Download Short-term stress experienced at time of immunization induces a

Am J Physiol Regul Integr Comp Physiol 289: R738 –R744, 2005.
First published May 12, 2005; doi:10.1152/ajpregu.00145.2005.
Short-term stress experienced at time of immunization induces a long-lasting
increase in immunologic memory
Firdaus S. Dhabhar1,2,3 and Kavitha Viswanathan1
1
Department of Oral Biology, College of Dentistry, 2Department of Molecular Virology, Immunology and Medical Genetics,
College of Medicine, and 3Institute of Behavioral Medicine Research, The Ohio State University, Columbus, Ohio
Submitted 28 February 2005; accepted in final form 9 May 2005
hormone; Langerhans cell; skin diseases; surveillance; leukocyte
trafficking
“STRESS” IS DEFINED as a constellation of events comprising a
stimulus (stressor), which precipitates a reaction in the brain
(stress perception), which activates physiological fight/flight
systems in the body (stress response) (14). It is often overlooked that an acute (lasting for minutes to hours) stress
response has salubrious effects in the short run (12, 16, 32, 42),
although chronic stress can be harmful (4, 14, 33, 37, 41). An
acute stress response is an evolutionarily adaptive psychophysiological survival mechanism (12, 15, 18). Most selection
pressures, the chisels of evolution, are physiological (scarcity
of food, salt, or water), physical (wounding, infection), or
psychological (threat, aggression) stressors. The brain perceives stressors, warns the body of danger, and promotes
survival (e.g., when a gazelle sees a charging lion, the gazelle’s
Address for reprint requests and other correspondence: F. S. Dhabhar, 4179
Postle Hall, 305 West 12th Ave., Columbus, OH 43210 (e-mail:
[email protected]).
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brain detects a threat and orchestrates a physiological response
that enables the gazelle to flee). Because stressful experiences
often result in wounding or infection, immunoenhancement
rather than immunosuppression would be adaptive during acute
stress, since it is unlikely that eons of evolution would select
for a system exquisitely designed to escape the jaws and claws
of a lion only to succumb to wounds and pathogens.
Previous studies have shown that whereas chronic stress is
immunosuppresive, acute stress experienced at the time of
secondary antigen exposure has potent immunoenhancing effects with respect to skin immune function (14). An acute
stress-induced enhancement of skin cell-mediated immunity
(CMI) is partially mediated by a stress-induced trafficking of
leukocytes to skin and skin-draining lymph nodes (16, 44).
Stress-induced enhancement of skin CMI is eliminated after
adrenalectomy and restored by acute administration of physiological stress levels of corticosterone and epinephrine, indicating that the immunoenhancing effects of stress are mediated
by physiological concentrations of these principal stress hormones (13–16, 18). In contrast, chronic exposure to endogenous glucocorticoids or to synthetic glucocorticoid hormones
such as dexamethasone is potently immunosuppressive, as
might be expected given their clinically well-known antiinflammatory effects (15).
In this study we show that acute stress experienced during
primary immunization enhances antigen-specific memory for a
significant portion of the animal’s life span. We identify a key
immunologic mechanism, namely, an acute stress-induced increase in effector and memory helper T cells at the time of
primary immunization, as a mediator of these novel and longlasting adjuvant-like effects of the psychophysiological stress
response. It is important to note that such stress-induced
immunoenhancement may promote immunoprotection in case
of wounding, infection, or vaccination. However, such immunoenhancement also may contribute to stress-induced exacerbation of inflammatory and autoimmune diseases.
MATERIALS AND METHODS
Animals. Young (8 –10 wk at the beginning of the study), male
C57BL6 mice (Taconic, Germantown, NY) were housed in groups of
five on corn cob bedding in plastic cages in the accredited (American
Association of Accreditation of Laboratory Animal Care) Postle Hall
vivarium at The Ohio State University. Experiments were conducted
according to protocols approved by the Ohio State University Institutional Laboratory Animal Care and Use Committee. The animal
room was maintained on a 12:12-h light-dark cycle (lights on at 6:00
AM). Animals were given food and water ad libitum.
