsis, suggesting that proinflammatory cytokines play a role in heatstroke pathophysiology. Therefore, we sought to determine whether heat acclimation (HA) could be used to "precondition" humans and improve thermotolerance as manifest by an increase in HSP72 and a decrease in proinflammatory cytokine response. Since plasma TNF-{alpha} levels were undetectable across HA (data not shown), it was unrealistic to determine whether TNF-{alpha} decreased during HA and therefore, IL-10, an anti-inflammatory cytokine was measured. Our first purpose was to assess the changes in IC HSP72 in response to an acute bout of exercise in the heat. Since HSP72 is involved with increased cellular protection and tolerance, we sampled peripheral blood mononuclear cells (PBMCs) because they are directly related to immunity. We hypothesized that IC HSP72 would increase from pre- to postexercise because earlier studies showed that IC HSP72 increases in response to exercise (11, 40, 43). Our second purpose was to determine whether resting IC HSP72 levels changed across 10 days of HA. We hypothesized that resting levels would progressively increase throughout HA, as previously shown (31a). Our third purpose was to determine whether basal serum (EC) HSP72 would decrease from pre- to post-HA. Since Kresfelder et al. (23) showed that basal serum HSP72 levels decreased in 14 subjects who successfully completed 5 days of HA, we hypothesized that basal serum HSP72 levels would decrease after 10 days of HA. We also hypothesized that serum HSP72 will increase from pre- to postexercise (after an acute exercise bout), as other studies have shown (9, 12, 30). In vitro experiments show that IC HSP72 suppresses mRNA transcription of proinflammatory cytokines IL-1 and TNF-{alpha} (22). Since intense endurance exercise suppresses T-helper-1 immunity (41) and the production of anti-inflammatory cytokines balances proinflammatory cytokines (8), we postulated that the anti-inflammatory cytokines (i.e., IL-4, IL-10, IL-1ra) would be upregulated. Therefore, to test this hypothesis, our fourth purpose was to determine plasma IL-10 levels from pre- to postexercise on the 1st and 10th day of HA. We hypothesized that IL-10 would be upregulated from pre- to postexercise and from the 1st to 10th day of HA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Twelve (10 men, 2 women) healthy, nonsmoking subjects [mean ± SD, age: 24 ± 4 yr, maximum O2 uptake (VO2 max): 54.5 ± 9.1 ml·kg–1·min–1, body fat: 13.6 ± 7.6%] participated in this study. Before testing, the study was approved by the Institutional Review Board. All testing took place at an altitude of 1,572 m (barometric pressure = 635 mmHg) and during February through April, when the average maximal outdoor temperature in April was 23.3°C (74°F). All subjects had less than two positive cardiovascular risk factors, as outlined by the American College of Sports Medicine (1). Subjects were excluded from the study if they had a history of heat illness or gastrointestinal ulcers, known diseases or viral infections, or were taking vitamin supplements or other medications that would affect HSP72, exercise, or thermoregulatory responses. They were also excluded if they traveled to a warm climate or had visited a sauna or hot tub the month before testing.

Baseline measurements.   After the initial screening, body density was determined using the sum of three sites and the Jackson and Pollack equation (1); ethnic-specific equations were used to calculate percentage of body fat from body density (1). All subjects completed a graded treadmill VO2 max test in a thermoneutral environment. The VO2 max test consisted of 1-min stages with incremental increases in speed and grade, and it was terminated at volitional fatigue. VO2 max was defined as the highest 30-s average, and the maximal heart rate (HRmax) attained was recorded using a telemetric heart rate (HR) transmitter strap and watch (S810i series, Polar). Breath-by-breath O2 uptake was measured with a fast-response turbine flow transducer (K. L. Engineering model S-430, Van Nuys, CA) and custom-developed software (LabVIEW, National Instruments, Austin, TX) with O2 and CO2 electronic gas analyzers (AEI Technologies, model S-3A and model CD-3H, Pittsburgh, PA).

HA protocol.   No sooner than 2 days after the VO2 max test, subjects completed a 10-day HA protocol (HAP), where they exercised in the heat during each session. Subjects were instructed to eat a high-carbohydrate meal (they were given a list of foods high in carbohydrate). They were instructed to avoid exercise, caffeine, alcohol, and diuretics 24 h before testing. All trials were completed at the same time of day and within 14 days. Each night before the HA session, subjects were instructed to swallow a telemetric temperature sensor capsule (Jonah Ingestible Core Temperature Capsule, Mini Mitter, Bend, OR) that was used to measure Tc. Ingesting the capsule the previous night ensured that it would be emptied from the stomach and be unaffected by water ingestion during the HAP. Before ingestion, each capsule was calibrated against a Fisherbrand factory-calibrated thermometer (National Institute of Standards and Technology traceable, Fisher Scientific, 15-041A).

