People who exercise regularly report fewer upper respiratory tract infections (URTI) than their sedentary peers (12). Data from three randomized studies support this viewpoint that near-daily physical activity reduces the number of days with sickness (14,19,20). In these studies, women in the exercise groups walked briskly 35–45 min, 5 d·wk−1, for 12–15 wk during the winter/spring or fall, while the control groups remained physically inactive. Walkers experienced about half the days with URTI symptoms of the sedentary controls. Epidemiologic research also supports a reduction in URTI risk for those engaging in regular versus irregular moderate-to-vigorous physical activity (7), in contrast to an elevated URTI risk in athletes training heavily or competing in marathon race events (16).
The exercise link with lowered URTI risk suggests that positive immune changes occur during physical activity. In contrast to animal studies (3,6), randomized exercise training human studies have failed to demonstrate that immune function measured in the resting state is altered after 12–15 wk of near-daily moderate physical activity (14,19,20). For these reasons, we have hypothesized that the acute immune changes occurring during and shortly after the moderate exercise bout itself may explain the lowered risk of URTI (12). In other words, host protection against pathogens is improved through a summation effect of improved immunosurveillance that occurs acutely with each moderate exercise bout. This link, however, has not yet been established and will require periodic immune testing before and after walking in a large cohort of individuals randomized to moderate exercise and sedentary groups and followed for URTI incidence.
In comparison with high-intensity, long-duration exercise (10,11,15), a dearth of published information exists on the acute immune response to 30 min of brisk walking (or similar activity), a commonly prescribed exercise (12,17,18). Several studies suggest positive immune changes during moderate activity (8,9,18,20), but only a few different types of immune measures have been conducted, and a resting control condition has often been lacking. Exercise assist devices such as walking poles or hand weights are often used by walkers (13), but no information exists regarding the effect of using exercise assist devices on immune responses to walking. The purpose of this study was to a measure a wide range of immune changes in response to walking for 30 min with or without an exercise assist device compared with sitting in female subjects accustomed to walking. We hypothesized that walking with the exercise assist device would increase perturbations in several components of immunity measured during walking alone.
Fifteen female subjects between the ages of 20 and 55 were recruited. Recruitment into the study was contingent upon several characteristics: body mass index (BMI) of less than 35 kg·m−2, healthy (with no known disease or medication use), and accustomed to walking (history of walking at least 20–30 min, 2–7× wk−1, for the previous 3 months or longer). Informed consent was obtained from each subject, and the experimental procedures were in accordance with the policy statements of the institutional review board of Appalachian State University.
In the first test session, subjects filled out a medical/health questionnaire and were tested for body composition and maximal aerobic fitness. Maximal aerobic power (V̇2max) and other metabolic measures were determined during the Bruce graded maximal treadmill protocol with the MedGraphics CPX Express metabolic system (MedGraphics Corporation, St. Paul, MN) (2). Submaximal and maximal heart rates were measured using a chest heart rate monitor (Polar Electro Inc., Woodbury, NY). V̇2max was achieved when subjects achieved two of three criteria: respiratory exchange ratio (RER) ≥1.1, oxygen consumption increased <2 mL·kg−1·min−1 during the final minute, or the subject’s heart rate increased to within 10 beats of the age-predicted maximum. Body composition was estimated using a three-site skinfold test and equation from Jackson et al. (5).
Subjects next reported back to the Human Performance Laboratory for three test sessions separated by 1–2 wk; these were randomly assigned and conducted from 3:00 to 5:00 p.m. to control for diurnal variation. During the test sessions, subjects sat in the laboratory for 30 min, or walked for 30 min with or without an exercise assist device called the BODY BAT® Aerobic Exerciser (13). The walking speed was the same for both walking sessions and adjusted to correspond to approximately 60% V̇2max while walking without the exercise assist device. The actual workload achieved during walking without the exercise assist device was 58.5 ± 0.5% V̇2max compared with 64.7 ± 1.2% V̇2max with the exercise assist device. In all three test sessions, subjects were 3–4 h postprandial and sat quietly for 15 min before giving the first blood and saliva samples. Additional blood and saliva samples were collected immediately postexercise/sitting, and 1 h postexercise/sitting.