The costs of publication of this article were defrayed in part by the payment
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0363-6119/05 $8.00 Copyright © 2005 the American Physiological Society
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Dhabhar, Firdaus S., and Kavitha Viswanathan. Short-term
stress experienced at time of immunization induces a long-lasting
increase in immunologic memory. Am J Physiol Regul Integr Comp
Physiol 289: R738 –R744, 2005. First published May 12, 2005;
doi:10.1152/ajpregu.00145.2005.—It would be extremely beneficial if
one could harness natural, endogenous, health-promoting defense
mechanisms to fight disease and restore health. The psychophysiological stress response is the most underappreciated of nature’s survival
mechanisms. We show that acute stress experienced before primary
immunization induces a long-lasting increase in immunity. Compared
with controls, mice restrained for 2.5 h before primary immunization
with keyhole limpet hemocyanin (KLH) show a significantly enhanced immune response when reexposed to KLH 9 mo later. This
immunoenhancement is mediated by an increase in numbers of
memory and effector helper T cells in sentinel lymph nodes at the time
of primary immunization. Further analyses show that the early stressinduced increase in T cell memory may stimulate the robust increase
in infiltrating lymphocyte and macrophage numbers observed months
later at a novel site of antigen reexposure. Enhanced leukocyte
infiltration may be driven by increased levels of the type 1 cytokines,
IL-2 and IFN-␥, and TNF-␣, observed at the site of antigen reexposure in animals that had been stressed at the time of primary immunization. In contrast, no differences were observed in type 2 cytokines,
IL-4 or IL-5. Given the importance of inducing long-lasting increases
in immunologic memory during vaccination, we suggest that the
neuroendocrine stress response is nature’s adjuvant that could be
psychologically and/or pharmacologically manipulated to safely increase vaccine efficacy. These studies introduce the novel concept that
a psychophysiological stress response is nature’s fundamental survival mechanism that could be therapeutically harnessed to augment
immune function during vaccination, wound healing, or infection.
ACUTE STRESS ENHANCES FORMATION OF IMMUNOLOGIC MEMORY
AJP-Regul Integr Comp Physiol • VOL
analyzed using CBA software (BD-Pharmingen). Standard curves for
each cytokine were generated, and the concentration of each cytokine
in each assay sample from each animal was determined.
Preparation lymph node cell suspension. In a separate experiment,
animals belonging to the three treatment groups were euthanized at
72 h after primary immunization, and scapular lymph nodes draining
the site of immunization were collected. Briefly, cell suspensions were
prepared by squeezing lymph nodes between the frosted ends of glass
slides. Cells were then filtered through a 70-␮m nylon sieve. The cell
suspension thus obtained was washed twice and resuspended in
Hanks’ buffered salt solution (Sigma Aldrich). Cell numbers were
counted on a hematology analyzer (Hemavet, Oxford, CT), viability
was tested using Trypan blue (Sigma Aldrich) exclusion, and remaining cells were stained for flow cytometric analysis.
Flow cytometry. Scapular lymph node lymphocyte subtypes were
measured using immunofluorescent antibody staining and analyzed
using multicolor flow cytometry (FACSCalibur). Cell suspensions
were incubated with Fc Block (BD-Pharmingen) for 15 min on ice to
block nonspecific binding. Cells were then incubated with specific
monoclonal antibodies for 30 min at room temperature, washed with
PBS, and analyzed on the FACSCalibur. Each panel consisted of
either single or multiple antibodies. Fluorochrome-conjugated rat
anti-mouse CD3 (clone 145–2C11), CD4 (RM4 –5), CD44 (IM7),
CD45RA (14.8), and CD62L (MEL-14) (BD-Pharmingen) were used.
Approximately 10,000 events were counted per sample. Control
samples matched for each fluorochrome and antibody isotype were
used to set negative staining criteria. Data were analyzed using
CellQuest software (Becton Dickinson). Lymphocytes were identified
and gated using forward- vs. side-scatter characteristics. CD4⫹CD3⫹
cells were identified as helper T (Th) cells.
Data analysis and statistics. One-way ANOVA was used to test for
differences in pinna thickness (Fig. 1), lymphocyte and macrophage
numbers in antigen-exposed pinnae (see Fig. 3), and T cell phenotype
analysis in draining lymph nodes (see Fig. 5). Tukey’s honestly
significant difference (HSD) or Tamhane’s post hoc test was used to
Fig. 1. Acute stress experienced at the time of immunization induces a
long-lasting enhancement of antigen-specific immunity. Nonstressed (NS)
or acutely stressed (STR) mice were immunized with keyhole limpet
hemocyanin (KLH; 100 ␮g, subcutaneous, dorsum) while nonimmunized
controls remained in their home cages (n ⫽ 10 mice per treatment group).