In the morning of the session, subjects were instructed to drink 500 ml of water before reporting to the laboratory. Subjects were asked to collect a urine sample and if a subject was deemed dehydrated (specific gravity >1.030, or color >6) (2), they ingested an additional 200 ml of water. Baseline clothed and nude body weights were measured, and subjects dressed in a t-shirt, shorts, socks, and athletic shoes.

Blood samples were obtained on days 1, 2, 4, 6, 8, and 10 through venipuncture of the antecubital vein. Subjects sat for 20 min before the initial blood draw, and during this period they were instrumented with a HR transmitter strap (S810i series, Polar); skin thermistors (Grant, UK, EUS-U-VL5-0) attached to the right side of the body: chest (~5 in. below midclavicle), lateral arm (midpoint between olecranon and acromion processes), lateral thigh (6 in. above the superior patella border), and lateral calf (midpoint between the mallelous and the bottom border of patella). The signal from the temperature capsule was detected with an external data logger (VitalSense, Mini Mitter), and baseline Tc was recorded. A baseline blood sample was drawn in thermoneutral conditions: 5 ml of blood were allowed to clot to determine serum osmolality and serum HSP72; 1 ml of heparinized blood was aliquoted for the measurement of hemoglobin (Hb) and hematocrit (Hct); on days 1, 6, and 10, an additional 12 ml of heparinized blood were collected for the separation of PBMC. Postexercise, another blood sample was drawn from the antecubital vein and aliquoted as outlined above. Figure 1 outlines the procedures during a HA session.


Figure 1
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 1. Procedures for each heat acclimation (HA) session. VO2, O2 uptake; HR, heart rate.

 
Subjects then entered the environmental chamber (mean ± SD, 42.5 ± 0.1°C DB, 25.9 ± 0.4°C WB, 30.6 ± 0.3°C WBGT, 41.9 ± 0.6°C globe, 27.9% RH, 0.3 m/s wind speed) and warmed up by walking for 5 min at 3.5 mph, 0% grade, and then exercised at 56% of their VO2 max. Depending on their fitness level, subjects either walked or ran for 100 min (two 50-min work bouts), separated by 15 min of rest. Treadmill speed and grade ranged from 3.8 to 6.3 mph and 0 to 4%, respectively. Termination criteria included the following: completion of the protocol, a Tc of 39°C, 95% of HRmax, the subject's request to stop, or symptoms of exertional heat illness. During the protocol, subjects were allowed to drink water ad libitum.

During the test, a data logger (Squirrel 2020 series, Grant Instruments, Cambridgeshire, UK) recorded skin temperatures at 15-s intervals. Tc was monitored and recorded by the external data logger (VitalSense, Mini Mitter). HR was recorded every 5 s by the HR watch. Mean skin temperature (MST) was calculated with the following formula: MST = 0.3 * (Tchest + Tarm) + 0.2 * (Tcalf + Tthigh), where Tchest, Tarm, Tcalf, and Tthigh are skin temperatures at the specified site (37).

Hb, Hct, serum osmolality, and cell count.   Heparinized blood was used to measure Hct in triplicate and Hb in duplicate. Microcapillary tubes were filled with heparinized blood and spun in a centrifuge for 5 min (Damon/IEC Division), and then Hct was immediately recorded as a percentage using a Hct reader. For the measurement of Hb, 20 µl of whole blood were added to 5 ml of Drabkins solution, and absorbance was read at 540 nm (Thermospectronic 401, Milton Roy, Louisville, CO). Whole blood was allowed to clot, and the serum was removed. Serum osmolality was measured using freezing point depression in duplicate (model 3D3, Advanced Instruments, Norwood, MA). Plasma volume (PV) expansion from the 1st to 10th day of HA was calculated from preexercise Hct and Hb measurements (6).

PBMC HSP72.   PBMC were isolated on days 1, 6, and 10 (Histopaque 1077, Sigma-Aldrich, St. Louis, MO). The cell pellet was resuspended in warmed RMPI-1640 (Roswell Park Memorial Institute, 37°C) supplemented with 10% fetal bovine serum (GIBCO, Invitrogen, Carlsbad, CA) and 50-µl penicillin streptomycin (100 U/ml, GIBCO, Invitrogen). The cells were washed three times and during the second wash, cells were counted on a hemacytometer using 0.4% Trypan blue (Sigma); viability was expressed as a percentage (97%). The pellet was stored at –20°C.

