Download Exercise and Psychosocial Factors Modulate Immunity to Influenza

Journal of Gerontology: MEDICAL SCIENCES
2002, Vol. 57A, No. 9, M557–M562
Copyright 2002 by The Gerontological Society of America
Exercise and Psychosocial Factors Modulate Immunity
to Influenza Vaccine in Elderly Individuals
Marian L. Kohut,1 Megan M. Cooper,1 Michael S. Nickolaus,1 Dan R. Russell,2 and Joan E. Cunnick3
Departments of 1Health and Human Performance, 2Psychology, and 3Microbiology and Immunology, Iowa State
University, Ames.
Background. Decreases in immune responsiveness with age contribute to the increased incidence and severity of infectious disease among elderly adults. The immune response to immunization also decreases with advancing age. Lifestyle factors (exercise, diet) have been established to play an important role in immunosenescence, and the practice of
“healthy” behavior may minimize the age-associated decline of immune function. The objective of this study was to determine whether exercise, diet, and psychosocial factors were associated with altered immune response to influenza vaccine.
Methods. Adults aged 62 years and older were categorized into one of three groups: active (20 min vigorous exercise three or more times per week), moderately active (regular exercise but with less intensity, frequency, and/or duration), or sedentary (no exercise). Two weeks postimmunization, serum was frozen for antibody analysis, and peripheral
blood mononuclear cells (PBMC) were cultured in vitro with influenza vaccine to elicit antigen-specific responses (proliferation and cytokine [IL-2, IFN-, IL-10] production). Cytokines and antibody were measured by enzyme-linked immunosorbent assay.
Results. The results demonstrated that anti-influenza IgG and IgM were greater in active as compared with moderately
active or sedentary participants. PBMC proliferation was lowest in sedentary subjects. Perceived stress was a significant
predictor of IL-2. Greater optimism and social activity were associated with greater IL-10. Daily multivitamin intake
was significantly correlated with IL-2.
Conclusions. These results suggest that lifestyle factors including exercise may influence immune response to influenza immunization. The practice of regular, vigorous exercise was associated with enhanced immune response following influenza vaccination in older adults.
N
UMEROUS changes in immune responsiveness occur
with age including diminished T-cell proliferation, reduced IL-2 production, and decreased antibody production
(1–4). The age-associated decline of immune function may
contribute to an increased susceptibility to infectious disease. For example, in 1997, pneumonia/influenza was the
fifth leading cause of mortality among those 65 years of age
and older, but the 10th leading cause of death for adults
aged 25–44 (5). Influenza vaccine efficacy estimates for elderly adults range from 31% to 65% in preventing influenza
(6), whereas vaccine efficacy ranges from 68% to 88%
among younger adults (7,8). A significant number of older
adults may not be fully protected against influenza even
though they have been immunized. Upon infection with influenza virus, cell-mediated immune responses are important in clearing the infection; however, cell-mediated responses are also impaired among elderly people (9–12).
The degree of immune decline varies widely among older
adults and may be related to health behaviors. Nutrient intake and psychosocial factors can affect resistance to infection as well as the immune response to influenza immunization (13–16). Exercise may also alter immune function in
older adults (17–19). Nutritional and psychosocial factors
can modulate antigen-specific responses among older adults
(13,16,20); however, very little data exist regarding the potential effect of exercise on influenza-specific immunity.
Influenza is a significant cause of mortality, and vaccination does not always provide adequate protection in older
adults. Therefore, it is worthwhile to evaluate the potential
role of exercise as a method of enhancing the immune response to influenza immunization. The purpose of this study
was to test the hypothesis that exercise is associated with
enhanced influenza-specific immune responses following
immunization. Antibody (IgG, IgM) and cell-mediated immune responses (influenza-specific lymphocyte proliferation and IL-2, IL-10, IFN production) were evaluated in
older adults of differing physical activity levels.
METHODS
Subjects
Fifty-six adults aged 62 years and older participated in the
study. Individuals suffering from untreated chronic disease,
autoimmune disease, cancer, or any other disease known to
alter immunity were excluded. All subjects completed a
written informed consent, and the project was approved by
the Iowa State University Human Subjects review board.
