Effects of stress on immune cell distribution.

Dynamics and hormonal mechanisms.

F S Dhabhar, A H Miller, B S McEwen and R L Spencer
This information is current as J Immunol 1995; 154:5511-5527; ;
of July 1, 2017. http://www.jimmunol.org/content/154/10/5511

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Copyright 1995 by American Association of Immunologists
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Effects of Stress on Immune Cell Distribution
Dynamics and Hormonal Mechanisms

Firdaus S. Dhabhar,2* Andrew H. Miller,3t Bruce S. McEwen,* and Robert L. Spencer4*

*Laboratory of Neuroendocrinology, The Rockefeller University, New York, NY 10021; and the Department of Psychiatry,
Mt. Sinai School of Medicine, New York, N Y 10029

Immune celltrafficking is crucial tothe performance of the surveillance as well as effector functions of the immune
system. Because immune cells travel between tissues through the bloodstream, the numbers and proportions of
leukocytes in the circulation providean important representation of the state of leukocyte distribution inthe body.

Downloaded from http://www.jimmunol.org/ by guest on July 1, 2017

The studies described here examine significant and selective changes in numbers and percentages of peripheral
blood leukocyte subpopulations in the rat. These changes were rapidly induced under conditions of mild acute
stress. Stress-induced increases in plasma corticosterone were accompanied by a significant decrease in numbers
and percentages of lymphocytes, and by an increase in numbers and percentages of neutrophils. Flow cytometric
analysis revealed that B cell, N K cell, and monocyte numbers showed a greater stress-induced decrease than did
T cells. All stress-induced changes were observed during the light (inactive) as well as the dark (active) period of
the animals diurnal cycle. Importantly, the stress-induced changes in leukocyte numbers and percentages were
rapidly reversed upon the cessation of stress. Furthermore, the effects of stress were largely dependent on adrenal
hormones, because the magnitude of the stress-induced changes was significantly reduced in adrenalectomized
animals. Moreover, administration of corticosterone to adrenalectomized animals resulted in a close replication
of stress-induced changes observed in adrenal-intact animals. These results suggest that endocrine factors released
during stress modulate leukocyte trafficking and result in the redistribution of leukocytes between the blood and
other immune compartments. Such a redistribution may significantly affect the ability of the immune system to
respond to potential or ongoing immune challenge. The journal of Immunology, 1995, 154: 551 1-5527.

T
heappropriate distribution of immunecellsbe-
tween different tissues in the body is crucial to the
performance of the surveillance as well asthe ef-
fector functions of the immune system. The continuous
circulation of immune cells from the blood, into various
immune compartments, and back into the blood, is essen-
tial for the maintenance of an effective immune defense
network (1).The numbersand proportions of leukocytes in
the blood provide an important representation of the state
Received for publication December14, 1994. Accepted for publication March
2, 1995.
The costs of publication of this article were defrayedin part by the payment of
page charges. This article must therefore he hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by the Health and Behavior Network of the John D. and Catherine
T. MacArthur Foundation, and by National Institutes of Mental Health Grant
MH-47674.
* Address correspondence and reprint requests to Dr. Firdaus S . Dhabhar, The
Rockefeller University, 1230 York Ave, Box 134, New York, NY 10021.
Current address: Department of Psychiatry, Emory University, Atlanta, C A
30322.
Current address: Department of Psychology, University of Colorado, Boul-
der, GO 80309.
of distribution of leukocytes in the body and of the stateof
activation of the immune system. We have established a
model in which relatively mild acute stress producesrapid
andselective changes in numbers andpercentages of
bloodleukocyte subpopulations (2). The magnitudeand
specificity of these changes make this a very useful model
for studying the kinetics, hormone dependency, and con-
sequences of an effect registered by the immune systemin
response to acute stress.
Numerous studies have investigated the effects of envi-
ronmental and psychologic stress on alterations in immune
parameters, and in susceptibility to disease (3, 4). How-
ever, the majority of these studies have focused on stress-
induced changes in immune cell function as opposed to
changes in immune cell distribution as a possible mecha-
nism for the effects of stress on the immune system. We
have previously hypothesized that an important effect of
acute stress may be the regulation of immune cell distri-
bution between different body compartments and that such
a redistribution may have significant consequences for the
immuneresponse (2). Supportfor thishypothesis also

Copyright 0 1995 by The American Association of Immunologists 0022-1 767/95/$02.00