The pellet was homogenized, and the supernatant was used in SDS-PAGE. Total protein concentration of the supernatant was measured using the Bradford assay. Cell lysates were separated on a 12% polyacrylamide gel by electrophoresis. Care was taken to load 14 µg of total protein per lane. Five microliters of a protein standard were used to identify HSP72 (Precision Plus Protein Standard, BioRad, Hercules, CA). Protein was transferred to a nitrocellulose membrane (Hybond-C Extra, Amersham Biosciences, catalog no. RPN303E), and then nonspecific binding sites were blocked with 8% milk. To detect HSP72, the primary antibody (mouse anti-HSP70 SPA810, Stressgen, Assay Designs, Ann Arbor, MI) was applied, and the appropriate secondary antibody was applied (horseradish peroxidase rabbit anti-mouse IgG1, Invitrogen). The proteins were developed using a chemiluminescent reagent specific for horseradish peroxidase-conjugated secondary antibodies (Western blotting luminol reagent, Santa Cruz Biotechnology). The blot was scanned into the computer and printed (GeneSnap version 6.05, SynGene, Beacon House, Cambridge, UK). The bands were then scanned (HP Precision Scan Pro 2.5) into Adobe Photoshop (version 7.0), and luminosity was measured and then multiplied by the number of pixels (intensity). Ratios were used to express differences in bands, with the first band being the control, or 1. The intensities for subsequent bands were divided by the intensity of the first sample (preexercise, day 1 control), and expressed as a ratio.

Serum HSP72 and plasma IL-10.   A commercially available ELISA (EKS-700A, Assay Designs, Ann Arbor, MI) was used to determine serum HSP72 on the 1st and 10th day of HA. Serum dilutions of 1:2 and a standard curve ranging from 0.78 to 12.5 ng/ml (r2 = 0.999) were used to determine HSP72 concentration. The ELISA sensitivity is reported to be 0.2 ng/ml for HSP72, and intra-assay coefficient of variance was 4.7%. A commercially available ELISA with a sensitivity of 4 pg/ml was used to determine plasma IL-10 concentrations, and intra-assay coefficient of variance was 4.6%. A 1:1 plasma dilution and a standard curve ranging from 2 to 125 pg/ml (r2 = 0.993) were used to determine plasma IL-10 concentration on the 1st and 10th day of HA (BD OptEIA no. 555157, BD Biosciences, San Diego, CA). All samples were analyzed in duplicate, and absorbance was read at 450 nm (Molecular Devices, Versamax Tunable Microplate Reader, Sunnyvale, CA). Preexercise plasma IL-10 and serum HSP72 values on day 10 were corrected for changes in PV. Data are presented in the uncorrected and corrected form when applicable.

Lactate dehydrogenase.   Lactate dehydrogenase (LDH) was measured (CytoTox96 Non-radioactive Cytotoxicity Assay, Promega, Madison, WI) in serum samples obtained preexercise on days 1, 2, and 10 of HA. The assay was conducted as specified by manufacturer's guidelines. The absorbance on day 1 was set as the control, or 1, and the two remaining samples were expressed as a ratio of the control.

Statistical analyses.   Dependent t-tests (Statisitica, version 5, Tulsa, OK) were used to determine whether significant differences existed between days 1 and 10 of HA; dependent variables were duration, peak HR expressed relative to HRmax, peak Tc, and peak MST. These methods were used to confirm that subjects were heat acclimated.

One-way repeated-measures ANOVA were used to determine the following: 1) the change in pre- to postexercise PBMC HSP72 on days 1, 6, and 10; 2) the change in basal PBMC HSP72 levels on days 1, 6, and 10 of HA; 3) changes in plasma IL-10 and serum HSP72 pre- to postexercise on the 1st and 10th days of HA; and 4) changes in LDH during HA. If there was a significant main effect, Tukey's post hoc test was used. If data were missing, Tukey's honestly significant difference post hoc test for unequal N was used. Based on a priori power analyses and similar studies, seven subjects would result in a 99% probability of detecting a difference in PBMC HSP72 (40), incorporating eight subjects would result in a 93% probability of detecting differences in serum HSP72 (42), and 10 subjects would provide sufficient power (100%) to detect a difference in plasma IL-10 levels (35). Dependent t-tests were used to compare preexercise serum HSP72 and plasma IL-10 levels on days 1 and 10. Lastly, correlation analyses were used to determine the relationship between serum and PBMC HSP72 pre- and postexercise on the 1st and 10th day of HA for each subject (Graphpad Prism V.4, San Diego, CA). Figures and data represent means ± SD; statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
This study initially involved 12 subjects; however, subject 3 was unable to complete the study due to hip pain. Subject characteristics are outlined in Table 1.


View this table:
[in this window]
[in a new window]

  		Table 1. Subject characteristics

 
HA.   Subjects were heat acclimated, as they were able to exercise longer with lower Tc and HRs on day 10 compared with day 1 (see Fig. 2). Furthermore, subjects exhibited a PV expansion of 9.25 ± 3.25%. The PV expansion in the present study was mirrored by a significant decrease in preexercise Hct and Hb concentration from days 1 to 10 (47.6 ± 2.6 vs. 46.1 ± 2.5%, 15.3 ± 1.0 vs. 14.4 ± 0.9 g/dl, respectively, P < 0.05). Subjects were hydrated before each HA session, as urine color and specific gravity were 2 ± 0 and 1.012 ± 0.002, respectively.


Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 2. Physiological measurements during HA. A: mean duration; B: peak HR expressed as a percentage of maximal HR (HRmax); C: peak core body temperature (Tc); D: peak mean skin temperature (MST). *Significant change between the 1st and 10th day of HA, P < 0.01.

 
PBMC HSP72.   SDS-PAGE was used to detect PBMC HSP72, but due to technical difficulties, we only had samples for eight subjects. Figure 3 outlines the individual data and mean changes in PBMC HSP72 during HA. Subjects 10 (preexercise, day 10) and 6 (day 6) did not have sufficient sample, and therefore data are not shown. The Western blot did not detect any HSP72 preexercise on day 1 for subject 12. Therefore, the group's mean ratio of pre- to postexercise on day 1 was applied for this subject, and the theoretical change was used in the ratio calculations.


Figure 3
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 3. Peripheral blood mononuclear cell (PBMC) heat shock protein (HSP) 72 during HA (n = 7). A: pre- and postexercise PBMC HSP72 on days 1, 6, and 10 of HA. Open bars, preexercise; solid bars, postexercise. Ratios are presented in relation to preexercise day 1. B: individual subject (S) data. Legend identifies subjects. Significant increase compared with preexercise day 1: *P < 0.01, **P < 0.05.

 
A significant main effect for HSP72 during HA [F(5,25) = 4.78, P = 0.003] was found. Tukey's post hoc test (for unequal N) revealed that basal HSP72 increased from days 1 to 6 (P = 0.009) and from days 1 to 10 (P = 0.043); there were no differences between basal levels on days 6 and 10 (P = 0.343, Fig. 3A). Figure 3 illustrates the blot from subject 8, which represents the sample's mean HSP72 response to HA. To summarize, PBMC HSP72 significantly increased from preexercise on day 1 to preexercise on the 6th day and remained elevated thereafter. HSP72 plateaued from preexercise on day 6 and did not increase from pre- to postexercise on day 6 or 10.

EC HSP72, IL-10, and LDH.   Plasma IL-10 was measured in 10 subjects because one plasma sample was missing for subject 10. Figures 4 and 5 show that there were no significant differences among serum HSP72 and IL-10 means from pre- to postexercise on days 1 and 10, even when adjusted for PV expansion. Figure 5, C and D, illustrates uncorrected and corrected individual serum HSP72 data, respectively.


Figure 4
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 4. IL-10 during HA (n = 10). A: data uncorrected; no significant differences (ANOVA). B: preexercise day 10 values are corrected for changes in plasma volume (PV); no significant differences (t-test). Open bars, preexercise; solid bars, postexercise.

 

Figure 5
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 5. Serum HSP72 during HA (n = 8). A: no differences among means (ANOVA). Data are uncorrected for PV changes. Open bars, preexercise; solid bars, postexercise. B: no difference between preexercise means (t-test). Data are corrected for PV changes. C: individual subject data. D: individual subject data. Preexercise day 10 data are corrected for PV changes.

 
Correlations were determined for seven subjects, as serum HSP72 was below detection limits for subject 8. The Pearson's r was 0.64 ± 0.12 and 0.56 ± 0.39 when uncorrected and corrected for changes in PV, respectively (Figs. 6 and 7). Pearson's r was >0.95 for subjects 2 and 12. There were no mean differences in serum LDH among days 1, 2, and 10 of HA (1 ± 0, 0.97 ± 0.08, 1.00 ± 0.10, respectively).


Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 6. Correlation between serum and PBMC HSP72 in samples obtained pre- and postexercise on days 1 and 10 of HA. Data are uncorrected for changes in PV.

 

Figure 7
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 7. Correlation between serum and PBMC HSP72 in samples obtained pre- and postexercise on days 1 and 10 of HA. Preexercise day 10 data are corrected for changes in PV.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The major finding of this study was that IC HSP72 increased within the first 6 days of HA. The elevated IC HSP72 may confer protection from heat illness by maintaining cellular protein homeostasis (33), or reducing the risk of endotoxemia by preserving epithelial tight junctions of the gut (7). We did not see changes in EC HSP72 or EC IL-10 during HA.