Exercise Group Assignment
A phone interview was used to assess the level of physical activity. Subjects reported the type(s) of exercise they
participate in, described how often and how many minutes
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they typically exercise, and rated the intensity of exercise as
moderate or vigorous. Vigorous was defined as “exercise
that is intense enough to cause large increases in heart rate,
breathing, sweating, at a level that makes it somewhat difficult to carry on a normal conversation.” Moderate was defined as “exercise that is not intense enough to cause large
increases in heart rate, breathing, sweating, at a level that
makes it possible to carry on a normal conversation.” Participants were then classified as sedentary, moderately active,
or active. Active people (n 16; nine women and seven
men) participated in aerobic exercise at a “vigorous” intensity 20 minutes or longer three or more times per week for
the previous year. Moderately active people (n 25; 17
women, and 8 men) participated in aerobic exercise at a
“moderate” intensity one or more times per week over the
previous year, but did not meet criteria for inclusion in the
active group. Sedentary people (n 15; nine women, six
men) did not exercise or participated in aerobic exercise less
than once per week.
Diet and Psychosocial Questionnaires
Diet was assessed by the Block 1998 food frequency
questionnaire. The Perceived Stress Scale (PSS) (20) and
Life Orientation Test (LOT), a measure of optimism (21),
were administered. Subjects also reported the number of
different social activities they participated in on a monthly
basis.
Immunization
All participants were immunized during the first 2 weeks
of October, 1999. The trivalent Influenza Type A and B vaccine (Flushield, Wyeth-Ayerst, Marietta, PA) was administered to all subjects and contained 15 g HA of A/Beijing/
262/95 (H1N1), 15 g HA of A/Sydney/5/97 (H3N2), and
15 g HA of B/Yamanashi/166/98 (B/Beijing 184/93-like).
Peripheral Blood Mononuclear Cell (PBMC) Isolation
Blood was collected on day 14 postimmunization. PBMC
were isolated by centrifugation over Ficoll-Paque plus (Amershan Pharmacia Biotech, Piscataway, NJ) and were adjusted to 4 106 cells/ml in RPMI media (Life Technologies, Grand Island, NY) plus 5% fetal bovine serum
(Hyclone Laboratories, Logan, UT) and 100 U/ml penicillin, 100 g/ml streptomycin sulfate (Sigma Chemicals, St.
Louis, MO). Cells were incubated in vitro with influenza virus (Flushield) at 0.18 g/ml to stimulate antigen-specific
responses.
Proliferation Assay
To assess virus-specific lymphocyte proliferation, 100 l
of cells were plated in triplicate with or without inactivated
influenza virus for 5 days at 37C in 5% CO2. Ten microliters of 5 mg/ml of MTT (Sigma Chemical Co., St. Louis,
MO) was added for the last 4 hours of incubation, followed
by the addition of 0.04 N HCl in isopropanolol. Absorbance
was read at a dual wavelength of 570 and 630 nm.
Assay for Influenza-Specific Cytokine Production
Lymphocyte influenza-specific cytokine production (IL2,
IL-10, IFN-) was measured in cell supernatants. PBMC
were incubated with 0.18 g/ml of inactivated influenza virus in vitro at 37C, and 5% CO2 and cell supernatants were
collected. Enzyme-linked immunosorbent assay (ELISA)
kits were used to measure cytokine (PharMingen, San Diego, CA).
Anti-influenza Antibody ELISA
Influenza-specific antibody (IgG, IgM) in serum was determined by ELISA. Plates were coated overnight with 0.18
g/ml of influenza virus. Sera was diluted in phosphatebuffered saline–Tween containing 1M NaCl, added to plates
and incubated for several hours at 37C. Alkaline-phosphatase-conjugated mouse, antihuman IgM, and IgG were
added, and the plates were incubated overnight at 4C. The
substrate was added (p-nitrophenyl phosphate). Absorbance
at 405 nm was measured on a microplate reader.