551 2
comes from studies that suggest that stress-induced sup-
pression of splenic and peripheral blood NK activity may
be related to stress-induced migration of NK cells out of
these compartments ( 9 , and studies that havedemon-
strated that a decrease in peripheral blood NK activity af-
ter examination stress may bemediated by astress-in-
duced decrease in blood NK cell number (6).
The experimentsdescribed here were carefully designed
to study the dynamics and hormonal mechanisms of acute
stress-induced changes in leukocyte distribution. A within-
animal design was used in whichthe numbers and per-
centages of peripheralbloodleukocyte subpopulations
were examined repeatedly throughout exposure to stress,
as well as duringthe recovery period after stress. For each
time point, an entire panel of blood leukocyte subpopula-
tions(lymphocytes,neutrophils, Th cells,cytotoxic/sup-
pressor T cells (CTLDs), B cells, NK cells, and mono-
cytes) was examined. A single session of mild acute stress
induced rapid and large-magnitude changes in all leuko-
cyte subpopulations. Moreover, these changes were rap-
idly reversed upon the cessation of stress.
Our results suggest that corticosterone released acutely
during physiologicly relevant conditions plays an impor-
tant role in regulating the traffickinghedistribution of leu-
kocytes between thebloodand other immune compart-
ments. Such redistribution
a may have significant
consequences for the ability of the immune system to re-
spond to potential or ongoing immune challenge.
Materials and Methods
Animals
Young adult, male Sprague-Dawley rats (200-300 g) (Harlan Sprague-
Dawley, Indianapolis, IN) were used in all studies. Animals were housed
in hanging wire mesh cages in the accredited (American Association of
Accreditation of Laboratory Animal Care) animal facilities of The Rock-
efeller University (New York, NY). The animal room was maintained on
a 12-h light-dark cycle. Lights went on at 7 am and off at 7 pm for
experiments performed during the light (inactive) period of the diurnal
cycle. Lights went on at 7 pm and off at 7 am for experiments performed
during the dark (active) period of the diurnal cycle. All animals were
given rat chow and tap water ad libitum.
Experimental procedures
It is important to note that all the experiments described below involved
repeated hormonal, hematologic, and flow cytometric measures on each
of six animals within a treatment group. Because of the sensitivity of the
hormonal and leukocyte measures to stress, blood samples were obtained
as rapidly as possible, and all animals were sampled simultaneously and
for the same duration of time. These considerations necessitated the anal-
ysis of large numbers of samples. However, it was important to conduct
the experiments in this manner to accurately establish the kinetics, sub-
oooulation wecificitv.
I , -
,, magnitude. and hormone dependency of the ob-
served effects of stress on leukocyte distribution in the blood. Further-
more, repeated measures within the same animal allowed us to make
statistically powerful within-animal comparisons (using each animal as
its own control) in addition to comparing means of treatment groups.
i Abbreviations used i n t h i s paper: CTL/T~,cytotoxic/suppressor T cell; WBC,
white blood cell; LDH, lactatedehydrogenase; ADX, adrenalectomy; NAD,
nicotlnamide adenine dinucleotide.
EFFECTS OF STRESS O N IMMUNE CELL DISTRIBUTION
Experiments examining stress-induced changes in peripheral blood
leukocytes. For each study, six animals were placed i n horizontal, cy-
lindrical, Plexiglass restrainers within one minute of the invcstigator en-
tering the animal enclosure. Blood samples (20WOO PI) were collected
immediately for baseline measures (0 min) by the tail clip method. An-
imals were then kept in the restrainers for the duration of the stress test
(2 h) and returned to their home cages at the end of the stress session.
During the stress session, blood samples were rapidly collected from
each animal at each of the timepoints shown. Tails were clipped only for
collection of the first blood sample. No further clipping is necessary for
subsequent sample collection. The stress of sample collection alone is not
sufficient to produce the observed effects on leukocyte distribution. As
seen in the recovery studies described here, the stress of blood collection
does not prevent the return of corticosterone and blood leukocyte num-
bers back to baseline in the absence of ongoing restraint stress. Plexiglass
restrainers provide ample ventilation for breathing, and are not believed
to cause pain or undue discomfort for the animal. This model of restraint
is believed to approximate stress that is largely psychologic rather than
physical in nature (7).
For studies investigating recovery from stress-induced changes, ani-
mals were returned to their home cages after 2 h of restraint stress. Sub-
sequently, at the first recovery time point (1 h after cessation of the stress)
each animal was rapidly placed in a restrainer and a blood sample was
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collected immediately. The animals were then placed back in their home
cages and exactly the same procedure was repeated to collect another
blood sample at the second recovery time point (3 h after cessation of
stress). Experiments were conducted during the light (inactive) as well as
the dark (active) phase of the animals diurnal cycle. For experiments
preformed during the dark phase, animals were sampled in an area with
the minimum amount of indirect light required to obtain samples.
Experiments examining the effects of adrenalectomy on stress-induced
changes in peripheral blood leukocytes. The effects of adrenalectomy
on stress-induced changes in peripheral blood leukocytes were investi-
gated in two experiments: The first experiment was performed during the
light (inactive) phase of the diurnal cycle. The second experiment was
performed during the dark (active) phase of the diurnal cycle. A total of
18 animals was used for each experiment. Six animals were adrenalec-
tomized, six animals were sham adrenalectomized, and six animals were
left intact for each experiment. Experiments were performed 6 days after
surgery. (In previous studies, we have determined that a period of 6 to 7
days after surgery is sufficient for relative normalization of leukocyte
numbers in peripheral blood (8)).
On the day of the experiment, six animals from each treatment group
were placed in Plexiglass restrainers within 1 minute of the investigator
entering the animal enclosure (as described above). Blood samples were
collected immediately from each animal for baseline measures (0 h) by
the tail clip method. Animals were then kept in the restrainers for 2 h, and
another blood sample was rapidly collected from each animal. This was
the 2-h stress time point. All animals were then returned to their home
cages.
Experiment examining the effects of corticosterone administration on
peripheral blood leukocytes in adrenalectomized animals. Five animals
were adrenalectomized as described below. Six days after adrenalectomy,
the animals were placed in Plexiglass restrainers and blood samples col-
lected as previously described. The animals were removed from the re-
strainers and rapidly injected (s.c.) with approximately 300 PI cortico-
sterone solution (5 mgiml corticosterone dissolved in propylene glycol to
yield an injection dose of 5 mgikg body weight). The animals were then
returned to their home cages. Blood samples were collected from each
animal (as described previously) at each of the timepoints shown in Fig-
ure 9. Because no significant differences were observed (in the studies
described above) between experiments performed during the light or dark
phase of the diurnal cycle, the corticosterone replacement experiment
