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J Appl Physiol 98: 663-671, 2005. First published September 24, 2004; doi:10.1152/japplphysiol.00510.2004
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Suppression of viral specific primary T-cell response following intense physical exercise in young but not old mice

Zoher F. Kapasi,1 Michael L. McRae,1 and Rafi Ahmed2

1Division of Physical Therapy, Department of Rehabilitation Medicine, and 2Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia

Submitted 14 May 2004 ; accepted in final form 22 September 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Intense exercise to exhaustion leads to increased susceptibility and severity of infections. T cells play an essential role in control of viral infections. Whereas immune suppression is considered as a likely mechanism for exhaustive exercise-induced susceptibility to infection, we know little about viral-specific T-cell response following exhaustive exercise in young or old mice. In this study, one group of female young (10–12 wk) and old (22–24 mo) C57BL/6 mice was exposed to a single bout of intense exercise to exhaustion and immediately infected with lymphocytic choriomeningitis virus (LCMV). Eight days later, at the peak of expansion phase of T-cell response, we used tetramers of MHC class I molecules containing viral peptides to directly visualize antigen-specific CD8 T cells and a sensitive functional assay measuring interferon-{gamma} production at the single-cell level to quantitate the CD8 and CD4 T-cell response. To evaluate the impact of intense exercise during both the initiation and evolution of the expansion phase of the T-cell response, a second group of young and old mice continued their daily bouts of intense exercise to exhaustion over the next 8 days. Our data show that, in young mice, LCMV infection following exhaustive exercise leads to suppression of LCMV-specific CD8 and CD4 T-cell responses, and this suppression effect occurs at the initiation of the expansion phase of viral-specific T cells. However, in old mice, unlike young mice, exhaustive exercise does not cause suppression of LCMV-specific T-cell responses.

stress; lymphocytic choriomeningitis virus; exhaustive exercise; T cells

T CELLS PLAY AN ESSENTIAL role in control of viral infections (1). Potent CD8 T-cell responses are induced during infections of humans with human immunodeficiency virus, Epstein-Barr virus, and cytomegalovirus and in mice following infection with lymphocytic choriomeningitis (LCMV), vesicular stomatitis virus, vaccinia virus, influenza virus, and Sendai virus. These virus-specific CD8 T cells control viral infections by killing infected cells and by secreting INF-{gamma} and other cytokines (8, 27, 33, 34). CD4 T cells contribute to viral clearance (37) by producing interleukin-2, which facilitates CD8 T-cell activation and expansion, and by secreting INF-{gamma} and tumor necrosis factor to activate macrophages and inhibit viral replication. CD4 T cells are also responsible for helping B cells to generate neutralizing antibodies, which are important for reducing the viral load by inhibiting infection of cells and by opsonizing virus-infected cells. These antibodies also play a role in protecting against reinfection.

Intense exercise to exhaustion leads to increased susceptibility and severity of infections (14, 21, 26, 28, 30). Increased susceptibility and severity of infections following exhaustive exercise have been shown for polio virus (21) and Coxsackie B virus (14). Monkeys subjected to exhausting exercise during the incubation period of experimental poliomyelitis developed a higher incidence and more severe paralysis than controls (21). In mice infected with Coxsackie B virus, enforced swimming increased mortality from 5 to 50%, and virus replication in the heart was markedly augmented (14). Whereas immune suppression is considered as a likely mechanism for exhaustive exercise-induced susceptibility to infection, to date, there are no studies that have examined viral-specific T-cell response following exhaustive exercise. Because intense exercise to exhaustion has been shown to decrease in vitro responses to T- and B-cell mitogens, natural killer cell response, and T-helper-to-suppressor cell ratio and cytokine responses (6, 7, 19, 23, 29), we hypothesized that viral infection following intense exercise to exhaustion causes a decrease in viral-specific T-cell response.