Flow Cytometry
PBMC were incubated with fluorescein-conjugated antiCD3 and PE-conjugated anti-CD4 or PE-conjugated antiCD8. Fluorescein- or PE-conjugated mouse antihuman
IgG1 (isotype controls) were diluted in phosphate-buffered
saline–1% bovine serum albumin. Cells were washed and
fixed in 1% paraformaldehyde. Cells were analyzed for fluorescent intensity on a Coulter XL flow cytometer (Coulter
Instruments, Hialeah, FL).
Influenza Symptom Incidence
All subjects reported influenza-like symptoms (high fever, aches, and fatigue, followed by respiratory symptoms
such as sore throat, stuffy/runny nose, cough) that occurred
from October through April. Subjects were contacted every
2 weeks to check the occurrence of any illness symptoms.
Analysis and Interpretation
Statistical analysis was performed using SPSS software
(SPSS Inc., Chicago, IL). A three-way analysis of covariance (activity, gender, day of blood draw) was used for each
of the dependent variables. If there was not a significant
main effect of gender or day of blood draw, the data were
combined for the analysis of activity. Dietary factors and
psychosocial variables were assessed as covariates. Linear
regressions were used to determine whether specific dietary
factors or psychosocial factors were predictive of the immune response to influenza immunization. A regression
analysis was also performed to assess the significance of activity level net of psychosocial factors and nutrient intake. A
test for interactions and a test for correlation were done between activity level and each psychosocial and diet factor.
RESULTS
Nutrient Intake
Nutrient intake did not differ between the three activity
groups for any of the nutrients measured with the exception
of carbohydrate intake. Carbohydrate intake was lower (p .007) in the moderately active group than either the sedentary or the active group. A significantly greater kcal intake
and protein intake was found in men than women (p .05).
EXERCISE AND INFLUENZA VACCINE IN ELDERLY INDIVIDUALS
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Table 1. Subject Age and Psychosocial Characteristics
Group
Age (SD)
Social (SD)
LOT (SD)
PSS (SD)
Sedentary
Moderately Active
Active
71.5 7.1
0.7 6.3
71.9 5.2
3.9 1.6
4.1 1.7
4.6 2.2
49.7 9.0
50.5 7.6
50.4 7.7
20.5 8.7*
15.7 5.2
14.9 6.1
Note: LOT Life Orientation Test; PSS Perceived Stress Scale.
*p .05, Sedentary Moderately Active and Active.
Psychosocial Survey Results
Social activity scores and LOT scores did not differ significantly between activity groups (Table 1). However, active
and moderately active subjects reported lower levels of perceived stress than the sedentary group ( p .043, Table 1).
Influenza-Specific Proliferation
Exercise was associated with improved in vitro proliferation to the influenza vaccine (Figure 1). PBMC from moderately active and active groups had greater proliferation than
the sedentary group ( p .05).
Anti-influenza IgM and IgG
IgG titer in serum was higher in active participants ( p .014) compared to moderately active and sedentary participants (Figure 2). PSS score and zinc supplementation were
associated with IgG titer, and these factors were included in
the model as covariates. IgM anti-influenza titer was also
greater in active than in moderately active or sedentary participants ( p .047, Figure 3).
Anti-influenza IL-2, IL-10, IFN-
Influenza-specific cytokine production was measured at
the time of peak level in culture, and this time varied according to cytokine (see figure legend). Cytokine production was not altered by physical activity (Figure 4).
Figure 1. Influenza-stimulated proliferation in peripheral blood
mononuclear cells (PBMC) from sedentary, moderately active, and
active. PBMC were collected 2 weeks postimmunization and were
cultured with influenza vaccine for 5 days. Results are expressed as
mean SEM. Asterisks indicate significant differences (p .05)
compared with all other treatment groups. Background proliferation
was determined from the wells containing cells and media without virus. The background was subtracted from the average reading obtained in the wells incubated with virus.