was performed only during the light phase. Only WBC, lymphocyte, and
neutrophil numbers and percentages were elucidated in this experiment.
Collection and treatment of blood samples
All blood samples were collected in heparinized microcentrifuge tubes
and immediately placed on a shaker to prevent clotting. An aliquot of
whole blood was removed for separation of plasma for hormone assays.
Plasma was frozen and stored at -20C. The remaining aliquot of blood
was kept on the shaker until it was used for the hematology and flow
cytometric analyses described below.
The Journal of Immunology
Table I. ldentification of leukocyte subpopulations
Total WBC Coulter counts
Total lymphocytes Modified Wright-Giemsa
Total neutrophils Modified Wright-Giemsa
Total T cells OX19+
Cytotoxic/Suppressor T cells OX19+ & OX8+, or
OX19+ & W3/25-
Helper T cells OX19+ & W3/25+
B cells OX33+
NK cells 0x19- & OX8+
Monocytes 0x19- & W3/25+
Adrenalectomy (ADX)
Bilateral adrenalectomy was performed using standard aseptic surgical
techniques on animals fully anesthetized with the inhalant, methoxyflu-
rane (Metofane; Pitman-Moore, Washington Crossing, NJ). Sham adre-
nalectomized animals went through exactly the same regimen as the adre-
nalectomized animals except that their adrenal glands were not removed.
Adrenalectomized animals were provided with 3% saline in addition to
tap water.
Analysis of leukocyte subtypes in peripheral blood
The white blood cell (WBC) count was obtained on a Coulter counter,
and the differential (lymphocytes and neutrophils) was determined by
microscopic examination of peripheral blood smears stained by a mod-
ification of the Wright-Giemsa staining procedure (Diff Quick, Baxter
Scientific Products, Miami, FL). At least 200 cells were counted to pro-
duce each differential. Specific subtypes of immune cells were measured
by immunofluorescent Ab staining of whole blood and subsequent anal-
ysis using two-color flow cytometry (EPICS-Profile, Coulter, Hialeah,
FL). One hundred microliters of whole blood was incubated with 100 pI
of PBS (pH 7.2) and 5 to 10 p1 of the appropriate mAb. In the case of the
directly labeled Abs, cells were incubated with Ab for 45 min at 4C and
then washed two times in PBS. Red blood cells were lysed, and cells
were fixed by adding 1 ml of Immunolyse (Coulter, Hialeah, FL) for 60
s followed by 250 p1 of fixative (Coulter, Hialeah, FL). Cells were then
washed two times in PBS and analyzed on the flow cytometer. In the case
of the indirectly labeled Abs, cells were incubated for 45 min with the
primary Ab followed by two PBS washes and a 45-min incubation with
the secondary Ab. Cells were then lysed and fixed as indicated above. For
analysis of lymphocyte subpopulations, bit-map gating was initially set
according to light scatter patterns and was subsequently adjusted to
achieve at least 90% positive staining for the panel of Abs listed below.
Numbers for specific subpopulations were derived from a multiplication
of the white blood cell count by the lymphocyte percentage and then by
the specific subpopulation percentage.
A bs
Lymphocyte subsets were defined using the following mAbs (Harlan,
Indianapolis, IN): OX-19 directed against the rat homologue of human
CD5, and mouse Ly-1, was used asa pan-T cell marker (9); OX-8,
directed against the rat homologue of the human CD8 Ag and mouse
Ly-2, was used as a marker for cytotoxicisuppressor T cells (CTL/Ts)
and NK cells (10, 11); W3/25, directed against the rat homologue of the
human CD4 Ag, was used as a marker for Th cells and macrophages/
monocytes (12); 0 x 3 3 , directed against the rat homologue of the B cell
leukocyte common Ag, was used as a B cell marker (13). All Abs were
directly conjugated with either phycoerythrin (0x19) or FITC (0x8,
W3/25), except OX33 which was detected by a secondary Ab, a donkey
anti-mouse FITC conjugate (Jackson Immunoresearch, West Grove, PA).
IgGl isotype controls were used to set the negative criterion for histo-
gram analysis. In summary, total and specific leukocyte subpopulations
were identified as indicated in Table 1.
Steroids
[1,2,6,7-'H(N)]corticosterone (72 Ciimmol) was obtained from New En-
gland Nuclear (Boston, MA). Unlabeled corticosterone was obtained
from Steraloids (Wilton, NH).
551 3
Total plasma corticosterone radioimmunoassay
Total plasma corticosterone was measured by radioimmunoassay using
rabbit antiserum raised against corticosterone-21-hemisuccinateBSA
(B21-42, Endocrine Sciences, Tarzana, CA) and [3H]corticosterone
(New England Nuclear). The antiserum has very low cross-reactivity
with other glucocorticoids and their metabolites. Assay sensitivity was 10
pg of corticosterone, and the within-assay coefficient of variability was
less than 4% ( n = 8). Total corticosterone values are expressed in units
of p g of corticosterone per 100 ml plasma.
Lactate dehydrogenate (LDH) enzyme assay
LDH was measured using a quantitative kinetic method for determining
enzyme activity in plasma (14). Plasma (50 pl) was added to 1 ml of
lactate dehydrogenase reagent (Sigma Chemical Co. Diagnostics, St.
Louis, MO) which consists of lactate (SO mmol/L), nicotinamide adenine
dinucleotide (NAD) (7 mmoliL), and sodium azide (0.05%). pH = 8.9.
LDH catalyzes the oxidation of lactate to pyruvate with concurrent re-
duction of NAD to NADH. The increase in NADH is directly propor-
tional to LDH activity in the sample, and is measured as an increase in
absorbance at 340 nm. Changes in absorbance were measured at 15-
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second intervals for 1 min at 340 nm (25"C, light path = 1 cm). One unit
of LDH activity is defined as the amount of enzyme that will catalyze the
formation of one micromole of NADH per minute under the conditions
of the assay procedure. Unit activity of plasma LDH was calculated ac-
cording to the following formula:
LDH activity (U/L) =
change in absorbanceimin at 340 nm X reaction volume X 1000
6.22 X sample volume X light path
6.22 = millimolar absorptivity of NADH at 340 nm
Data analysis and statistics
In all experiments, repeated measures were made on each animal. This
made it possible to use statistically powerful within-subject comparisons
for data analysis. For comparisons of stress-induced changes, and corti-
costerone-induced changes, repeated measures analysis of variance was
used to test for overall significance of changes observed across time.
Significant differences between timepoints within a specific leukocyte
subpopulation were analyzed using the Student's t-test for dependent
measures, as a test for significant differences between means. For the
adrenalectomy studies, one-way analysis of variance was used to test for
differences between treatment groups (Tables 11-IV), and the Tukey test
was used as a post hoc test of significant differences between means.
Means that differed significantly are indicated by symbols that are de-
fined in the figure legends. Data are expressed as mean ? SEM in all
figures. A computer statistics package was used for statistical analyses
(SYSTAT v5.2.1, Systat Inc., Evanston, IL).
Results
Stress-induced changes in distribution of peripheral
blood leukocytes observed during the light (inactive)
period of the diurnal cycle
Plasma corticosterone and leukocyte numbers were mea-
sured at 0 min (baseline) and at 10, 30, 60, and 120 min
duringstress.Figure 1A depictsa rapid and significant
increase in plasmacorticosteroneduringamild,acute
stress session (2 h of restraint), and Figure 1B shows cor-
responding changes in WBC, lymphocyte, and neutrophil
number. The changes in leukocyte numbers were charac-
terized by a rapid and significant decrease in WBC and
lymphocyte numbers, which was accompanied by a slight
increase in neutrophil numbers. The percent change in leu-
kocyte numbers after2 h of stress (relative to 0 h baseline)
551 4 EFFECTS OF STRESS O N IMMUNE CELL DISTRIBUTION

