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Research Reports |
ZF Kapasi, PT, PhD, is Assistant Professor, Division of Physical Therapy, Department of Rehabilitation Medicine, Emory University School of Medicine, 1441 Clifton Rd NE, Atlanta, GA 30322 (USA) (zkapasi{at}emory.edu). Address all correspondence to Dr Kapasi
PA Catlin, PT, EdD, is Professor and Director, Division of Physical Therapy, Department of Rehabilitation Medicine, Emory University School of Medicine
DR Joyner, PT, is Physical Therapist, Tooele Valley Healthcare System, Tooele, Utah
ML Lewis, PT, is Staff II Physical Therapist, Washington Hospital Center, Washington, DC
AL Schwartz, PT, is Physical Therapist, Center for Rehabilitation Medicine, Emory University Hospital
EL Townsend, PT, is Research Specialist, NICHD Study of Early Child Care and Youth Development, University of Virginia, Charlottesville, Va
Submitted September 13, 1999;
Accepted June 29, 2000
 | Abstract |
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Key Words: Intense exercise training • Old mice • Secondary antibody response
| Introduction |
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 TopAbstract Introduction Materials and MethodResultsDiscussionConclusionsReferences |
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Aging is associated with decline in both humoral and cellular immunity.5 Humoral immune responses are characterized by antibody production.6 Primary antibody response occurs following an initial exposure to an antigen (foreign substance). Subsequent exposure to the same antigen leads to a stronger secondary antibody response that results in long-lasting immunity. The secondary antibody response reaches a high level in mice at about 10 to 14 days following immunization.7,8 In old mice, the secondary antibody response is more profoundly depressed in comparison with the primary antibody response, resulting in a decreased capacity of old individuals to maintain long-term humoral immunity.8–10 Antibody production occurs as a result of interaction between the retained antigen on follicular dendritic cells, B lymphocytes, and T-helper lymphocytes.11 With aging, follicular dendritic cells atrophy and decrease in their capacity to trap antigens.10 A reduction in the number of memory B cells generated in old mice occurs after immunization, and T cell function also declines with aging.5,12 In this way, all 3 of the major, cellular components involved in antibody production are affected by aging.
Intense exercise has been defined as exercising at a minimum of 80% of maximum oxygen consumption (
O2max).13 The number of lymphocytes in circulation increases during exercise but decreases below the normal levels for several hours after intense exercise.3 Decreased numbers of lymphocytes are associated with decreased lymphocyte responsiveness and antibody response to several antigens after intense exercise.3,14 The effects of intense exercise on secondary antibody response in old human or animal subjects have not been documented. Because aging is associated with a decline in immune function, including secondary antibody response,5 intense exercise in old subjects may further suppress the secondary antibody response. Repeated bouts of intense exercise also could be detrimental to cardiopulmonary function and could theoretically lead to serious health hazards in elderly subjects. To avoid the possibility of serious health hazards and control for nutritional and environmental factors that influence immune function,15–17 we chose an animal model for this study on the effect of intense exercise.
| Materials and Method |
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Instrumentation
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 as follows. The treadmill was a manual human treadmill that was motorized to drive the treadmill belt and control for speed accuracy. The treadmill speed was calibrated (±0.25 m/min) 3 times during the experiment, using an electronic calibrator placed on the treadmill that measured the speed in meters per minute. The treadmill consisted of 6 lanes separated by aluminum partitions, 5 of which were used during each exercise session. The treadmill belt formed the floor of the lanes, and the roof of the lanes consisted of hinged Plexiglas.
Anti-HSA antibody levels were measured by assaying the serum using the enzyme-linked immunosorbent assay (ELISA) microplate reader.
The antibody level measurements included all immunoglobulins in the blood against HSA. The ELISA microplate reader was calibrated by the manufacturer. For ELISA, HSA in carbonate buffer (0.05 M NaHCO3, pH 9.6), at a concentration of 50 µg/mL, was adsorbed to the surface of polystyrene 96-well, flat-bottom plates (50 µL/well) by incubation overnight at 4°C. The plates were emptied and washed 3 times with distilled water. One hundred microliters of 1% bovine serum albumin (BSA) was added to the wells.
