The Effects of Intense Physical Exercise on Secondary Antibody Response in Young and Old Mice.

Conditioning exercise is an important intervention used by physical therapists in the treatment of patients with many clinical problems. The effects of exercise on the cardiopulmonary and musculoskeletal systems have been well characterized.[1] The effects of exercise on the immune system, however, are only beginning to be explored. Based largely on data from young animal and human subjects, intense physical exercise is believed to suppress several aspects of immune function, including susceptibility to infections, natural killer cell function, and immunoglobulin production.[2,3] The effect of intense exercise on immune function of old subjects remains to be investigated. We do not believe that physical therapists typically prescribe intense exercise to exhaustion in elderly people. However, due to decreases in cardiopulmonary reserve with aging,[4] exhaustion can occur in elderly patients while performing simple tasks such as getting out of bed and walking to the bathroom. Exhausting activities may predispose the elderly patient to infection due to immunosuppression.

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 ([VO.sub.2]max).[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

Design

The secondary antibody response in young and old mice was examined using a randomized, multiple-observation, control group design. To evaluate the impact of intense exercise during both the initiation and evolution phase of the secondary antibody response, mice in each of 2 age groups (ie, young and old) were randomly assigned to 1 of 3 exercise conditions: (1) control (no exercise), (2) 1 bout of intense exercise, or (3) 9 bouts of intense exercise. Immune response was measured 31 days after the initial HSA injection (primary antibody response -- 1), immediately prior to the booster HSA injection and immediately after 1 bout of exercise (primary antibody response -- 2), and 10 days after booster HSA injection (secondary antibody response) (Tab. 1).

Table 1. Time Line of Different Interventions in Young and Old Mice

Days                 No Exercise (Control)

After 5 days of      First injection of HSA(a)
  acclimatization

After 31 days        Bleed mice to assess primary
  of first             antibody response -- 1
  injection
  (24-36 hours
  before day 0)

Day 0                No exercise
                     Bleed mice to assess primary
                       antibody response -- 2
                     Booster injection of HAS

Day 1                No exercise

Day 2                No exercise

Day 3                No exercise

Day 4                No exercise

Day 5                No exercise

Day 6                No exercise

Day 7                No exercise

Day 8                No exercise

Day 9                No exercise

Day 10               Bleed mice to assess secondary
                       antibody response

Days                 1 Bout of Exercise

After 5 days of      First injection of HSA
  acclimatization

After 31 days        Bleed mice to assess primary
  of first             antibody response -- 1
  injection
  (24-36 hours
  before day 0)

Day 0                1 bout of exercise
                     Bleed mice to assess primary
                       antibody response -- 2
                     Booster injection of HSA

Day 1                No exercise

Day 2                No exercise

Day 3                No exercise

Day 4                No exercise

Day 5                No exercise

Day 6                No exercise

Day 7                No exercise

Day 8                No exercise

Day 9                No exercise

Day 10               Bleed mice to assess secondary
                       antibody response

Days                 9 Bouts of Exercise

After 5 days of      First injection of HSA
  acclimatization

After 31 days        Bleed mice to assess primary
  of first             antibody response -- 1
  injection
  (24-36 hours
  before day 0)

Day 0                1 bout of exercise
                     Bleed mice to assess primary
                       antibody response -- 2
                     Booster injection of HSA

Day 1                1 bout of exercise

Day 2                1 bout of exercise

Day 3                1 bout of exercise

Day 4                1 bout of exercise

Day 5                1 bout of exercise

Day 6                1 bout of exercise

Day 7                1 bout of exercise

Day 8                1 bout of exercise

Day 9                No exercise

Day 10               Bleed mice to assess secondary
                       antibody response

(a) HSA=human serum albumin.

Subjects

Animals were chosen by nonprobability, convenience sampling. The sample consisted of 30 young (6-8 weeks) and 30 old (22-24 months), syngeneic female C57BL/6 mice.(*) The inbred C57BL/6 mice (longevity of 794-818 days in males and 827-878 days in females) were chosen for our study because they have been well characterized for immunological and survival traits. Female mice were selected because they have a longer life span than male mice. Six- to eight-week-old mice correspond to teenaged to young adult humans, and 22- to 24-month-old mice correspond to humans in their early to mid 70s. Each group of mice was divided into 3 subgroups by random assignment to exercise condition. Each of the subgroups (n=10) was separated into 2 cages for housing, with 5 mice per cage, for a total of 12 cages. The mice were given a standard laboratory rodent diet and water ad libitum.

Instrumentation

Intense exercise consisted of running the mice on a Vitamaster Rhythm Walker Plus treadmill,([dagger]) 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 ([+ or -] 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.([double dagger])

Anti-HSA antibody levels were measured by assaying the serum using an enzyme-linked immunosorbent assay (ELISA) microplate reader.([sections]) 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 Na[HCO.sub.3], pH 9.6), at a concentration of 50 [micro]g/mL, was adsorbed to the surface of polystyrene 96-well, flat-bottom plates (50 [micro]L/well) by incubation overnight at 4 [degrees] 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').sub.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 [micro]L of substrate solution (1 mg of p-nitrophenyl phosphate per milliliter of substrate buffer, 48 mL of diethanolamine, 24.5 mg Mg[Cl.sub.2] [multiplied by] 6[H.sub.2], 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.([sections]) 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 [micro]g/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 [degrees] 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 nonexercising 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 mice[19] and 17 m/min for old mice,[20] speeds corresponding to greater than 90% of [VO.sub.2]max 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 [VO.sub.2]max. 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:

1. Among young mice, data obtained from 8 animals were excluded from analysis because the blood sample volumes drawn were insufficient for ELISA analysis.