LIGHT / INACTIVEPERIOD isalso indicated (Fig. 1B). After 2 h of stress, WBC and
lymphocyte numbers were reduced by 45% and 54% re-
spectively, whereas neutrophil numbers increased by 13%.
Figure 2 shows the leukocyte subpopulation specificity
45 - "Corticosterone
of the stress-induced decrease in blood leukocyte numbers.
Changes in numbers of T cells (Fig. a), B cells (Fig. 2 B )
40 - ** and NK cells and monocytes (Fig. 2C) shown.
are All
leukocyte subpopulations showed a significant reduction
35 -
in cell numbers during the stress session. B cell, NK cell,
30 - and monocyte numbers showed a greater reduction than
did T cell numbers after acute stress.
25 - Significantstress-induced changes in relativepercent-
ages of bloodleukocyte subpopulationswere also ob-
20 - served (data not shown). A stress-induced decrease (20%)
in the relative percentage of lymphocytes was accompa-
15- nied by an increase (113%) in the percentage of neutro-
phils. The percentage of CTL/Tscells was unchanged,
10-

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whereas the percentage of Th cells increased (48%) sig-
5- nificantly during the stress session. Percentages of B cells,
and NK cells and monocytesalso showedasignificant
0- decrease (16%, 35%,and 49% respectively) during the
stress session. stress
1 0 30 60 90 120
Stress-induced changes in numbers as well as in per-
centages of leukocytes were apparent within 10 min after
Time (min) the beginning of the stress session, reached statistical sig-
nificance at 1 h, and continued further until the end of the
-" WBC stress session (Figs. 1 and 2).
"-0".
Lymphocytes
12 Reversibility of stress-induced changes in distribution
of peripheral blood leukocytes observed during the
light (inactive) period of the diurnal cycle
10
Theexperiment described in Figures 1 and 2 showed
rapid, significant, and selective changes in the distribution
8
of leukocytes in the peripheral blood. The next step wasto
examine whether these changes were reversible upon ces-
45 %
6 sation of stress, i.e., whether leukocyte numbers and per-
centages returned to baseline levels after the stressor was
54 % removed. Figure 3 shows the kinetic profiles of changes in
4
plasmacorticosteroneandperipheralblood WBC, lym-
phocyte, and neutrophil numbers during 2 h of stress, and
recovery of these measures after the cessation of stress.
Plasmacorticosteronereachedpeaklevels after 1 h of
stress stress and returned to baseline 3 h after the cessation of
I ' I ' I ' I ' I ~ I stress (Fig. 3A). A significant decrease in WBC and lym-
~