Aging is associated with decline in both cell-mediated and humoral immunity (24), including viral-specific T-cell responses (17), and we hypothesized that exhaustive exercise would further suppress immune response. However, previously, we have shown that intense exercise to exhaustion did not suppress secondary antibody response to a protein antigen in old mice (16). Also, a 32-wk exercise intervention that caused fatigue did not bring about detrimental effects on immune parameters in the frail, elderly nursing home population (18). In this study, we examined whether exhaustive exercise suppressed viral-specific T-cell responses in old mice.

LCMV causes a natural infection in mice and provides a useful model for studying the interaction between a viral infection and host immunity (3). Intravenous or intraperitoneal injection of adult immunocompetent mice with the Armstrong strain of LCMV results in acute infection. After infection, the virus replicates rapidly in many tissues, such as the lung, liver, spleen, and lymph nodes. Levels of infectious virus peak ~3 days after the infection (35). By day 5, there is a rapid decline in virus titers, and the infection is completely resolved by day 8. Evaluation of the immune response shows that, around day 5, LCMV-specific cytotoxic T cells appear. The peak of CD8 and CD4 T-cell response occurs on days 8–9 of infection and falls off rapidly over the next 5–10 days. Synthesis of LCMV-specific antibody is detectable in the serum by day 4 after infection, reaches high levels between days 10 and 20, and is then maintained at high levels indefinitely (3).

Our data show that, in young mice, LCMV infection following exhaustive exercise leads to suppression of LCMV-specific CD8 and CD4 T-cell responses. In old mice, LCMV-specific CD8 and CD4 T-cell responses are significantly lower compared with those in young mice. However, unlike young mice, exhaustive exercise does not cause suppression of LCMV-specific T-cell responses in old mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Virus infection and mice.   Young (2–3 mo old) and old (22–24 mo old) female C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME) and NIA colony at Charles River Laboratories, respectively, and allowed to acclimate for at least 5 days in our vivarium before initiation of any experimental procedures. The cages were kept in a climate-controlled environment with 12-h light and dark cycles. All mice had continuous access to food and water. Mice were infected with 2 x 105 plaque-forming units (PFU) of LCMV Armstrong intraperitoneally (2). Virus stocks were grown and quantitated as described previously (2). Infectious LCMV was quantitated by plaque assay on Vero cell monolayers, as previously described (2). The study was approved by the Institutional Animal Care and Use Committee.

Intense exercise to exhaustion protocol.   To evaluate the impact of intense exercise during both the initiation and evolution phase of the primary response to LCMV infection, young (n = 18) and old mice (n = 18) were randomly assigned to one of the following three groups: control (no exercise, n = 6), one bout of exercise (n = 6), and seven bouts of exercise (n = 6). Young and old mice in the one bout of exercise group were immediately infected after the intense bout of exercise (day 0, see Table 1), and their response was evaluated on day 8 after infection. The one-bout group helped us to assess the effects of intense exercise on the initiation phase of primary response. Young and old mice in the seven bouts of exercise group were also immediately infected after the first bout of intense exercise but were then exposed every day to intense exercise until day 8. The seven-bout group helped us to assess the effects of intense exercise on the initiation and evolution phase of primary response. Note that exercise was stopped 48 h before mice were killed on day 8 to ensure that the changes detected in the primary response reflected the cumulative effect of multiple bouts of exercise and not acute changes in response to the last exercise session. Intense exercise consisted of running the mice on a Vitamaster Rhythm Walker Plus treadmill, modified for this experiment by the Emory University Medical Engineering Department. The treadmill was a manual human treadmill motorized to drive the treadmill belt and control for speed accuracy. The treadmill consisted of six lanes, separated by aluminum partitions. The treadmill belt formed the floor of the lanes, and the roof of the lanes consisted of hinged Plexiglas. The exercise sessions were always conducted at the end of the dark cycle of a 12:12-h light-dark cycle. For a given animal, an exercise session only occurred once in a 24-h period. For a given exercise session, each exercising mouse was randomly placed in a treadmill lane and allowed to groom for 5 min before exercise commenced. Only mice from the same group were placed on the treadmill during a given exercise session. The target speeds of 32 m/min for young mice (13) and 17 m/min for old mice (32), speeds corresponding to >90% maximum O2 consumption per age group, were achieved by gradually increasing the speed every minute. The animals started to run at the speed of 4 m/min, and the speed was increased by 4 m/min every minute until the mice reached their target speed. Young mice took 8 min and old mice took 5 min to reach their target speed. Each mouse continued to run at the target speed until exhaustion occurred. Exhaustion was the point in time when the mouse refused to run on the treadmill, even after two prods (gentle pushes) on the buttocks. The average time to exhaustion was 13.1 ± 4.6 min for young mice and 11.9 ± 7.2 min for old mice. Moreover, training adaptation did not occur for young and old mice (in the seven-bout group) between bouts of exercise conducted on days 0 and 6 (Table 1) because there was no difference in the average time to exhaustion for young mice on day 0 (13.4 ± 4.2 min) and day 6 (11.0 ± 5.4 min) and also for old mice on day 0 (15.2 ± 9.1 min) and day 6 (19.7 ± 14.2 min). At exhaustion, the mouse was removed from the treadmill. The duration of exercise for each mouse was measured and recorded. Immediately on removal from the treadmill after the first exercise session, mice were injected with the virus intraperitoneally. Similarly, control mice were infected immediately after exposure to the noise and vibration of the treadmill (see below).