2. Among old mice, data obtained from 12 animals were excluded because they died prior to the beginning of the exercise intervention (n=2; none of the animals died after the initiation of the exercise intervention), blood sample volumes drawn were insufficient for ELISA analysis (n=8), or the animals exhibited tumors on post-mortem analysis (n=2). Note that C57BL/6 mice exhibit a 30% to 40% incidence of spontaneous neoplasms (lymphomas) after about 22 months of age.[21] At the time mice were sacrificed, those with gross pathology (neoplasms) were excluded from the study. The number of mice remaining in each group after the exclusions is shown in Table 2.

Table 2. Frequency (n), Mean, Standard Deviation, and Minimum to Maximum Values of Primary and Secondary Anti-Human Serum Albumin Antibodies in Mice

                                   Primary Antibody
                                   Response - 1

                                   [bar]X [+ or -] SD
Group                          n   (Minimum-Maximum)

Young mice

  No exercise                  8   103.9 [+ or -] 88.5
                                       (25.1-274.7)

  One bout of exercise         6    61.8 [+ or -] 47.2
                                       (12.0-110.9)

  Multiple bouts of exercise   8    33.2 [+ or -] 8.3
                                       (24.4-48.7)

Old mice

  No exercise                  7    38.5 [+ or -] 29.9
                                       (13.8-98.0)

  One bout of exercise         4   116.5 [+ or -] 152.5
                                       (27.3-344.3)

  Multible bouts of exercise   7    19.9 [+ or -] 13.8
                                       (6.8-47.1)

                                   Primary Antibody
                                   Response - 2

                                   [bar]X [+ or -] SD
Group                          n   (Minimum-Maximum)

Young mice

  No exercise                  8   135.8 [+ or -] 129.7
                                       (27.1-375.3)

  One bout of exercise         6    87.2 [+ or -] 53.9
                                       (13.1-158.4)

  Multiple bouts of exercise   8    30.8 [+ or -] 5.6
                                       (23.5-38.4)

Old mice

  No exercise                  7    33.9 [+ or -] 27.8
                                       (8.8-79.0)

  One bout of exercise         4   118.7 [+ or -] 151.6
                                       (20.8-341.9)

  Multible bouts of exercise   7    17.7 [+ or -] 10.7
                                       (7.0-35.7)

                                   Secondary Antibody
                                   Response

                                   [bar]X [+ or -] SD
Group                          n   (Minimum-Maximum)

Young mice

  No exercise                  8   712.6 [+ or -] 226.0
                                       (276.9-998.0)

  One bout of exercise         6   668.1 [+ or -] 523.8
                                       (247.2-1,708.0)

  Multiple bouts of exercise   8   516.0 [+ or -] 273.1
                                       (151.6-936.1)

Old mice

  No exercise                  7   241.6 [+ or -] 282.6
                                       (36.3-827.0)

  One bout of exercise         4   968.5 [+ or -] 591.2
                                       (514.8-1,834.5)

  Multible bouts of exercise   7   250.0 [+ or -] 212.3
                                       (7.2-567.2)

The mean, standard deviation, and minimum and maximum values were calculated for anti-HSA antibody levels for the young mice subgroups (no exercise, 1 bout of intense exercise, 9 bouts of intense exercise) and the old mice subgroups (no exercise, 1 bout of intense exercise, 9 bouts of intense exercise). Normality of distribution and homogeneity of variance were tested per antibody sample for each subgroup, prior to statistical analysis, using the Shapiro-Wilk test and the sphericity test, respectively.

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 [is less than or equal to] .05 was used. Power of the statistical tests was .54 to .92[22] using 4 to 8 mice per group. The effect size of .80 was based on secondary antibody response after exposure to HSA.[7,8]

Results

Anti-HSA antibody level mean values for each group at each time of measurement are presented in Table 2. Comparisons of anti-HSA antibody levels among times of measurement, age groups, and exercise conditions were statistically significant (P [is less than or equal to] .05) for the main effects of exercise condition and time of measurement and for all exercise condition interactions (Tab. 3).