0 30 60 90 120 phocyte numbers was observed at 1 h and 2 hafterthe

Time (min)
FIGURE 1. Stress-induced changes in peripheral blood
leukocytedistribution. The plasma corticosterone response
during 2-h restraint stress ( A ) , and the time course of stress-
induced changes in WBC, lymphocyte, and neutrophil num-
bers ( B ) are shown. The experiment was conducted during
the light phase of the diurnal cycle. Total WBC counts were
obtained on a Coulter counter, andlymphocyteand neutro-
phi1 numbers were obtained from smears of peripheral blood
(modified Wright-Giemsa stain). The percentage change in
leukocyte numbers after 120 rnin stress relative to baseline (0
beginning of stress. However, WBCand lymphocyte num-
bers beganto increasewithin 1 hafter the cessation of
stress. Threehoursafter thecessation of stress, WBC
min), is shown ( B ) . Data are expressed as means 2 SEM.
Statistically significant differences are indicated: * p < 0.05;
** p < 0.005, significantly different from baseline (0 rnin)
(paired t-test).
The Journal of Immunology 551 5

LIGHT / INACTIVE PERIOD

5.0 -3
"Th
2.0 - " B cells 3.0- "
Monocytes
4.5-
"-A"
CTLITS 2B 2c "-0"
NK cells

4.0- 2.5 -
1.5-
3.5 -
-
h
-
h

3.0 -

2.5 -
&
g
v 2'o:

2.0 - - 34 % -
rn
U
1.0-
s
E
1.5-

1.5-
a
61 % =3 1.0-
U
0.5 -
1.0- -45%
0.5- 16 %
0.5 -
4" stress

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0.0 0.0 0.0 i
0 30 stress
60 90 1 2 0 7
96
0 30 60 90 120 0 30 60 90 1 2 0
Time (min) Time (min) Time (min)
FIGURE 2. Subpopulation specificity of stress-induced changes in peripheral blood leukocyte distribution. Changes in num-
bers of T cells ( A ) , B cells ( B ) and N K cells and monocytes (C) during a 2-h stress session are shown. The percentage change
in leukocyte numbers after 120 minutes of stress relative to baseline (0min), is indicated for each subpopulation in each panel.
The experiment was conducted during the light phase ofthe diurnal cycle. Leukocyte subpopulations were measured using two-color
flow cytometry with the following mAbs: Cytotoxic (CTL)/suppressor (Ts)T cells (OX19+, W3/25-), Th cells (OX19+, W3/25'), B
cells (OX33+), NK cells (OX19-, OX8+), and monocytes (OX19 -, W3/25+). Data are expressed as means i SEM. Statistically
significant differences are indicated: * p < 0.05; ** p < 0.005, significantly different from baseline (0 min) (paired t-test).