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  		Table 1. Timeline of different interventions in young and old mice

 
The control group of young and old mice was placed in an empty container on top of the Plexiglas on the treadmill, allowed to groom for 5 min, and exposed to the vibratory and noise effects caused by progressing the treadmill speed to speeds and duration corresponding to the exercising mice. Note that the mice in the control and one-bout group were exposed to the noise and vibration of the treadmill at every session that the mice in the seven-bout group were exercising.

Death of the mice occurred on day 8 with an overdose of the anesthetic metofane, and blood and organs were collected for analysis.

Cytotoxic T lymphocyte assay.   Cytotoxicity was assessed in a 5-h 51Cr-release assay, as described previously (27). Briefly, single-cell suspensions of spleens, free of erythrocytes, were prepared in complete RPMI medium and tested for cytotoxicity on uninfected and LCMV-infected MC57 (H-2b) targets.

Antibodies and MHC tetramers.   All of the antibodies used in these experiments were from Pharmingen (San Diego, CA) and have been described previously (27). The construction and purification of the MHC class I LCMV tetramers Db-NP(396–404) and Db-GP(33–41) have been described (27).

Flow cytometry and fluorescence-activated cell sorting analysis.   Single-cell suspensions of spleen were prepared, and 106 cells were stained in phosphate-buffered saline containing 1% bovine serum albumin and 0.02% sodium azide [fluorescence-activated cell sorting (FACS) buffer] for 30 min at 4°C followed by three washes in FACS buffer. Samples were acquired on either a FACScan flow cytometer or FACSCalibur instrument (Becton Dickinson, San Jose, CA). The data were analyzed by using CELLQuest software (Becton Dickinson Immunocytometry Systems). The numbers of specific cell populations were calculated by multiplying the percentage of specific cell populations with the total number of spleen cells for each animal.

Intracellular IFN-{gamma} staining.   Spleen cells were cultured for 5 h in 96-well flat-bottomed plates (Costar, Cambridge, MA) at a concentration of 1 x 106 cells/well in a volume of 0.2 ml of complete medium supplemented with 1 µl/ml Brefeldin A (Golgistop, Pharmingen), either with or without cytotoxic T lymphocyte (CTL) epitope peptides. The peptides were used at a concentration of 0.1 µg/ml. After 5 h of culture, the cells were harvested, washed once in FACS buffer, and surface stained in FACS buffer with phycoerythrin-conjugated monoclonal rat anti-mouse CD8a (clone 53–6.7) antibody. After the unbound antibody was washed, cells were subjected to intracellular cytokine stain by using the Cytofix/Cytoperm kit, according to the manufacturer's instructions (Pharmingen). For intracellular IFN-{gamma} staining, we used FITC-conjugated monoclonal rat anti-mouse IFN-{gamma} antibody (clone XMG 1.2) and its isotype control antibody (rat IgG1) (Pharmingen).