Nine months later, all mice were exposed to KLH at a novel site (25 ␮g,
intradermal, pinnae) under resting conditions. Pinna thickness was measured 24 h after antigen reexposure and compared with baseline thickness
measured before antigen reexposure. Animals that had been acutely
stressed before primary immunization showed significantly greater pinna
swelling than the other 2 groups. Results are expressed as means ⫾ SE.
†Significantly different from nonimmunized group; *significantly different
from NS before immunization group [P ⬍ 0.05, Tukey’s honestly significant difference (HSD)].
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Stress paradigm. Acute stress was administered by placing animals
(without squeezing or compression) in well-ventilated wire mesh
restrainers for a single session of 2.5 h beginning at 9:00 AM (lights
in the animal room were turned on at 6:00 AM). This procedure
mimics stress that is largely psychological in nature because of the
perception of confinement on the part of the animals (3, 20). The
psychological component of restraint stress is thought to arise from it
mimicking a collapsed tunnel that is intrinsically stressful for these
burrow-dwelling animals (9). It also has been suggested that restraint
stress may have a neurogenic component due to forced positioning
(2). This stressor activates the autonomic nervous system (24) and the
hypothalamic-pituitary-adrenal axis (17, 35) and results in the activation of adrenal steroid receptors in tissues throughout the body (17,
35). Antigen was administered at the end of restraint (⬃11:30 AM).
Treatment groups. Each experiment examined three treatment
groups (n ⫽ 10 per group) of mice. One group was not immunized but
was exposed to keyhole limpet hemocyanin (KLH) on the pinna for
the first time in parallel with the recall phase of the following two
groups. This group served as the nonimmunized control. The second
group of animals remained in their home cages before primary
immunization as the nonstressed immunized controls and were reexposed to KLH on the pinnae 9 mo later, also under resting conditions.
The third group was stressed before primary immunization and
reexposed to KLH on the pinnae 9 mo later under resting conditions.
This was the only group that was acutely stressed, and stress was
administered only once for 2.5 h before primary immunization.
Primary immunization. Nonstressed immunized and stressed immunized animals were given a subcutaneous injection of 100 ␮g of
KLH (Calbiochem, La Jolla, CA) in 0.1 ml of 0.9% sterile saline on
their dorsum.
Antigen reexposure and elicitation of CMI. Nine months after
primary immunization, all animals were exposed to KLH (25 ␮g of
KLH in 25 ␮l of saline) at a novel site (pinnae) under resting
conditions, to elicit a skin CMI response that is also known as a
delayed type hypersensitivity response. KLH was administered intradermally on the dorsal surface of the pinnae. At the same time, the
previously nonimmunized group was exposed to KLH for the first
time. The immune reaction at the site of antigen reexposure was
characterized by an increase in thickness of the pinnae, which was
measured using a constant-loading dial micrometer (Mitutoyo, Tokyo,
Japan) and by histological and cytokine analyses 24 h after KLH
administration.
Histological analyses. Animals were euthanized with carbon dioxide at 24 h after antigen reexposure. Pinnae were immediately collected, frozen, and stored at ⫺80°C. Sections (10 ␮m) were obtained
from each pinna, processed, and analyzed using hematoxylin (Sigma
Aldrich, St. Louis, MO) and eosin (Fisher Diagnostics, Middletown,
VA) staining. Stained sections from nonimmunized, nonstressed immunized, and stressed immunized groups were also analyzed for
enumeration of leukocyte subpopulations. Two sections were analyzed for every animal from each treatment group. Three representative regions (circles, 33-␮m radius) were identified for each section
analyzed, and the total number of macrophages and lymphocytes was
counted in a blinded fashion.
Cytokine analyses. The mouse Th1/Th2 cytokine cytometric bead
array (CBA; BD-Pharmingen, San Diego, CA) was used to detect
TNF-␣, IFN-␥, IL-2, IL-4, and IL-5 levels in KLH-challenged mouse
pinnae from nonstressed immunized and stressed immunized groups.