numbers were significantly higher than baseline, whereas
lymphocytenumbers had returned to baselinelevels. In
contrast, neutrophil numbers, increased significantly dur-
ing the stress session and continued to increase even after
the cessation of stress. Neutrophil numbers observed at 3
h afterthe cessation of stress weresignificantlyhigher
than baseline neutrophilnumbers. Thus, theincreasein
WBC numbers (over baseline) observed at 3 h after the
cessation of stress probably reflected the increase in neu-
trophil numbers observed at that time point.
Figure 4 shows the leukocyte subpopulation specificity
of recoveryfromstress-induced changes in leukocyte
numbers. The decrease in numbers of T cells (Fig. 4A), B
cells (Fig. 4B), NK cellsand monocytes (Fig. 4C) after 2 h
of stress was almost identical to that seen in Figure 2. All
leukocyte subpopulations showed asignificantreduction
in cell numbers after 2 h of stress. Importantly, all leuko-
cytesubpopulationsalsoshowed a complete return to
baseline levels upon the cessation of stress.
Figure 5 shows changes in relative percentages of blood
leukocyte subpopulations observed during 2 h of restraint
stress, and subsequent return to baseline levels after ces-
sation of stress. We observed a significant stress-induced
decrease (45%) in the relative percentage of lymphocytes,
which was accompanied by a significant increase (133%)
in the percentage of neutrophils (Fig. 5A). The percentage
of CTL/Ts cells wasunchanged, whereas the percentage of
Thcells increased (43%) significantly during the stress
session (Fig. 5B). Percentages of B cells (Fig. 5C), and
NK cells and monocytes (Fig. 5 D ) also showed a signif-
icant decrease (20%, 47%, and 42% respectively) during
the stress session. Importantly, relative percentages of all
leukocytesubpopulations (exceptneutrophils) showed a
complete return to baseline levels within 3 h after cessa-
tion of the stress session.
Rapid and reversible stress-induced changes in
distribution of peripheral blood leukocytes observed
during the dark (active) period of the diurnal cycle
The studies describedabove were conducted during the
light (inactive) phase of the rat's diurnal cycle. However,
the adrenal hormone corticosterone, which appears to be a
major mediator of the stress-induced changes in blood leu-
kocyte distribution, shows asignificantdiurnalrhythm.
Therefore, it was important (see Discussion) to determine
whether the effects of stress on leukocyte distribution dur-
ing the dark (active) phase of the diurnal cycle were dif-
ferentfromthoseobserved during thelight(inactive)
phase of the diurnal cycle.
Figure 6 shows thekineticprofiles of stress-induced
changes in plasmacorticosteroneandperipheralblood
WBC, lymphocyte,andneutrophil numbersduring the
551 6 EFFECTS OF STRESS ON IMMUNE CELL DISTRIBUTION

LIGHT / INACTIVE PERIOD dark (active) phase of the diurnal cycle. Plasma cortico-
sterone and leukocyte numbers were measured at 0 min
(baseline) and 10, 30, 60, and 120 min during stress. Re-
35 - "Corticosterone
covery from the stress-induced changes after the cessation
of the stress is also shown in Figure 6. Recovery measures
weremade at 1 h and 3 h after cessation of stress. In
30 - general, the pattern of stress-induced changes in leukocyte
numbers and percentages wasvery similar to that observed
duringthe light (inactive)period.However,during the
25 -
dark (active) period, the decrease in lymphocyte numbers
was of a larger magnitude and it occurred more rapidly
20 - than that observed during the light period.
Baseline (0 min) plasma corticosterone levels in Figure
15- 6 were higher than corresponding baseline levels during
the light (inactive) period. The higher baseline corticoste-
rone observed in Figure 6 is in keeping with the diurnal
10- corticosterone rhythm, according to which basal cortico-

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steronelevelsare higher at the beginning of the active
5- period than at the beginning of the inactive period. Plasma
corticosterone rose rapidly, peaked at 30 min after stress,
was elevated throughout the duration of the stress session,
0-