Statistical analysis.   Data were analyzed by using commercial software (SPSS, Chicago, IL). Various immune parameters were compared among age group and exercise conditions by using a two-way ANOVA. The factors were age (young or old) and exercise condition (no exercise, one bout, and seven bouts). CTL activity was analyzed by using a three-way repeated-measures ANOVA [age, exercise condition, and effector-to-target cell ratios (E/T)], with E/T being the repeated measure. Tukey's honestly significant difference post hoc test was performed, as indicated, for significant ANOVA findings. All statistical tests were two-tailed, and a criterion probability value of ≤0.05 was used.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Effects of exhaustive exercise on primary CD8 T-cell response and viral clearance in young and old mice.   To evaluate the effects of exhaustive exercise on immune response to acute LCMV infection in young and old mice, we first studied the development of primary CD8 T-cell response to LCMV in young and old mice following the exhaustive exercise. To assess CD8 T-cell activation, cells were double-stained for CD8 and CD44, an adhesion molecule whose expression goes up in activated T cells. Representative FACS analyses of the spleen cells from young and old mice are shown in Fig. 1A. Note that, as previously reported, the percentage of CD8CD44hi T cells is greater in uninfected old mice compared with uninfected young mice (17) (Fig. 1B). By day 8 post-LCMV infection, young and old mice show a significant increase in the percentage of CD8 T cells expressing the activated (CD44hi) phenotype; however, the percentage of CD8 T cells that are CD44hi is now greater in infected young mice compared with infected old mice and is reflected in greater numbers of activated splenic CD8 T cells in young mice compared with old mice (Fig. 1C). As reported previously, the expansion in the number of activated CD8 T cells in spleens of young mice is greater than that in old mice (17). Interestingly, neither single nor multiple bouts of exhaustive exercise had an effect on the percentage or numbers of activated splenic CD8 T cells in young or old mice. Clearly, activation of splenic CD8 T cells is not affected by exhaustive exercise in young and old mice.



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  		Fig. 1. CD8 T-cell activation in young and old mice following intense exercise to exhaustion. Young (2–3 mo old) and old (22–24 mo old) mice were infected intravenously with 2 x 105 plaque-forming units (PFU) of the Armstrong strain of LCMV following exercise or no exercise intervention. Spleen cells from uninfected and day 8 postinfection young and old mice were stained with anti-CD8 and anti-CD44. A: representative flow cytometric analysis for CD8 T cells. Numbers indicate the percentage of CD44hi or lo CD8 T cells. Percentage (B) and number (C) of CD44hi CD8 T cells are shown. No significant differences were found in the percentage or numbers of activated splenic CD8 T cells within young or old mice following single or multiple bouts of intense exercise. Values are means ± SD.