After incubation for 30 minutes at room temperature, the plates were emptied. The antibody standard was a pooled sample of mouse anti-HSA antiserum containing 1 mg/mL of anti-HSA, as determined by quantitative precipitation. Fifty microliters of an antibody standard and of each serum sample was diluted 6 times from 1:250 to 1:32,000 in 1% BSA solution in phosphate-buffered saline (PBS) in triplicate wells for each dilution. The solutions were incubated for 1 hour at room temperature and then washed 3 times in distilled water, as described. Fifty microliters of 1:5,000 1% BSA/PBS diluted alkaline phosphatase-conjugated Rabbit F(ab')2 anti-mouse IgH (H+L) was added to each well and incubated for 1 hour at ambient temperature. The plates were washed 3 times with distilled water, and 100 µL of substrate solution (1 mg of p-nitrophenyl phosphate per milliliter of substrate buffer, 48 mL of diethanolamine, 24.5 mg MgCl2·6H2, 400 mL of distilled water, pH 9.8) was added to each well, resulting in a yellow-colored reaction.
Thirty minutes after the reaction began, the optical density of each well was read at 405 nm, using a calibrated Vmax kinetic microplate reader with Softmax software. The number of anti-HSA antibodies (Ig) was determined by comparing the optical density of the mouse's anti-HSA antibodies with the optical density of the known anti-HSA antibody standard and expressed in milligrams per milliliter. The values from each set of triplicate wells from a given serum sample were averaged to give the value of anti-HSA antibodies for that particular mouse.
Interrater reliability for exercise duration and for ELISA microplate reader of antibody levels was maintained as exact agreement of values obtained by concurrent, independent measurement of 2 investigators. Two raters recorded the time of exercise duration from a single stopwatch for each mouse at every exercise session. Similarly, 2 raters recorded ELISA readings from the computer printout.
Procedure
The sex of the mice was determined, and then they were randomly assigned to an exercise condition, divided into respective cages, tagged, and labeled. Because transportation is a clear stressor of animals requiring a period of adaptation and restoration to homeostasis,18 the mice were acclimatized in our vivarium for at least 5 days after arrival. An antigen injection, consisting of a solution of HSA (200 µg/mL) in the adjuvant 9% potassium aluminum sulfate, was given at 2 different times to initiate primary and secondary antibody responses, respectively (Tab. 1). Human serum albumin is a potent protein antigen known to initiate antibody responses in young and old mice.8 The initial injection of 0.1 mL of antigen solution was given subcutaneously in the nape of the neck. A 31-day waiting period for primary antibody response was allowed. The second administration of antigen was given as 4 injections of 0.05 mL of antigen solution subcutaneously in the dorsum of each foot (total=0.2 mL). This second series of injections was administered on day 0 after the exercise or control (no exercise) session.
Anti-HSA antibody levels were measured from blood taken at 3 different times during the study (Tab. 1). The first 2 samples of blood were taken from the tail 24 to 36 hours before day 0 (primary antibody response – 1) and 10 to 15 minutes after each group's session at day 0 (primary antibody response – 2). The third blood sample was taken 40 to 50 hours after day 8 to ensure that the changes detected in the secondary antibody response reflected the cumulative effect of multiple bouts of exercise and not acute changes in response to the last exercise session. After collection, the blood was centrifuged, and the serum was extracted and stored at –70°C in Eppendorf tubes for later ELISA analysis.
The 12 cages of animals were separated into 4 sets (2 sets of young mice and 2 sets of old mice), with each set containing one of each of the following exercise conditions: no exercise, 1 bout of intense exercise, and 9 bouts of intense exercise. The sets of mice underwent all blood sampling and exercise or control sessions 2 days apart to stagger the interventions.
During the appropriate session for each set at day 0, animals in the 1-bout and 9-bout exercise groups underwent 1 bout of intense exercise to exhaustion. Exhaustion was the point in time when the mouse refused to run on the treadmill, even after 2 prods (gentle pushes) on the buttocks. Animals in the control group experienced nonexercising conditions. The exercise protocol was conducted before the control protocol to establish the average exhaustion time (ie, 20 minutes). Ten to fifteen minutes following the exercise or control session at day 0, the second blood sample was taken from the tail vein, and then the booster injection of 0.05 mL of antigen solution was administered to each footpad. The rationale for giving booster immunization immediately following the exercise session was to evaluate any detrimental effects of intense exercise during the initiation phase of the secondary antibody response.