Table 3. Three-Way, Repeated-Measures Analysis of Variance Comparing the Amount of Anti-Human Serum Albumin Antibodies in the Blood Among Age, Exercise Condition, and Measurement Time

Source                        df      SS          MS

Age                            1     103,612     103,612
Exercise condition             2     649,710     324,855
Time of measurement            2   6,125,708   3,062,854
Age x exercise condition       2     513,935     256,967
Age x time of measurement      2     104,444      52,222
Exercise condition x
  time of measurement          4     582,680     145,670
Age x exercise condition x
  time of measurement          4     427,462     106,865

Error
  Between                     34   2,088,290      61,420
  Within                      68   2,310,510      33,978

  Total                      102   4,398,800      43,125

Source                        F        P

Age                           1.7   .2027
Exercise condition            5.3   .0100(a)
Time of measurement          90.1   .0001(a)
Age x exercise condition      4.2   .0237(a)
Age x time of measurement     1.5   .2224
Exercise condition x
  time of measurement         4.3   .0037(a)
Age x exercise condition x
  time of measurement         3.2   .0197(a)

Error
  Between
  Within

  Total

(a) Statistical significance at P [is less than or equal to] .05.

Primary Antibody Responses 1 and 2

Within young and old mice among different exercise conditions. Anti-HSA antibody levels in young mice between primary antibody responses 1 and 2 were not different (P [is greater than] .05) among exercise conditions (Tab. 4, Figure). Similarly, anti-HSA antibody levels in old mice between primary antibody responses 1 and 2 were not different (P [is greater than] .05) among exercise conditions (Tab. 5, Figure).

[TABULAR DATA 4-5 NOT REPRODUCIBLE IN ASCII]

Between young and old mice among different exercise conditions. Anti-HSA antibody levels during primary antibody responses 1 and 2 were not different (P [is greater than] .05) between young and old mice in any exercise condition (Tab. 4, Figure).

Secondary Antibody Response

Within young and old mice among different exercise conditions. Secondary anti-HSA antibody levels in young mice were not different (P [is greater than] .05) among exercise conditions (Tab. 4, Figure). However, secondary anti-HSA antibody levels in old mice were greater (P [is less than or equal to] .05) in the 1-bout exercise group (968.5 [+ or -] 591.2 [micro]g/mL) than in the control (nonexercising) group (241.6 [+ or -] 282.6 [micro]g/mL) and the 9-bout exercise group (250 [+ or -] 212.3 [micro]g/mL) (Tabs. 2 and 5, Figure).

Between young and old mice among different exercise conditions. Secondary anti-HSA antibody levels were greater (P [is less than or equal to] .05) in young mice (712.6 [+ or -] 226 [micro]g/mL) than in old mice (241.6 [+ or -] 282.6 [micro]g/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 [+ or -] 591.2 [micro]g/mL) were greater than secondary anti-HSA antibody levels in young mice in the 9-bout exercise group (516 [+ or -] 273.1 [micro]g/mL) and equivalent to those levels in the young mice receiving no exercise (712.6 [+ or -] 226 [micro]g/mL) or 1 bout of exercise (668.1 [+ or -] 523.8 [micro]g/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 [is less than or equal to] .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

The purpose of our study was to determine the effect of intense exercise on the secondary antibody response in young and old mice. Secondary antibody response appears to be age dependent, because young nonexercising animals had higher anti-HSA antibody levels than old nonexercising animals, confirming the results of previous studies[8,9] showing an age-related decline in secondary antibody response. Secondary antibody response also appears to be exercise dependent, because old mice that received 1 bout of intense exercise demonstrated increased anti-HSA antibody levels compared with old nonexercising mice or old mice that received 9 bouts of intense exercise. Moreover, the old mice that received the booster immunization after undergoing 1 bout of intense exercise attained levels of anti-HSA antibodies comparable to those seen in the young mice (Figure).

[Figure ILLUSTRATION OMITTED]

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 [VO.sub.2]max than at 70% of [VO.sub.2]max.[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 [Beta]-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.

Conclusions

Our findings provide initial information for examining the effects of intense exercise on antibody response in young and old mice. Old mice, when subjected to one bout of intense exercise, appear to have an enhanced secondary antibody response to an antigen. However, with multiple bouts of intense exercise, secondary antibody response remained at levels comparable to those of old nonexercising mice. Intense exercise does not seem to adversely affect the secondary antibody response in old mice, although intense exercise may suppress the immune system in young mice. The physical therapist's goals for patients, in our opinion, should include maximizing cardiopulmonary and musculoskeletal function, without impairing immune function. Clearly, if intense exercise is shown not to cause detrimental effects on aspects of immune function in elderly humans, then physical therapists can prescribe and implement exercise programs without adversely affecting immune function. The relationship between intense exercise and changes in immune system needs to be established in humans and may be meaningful when developing exercise programs for elderly people.

(*) Charles River Laboratories, 251 Ballardvale St, Wilmington, MA 01887.

([dagger]) Road Master Corp, 4501 Old Troup Hwy, Tyler, TX 75707.

([double dagger]) Rohm & Haas Co, Independence Mall W, Philadelphia, PA 19105.

([sections]) Molecular Devices Corp, 1311 Orleans Ave, Sunnyvale, CA 94089-1136.

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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) ([e-mail]). 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.

Mr Joyner, Ms Lewis, Ms Schwartz, and Ms Townsend were graduate students, Division of Physical Therapy, Department of Rehabilitation Medicine, Emory University, during this study, which was undertaken in partial fulfillment of the requirements for their Master of Physical Therapy degrees.

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.

This article was submitted September 13, 1999, and was accepted June 29, 2000.