 
Next we quantitated the effect of exhaustive exercise on the size of the primary virus-specific CD8 T-cell response in young and old mice. C57BL/6 (H-2b) young and old mice underwent exhaustive exercise or control condition and were infected with LCMV, and, on day 8 of the primary response, the frequency of CD8 T cells specific for the different LCMV CTL epitopes was determined by means of MHC class I tetramer binding and intracellular cytokine staining (functional assay). We did not see a difference in frequency of Ag-specific CD8 T cells detected by MHC class I tetramer staining or intracellular cytokine staining for young or old mice (data not shown), suggesting that cells detected by MHC class I tetramer staining are also functional cells. However, we found significant differences in the size of the virus-specific response between young and old mice following exhaustive exercise. Infection following a single bout of exhaustive exercise caused a significant suppression of virus-specific CD8 T-cell response in young mice for the two immunodominant epitopes, NP396–404 and GP33–41/GP34–41 (Fig. 2A), and the subdominant epitopes, GP276–286, NP205–212, and GP92–101 (Fig. 2B). Moreover, young mice subjected to additional bouts of exhaustive exercise during the evolution of the expansion phase showed a similar suppression of the response as young mice exposed to a single bout of exhaustive exercise for all of the LCMV epitopes (Fig. 2, A and B, significant age x exercise interaction effect, P < 0.05, for all epitopes). Clearly, in young mice, additional bouts of exhaustive exercise did not cause further suppression of the virus-specific CD8 T-cell response beyond that caused by the first bout of exhaustive exercise just before infection. Overall, there was a 6- to 10-fold suppression in the numbers of virus-specific CD8 T cells in young mice exposed to single or multiple bouts of exhaustive exercise (Fig. 2, C and D, significant age x exercise interaction effect, P < 0.05, for all epitopes). In contrast to young mice, old mice exposed to single or multiple bouts of exhaustive exercise did not show suppression of virus-specific CD8 T-cell response for any of the immunodominant or subdominant epitopes (Fig. 2, A–D). Aging is accompanied by a decline in virus-specific responses (17), and here we show that exhaustive exercise does not cause further suppression of the already diminished CD8 T-cell response. A representative set of actual data along with specificity controls is shown in Fig. 2E. Spleen cells from LCMV-infected young and old mice made no IFN-{gamma} in the absence of stimulation. In addition, spleen cells from uninfected young and old C57BL/6 mice did not produce any IFN-{gamma} after stimulation with any of the peptides (data not shown).



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  		Fig. 2. Frequency and number of virus-specific CD8 T cells during primary lymphocytic choriomeningitis (LCMV) infection in young and old mice following intense exercise to exhaustion. Spleen cells from young (2–3 mo old) and old (22–24 mo old) C57BL/6 mice on day 8 post-LCMV infection were cultured in vitro for 5 h, either with or without the indicated MHC class I-restricted LCMV peptides, surface stained with anti-CD8, and intracellularly stained with anti-IFN-{gamma}. Data are presented as the percentage (A and B) and number (C and D) of CD8 T cells that make IFN-{gamma} on stimulation with each of the indicated epitopes. E: representative flow cytometric analysis. The numbers shown in the plot indicate the percentage of CD8 T cells that are positive for the intracellular IFN-{gamma} stain. Values are means ± SD. * Significant difference within young mice between no exercise and 1- and 7-bout groups, P < 0.05.

 
We next performed cytotoxicity assays to compare the direct ex vivo CTL responses in both young and old mice following the exhaustive exercise. A three-way repeated-measures ANOVA showed a significant age x exercise condition x E/T interaction effect (P = 0.001). As shown in Fig. 3A, and as previously reported, the CTL activity, on day 8 postinfection, was higher in young mice compared with old mice at all E/T. Single or multiple bouts of exhaustive exercise cause significant suppression of CTL activity in young mice. In contrast to young mice, exhaustive exercise did not cause suppression of CTL activity in old mice.



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  		Fig. 3. Direct ex vivo cytotoxic T lymphocyte activity and level of virus in serum and spleen of young and old mice following intense exercise to exhaustion. A: young (2–3 mo old) and old (22–24 mo old) female C57BL/6 mice were infected intravenously with 2 x 105 PFU of the Armstrong strain of LCMV, and direct ex vivo cytotoxic T lymphocyte activity in the spleens 8 days postinfection was measured by 5-h 51Cr-release assay. Percent specific killing of infected targets [(%51Cr release from LCMV-infected targets) – (%51Cr release from uninfected targets)] is shown for the indicated effector-to-target cell ratios. Values are means ± SD. * Significant difference within young mice between no exercise and 1- and 7-bout groups, P < 0.05. B: virus titers were checked 8 days after infection. Each value represents viral titer in serum or spleen of an individual animal. No significant differences were found in the viral titers in serum or spleen within young or old mice following single or multiple bouts of intense exercise.