Figure 2. Serum IgG anti-influenza antibody response in sedentary,
moderately active, and active at 1:25 and 1:125 dilution. Values are
means SEM. Asterisks indicate significant differences (p .05)
compared with all other treatment groups. indicates active significantly greater than moderately active.
CD3CD4 and CD3CD8 Cell Percentage
There was not a significant effect of activity on the percentage of CD3CD4 cells or CD3CD8 cells (Figure
5). However, women had a greater percentage of CD3
CD4 cells than men (women 56.8%, men 41.9%, p .001), and, conversely, men had a significantly greater percentage of CD3CD8 cells (women 12.3%, men 19.2%, p .003).
Influenza Symptom Incidence
In the sedentary group, one of 15 subjects reported flu
symptoms; three of 25 subjects in the moderately active
group reported flu symptoms; and 0 of 16 subjects in the ac-
Figure 3. Serum IgM anti-influenza antibody response in sedentary,
moderately active, and active at 1:25 and 1:125 dilution. Values are
means SEM. Asterisks indicate significant differences (p .05)
compared with all other treatment groups. indicates active significantly greater than moderately active.
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KOHUT ET AL.
Figure 5. Percentage of CD3 CD4 and CD3 CD8 cells in
peripheral blood mononuclear cells of sedentary, moderately active,
and active subjects. Cells were collected 2 weeks postimmunization.
Although perceived stress score was associated with physical activity (see Table 1), including stress as a factor in the
model did not decrease the amount of variance explained by
activity, suggesting that activity and stress may indepenFigure 4. In vitro influenza-stimulated IL-2 at 48 hours in vitro
(A); and at 96 hours in vitro IFN- (B) and IL-10 (C) production by
peripheral blood mononuclear cells (PBMC) from sedentary, moderately active, or active subjects. PBMC were collected 2 weeks postimmunization and were restimulated in vitro with influenza vaccine.
tive group reported flu symptoms. There was not a significant difference between the groups regarding symptom incidence.
Nutrient Intake, Psychosocial Factors, and InfluenzaSpecific Immune Response
Subjects consuming a daily multivitamin produced
greater amounts of IL-2 (see Figure 6). There was also a
trend (p .08) toward increased influenza-specific antigenproliferation in subjects consuming a daily multivitamin.
Greater zinc intake and lower perceived stress were both
significant predictors of serum anti-influenza IgG titer and,
along with activity, accounted for 27.8% of the variance.
Figure 6. In vitro influenza-stimulated IL-2 production by peripheral
blood mononuclear cells from subjects consuming a daily multivitamin
compared to subjects not consuming a daily multivitamin. The asterisk
indicates significant differences (p .05) between treatment groups.
EXERCISE AND INFLUENZA VACCINE IN ELDERLY INDIVIDUALS
dently influence anti-influenza IgG. The perceived stress
score was also a statistically significant predictor (p .042)
of influenza-specific IL-2, accounting for 7.8% of the variance (lower perceived stress was associated with greater IL-2
production). LOT and social activity together accounted for
13.7% of the variance in influenza-specific IL-10. Social
activity was a statistically significant predictor (p .031)
whereas the LOT score approached statistical significance
(p .061). Greater social activity and higher optimism
scores were associated with greater IL-10 production.
DISCUSSION
The findings from this study suggest that there is an association between physical activity, diet, psychosocial factors,
and the immune response to influenza immunization in
older adults. This is the first study, to our knowledge, that
observed an association between physical activity and immune response to influenza immunization. Older adults
classified as active had greater anti-influenza IgG, anti-influenza IgM, and greater influenza-specific lymphocyte proliferation than sedentary individuals. Older adults classified as
moderately active had greater influenza-specific lymphocyte proliferation than sedentary subjects, but did not demonstrate greater antibody titer.
Serum IgG is important in protection against influenza
infection and is considered to be a good predictor of resistance to infection (22). In our study, the subjects who exercised vigorously had a higher concentration of both IgG and
IgM, suggesting a greater degree of protection, and our data
on symptom incidence are consistent with this possibility.