Because mice are nocturnal animals, the exercise sessions were always conducted in the dark cycle of a 12-hour light/dark cycle. For a given animal, an exercise session occurred only once in a 22-hour period. During the day 1 session, animals in the 9-bout exercise groups underwent 1 bout of intense exercise, and animals in the 1-bout exercise and control groups experienced nonexercising conditions. The procedure for the day 1 session was repeated every day for an additional 7 consecutive days, such that mice in the 9-bout exercise groups exercised a total of 9 days, mice in the 1-bout exercise groups experienced 1 day of exercise followed by non-exercising conditions for 8 days, and mice in the control groups experienced nonexercising conditions for a total of 9 days.
For a given exercise session, each exercising mouse was randomly placed in a treadmill lane and allowed to groom for 5 minutes before exercise commenced. Only mice from the same cage were placed on the treadmill during a given exercise session. The target speeds of 32 m/min for young mice19 and 17 m/min for old mice,20 speeds corresponding to greater than 90% of O2max per age group, were achieved by gradually increasing the speed every minute for 23 minutes. After 5 to 6 minutes of treadmill running, the mice were running at speeds corresponding to 80% of O2max. Each mouse continued to run at the target speed until exhaustion occurred. After exhaustion, the mouse was removed from the treadmill. The duration of exercise for each mouse was measured and recorded. For the nonexercising conditions, each mouse was placed, with cage members of its respective group (control or 1-bout exercise group), in an empty container on top of the Plexiglas on the treadmill, allowed to groom for 5 minutes, and exposed to the vibratory and noise effects caused by progressing the treadmill speed for 20 minutes, the average time to exhaustion.
The mice were sacrificed with an overdose of the anesthetic metofane 40 to 50 hours after the final session at day 8 (day 10 after the booster immunization), and the third blood sample (for analysis of secondary antibody response) was taken immediately intracardially and stored for later ELISA analysis.
Data Analysis
Some data obtained from the mice were not represented for one of the following reasons:
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The amount of anti-HSA antibodies in the blood was compared among age groups, exercise conditions, and times of measurement using a 3-way, repeated-measures analysis of variance (ANOVA). The factors were age (young or old), exercise condition (no exercise, 1 bout of intense exercise, 9 bouts of intense exercise), and time of measurement (primary antibody response – 1, primary antibody response – 2, and secondary antibody response); time of measurement was the repeated measure. If a difference from ANOVA testing was found, a Tukey Honestly Significant Difference (HSD) post hoc test was used, as appropriate. All statistical tests were 2-tailed, and a criterion probability value of 
.05 was used. Power of the statistical tests was .54 to .9222 using 4 to 8 mice per group. The effect size of .80 was based on secondary antibody response after exposure to HSA.7,8
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TopAbstractIntroductionMaterials and MethodResultsDiscussionConclusionsReferences |
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.05) for the main effects of exercise condition and time of measurement and for all exercise condition interactions (Tab. 3).
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Secondary Antibody Response
Within young and old mice among different exercise conditions.
Secondary anti-HSA antibody levels in young mice were not different (P>.05) among exercise conditions (Tab. 4, Figure). However, secondary anti-HSA antibody levels in old mice were greater (P .05) in the 1-bout exercise group (968.5±591.2 µg/mL) than in the control (nonexercising) group (241.6±282.6 µg/mL) and the 9-bout exercise group (250±212.3 µg/mL) (Tabs. 2 and 5, Figure).
Between young and old mice among different exercise conditions.
Secondary anti-HSA antibody levels were greater (P .05) in young mice (712.6±226 µg/mL) than in old mice (241.6±282.6 µg/mL) for the control groups, but not for the 1-bout and 9-bout exercise groups (Tabs. 2 and 4, Figure). Secondary anti-HSA antibody levels in old mice in the 1-bout exercise group (968.5±591.2 µg/mL) were greater than secondary anti-HSA antibody levels in young mice in the 9-bout exercise group (516±273.1 µg/mL) and equivalent to those levels in the young mice receiving no exercise (712.6±226 µg/mL) or 1 bout of exercise (668.1±523.8 µg/mL) (Tabs. 2 and 4, Figure).