IC HSP72.   Since subjects did not perform a separate exercise bout in thermoneutral conditions, it is not possible to separate the effects of heat and exercise on HSP72; only the combined effects of exercise in the heat on HSP72 will be discussed below. Although not significant, there was a tendency (P = 0.34) for HSP72 to increase from pre- to postexercise on the 1st day of HA. On average, the exercise duration on the 1st day of HA was 59.3 min. This is in accordance with Shin et al. (40), who showed that leukocyte HSP72 increased in 10 trained and 10 sedentary subjects immediately post- and 30-min postexercise (trained: 127 and 141%; sedentary: 127 and 139% from baseline, respectively). The authors reported that the subjects ran at 70% of HR reserve for 1 h (24°C, 60% relative humidity). The present study used a lower exercise intensity and a higher ambient temperature, while the Shin et al. (40) study used a higher exercise intensity and lower ambient temperature. Although the two studies used different exercise conditions, both provided the necessary stimulus to elicit the HSP72 response, with the present study showing a larger increase.

As subjects became heat acclimated, they exhibited PV expansion and a decrease in peak HR, Tc, and MST, decreasing the relative stress over HA. Despite this decrease in relative stress, the level of PBMC HSP72 remained elevated in the group. Furthermore, PBMC HSP72 remained elevated, despite the fact that 1) exercise durations were not the same for each subject because exercise was terminated when HR or Tc reached critical levels; 2) not all subjects terminated exercise due to a critical Tc of 39°C, thus levels of "peak heat stress" were different; 3) the decrease in relative stress during HA probably differed among subjects, causing subjects to receive different levels of stress; 4) the physiological and psychological response to exercise and heat stress probably differed among subjects; and 5) although all subjects exercised at 56% of VO2 max, some subjects ran while others walked. This is a very interesting observation, as HA is capable of increasing and maintaining PBMC HSP72 levels throughout the entire HAP, despite varying levels of exercise and heat stress among subjects. Since the protocol required subjects to complete 10 days of HA within a 14-day period, each subject had different schedules, which consisted of different consecutive days. Interestingly, subjects 2, 4, 5, and 12 had at least 48 h of rest before the 6th day of HA. The results show that the HSP72 response was elevated (or maintained), even in the absence of exercise the previous day. These results are consistent with Fehrenbach et al. (14), who found that PBMC HSP72 levels remained elevated 24 h after a 2.5- to 3.5-h marathon in 12 well-trained male athletes.

As HA decreases the set point for onset of sweating, HA may also decrease the stress threshold necessary for HSP72 upregulation, providing enhanced cellular protection. Maloyan et al. (28) reported that HA rats exhibited a shorter time to peak HSP72 mRNA levels compared with control rats. They concluded that HA increased transcription rates in the acclimated state, with HSP72 accumulation being faster with increased levels of stress. HA may also affect posttranscriptional activities; however, this study did not investigate this potential acclimatory phenomenon.

EC HSP72.   Even when data were corrected for PV expansion, there was no mean change in serum HSP72 and plasma IL-10 levels. However, four subjects exhibited an increase, while the remaining four showed no change in preexercise serum HSP72 from day 1 to 10 when corrected for PV expansion. The significance of a postexercise increase in EC HSP72 remains unclear. Pittet et al. (36) reported that higher serum HSP72 levels were correlated with survival in trauma patients, whereas da Rocha et al. (5) reported that lower serum HSP72 levels correlated with survival in patients with traumatic brain injury. Increased serum HSP72 may indicate that the subject's immune system was preparing for a pathogenic challenge or initiating an immune response.

It is difficult to explain why only two of seven subjects had strong, positive relationships because little is known about the signals that stimulate the in vivo release of HSP72 into the EC space or the cells that release it (21). However, previous studies have shown in vivo HSP72 release from the human liver (10) and the human brain (26). HSP72 may also be released through exocytosis by glial cells (19), B cells (4), PBMC (25), lipid rafts (2a), or exosomes (4, 25), or released as a result of cell necrosis (17). Furthermore, there is no obvious commonalities between these two subjects, as they were different sexes, have very different fitness levels, and had different changes in Tc throughout exercise (as the female subject started with a higher Tc, resulting in a smaller change in Tc).

Since subjects 2 and 12 had increased serum HSP72 levels during HA, but no change in serum LDH, a marker of cell damage, this suggests that EC HSP72 release did not occur through muscle damage or cellular necrosis associated with the daily exercise program. Johnson and Fleshner (21) suggest that an increase in EC HSP72 is due to sympathetic nervous system stimulation, which causes norepinephrine to bind to {alpha}1-adrenoreceptors, causing an increase in IC calcium, which results in the release of HSP72-filled exosomes. Thus previous studies that observed an increase in serum HSP72 postexercise may have caused sufficient skeletal muscle damage to induce necrosis and release of HSP72 into the EC space or had greater HSP72 release through exocytosis or exosomes.