Skin punches from each individual animal’s pinna were homogenized
in a PBS buffer containing phenylmethylsulfonyl fluoride (Sigma
Aldrich). Tissues from different animals were run separately. The
homogenates were centrifuged (12,000 rpm, 15 min, 4°C), and the
resulting supernatant was used for cytokine analysis. Each sample was
mixed with cytokine capture beads and phycoerythrin detection reagent, and the mixture was incubated for 2 h at room temperature
according to the manufacturer’s instructions. Data were acquired on a
FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) and
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ACUTE STRESS ENHANCES FORMATION OF IMMUNOLOGIC MEMORY
Fig. 2. Acute stress experienced during primary immunization enhances leukocyte infiltration at the site of antigen reexposure 9 mo later. Pinna sections from
nonimmunized controls and animals that were not stressed (NS) before immunization or acutely stressed (STR) before immunization are shown. Nine months
after immunization, pinnae from all 3 groups were exposed to KLH. Pinnae were collected 24 h after exposure and processed for hematoxylin an eosin staining
and analyses. Mice that had been acutely stressed before immunization showed significantly greater leukocyte infiltration than mice in the other 2 groups. Scale
bar, 100 ␮m.
RESULTS
Acute stress experienced during primary immunization enhances recall response upon antigen reexposure 9 mo later.
Nine months after primary immunization with KLH, animals
were tested under resting conditions for a recall response
following antigen reexposure at a novel site (pinna) (Fig. 1).
ANOVA revealed a significant difference between groups in
the magnitude of the immune response to KLH [F(2, 37) ⫽
13.15, P ⬍ 0.01]. Tukey’s HSD further showed that animals
acutely stressed at the time of primary immunization had a
significantly larger immune response compared with animals
that had not been stressed at the time of primary immunization
(P ⬍ 0.01). Importantly, this stress-induced immunoenhancement was observed 9 mo after acute stress administration and
primary immunization.
Acute stress experienced during primary immunization enhances macrophage and lymphocyte infiltration at site of
antigen reexposure 9 mo later. Histological changes were
analyzed by examining hematoxylin and eosin-stained pinna
sections. Compared with nonimmunized controls, both groups
of previously immunized animals showed more leukocyte
infiltration, as would be expected (Figs. 2 and 3). Moreover,
ANOVA revealed a significant difference among groups in the
numbers of infiltrating macrophages [F(2, 19) ⫽ 7.63, P ⬍
0.01] and lymphocytes [F(2, 19) ⫽ 17.86, P ⬍ 0.01] (Fig. 3).
Specifically, acutely stressed animals showed significantly
higher macrophage numbers compared with both nonimmunized (P ⬍ 0.01, Tamhane’s) and nonstressed immunized (P ⬍
0.05, Tamhane’s) animals, as well as significantly higher
lymphocyte numbers compared with the other two groups (P ⬍
0.01 for both, Tukey’s HSD).
Acute stress experienced during primary immunization enhances IL-2, IFN-␥, and TNF-␣ levels at a novel site of antigen
reexposure. Compared with nonstressed animals, animals
acutely stressed at the time of primary immunization showed
higher levels (P ⬍ 0.05, unpaired t-test) of the type 1 cytokines
AJP-Regul Integr Comp Physiol • VOL
IL-2 and IFN-␥ and the proinflammatory cytokine TNF-␣ after
reexposure to antigen at a novel site 9 mo later (Fig. 4).
Interestingly, no differences were observed between nonstressed and acutely stressed animals in levels of the type 2
cytokines IL-4 and IL-5 (Fig. 4). These results, taken together
with the finding that memory lymphocytes mediate protection
in peripheral tissues by rapidly producing type 1 cytokines
(19), suggest that acute stress experienced during primary
immunization increased the numbers and/or activity of memory T cells. This enhanced antigen-specific immunity over a
time period that encompasses a major portion of the animals’
life span.
Acute stress experienced at the time of primary immunization increases numbers of effector and central memory-like T
cells in lymph nodes draining the site of immunization. Longterm immunoenhancement induced by a single acute stressor
experienced before primary immunization suggested that stress
Fig. 3. Acute stress administered at the time of immunization increases
macrophage and lymphocyte infiltration at the site of antigen reexposure. NS
or STR mice were immunized with KLH (100 ␮g, subcutaneous, dorsum)
while nonimmunized controls remained in their home cages (n ⫽ 10 mice per
treatment group). Nine months later, all mice were exposed to KLH at a novel
site (25 ␮g, intradermal, pinnae) under resting conditions. STR immunized
mice showed significantly higher numbers of macrophages (M⌽) and lymphocytes (LY) compared with nonimmunized and NS immunized mice. Results
are expressed as means ⫾ SE. †Significantly different from nonimmunized
group; *significantly different from NS before immunization group [P ⬍ 0.05,
Tukey’s HSD (for lymphocytes), Tamhane’s test (for macrophages)].