0
I
- '
stress
I

60
. I

120
'
no stress
l . I

180 240 300

. I '
and decreased to lower than baseline levels 3 h after the
cessation of stress (Fig. 6A). A significant decrease in
WBC, lymphocyte,
. . and neutrophil numberswasobserved
within 30 min after the beginning of stress. However,
Time
(min) whereas WBC and lymphocyte numbers continued to de-
crease at l h and 2 h into the stress session, neutrophil
numbers showed a slight increase. Compared with the 2-h
20 - "WBC stress levels, WBC and lymphocyte numbers signif-
were
icantly higher at 1 h and 3 h after the cessation of stress.
18-
*0 However, compared with baseline (0 h) levels, WBC, lym-
phocyte, and neutrophil numbers were still lower after 3 h
16-
of recovery after cessation of stress.
Figure 7 shows the leukocyte subpopulation specificity
14-
of recovery from stress-induced changes in leukocyte
12-
,g 0 numbers during the active (dark) period. The kinetics and
magnitude of the reduction in numbers of T cells (Fig.
10- 7 A ) , B cells (Fig. 7B), and NK cells and monocytes (Fig.
7C) during 2 h of stress were almost identical to those seen
8- in Figures 2 and 4. All leukocyte subpopulations (except
neutrophils) showed a significant reduction in cell num-
6- bers after 2 h of stress. However, all leukocyte subpopu-"

lations (withthe exception of Th cellsand monocytes) also

4-

2-

O ~ .
-' l
stress
'

6o
l .

2o
I
no stress
' l '

8o 240 300
l '
Time
(min)
FIGURE 3. Recovery from stress-induced changes in pe-
ripheral blood leukocyte distribution. The plasma corticoste-
rone response during 2-h restraint stress, and subsequent re-
turn of hormone levels to baseline after cessation of stress is
shown ( A ) . The time course of stress-induced changes in
WBC, lymphocyte and neutrophil numbers, and recovery af-
l '
ter cessation of stress is also shown (6).The experiment was
conducted during the light phase of the diurnal cycle. Total
WBC counts were obtained on a Coulter counter, and lym-
phocyte and neutrophil numbers were obtained from smears
of peripheral blood (modified Wright-Giemsa stain). The per-
centage change in leukocyte numbers after 120 rnin of stress
relative to baseline (0 min), is shown in ( B ) . Data are ex-
pressed as means t SEM. Statistically significant differences
are indicated: * p < 0.05; ** p < 0.005, significantly different
from 0 h baseline (paired t-test). 5 p < 0.05, significantly
different from 2-h stress time point (paired t-test).
The Journal of Immunology 551 7

LIGHT I INACTIVE PERIOD

"B cells
3.5 1 2.51 " NK cells

4A 4.5- 4B 4c
3.0 -
4.0 - 2.0 -
-
3.5 -
- 2.5 h
I

s
g. 2
h

2.0-
8 1.5-

e 2.5 3
5
\** =8
v1
n
4
1.5-
5 1.0-

1.5
E4
1.o - -E:
4

0.5 -
0.5 -

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stress no stress no stress stress
0 60 120
180
240
300 0 60240
180
120 300 0 60
300
240
180
120
Time (min) Time (min) (min) Time
FIGURE 4. Subpopulationspecificity of stress-induced changes in peripheral blood leukocytedistribution,and recovery
following cessation of stress. Changes in numbers of T cells ( A ) , B cells ( B ) ,and NK cells and monocytes ( C )during 2-h stress
and recovery after cessation of stress are shown. The percentage change in leukocyte numbers after 1 2 0 min stress relative to
baseline (0 min) is indicated for each subpopulation in each panel. The experiment was conducted during the light phase of
the diurnal cycle. Leukocyte subpopulations were measured using two-color flow cytometry with the following rnAbs: Cyto-
toxic (CTL)/suppressor (Ts) T cells (OX19+, W3/25-), Th cells (OX19', W3/25+), B cells (OX33+), N K cells (OXlS-, OX8+),
and monocytes (OX19T, W3/25+). Data are expressed as means +- SEM. Statistically significant differences are indicated: * p <
0.05; ** p < 0.005, significantly different from 0-h baseline (paired t-test). 5 p < 0.05, significantly different from 2-h stress time
point (paired t-test).