Resting serum HSP72 values in the present study fall in the relatively large range (0.1–4.0 ng/ml) reported in previous studies (12, 30, 42). The results of previous studies indicate that serum HSP72 levels usually increase after exercise (12, 30, 42); however, we did not show this response. Specifically, Marshall et al. (30) demonstrated an increase in serum HSP72 levels from pre- to postexercise in subjects who cycled on 2 consecutive days and a decrease in basal levels of serum HSP72. Also, Kresfelder et al. (23) showed that basal serum HSP72 levels decreased after 5 days of HA. The reason for this discrepancy is unclear. Since the exercise protocols used in previous studies (12, 30) were similar in intensity as that in the present study, the exercise protocol does not explain the discrepancy. The insensitivity of ELISA may account for the discrepancy. The assay sensitivity is 0.2 ng/ml, which shows that resting values of 0.1 ng/ml are theoretical. Furthermore, postexercise values of previous research fall on the lower 1/10th of the standard curve (12, 30, 42), making the detection of small changes in serum HSP72 values difficult.

Plasma IL-10.   The lack of change in IL-10 levels suggests that the HAP did not provide sufficient stress, because previous studies that showed postexercise increases in IL-10 incorporated higher intensities [i.e., running at 85% of VO2 max (35), a long distance triathlon, and a 100-km run (18)]. If the HAP provided greater exercise stress, or cells were stressed in vitro with endotoxins, different IL-10 responses may have been observed. Alternatively, plasma may also have been sampled too soon, as cytokine levels have been shown to peak 30 min postexercise (38).