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identify statistically significant differences among the nonimmunized,
nonstressed immunized, and stressed immunized groups. For data
shown in Fig. 4, where only two groups are compared, the unpaired
Student’s t-test was used to test for significant differences in cytokine
levels between the nonstressed immunized and stressed immunized
groups. Data are expressed as means ⫾ SE. Differences are considered significant when P ⬍ 0.05; means that differ significantly are
indicated. The computer statistics package SPSS (SPSS, Chicago, IL)
was used for statistical analyses.
ACUTE STRESS ENHANCES FORMATION OF IMMUNOLOGIC MEMORY
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CD44hi, and CD45RA⫺CD62L⫹, phenotypes, suggesting
that the observed changes in the draining lymph nodes of
immunized mice were caused by antigen exposure. These
results suggest that acute stress experienced during primary
immunization increased the numbers of effector and Tcmlike Th cells in sentinel lymph nodes during the early phase
of the primary immune response.
DISCUSSION
influenced the formation of memory T cells. Therefore, in a
separate experiment, we examined the effects of stress on Th
cell phenotype changes 72 h after primary immunization,
which provides sufficient time for important early phenotypic
changes to register in lymph nodes draining the site of antigen
exposure (8). Downregulation of CD62L and upregulation of
CD44 corresponds to a change from a naive to effector phenotype (6, 38) with associated cytoskeletal and proliferative
changes that allow accelerated movement of T cells toward a
target site (31). ANOVA [F(2, 11) ⫽ 8.03, P ⬍ 0.01] showed
that compared with nonimmunized controls, both nonstressed
and acutely stressed immunized animals showed a robust
increase in lymph node cellularity (nonimmunized, 2.91 ⫾
0.35 ⫻ 106 cells; nonstressed immunized, 7.62 ⫾ 1.59 ⫻ 106
cells; stressed immunized, 8.91 ⫾ 0.59 ⫻ 106 cells; P ⬍ 0.01,
Tamhane’s). ANOVA revealed that there were significant
differences among groups in the numbers of CD62Llow [F(2,
11) ⫽ 7.51, P ⬍ 0.01], CD44hi [F(2, 11) ⫽ 32.01, P ⬍ 0.01],
and CD45RA⫺CD62L⫹ [F(2, 11) ⫽ 13.94, P ⬍ 0.01] Th
cells in draining lymph nodes after immunization. Furthermore, stressed immunized animals had higher numbers of
CD62Llow Th cells than nonimmunized (P ⬍ 0.01, Tukey’s
HSD) but not nonstressed immunized animals. Stressed immunized animals also had significantly higher numbers of CD44hi
Th cells than both nonimmunized (P ⬍ 0.01, Tukey’s HSD)
and nonstressed immunized animals (P ⬍ 0.05, Tukey’s HSD).
This suggests that acute stress induces an increase in effector
Th cell numbers in sentinel lymph nodes (Fig. 5). Central
memory (Tcm)-like Th cells express a CD45RA⫺CD62L⫹
phenotype (6, 43). Stressed immunized animals also showed
significantly higher numbers of CD45RA⫺CD62L⫹ Tcmlike Th cells (P ⬍ 0.05, Tukey’s HSD) than nonstressed
immunized animals. Overall, the nonimmunized controls
had significantly lower numbers of activated CD62Llow,
AJP-Regul Integr Comp Physiol • VOL
Fig. 5. Acute stress experienced at the time of primary immunization increases
the numbers of effector and central memory-like T cells in lymph nodes
draining the site of immunization. NS or STR mice were immunized with KLH
(100 ␮g, subcutaneous, dorsum) while nonimmunized controls remained in
their home cages (n ⫽ 5 mice per treatment group). Seventy-two hours after
immunization, scapular lymph nodes draining the site of antigen exposure were
removed and CD4⫹ helper T (Th) cell phenotypes were analyzed using flow
cytometry. Animals that had been acutely stressed at the time of primary
immunization showed significantly higher numbers of CD62Llow and CD44hi
effector and CD45RA⫺CD62L⫹ central memory-like Th cells compared with
the other 2 groups. Results are expressed as means ⫾ SE. †Significantly
different from the nonimmunized group; *significantly different from the NS
before immunization group (P ⬍ 0.05, Tukey’s HSD).