The BODY BAT® Aerobic Exerciser is an exercise assist device resembling a pair of baseball bats seamlessly joined together end to end with the handles and the end knobs facing in opposite directions (13). The BODY BAT® comes in three sizes and is based on the distance from the sternum to the hand with the arm held straight to the side at shoulder height. Subjects held the implement with both hands while simultaneously swinging it across their bodies in a side-to-side pendulum motion in synchronism with each step when walking (i.e., the implement is swung to the right as the right foot moves forward and swung to the left as the left foot moves forward). The BODY BAT has a symmetrical configuration and is an inherently balanced mass when held in the prescribed manner. The implement is completely illustrated in U.S. Design Patent No. 372,748. The swing rate equaled the step rate, and was self-selected by the subjects. Expired gases, heart rate, and RPE were recorded every 5 min, and the values averaged to represent the metabolic demands of the 30-min walk with or without the BODY BAT.
Blood cell counts.
Blood samples were drawn from an antecubital vein with subjects in the seated position. Routine complete blood counts (CBC) were performed by a clinical hematology laboratory. Other blood samples were centrifuged in sodium heparin tubes, and plasma was aliquoted and then stored at −80°C. Plasma cortisol concentration was assayed in duplicate using a competitive enzyme immunoassay kit provided by R&D Systems, Inc. (Minneapolis, MN) with a minimum detectable cortisol concentration of 1.6 nmol·L−1. The intraassay coefficient of variation (CV) was less than 10%. Plasma volume changes were estimated using the method of Dill and Costill (4).
The proportions of T cells (CD3+), B cells (CD19+), natural killer (NK) cells (CD3−CD16+ CD56+), and activated T-cells (CD69+) were determined in whole blood preparations and absolute numbers calculated using CBC data to allow group comparisons of lymphocyte subset concentrations. Lymphocyte phenotyping was accomplished by two-color fluorescent labeling of cell surface antigens with mouse antihuman monoclonal antibodies conjugated to fluoresceinisothiocyanate (FITC) and phycoerythrin (PE) using Simultest™ reagents (Becton Dickinson Immunocytometry Systems, San Jose, CA). For immunophenotyping, 60-μL aliquots of heparinized whole blood from each sample were added to five wells of a 96-well plate; 60 μL of CD3/CD16 + CD56 (BD, cat. no. 340042), CD3/CD19 (BD, cat. no. 349211), CD45/CD14 (BD, cat. no. 340040), CD69 (BD, cat. no. 341652), or γ1γ2a isotype control (BD, cat. no. 340041) were added to the wells, and the samples were incubated in the dark for 20 min on ice with orbital shaking (170 rpm). The cells were then washed with 130 μL of phosphate-buffered saline (PBS), and the plate was centrifuged for 5 min at 1500 × g. The red blood cells were lysed by adding 200 μL of 1X FACS lysing solution (BD, cat. no. 349202) for 10 min in the dark, and the cells were pelleted by centrifugation. The lysing step was repeated, the cells washed in 200 μL of PBS, and the resulting cell pellet was fixed in 200 μL of Cytofix™ buffer (BD, cat. no. 554655). Samples were kept at 4°C in the dark until analyzed by flow cytometry at the Immunogenetics Laboratory with the VA–San Diego Health Care System.
PHA-induced lymphocyte proliferation.
The mitogenic response of lymphocytes was determined in whole blood culture using phytohemagglutinin (PHA) at optimal and suboptimal doses previously determined by titration experiments. Heparinized venous blood was diluted 1:10 with complete media consisting of RPMI-1640 supplemented with 5% heat-inactivated fetal bovine serum, penicillin, streptomycin, L-glutamine, β2-mercaptoethanol, and Mito+™ Serum Extender (BD Labware, cat. no. 355006). PHA was prepared in RPMI-1640 media, at a concentration of 1 mg·mL−1, and then further diluted with complete media to the optimal and suboptimal working concentrations (12 μg·mL−1 and 6 μg·mL−1, respectively). A 100-μL aliquot of the diluted blood was dispensed into each of triplicate wells of a 96-well flat-bottom microtiter plate. To each well, 100 μL of the appropriate mitogen dose was added. Control wells received complete media instead of mitogen. After a 72-h incubation at 37°C and 5% CO2, the cells were pulsed with 1 μCi of thymidine (methyl)-3H (New England Nuclear, Boston, MA) prepared with RPMI-1640. After pulsing, cells were incubated for an additional 4 h before harvesting. The radionucleotide incorporation was assessed by liquid scintillation counting with the results expressed as experimental minus control counts per minute (cpm). The intraassay CV was less than 10%. PHA-induced proliferation expressed on a “per T-cell” basis was calculated by dividing the cpm data by the number of T-cells in the assay wells.