 
Finally, we examined the kinetics of viral clearance in young and old mice following exhaustive exercise. Young mice clear LCMV virus by day 8 postinfection (27). In old mice, LCMV clearance in the early stages of infection (until day 3 postinfection) is delayed compared with that in young infected mice such that not all old mice clear infection by day 8 postinfection (17). The age x exercise interaction and the main effect of exercise intervention were not significant for viral titers in spleen and serum; however, the main effect of age was significant for viral titers in spleen (P = 0.01) but not serum (P = 0.08). Despite suppression of virus-specific CD8 T-cell response, young mice, exposed to single or multiple bouts of exhaustive exercise, cleared virus from serum and spleen by day 8 postinfection. There was no difference in the viral titers between old mice exposed to single or multiple bouts of exhaustive exercise and control old mice (Fig. 3B). Moreover, the immune response was not different between old mice that cleared the virus and old mice that did not clear the virus by day 8 in control, single-bout, and multiple-bout groups (data not shown). LCMV is a noncytocidal virus, and intraperitoneal injection of young and old mice with 2 x 105 PFU of Armstrong strain of LCMV causes the host immune response to quickly control the spread of the virus, and thus there is no significant tissue damage (3). We did not see any illness response in young and old mice exposed to control, single bouts, or multiple bouts of exercise.

Effects of exhaustive exercise on primary CD4 T-cell and antibody response in young and old mice.   We next investigated whether exhaustive exercise also caused suppression of virus-specific CD4 T-cell response in young mice with no effect on virus-specific CD4 T-cell response in old mice. The LCMV-specific CD4 response was also characterized by intracellular staining for IFN-{gamma} (following restimulation with NP309–328 and GP61–80). Infection following a single bout of exhaustive exercise caused a significant suppression of virus-specific CD4 T-cell response against epitopes NP309–328 and GP61–80 in young mice (Fig. 4A, significant age x exercise interaction effect, P = 0.0002). Moreover, young mice subjected to additional bouts of exhaustive exercise during the evolution of the expansion phase showed a similar suppression of the virus-specific CD4 T-cell response against epitopes NP309–328 and GP61–80 as young mice exposed to a single bout of exhaustive exercise (Fig. 4A). Overall, there was two- to threefold suppression in the numbers of virus-specific CD4 T cells in young mice exposed to single or multiple bouts of exhaustive exercise (Fig. 4B, significant age x exercise interaction effect, P = 0.03). In contrast to young mice, old mice exposed to single or multiple bouts of exhaustive exercise did not show suppression of virus-specific CD4 T-cell response against epitopes NP309–328 and GP61–80 (Fig. 4, A and B). Thus similar to the virus-specific CD8 T-cell response, exhaustive exercise also caused suppression of virus-specific CD4 T-cell response in young mice with no effect on virus-specific CD4 T-cell response in old mice.



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  		Fig. 4. Frequency and number of virus-specific CD4 T cells and antibody response during primary LCMV infection in young and old mice following intense exercise to exhaustion. Spleen cells from young (2–3 mo old) and old (22–24 mo old) C57BL/6 mice on day 8 post-LCMV infection were cultured in vitro for 5 h either with or without the MHC class II-restricted LCMV peptides (NP309–328 and GP61–80) and surface stained with anti-CD4 and intracellularly stained with anti-IFN-{alpha}. Data are presented as the percentage (A) and number (B) of CD4 T cells that make IFN-{alpha} on stimulation with both of the peptides (NP309–328 and GP61–80). C: LCMV-specific antibody response. Serum from young and old mice was taken on day 8 post-LCMV infection, and the level of LCMV-specific IgG antibodies in the serum was measured by ELISA. Values are means ± SD. * Significant difference within young mice between no exercise and 1- and 7-bout groups, P < 0.05. {dagger}Significant difference within young mice between no exercise and one-bout group, P < 0.05.