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Fig. 4. Acute stress experienced during primary immunization enhances
TNF-␣ and type 1 cytokine levels at a distal site of antigen reexposure. NS or
STR mice were immunized with KLH (100 ␮g, subcutaneous, dorsum). Nine
months later, all animals were reexposed to KLH at a novel site (25 ␮g,
intradermal, pinnae) under resting conditions. Cytokine levels were measured
in pinnae collected 24 h after KLH reexposure. Animals that had been acutely
stressed at the time of primary immunization showed higher levels of TNF-␣,
IFN-␥, and IL-2 but not IL-4 or IL-5 at the site of antigen reexposure. Results
are expressed as means ⫾ SE. Statistically significant differences are indicated
(*P ⬍ 0.05, unpaired Student’s t-test).
The studies described introduce a novel concept, namely,
that a psychophysiological stress response is nature’s fundamental survival mechanism that could be therapeutically harnessed to augment immune function during vaccination,
wound healing, or infection. We report the novel finding that
short-term stressors experienced at the time of primary immunization can induce long-lasting increases in immunological
memory that may be mediated by increased numbers of memory T cells.
These studies are important because they are specifically
designed to model naturally experienced temporal relationships
between stressor and antigen exposure. For example, when a
gazelle escapes from a charging lion, the gazelle is acutely
stressed during the attack, after which it is exposed to antigens
and pathogens that enter wounds that are likely to arise during
the stressful chase and frantic scuffle that enable the gazelle to
escape. Similarly, when a child sees a nurse approaching with
a vaccine-loaded syringe, it remembers a prior painful injection
and mounts a full-blown stress response before and during the
process of being immunized. Adults also may mount similar
psychophysiological stress responses before and/or during
medical or surgical procedures. Stressors experienced during
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AJP-Regul Integr Comp Physiol • VOL
memory T cells at the time of primary immunization translated
into an enhanced effector response driven by IL-2, IFN-␥, and
TNF-␣ and mounted by macrophages and T cells at the time of
antigen reexposure at a novel site.
Because the magnitude of an immune response is known to
decline with age (28), it is possible that the enhancement of the
recall response observed 9 mo after acute stress preceding
primary immunization may reflect an acute stress-induced
maintenance of immunological memory that resists an agerelated decline. Such a memory-maintaining effect of acute
stress would counter an age-related decrease in the primary
immune response that may be present in animals that were not
stressed at the time of primary immunization.
One important weakness of these studies is that we did not
directly enumerate KLH-specific T cells. It will be necessary to
examine the effects of acute stress on formation of antigenspecific T cells in future studies. However, the observation that
the nonimmunized animals failed to show the increase in
effector and Tcm-like lymph node Th cell numbers that was
seen in immunized animals (Fig. 5) suggests that the changes
seen in immunized animals were a direct result of antigen
stimulation. Moreover, when animals were exposed to antigen
9 mo after primary immunization, the previously immunized
groups showed more leukocyte infiltration at the site of reexposure than did the nonimmunized group (Fig. 2). This
strongly indicates that the recall response was driven by antigen-specific T cells. Importantly, animals that were acutely
stressed before primary immunization showed a significantly
larger recall response than nonstressed immunized controls
(Figs. 1–3). Because the two groups of previously immunized
animals were reexposed to KLH under identical resting conditions, the only difference between them was that the stressed
immunized group had been acutely stressed at the time of
immunization 9 mo earlier. The fact that the acutely stressed
group showed an enhanced recall response upon antigen reexposure 9 mo after stress and immunization strongly suggests
that stress experienced at the time of primary immunization
resulted in the formation of higher numbers of antigen-specific
memory T cells that were able to orchestrate a more robust
immune response at the time of antigen reexposure.
We recognize that it is important to further elucidate the
mechanisms by which acute stress enhances a primary immune
response. We suggest that macrophages and dendritic cells that
lie at the nexus between innate and adaptive immune responses, as well as skin surveillance T cells, may contribute to
a stress-induced enhancement of a primary immune response
(44a). It has been shown that acute stress enhances innate and
adaptive immune responses (7, 11, 16, 18, 25, 29, 39, 46, 47).