Conclusions.   In conclusion, this is a unique study, as it is the first to measure PBMC HSP72 during 10 days of HA. Physiological adaptations of HA traditionally include whole body adaptations. This study shows that PBMC HSP72 increases in response to HA, which may illustrate a cellular adaptation of HA in humans. It is also interesting to note that although the relative stress decreased during the 10 days of HA, HSP72 remained elevated, perhaps exhibiting a lower threshold for HSP72 production. Whether PBMC HSP72 is upregulated during HA in response to a lower "stress threshold" or whether the upregulation is due to an alteration of posttranscriptional regulation needs to be elucidated.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was funded by Defense Advanced Research Projects Agency Grant W911NF-06-1-0025 and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR40771.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Lou Stevenson for providing a Vitalsense; Brad Laprise, Gary Proulx, and Walter Teal for their support; Dr. Mold and Carol Morris for sharing their plate reader and expertise; Walker Wharton and Rebecca Lobb for helpful tips; and Dr. Ma and Karol Dokladny for providing chemiluminescent and assay materials and invaluable knowledge.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Yamada, Dept. of Pediatrics, Division of Endocrinology, 10833 Le Conte Ave., Rm. 22-315 MDCC, Los Angeles, CA 90095 (e-mail: pyamada{at}mednet.ucla.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. American College of Sports Medicine. ACSM's Guidelines for Exercise Testing and Prescription. New York: Lippincott Williams & Wilkins, 2000.
  2. Armstrong LE. Performing in Extreme Environments. Champaign, IL: Human Kinetics, 2000.
  3. Broquet AH, Thomas G, Masliah J, Trugnan G, Bachelet M. Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release. J Biol Chem 278: 21601–21606, 2003.[Abstract/Free Full Text]
  4. Byrne CL, Lee JK, Chew S, Lim C, Tan E. Continuous thermoregulatory responses to mass-participation distance running in heat. Med Sci Sports Exerc 38: 803–810, 2006.
  5. Clayton A, Turkes A, Navabi H, Mason MD, Tabi Z. Induction of heat shock proteins in B-cell exosomes. J Cell Sci 118: 3631–3638, 2005.[Abstract/Free Full Text]
  6. da Rocha AB, Zanoni C, de Freitas GR, Andre C, Himelfarb S, Schneider RF, Grivicich I, Borges L, Schwartsmann G, Kaufmann M, Regner A. Serum Hsp70 as an early predictor of fatal outcome after severe traumatic brain injury in males. J Neurotrauma 22: 966–977, 2005.[CrossRef][Web of Science][Medline]
  7. Dill DB, Costill DL. Calculation of percentage changes in volumes of blood, plasma, and red cells in dehydration. J Appl Physiol 37: 247–248, 1974.[Free Full Text]
  8. Dokladny K, Moseley PL, Ma TY. Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability. Am J Physiol Gastrointest Liver Physiol 290: G204–G212, 2006.[Abstract/Free Full Text]
  9. Elenkov IJ, Chrousos GP. Stress hormones, proinflammatory and antiinflammatory cytokines, and autoimmunity. Ann NY Acad Sci 966: 290–303, 2002.[Web of Science][Medline]
  10. Febbraio MA, Mesa JL, Chung J, Steensberg A, Keller C, Nielsen HB, Krustrup P, Ott P, Secher NH, Pedersen BK. Glucose ingestion attenuates the exercise-induced increase in circulating heat shock protein 72 and heat shock protein 60 in humans. Cell Stress Chaperones 9: 390–396, 2004.[CrossRef][Web of Science][Medline]
  11. Febbraio MA, Ott P, Nielsen HB, Steensberg A, Keller C, Krustrup P, Secher NH, Pedersen BK. Exercise induces hepatosplanchnic release of heat shock protein 72 in humans. J Physiol 544: 957–962, 2002.[Abstract/Free Full Text]
  12. Fehrenbach E, Niess AM, Schlotz E, Passek F, Dickhuth HH, Northoff H. Transcriptional and translational regulation of heat shock proteins in leukocytes of endurance runners. J Appl Physiol 89: 704–710, 2000.[Abstract/Free Full Text]
  13. Fehrenbach E, Niess AM, Voelker K, Northoff H, Mooren FC. Exercise intensity and duration affect blood soluble HSP72. Int J Sports Med 26: 552–557, 2005.[CrossRef][Web of Science][Medline]
  14. Fehrenbach E, Northoff H. Free radicals, exercise, apoptosis, and heat shock proteins. Exerc Immunol Rev 7: 66–89, 2001.[Web of Science][Medline]
  15. Fehrenbach E, Passek F, Niess AM, Pohla H, Weinstock C, Dickhuth HH, Northoff H. HSP expression in human leukocytes is modulated by endurance exercise. Med Sci Sports Exerc 32: 592–600, 2000.
  16. Fleshner M, Johnson JD. Endogenous extra-cellular heat shock protein 72: releasing signal(s) and function. Int J Hyperthermia 21: 457–471, 2005.[CrossRef][Web of Science][Medline]
  17. Fox SH, DuBois AB. The effect of evaporative cooling of respiratory protective devices on skin temperature, thermal sensation, and comfort. Am Ind Hyg Assoc J 54: 705–710, 1993.[Web of Science][Medline]
  18. Gallucci S, Lolkema M, Matzinger P. Natural adjuvants: endogenous activators of dendritic cells. Nat Med 5: 1249–1255, 1999.[CrossRef][Web of Science][Medline]
  19. Gomez-Merino D, Drogou C, Guezennec CY, Burnat P, Bourrilhon C, Tomaszewski A, Milhau S, Chennaoui M. Comparison of systemic cytokine responses after a long distance triathlon and a 100-km run: relationship to metabolic and inflammatory processes. Eur Cytokine Netw 17: 117–124, 2006.[Web of Science][Medline]
  20. Guzhova I, Kislyakova K, Moskaliova O, Fridlanskaya I, Tytell M, Cheetham M, Margulis B. In vitro studies show that Hsp70 can be released by glia and that exogenous Hsp70 can enhance neuronal stress tolerance. Brain Res 914: 66–73, 2001.[CrossRef][Web of Science][Medline]
  21. Holtzhausen LM, Noakes TD, Kroning B, de Klerk M, Roberts M, Emsley R. Clinical and biochemical characteristics of collapsed ultra-marathon runners. Med Sci Sports Exerc 26: 1095–1101, 1994.
  22. Johnson JD, Fleshner M. Releasing signals, secretory pathways, and immune function of endogenous extracellular heat shock protein 72. J Leukoc Biol 79: 425–434, 2006.[Abstract/Free Full Text]