 
Finally, we examined the anti-LCMV-specific antibody response. LCMV-specific antibodies are detectable in the serum by day 4 after infection and reach high levels between days 10 and 20 (3). The age x exercise interaction and the main effect of exercise intervention were not significant; however, the main effect of age was significant (P = 0.00001) for serum anti-LCMV antibody response. Although there was a decrease in the number of viral specific-CD4 T cells following intense exercise to exhaustion in young mice, the residual CD4 T cells were adequate in generating anti-LCMV-specific antibody response. Moreover, there was no suppression of anti-LCMV-specific antibody response on day 8 following infection in young mice exposed to single or multiple bouts of intense exercise to exhaustion. It is possible that suppression of anti-LCMV antibody response following exhaustive exercise would have been detectable at the peak of anti-LCMV antibody response between days 10 and 20 following LCMV infection. Furthermore, in contrast to our data, chronic activation of the stress response by forced treadmill running has been shown to suppress antigen-specific IgM during primary and secondary antibody response in young Sprague-Dawley rats (25). With aging, there is suppression of antibody response, and old mice in the control group had diminished anti-LCMV antibody response compared with young mice in the control group (P < 0.05). However, similar to the young mice, old mice exposed to single or multiple bouts of intense exercise to exhaustion did not show suppression of anti-LCMV-specific antibody response compared with old mice in the control group (Fig. 4C).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In this study, we show for the first time that intense exercise to exhaustion causes suppression of viral-specific T-cell response and may, in part, be responsible for the increased susceptibility and severity of infections in individuals who engage in exhaustive exercise. In young mice, infection following a single bout of intense exercise to exhaustion caused significant suppression of virus-specific CD8 and CD4 T-cell response, and additional bouts of exhaustive exercise do not cause further suppression of the virus-specific T-cell response beyond that caused by the first bout of exhaustive exercise. Our data indicate that intense exercise to exhaustion may have a suppression effect on the early events related to the expansion of viral-specific T cells during the primary response. The early events in the expansion phase of viral-specific T-cell response include recruitment of naive T cells in the immune response and receiving appropriate antigenic and costimulatory signals to become activated and committed to clonal expansion. During the first 24 h of stimulation, CD8 and CD4 T cells prepare for clonal expansion and increase in size, but no cell division is observed. Soon after, CD8 T-cell division commences at a rapid rate (~6–8 h per cell division), whereas CD4 T-cell division is typically delayed for another 12–24 h (36–48 h after the initial stimulus) and then occurs at a slightly slower rate (~10 h per cell division) (reviewed in Ref. 15). Intense exercise to exhaustion in rodents has been associated with reactive oxygen species (4, 22) or glucocorticoid-mediated (9) lymphocyte apoptosis within 24 h following the exercise and may reduce the availability of viral-specific naive T-cell precursors for clonal expansion. As naive T cells receive appropriate antigenic and costimulatory signals, they become activated and committed to clonal expansion. The possibility also exists that the stress associated with intense exercise to exhaustion could affect the antigenicity (virulence) of LCMV, thereby reducing the numbers of antigen-specific T cells to LCMV. However, mice exposed to cold or isolation stress resulted in increased neuroinvasiveness and virulence of an attenuated variant of West Nile virus (5). Moreover, intense exercise to exhaustion did not cause suppression of antibody response to LCMV infection, suggesting that the stress did not alter the antigenicity of LCMV virus. Finally, the effects of stress on costimulation have not been investigated in the context of viral-specific T-cell responses. Costimulatory requirements seem to be different for the activation of naive CD8 and CD4 T cells (36). LCMV-specific CD8 T-cell responses are efficiently induced in mice deficient in costimulatory molecules CD40L, CD28, or OX-40, whereas CD4 T-cell responses were severely compromised. In contrast, 41BB-deficient mice showed normal LCMV-specific CD4 T-cell response and a slight reduction in CD8 T-cell response (36). Because, naive CD8 T cells are minimally dependent on costimulation for activation following acute LCMV infection, we believe that stress caused by intense exercise to exhaustion may play a minimal role, if any, in the diminished generation of LCMV-specific CD8 T cells. Clearly, these are testable hypotheses that can be addressed in future studies.