The findings described in this study set the stage for future
studies that must specifically examine the effects of stress on
macrophages, dendritic cells, and effector and memory subtypes of Th cells and cytolytic T cells across a time frame that
examines multiple time points after immunization. Studies also
are needed to examine the mechanisms by which memory
function enhanced by stress at the time of primary immunization translates into greater T cell and macrophage effector
function during a recall CMI response.
These studies suggest that it may be possible to design
therapeutic interventions that involve behavioral manipulations, and/or administration of cocktails of physiological mediators, to maximally recruit the body’s natural army (the
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many such conditions are acute and temporally proximal to
accidental or surgical wounding and antigen exposure. Our
experiments, which are designed to mimic such temporal
relationships between stressor and immune challenge, suggest
that the physiological changes accompanying an acute stress
response may have long-lasting immunoenhancing effects that
are established early during the development of a primary
immune response.
These results show that acute psychophysiological stress
exerts adjuvant-like effects on the immune system. Animals
acutely stressed before immunization showed increased numbers of activated-effector (CD62Llow and CD44hi) and Tcmlike (CD45RA⫺CD62L⫹) Th cells during the early phase of
the primary immune response (Fig. 5). Although several weeks
may be required to generate a full repertoire of memory
lymphocytes (38), phenotypic changes that occur soon after
antigen exposure are critical for determining the ultimate
number and efficacy of the memory pool (23, 34). Effector
cells that subsequently transform into Tcm cells are conferred
with an ability to persist long term and offer better protection
during recall (40, 45). Thus stress-induced increases in numbers of effector and Tcm-like Th cell populations soon after
antigen exposure may mediate the long-lasting enhancement of
immunity.
The only stress manipulation that was performed in these
experiments occurred 9 mo before the recall CMI response was
measured. Interestingly, acutely stressed animals mounted a
significantly larger recall response that was marked by a robust
increase in macrophages and lymphocytes infiltrating the novel
site of antigen reexposure (Figs. 1–3). The enhanced CMI
response was accompanied by increased levels of type 1
cytokines IL-2 and IFN-␥ and the proinflammatory cytokine
TNF-␣ at the site of antigen reexposure (Fig. 4). Interestingly,
we did not see a difference between nonstressed and acutely
stressed animals in levels of the type 2 cytokines IL-4 and IL-5,
indicating that acutely stressed animals showed a shift toward
type 1 cytokine production. IFN-␥, IL-2, and TNF-␣ are
important cytokine mediators (18, 19), and macrophages and T
cells are important effector cells for CMI (5, 10). Memory
lymphocytes mediate protection in peripheral tissues by rapidly
producing type 1 cytokines (19). Our results suggest that acute
stress experienced during primary immunization induces a
long-term increase in memory T cells that results in an enhanced secondary cell-mediated immune response upon antigen reexposure. Memory T cells are likely to be the source of
the enhanced IL-2 and IFN-␥ levels observed at the site of
antigen reexposure (Fig. 4), although intracellular cytokine
staining coupled with surface marker flow cytometry is required to provide definitive evidence for this. IL-2 is a principal T cell growth factor that, together with IFN-␥, promotes
CMI (1, 27), although its crucial role in T cell tolerance is
increasingly being appreciated (30). IFN-␥ is a potent activator
of macrophages and effector CD8 T cells (1, 27). Th1 T cells
(26) and activated macrophages (1) are the major sources of
TNF-␣ that was also observed in higher levels in acutely
stressed animals. TNF-␣ is crucial for inducing endothelial cell
activation, vasodilation, leukocyte recruitment, and Langerhans cell activation and migration during the primary and
secondary phases (48) of a cell-mediated immune response (1,
22) and for promoting effector cell function (36). Therefore,
our results suggest that the acute stress-induced increase in
ACUTE STRESS ENHANCES FORMATION OF IMMUNOLOGIC MEMORY
ACKNOWLEDGMENTS
We thank Dr. Ning Quan, Alison Saul, and Dr. Hari Krishna Perali for help
with histological analyses and Cynthia Walter for help with statistical analyses.
GRANTS
This work was supported by National Institute of Allergy and Infectious
Diseases Grant R01 AI-48995 and the Dana Foundation.
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