Total plasma concentrations of interleukin-1 receptor antagonist (IL-1ra) and interleukin-6 (IL-6) were determined using quantitative sandwich ELISA kits provided by R&D Systems, Inc. (Minneapolis, MN). All samples and provided standards were analyzed in duplicate. A standard curve was constructed using standards provided in the kits, and the cytokine concentrations were determined from the standard curves using linear regression analysis. The assays were a two-step “sandwich” enzyme immunoassay in which samples and standards were incubated in a 96-well microtiter plate coated with polyclonal antibodies for the test cytokine as the capture antibody. After the appropriate incubation time, the wells were washed, and a second detection antibody conjugated to either alkaline phosphatase (IL-6 high sensitivity) or horseradish peroxidase (IL-1ra) was added. The plates were incubated and washed, and the amount of bound enzyme-labeled detection antibody was measured by adding a chromogenic substrate. The plates were then read at the appropriate wavelength. The minimum detectable concentration was <22 pg·mL−1 for IL-1ra and <0.039 pg·mL−1 for IL-6. The intraassay CV was less than 5% for both cytokines.
Unstimulated saliva was collected for 4 min by expectoration into 15-mL plastic, sterilized vials. Participants were urged to pass as much saliva as possible into the vials during the 4-min timed session. The saliva samples were stored at −80°C until analysis. Saliva volume was measured to the nearest 0.1 mL, and saliva total protein was quantified using the Coomassie® protein assay reagent, a modification of the Bradford (1) Coomassie® dye-binding colorimetric method. Salivary IgA (sIgA) was measured by enzyme linked immunosorbent assay according to the procedures of the Hunter Immunology Unit (Royal Newcastle Hospital, Newcastle, NSW, Australia) (personal communication). The data were expressed as concentration of sIgA (μg·mL−1), concentration of sIgA relative to total protein concentration (μg·mg−1), and sIgA secretion rate (μg·min−1). The intraassay CV was less than 10% for sIgA concentration.
Results are expressed as mean ± SE. The performance data in Table 2 comparing walking with or without the exercise assist device were compared using paired t-tests. All other data were analyzed using a 3 × 3 repeated measures ANOVA with two between-conditions factors (sitting compared with walking with or without an exercise assist device) and one within-subject factor (time of blood sample collection). Differences in change from preexercise between conditions were analyzed using paired t-tests with trends noted as P < 0.05 and significance set at P < 0.01 (to adjust for multiple comparisons).
Subject characteristics are summarized in Table 1. These data indicate that the subjects as a group were of average aerobic fitness and body composition for their age group. All subjects indicated that they were accustomed to walking.
The 30-min performance data while walking with or without the exercise assist device are summarized in Table 2. The treadmill speed while walking with or without the exercise assist device was 6.3 ± 0.4 km·h−1. Walking with the exercise assist device increased oxygen consumption 11 ± 2% and heart rate 8 ± 2 beats·min−1 (P < 0.001) but did not alter the respiratory exchange ratio or the rating of perceived exertion.
Changes in blood counts of total leukocytes, leukocyte subsets, and lymphocyte subsets are summarized in Table 3 and Figures 1 and 2. Plasma volume change was less than 1%, and did not differ between conditions. The pattern of increase in blood counts for total leukocytes, neutrophils, lymphocytes, monocytes, natural killer cells (CD3−CD16+CD56+), and activated T lymphocytes (CD69+) differed significantly when comparing walking with sitting, but no differences were found between walking with or without the exercise assist device. Changes in blood counts for total leukocytes were modest during walking with (19%) and without (24%) the exercise assist device, and were short-lived, with no significant differences compared with sitting at 1 h postexercise (Fig. 1) (time and interaction effects, P < 0.001 and P = 0.002, respectively). The increase in total leukocytes immediately postexercise was primarily neutrophils (51–66%), with about 40% of the increase comprised of lymphocytes and 5–8% monocytes. The increase in total lymphocytes immediately postexercise was primarily natural killer cells (54–60%), with 25–43% of the increase comprised of T cells. Natural killer cells increased 60–63% above preexercise levels after exercise, but then fell 17–35% 1 h postexercise compared with little change in the sitting condition (Fig. 2) (time and interaction effects, P < 0.001).