Cell-mediated responses to influenza virus are essential
for viral clearance and may also provide cross-reactive protection from strains of influenza that may not have been included in the annual influenza vaccine (23,24). With respect
to cell-mediated response, we observed greater influenzaspecific proliferation among the active and moderately active groups compared with the sedentary group.
We did not find any association between activity level
and cytokine production. This was surprising considering
that IL-2 promotes cell proliferation, and cell proliferation
was higher in the active and moderately active groups.
However, we measured cytokine concentration only at one
time (peak level of production in vitro). Our most recent
data suggest that vigorous activity is associated with a more
rapid production of IL-2. Therefore, the greater PBMC proliferation observed among active and moderately active individuals may reflect a shift in the kinetics of IL-2 production rather than higher peak levels of IL-2. Also, it is
possible that cytokine production varies by individual viral
strain (25), and, in future studies, we will evaluate cytokine
response to each individual viral strain.
Overall, our findings are consistent with other investigators who have reported improved immune function in wellconditioned elders (17–19,26). However, it is important to
note that physical activity may not be strongly associated
with enhanced immune function among older adults (27).
Instead, it is possible that overall health status impacts immunosenescence to a greater degree. For example, when
only “healthy” elderly subjects were included, there was no
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difference in natural killer cell function or response to exercise between young and old subjects (27).
In this investigation, our findings are new in that we evaluated the effect of exercise on the immune response to an
antigen (influenza virus), whereas other human studies have
examined nonspecific immune defenses and/or mitogenstimulated immune response (17–19,26). With an animal
model (28), the role of exercise training in modulating antigen-specific immunity was examined, but no effect of exercise on IgM or IgG production in response to challenge with
the antigen Keyhole Limpet Hemocyanin was found. Exercise may impact primary antibody and recall antibody responses in a different manner. In our study, we examined a
recall response, whereas the animal study evaluated the primary antibody response.
The role of exercise in modulating antibody response to
influenza immunization has been examined in college-aged
students, but no effect of exercise was found (29). It is possible that the immunomodulatory effects of exercise may be
greater in aged populations than in younger populations
(30,31) or that the use of differing techniques yielded different results. In our study, ELISA, which exhibits greater sensitivity (32,33) was used to detect anti-influenza IgG and
IgM, whereas hemagglutination inhibition was used in the
other study.
Psychosocial factors have been shown to influence immune response and alter susceptibility to infection (34).
Chronic stress was related to reduced antibody titer and influenza-specific IL-2 following influenza vaccination in
older adults (16,35). We also found that high stress was a
significant predictor of reduced anti-influenza IgG and influenza-specific IL-2. In our study, greater optimism and a
greater number of social interactions were associated with
higher levels of influenza-specific IL-10. Individuals with a
greater number of social ties may have decreased susceptibility to infection (36), and optimism has been linked to improved immune status (37).
We observed a higher IL-2 and a trend toward enhanced
proliferation in subjects consuming a daily multivitamin.
Also, higher anti-influenza IgG titers were associated with
greater zinc intake. Others have found a positive correlation
between zinc selenium intake, or vitamin E intake and
antibody titer following influenza immunization (38,39),
but these findings are not consistent (40). Given the small
number of subjects in our study, it is premature to make any
dietary supplement recommendations, rather, it is important
to consider nutrient intake as a factor when analyzing immune function in older adults given the possibility that malnutrition exists in this population.
In summary, many older adults receive the influenza vaccine annually, yet vaccine efficacy is reduced among this
population. Therefore it is important to identify specific lifestyle factors that may enhance vaccine efficacy. This is the
first study to suggest that the practice of regular, vigorous
exercise for at least 1 year may contribute to an enhanced
immune response to influenza immunization in older adults.
A follow-up randomized controlled trial is now being
planned to replicate these findings and explore mechanisms
that may account for exercise-induced alterations of immunity.
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Acknowledgment
Address correspondence to Marian L. Kohut, PhD, Department of
Health and Human Performance, Iowa State University, 235 Forker Building, Ames, IA 50011. E-mail: [email protected]
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Received March 20, 2002
Accepted April 1, 2002