Comparison of Secondary Antibody Response to Primary Antibody Response Within Young and Old Mice
Secondary anti-HSA antibody levels were greater (P .05) than anti-HSA levels during primary antibody responses 1 and 2 for all young mice and old mice in the 1-bout exercise groups, but not for old mice in the control and 9-bout exercise groups (Tabs. 4 and 5, Figure).
| Discussion |
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TopAbstractIntroductionMaterials and MethodResultsDiscussionConclusionsReferences |
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The secondary antibody response reaches a high level 10 to 14 days after exposure to an antigen.7,8 In our study, measurements of secondary antibody response were taken at the 10-day mark. Young mice in the 9-bout exercise group did not show decreased mean anti-HSA antibody levels after 10 days compared with young nonexercising mice and young mice in the 1-bout exercise group. We believe that the decrease might have become statistically significant if measurements had been taken at the 14-day mark.
Anti-HSA antibody levels in our study showed considerable minimum-to-maximum value ranges (Tab. 2), resulting in relatively high standard deviations for some groups. The variability reflected in the standard deviations of some groups was, generally, not due to 1 or 2 outliers in the groups, but rather was due to overall variability in antibody responses. The mean secondary anti-HSA antibody level in the old mice in the 1-bout exercise group (n=4), which was found to be greater than other secondary anti-HSA level means, included data from one mouse with more than twice the antibody level of other mice in the group. However, there was still a difference compared with other old mice during secondary antibody response with data from this mouse excluded. Our findings of high variability in antibody responses between mice are consistent with the literature.8
Secondary anti-HSA antibody levels were greater than anti-HSA levels during primary antibody responses 1 and 2. This effect occurred for old mice that received 1 bout of exercise and all young mice, but not for old nonexercising mice and old mice that received 9 bouts of exercise. The marked increase in antibody levels during secondary antibody response compared with primary antibody response is characteristic of immune response because memory B cells generated during primary antibody response allow enhanced antibody production with a second exposure to an antigen. The lesser effects in old mice in the control (nonexercising) and 9-bout exercise groups confirm the results of previous studies showing an age-related decline in secondary antibody response and may reflect age-related decline in function of all the major cells (follicular dendritic cells, B and T lymphocytes) that take part in the antibody response.9,10
One bout of intense exercise, just before the second exposure to an antigen, enhanced secondary anti-HSA antibody response in old mice, but not in young mice. The differential secondary anti-HSA antibody response in old mice compared with young mice is new information and is contrary to the expectation that intense exercise suppresses immune function. The mechanism underlying the increase in secondary anti-HSA antibody response in this group of old mice is not clear. Antibody production requires the coordination of B cells, T-helper cells, and follicular dendritic cells.11 Two-way communication is thought to occur between the neuroendocrine and immune systems, with both systems being capable of synthesizing and sharing many of the same messenger molecules (eg, stress hormones, cytokines).23 The neuroendocrine system can influence the antibody response both directly (via an influence upon B cell function) and indirectly (via actions on regulatory cells such as T-helper and follicular dendritic cells).23 Endogenous opioids enhance antibody response in moderate doses,24–26 but they suppress antibody response in high or low concentrations.26 The mechanism most likely involves opioid binding to specific receptors located on B and T cells.27 Serum concentrations of endogenous opioids increase in response to exercise and are greater at 80% of O2max than at 70% of O2max.28 Intense exercise may cause endorphin levels to rise beyond immuno-enhancing levels, actually resulting in suppression of B cell production of antibodies.
An endorphin-mediated suppression of antibody production with intense exercise, exacerbated by consecutive bouts of intense exercise, might explain the following trend: anti-HSA antibody levels in the young mice at the height of secondary antibody response were greatest in the control (nonexercising) group and least in the 9-bout exercise group; anti-HSA antibody levels of young mice in the 1-bout exercise group fell in the middle. Thus, the more bouts of intense exercise, the more immunosuppression in young animals. In our study, a decrease in secondary antibody production with increasing intense exercise was not evident in the old mice.
A single bout of exercise, just before booster (secondary) injection, enhanced secondary antibody response in the old mice. Intense exercise may not have caused elevation of opioid levels into immunosuppressive ranges in the old mice. Opioid peptide levels in the brains of rodents decrease with age.29 In older humans, a blunting of the circadian rhythm of beta-endorphin secretion also occurs. Although no age-related differences in endorphin production have been demonstrated with moderate exercise,30 intense exercise may elevate endorphin levels in young mice, but not in old mice. Thus, intense exercise may cause production of large amounts of endorphins in young mice, resulting in a decreased antibody response, and moderate amounts of endorphins in old mice, leading to an enhanced antibody response.