It is interesting that, despite a significant decrease in the numbers of LCMV-specific CD8 and CD4 T cells, we did not observe a decrease in the numbers of CD44-positive CD8 or CD4 T cells. CD44 expression is upregulated on lymphocytes following TcR engagement by antigen and also during inflammation (31). Inflammation associated with intense running to exhaustion may be responsible for the upregulation of CD44 on T cells.

Intense exercise is considered immunosuppressive. However, this general hypothesis is not true for old individuals. Previously, we have shown that intense exercise does not cause suppression of antibody response in old mice (16) and a variety of immune parameters in frail elderly (18), and here we show that intense exercise causes no suppression of viral-specific T-cell response in old mice. Clearly, in old mice, LCMV-specific CD8 and CD4 T-cell responses are significantly lower compared with those in young mice (17). However, unlike young mice, single or multiple bouts of exhaustive exercise do not cause suppression of LCMV-specific T-cell responses in old mice. The data from young mice exposed to single or multiple bouts of intense exercise to exhaustion show that intense exercise to exhaustion may have a suppression effect on the early events related to the expansion of viral-specific T cells during the primary response. In old mice, there is increased representation of T cells expressing determinants typical of memory cells with a concomitant decline in the proportion of T cells expressing determinants typical of naive cells (10, 11, 20). Thus reactive oxygen species (4, 22) or glucocorticoid-mediated (9) lymphocyte apoptosis that occurs within 24 h following the exercise may not further reduce the availability of viral-specific naive T-cell precursors for clonal expansion in old mice. Consequently, there was no difference in viral-specific T cells between control and exercised old mice. Alternatively, the possibility exists that intense exercise to exhaustion caused a decrease in viral-specific T-cell responses in old mice that were too small for our assays to detect. However, use of MHC class I tetramers complexed with defined viral peptide epitopes and measurement of the production of cytokines in response to stimulation with synthetic peptides employed in this study has allowed CD8 T-cell responses to be quantitated with great accuracy and are some of the most sensitive assays available to detect viral-specific T-cell responses (27). Finally, in contrast to viral-specific T-cell response, we did not investigate innate immune responses to viral infection, and it is possible that intense exercise to exhaustion may suppress innate immune responses in old mice.

Typically, physical therapists and other healthcare workers do not prescribe intense exercise to exhaustion in elderly people. However, due to the decrease in cardiopulmonary reserve with aging (12), exhaustion can occur in elderly patients while performing simple tasks, such as getting out of bed and walking to the bathroom. In a hospital or nursing home setting, exhausting activities may predispose the elderly patient to infection. However, based on this study and other studies in old animals (16) and humans (18), intense exercise may not have a detrimental effect on immune function in the elderly. Thus relatively intense exercise programs may be prescribed that could maximize cardiopulmonary and musculoskeletal function without impairing immune function in elderly people.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by National Institutes of Health Research Grants AG-17754 (to Z. F. Kapasi) and AI-30048 (to R. Ahmed).


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Patryce Mahar, Kaja Madhavi-Krishna, and Safronia Jenkins for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. F. Kapasi, Dept. of Rehabilitation Medicine, Division of Physical Therapy, Emory Univ. School of Medicine, 1441 Clifton Road, N.E., Atlanta, GA 30322 (E-mail: zkapasi{at}emory.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
 

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