  23. Kluger MJ, Rudolph K, Soszynski D, Conn CA, Leon LR, Kozak W, Wallen ES, Moseley PL. Effect of heat stress on LPS-induced fever and tumor necrosis factor. Am J Physiol Regul Integr Comp Physiol 273: R858–R863, 1997.[Abstract/Free Full Text]
  24. Kresfelder T, Claassen N, Cronje M. Hsp70 induction and hsp70 gene polymorphisms as indicators of acclimatization under hyperthermic conditions. J Therm Biol 31: 406–415, 2006.[CrossRef][Web of Science]
  25. Lambert G. Role of gastrointestinal permeability in exertional heatstroke. Exerc Sport Sci Rev 32: 185–190, 2004.[Web of Science][Medline]
  26. Lancaster GI, Febbraio MA. Exosome-dependent trafficking of HSP70: a novel secretory pathway for cellular stress proteins. J Biol Chem 280: 23349–23355, 2005.[Abstract/Free Full Text]
  27. Lancaster GI, Moller K, Nielsen B, Secher NH, Febbraio MA, Nybo L. Exercise induces the release of heat shock protein 72 from the human brain in vivo. Cell Stress Chaperones 9: 276–280, 2004.[CrossRef][Web of Science][Medline]
  28. Lim CL, Mackinnon LT. The roles of exercise-induced immune system disturbances in the pathology of heat stroke: the dual pathway model of heat stroke. Sports Med 36: 39–64, 2006.[CrossRef][Web of Science][Medline]
  29. Maloyan A, Palmon A, Horowitz M. Heat acclimation increases the basal HSP72 level and alters its production dynamics during heat stress. Am J Physiol Regul Integr Comp Physiol 276: R1506–R1515, 1999.[Abstract/Free Full Text]
  30. Maron M, Wagner J, Horvath S. Thermoregulatory responses during competitive marathon running. J Appl Physiol 42: 909–914, 1977.[Abstract/Free Full Text]
  31. Marshall HC, Ferguson RA, Nimmo MA. Human resting extracellular heat shock protein 72 concentration decreases during the initial adaptation to exercise in a hot, humid environment. Cell Stress Chaperones 11: 129–134, 2006.[CrossRef][Web of Science][Medline]
  32. Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol 12: 991–1045, 1994.[Web of Science][Medline]
  33. McClung JP, Montain SJ, Cheuvront SN, He J, Hasday JD, Singh S. Differential responsiveness to heat shock protein (HSP) induction following heat acclimation (Abstract). FASEB J 20: A1247, 2006.[Free Full Text]
  34. Moseley PL. Exercise, heat and thermotolerance: molecular mechanisms. In: Perspectives in Exercise Science and Sports Medicine: Exercise, Heat, and Thermoregulation, edited by Gisolfi C, Lamb D, and Nadel E. Traverse City, MI: Cooper, 2001, p. 305–334.
  35. Moseley PL. Heat shock proteins and heat adaptation of the whole organism. J Appl Physiol 83: 1413–1417, 1997.[Abstract/Free Full Text]
  36. Noakes TD. Exercise in the heat: old ideas, new dogmas. Int Sport Med J 7: 58–74, 2006 [www.ismj.com].
  37. Peake JM, Suzuki K, Hordern M, Wilson G, Nosaka K, Coombes JS. Plasma cytokine changes in relation to exercise intensity and muscle damage. Eur J Appl Physiol 95: 514–521, 2005.[CrossRef][Web of Science][Medline]
  38. Pittet JF, Lee H, Morabito D, Howard MB, Welch WJ, Mackersie RC. Serum levels of Hsp 72 measured early after trauma correlate with survival. J Trauma 52: 611–617, 2002.[Web of Science][Medline]
  39. Ramanathan NL. A new weighting system for mean surface temperature of the human body. J Appl Physiol 19: 531–533, 1964.[Abstract/Free Full Text]
  40. Rhind SG, Gannon GA, Shephard RJ, Buguet A, Shek PN, Radomski MW. Cytokine induction during exertional hyperthermia is abolished by core temperature clamping: neuroendocrine regulatory mechanisms. Int J Hyperthermia 20: 503–516, 2004.[CrossRef][Web of Science][Medline]
  41. Ryan AJ. Heat stroke and endotoxemia: sensitization or tolerance to endotoxins? In: Perspectives in Exercise Science and Sports Medicine: Exercise, Heat and Thermoregulation, edited by Gisolfi CV, Lamb DR, and Nadel E. Traverse City, MI: Cooper, 2001, version 6, p. 335–380.
  42. Shin Y, Oh J, Sohn H, Bae J, Lee M, Lee J, Yang H, Min Y, Song H, Ko K, Matsumoto T. Expression of exercise-induced HSP 70 in long-distance runner's leukocytes. J Therm Biol 29: 769–774, 2004.[CrossRef][Web of Science]
  43. Steensberg A, Toft AD, Bruunsgaard H, Sandmand M, Halkjaer-Kristensen J, Pedersen BK. Strenuous exercise decreases the percentage of type 1 T cells in the circulation. J Appl Physiol 91: 1708–1712, 2001.[Abstract/Free Full Text]
  44. Walsh RC, Koukoulas I, Garnham A, Moseley PL, Hargreaves M, Febbraio MA. Exercise increases serum Hsp72 in humans. Cell Stress Chaperones 6: 386–393, 2001.[CrossRef][Web of Science][Medline]
  45. Whitham M, Halson SL, Lancaster GI, Gleeson M, Jeukendrup AE, Blannin AK. Leukocyte heat shock protein expression before and after intensified training. Int J Sports Med 25: 522–527, 2004.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
Y. Ogura, H. Naito, S. Akin, N. Ichinoseki-Sekine, M. Kurosaka, R. Kakigi, T. Sugiura, S. K. Powers, S. Katamoto, and H. A. Demirel
Elevation of body temperature is an essential factor for exercise-increased extracellular heat shock protein 72 level in rat plasma
Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2008; 294(5): R1600 - R1607.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
J. P. McClung, J. D. Hasday, J.-r. He, S. J. Montain, S. N. Cheuvront, M. N. Sawka, and I. S. Singh
Exercise-heat acclimation in humans alters baseline levels and ex vivo heat inducibility of HSP72 and HSP90 in peripheral blood mononuclear cells
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2008; 294(1): R185 - R191.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
103/4/1196    most recent
00242.2007v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamada, P. M.
Right arrow Articles by Schneider, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamada, P. M.
Right arrow Articles by Schneider, S. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.