If the ability to produce endogenous opioids decreases with age, then repeated bouts of intense exercise may stress the systems of old mice such that even moderate levels of endogenous opioid production cannot be maintained during intense exercise. This phenomenon may explain why the old mice that received 9 bouts of intense exercise had lower anti-HSA antibody levels during secondary antibody response compared with mice that received 1 bout of intense exercise in our study.
Similar, dose-dependent changes in immune response have been reported for other neuroendocrine hormones.16 For example, B cells express ß-adrenergic receptors, and adrenergic innervation influences antibody synthesis.31 Norepinephrine enhances specific antibody synthesis in response to an antigen by increasing the number of antigen-specific B cells that differentiate into antibody-secreting plasma cells.31 Norepinephrine appears to mediate both suppression and stimulation of antibody synthesis, depending on the dosage and timing of administration in relation to antigen exposure.32 Exposure to norepinephrine early in the antibody response appears to enhance antibody synthesis, whereas later administration is associated with suppression of antibody synthesis. In old mice, immunization after one bout of intense exercise may result in the presence of norepinephrine during the early part of the antibody response, resulting in an enhancement of the antibody response. However, with multiple bouts of exercise, norepinephrine also would be present in the latter part of the antibody response, causing a suppression of the antibody response in both young and old mice. Because young mice are at the height of their antibody response capability, the presence of norepinephrine after one bout of intense exercise in young mice might not further increase the antibody response. Thus, the secondary antibody response of the young exercising mice was not different from that of the young nonexercising mice. Study of hormone-receptor antagonists may elucidate the mechanisms involved in exercise-mediated modulation of antibody responses to booster immunizations.
Primary antibody response against HSA was initiated prior to the exercise intervention. Clearly, anti-HSA antibody levels immediately following the one bout of intense exercise (primary antibody response – 2) were not different from the anti-HSA levels seen before exercise (primary antibody response – 1) in all groups. Previous findings showed that serum concentrations of immunoglobulins change slightly, if at all, after acute exercise.14 Furthermore, because anti-HSA antibodies are a small fraction of the total immunoglobulins present in circulation, no difference in the primary anti-HSA levels after one bout of exercise was observed in young and old animals.
Although we explored the potentially immunosuppressive effects of intense exercise, moderate exercise is believed to enhance immune function.2,33 Because immune function declines with advancing age,5 moderate exercise may be used as a therapeutic tool to enhance immune function in elderly people. Using a mouse model of viral infection, we are currently investigating the effects of moderate exercise on immune response in young and old mice.
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| Footnotes |
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All authors provided concept/research design, writing, data collection and analysis, project management, and consultation (including review of manuscript before submission). Dr Kapasi and Dr Catlin also provided subjects, fund procurement, facilities and equipment, and institutional liaisons.
This study was approved by the Institutional Animal Care and Use Committee of Emory University School of Medicine.
* Charles River Laboratories, 251 Ballardvale St, Wilmington, MA 01887. 
Road Master Corp, 4501 Old Troup Hwy, Tyler, TX 75707.
Rohm & Haas Co, Independence Mall W, Philadelphia, PA 19105.
Molecular Devices Corp, 1311 Orleans Ave, Sunnyvale, CA 94089-1136.
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  Z. F. Kapasi, M. L. McRae, and R. Ahmed Suppression of viral specific primary T-cell response following intense physical exercise in young but not old mice J Appl Physiol, February 1, 2005; 98(2): 663 - 671. [Abstract] [Full Text] [PDF]
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 Z. F Kapasi, P. A Catlin, M. A Adams, E. G Glass, B. W McDonald, and A. C Nancarrow Effect of Duration of a Moderate Exercise Program on Primary and Secondary Immune Responses in Mice Physical Therapy, July 1, 2003; 83(7): 638 - 647. [Abstract] [Full Text] [PDF]
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 Z. F. Kapasi, J. G. Ouslander, J. F. Schnelle, M. Kutner, and J. L. Fahey Effects of an Exercise Intervention on Immunologic Parameters in Frail Elderly Nursing Home Residents J Gerontol A Biol Sci Med Sci, July 1, 2003; 58(7): M636 - M643. [Abstract] [Full Text] [PDF]
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Z. F Kapasi, P. A Catlin, J. Beck, T. Roehling, and K. Smith The Role of Endogenous Opioids in Moderate Exercise Training-Induced Enhancement of the Secondary Antibody Response in Mice Physical Therapy, November 1, 2001; 81(11): 1801 - 1809. [Abstract] [Full Text] [PDF]
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