The pattern of increase in PHA-induced lymphocyte proliferation (12 μg·mL−1) differed significantly when comparing walking with sitting, but no differences were found between walking with or without the exercise assist device (Fig. 3) (time and interaction effects, P < 0.001). When adjusted for changes in T cell counts, the exercise-induced increase in PHA-induced lymphocyte proliferation (18–26%) compared with the sitting condition was removed (time and interaction effects, P = 0.282 and P = 0.162, respectively). No significant interaction effect was measured for PHA-induced lymphocyte proliferation at a mitogen concentration of and 6 μg·mL−1 (P = 0.107) (data not shown).
No significant time or interaction effects were measured for salivary IgA concentration or secretion rate (Table 4) or IL-1ra (Table 5). Plasma cortisol concentration dropped over time for all three conditions, and the pattern of decrease did not differ between conditions (interaction effect, P = 0.797) (Table 5). The pattern of increase in plasma IL-6 concentration differed significantly when comparing walking with sitting, but no differences were found between walking with or without the exercise assist device (Fig. 4) (time and interaction effects, P < 0.001 and P = 0.008, respectively).
These data indicate that the use of an exercise assist device increased oxygen consumption 11% during walking but did not alter the pattern of change in several components of immunity measured during walking alone in comparison with sitting. Walking for 30 min at 60–65% V̇2max with or without an exercise device compared with sitting caused modest and short-lived increases in several leukocyte subsets, primarily neutrophils, and natural killer cells. Mitogen-induced lymphocyte proliferation was elevated immediately after walking compared with sitting, but this increase was due to the influx of T lymphocytes into the blood compartment. No increase in the plasma concentration of the anti-inflammatory cytokine IL-1ra was measured during walking compared with sitting, and plasma IL-6 concentration was increased slightly but significantly. Walking did not cause an increase in cortisol. Subjects in our study were trained walkers, and these data may not be applicable to sedentary individuals.
Walking briskly with the exercise device only increased oxygen consumption 11%, and this was not enough to alter the immune response. In a previous study, we showed that when walking 4.0 and 4.8 km·h−1, use of the BODY BAT increased the heart rate by about 20 beats·min−1, and augmented oxygen consumption 32–42% (13). In the present study, subjects walked more briskly (∼ 6.3 km·h−1), and this resulted in a smaller metabolic difference between walking with or without the BODY BAT.
In contrast to moderate exercise such as 30 min of brisk walking, intense and prolonged exertion causes large increases in blood leukocyte, neutrophils, and monocytes counts, plasma cortisol concentration, plasma IL-6 and IL-1ra concentrations, and large postexercise decreases in sIgA secretion rate, natural killer cell counts and activity, and PHA-induced lymphocyte proliferation (10,11,15). These immune changes, measured after heavy exertion, have generally been interpreted as immunosuppressive, but a link with increased URTI risk has not yet been consistently established (10,11).
In the same way, the mild and transient immune perturbations measured after walking have been described as beneficial to immunosurveillance (3,6,8,9,18,21), but a link with decreased URTI risk has not yet been measured, and this was not the aim of the present study. Some data suggest that the benefits of moderate exercise on immunity may be related just as much to the absence of negative changes (e.g., large increases in stress hormones, cytokines, and apoptosis) as to improved immune recirculation. For example, Mooren et al. (8) showed that lymphocyte apoptosis occurs after intensive and exhaustive exercise but not during moderate-intensity exercise.
The acute immune changes after moderate compared with intensive and prolonged exercise are widely disparate, and further research is warranted to determine whether or not URTI risk can be linked to exercise workload in humans. In mice, Davis et al. (3) has shown that 1 h of moderate exercise per day for 6 d in a row improved macrophage resistance to herpes simplex virus type 1 and reduced URTI risk.
In summary, walking with or without an exercise assist device at 60–65% V̇2max compared with sitting was associated with modest and transient changes in blood cell counts (especially neutrophils and natural killer cells), PHA-induced lymphocyte proliferation, and plasma IL-6 concentration, and no changes in plasma cortisol concentration, salivary IgA output, or plasma IL-1ra concentration. These changes contrast sharply with the much larger perturbations we have reported following prolonged and intensive exercise such as marathon running (10,11,15). Further research is warranted to establish a link between the acute immune changes occurring during each walking bout with improved host protection against pathogens and lowered URTI risk.
This study was funded by a grant from Body Bat, Inc., P.O. Box 66720, St. Petersburg, FL 33736-6720.
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