showed a complete return to baseline levels upon the ces-
sation of stress.
We also observed significant stress-induced changes in
relativepercentages of bloodleukocytesubpopulations,
and their subsequentreturn to baseline levelsafter the ces-
sation of stress. These changes weresimilar to the changes
in relative percentages of leukocytes observed during the
lightphase of thediurnal cycle. We observed astress-
induced decrease (17%) in the relative percentage of lym-
phocytes, which was accompaniedby an increase (60%) in
the percentage of neutrophils. The percentage of CTL/Ts
cells was unchanged, whereas the percentage of Th cells
increasedsignificantly (43%) during thestresssession.
Percentages of B cells, and NK cells and monocytes also
showed a significant decrease (18%, 48%, & 35% respec-
tively) during the stress session. Relative percentages of
all leukocyte subpopulations (with the exception of NK
cells and monocytes) showed a return to baseline levels
within 3 h after cessation of the stress session.
Stress-induced changes in the CD4/CD8 ratio
Changes in the CD4/CD8 ratio (Th cell, to cytotoxic:sup-
pressor T cell ratio) observed during the experiments de-
scribed above are shown in Figure 8. There was a rapid
and significantstress-inducedincrease in the CD4/CD8
ratio during the light (inactive) as well asthe dark (active)
phases of the diurnal cycle. The CD4/CD8 ratio rapidly
returned to pre-stressbaselinelevelsuponcessation of
stress (Fig. 8, E and C).
Effects of adrenalectomy on stress-induced changes
in leukocyte distribution in the peripheral blood
Table I1 shows stress-induced changes in leukocyte num-
bers in intact, sham adrenalectomizedandadrenalecto-
mized animals observed during the light (inactive) period
of the diurnal cycle. A prestress baseline value (0 h base-
line) and a poststress value (2 h of stress) for leukocyte
numbers (1000/~1)is shown for each leukocyte subpopu-
lation in each treatment group. Compared with intact an-
imals, sham adrenalectomized and adrenalectomized ani-
malsshowed slightlylower baselinenumbersfor all
lymphocyte subpopulations,andslightlyhigherbaseline
numbers of neutrophils.However,thesedifferencesbe-
tweentreatment groups reachedstatisticalsignificance
only in the case of neutrophil, total lymphocyte, and Th
cell numbers. Table I1 shows that intact and sham adrena-
lectomized animals demonstrated a large decrease in num-
bers of all leukocyte subpopulations (except neutrophils)
after 2 h restraint stress. In contrast, adrenalectomized ani-
mals failed to show a stress-induced decrease in B cell and
551 8 EFFECTS OF STRESS ON IMMUNE CELL DISTRIBUTION

LIGHT / INACTIVE PERIOD animals observed during the dark (active) period of the diur-

5.4 ,,I
loo
"o"
%Lymphocytes
%Neutrophils 5B "1 -
"*"
%Th
%CTUs
nal cycle. No significant differences in baseline (Oh) leuko-
cyte numbers were observed between intact, sham adrena-
lectomized, and adrenalectomized animals. However, after
stress, intact, and sham adrenalectomized animals showed a
large decrease in numbers of all leukocyte subpopulations
(except neutrophils). Intact animals showed a significant in-
crease, and sham animals showed a significant decrease in
neutrophil numbers after stress. The effect of stress on leu-
kocyte numbers was eliminated in adrenalectomized animals
(except for a reduction in the CTUTs number).
lo]
0
, :s,ty,,n;;ys,s, , stress no stress Table IV shows stress-induced changes in relative per-
0 60 120180240300 0 60 120 180 300
240 centages of blood leukocyte subpopulationsin intact, sham
Time (min) (min) Time
adrenalectomized, and adrenalectomizedanimalsduring
25, % B cells 30, % NKcells
the dark (active) phase of the diurnal cycle. There were no
differences between the three treatment groups in baseline
relative percentages of any of the leukocyte subpopula-

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stress no stress
.0- , . (.. I 0 . -,.
0 60 120
180
240 300 0 60 120
Time (min)
FIGURE 5. Stress-inducedchanges in relative percentages of
peripheral blood leukocytes,and recovery aftercessation of
stress. Changes in percentages of lymphoyctes and neutrophils
(A), T cells (B), B cells (C),and NK cells and monocytes ( D )
during a 2-h stress session, and subsequent recovery after ces-
sation of stressare shown. The percentage change in relative
percentages after120 rnin of stress relative to baseline (0 min) is
indicated for each subpopulation in eachpanel.The experi-
ment was conducted during the light phase of the diurnal cycle.
Lymphocyte and neutrophil percentages were obtained from
smears of peripheral blood (modified Wright-Giemsa stain).
Leukocyte subpopulations were measured using two-color flow
cytometry with the following rnAbs: Cytotoxic (CTL)/suppressor
(Ts) T cells (OX19+, W3/25F), Th cells (OX19+, W3/25+), B
cells (OX33+), NK cells (OX19-, OX8'), and monocytes
(OX19-, W3/25+). Data are expressed as means ? SEM. Sta-
tistically significant differences are indicated: * p < 0.05; **p <
0.005, significantly different from 0-h baseline (paired t-test).
Sp < 0.05, significantly different from 2-h stress time point
(paired t-test).
NK cell number. Furthermore, for those leukocyte subpopu-
lations that did show a stress-induced decrease in cell number
in adrenalectomized animals, the magnitude of decrease was
significantly lower than the magnitude of the stress-induced
decrease in intact and sham adrenalectomized animals. The
basic pattern of stress-induced changes in leukocyte percent-
ages during the inactive period was similar to that described
below for the active period.
Table 111 shows stress-induced changes in leukocyte num-
bers in intact, sham adrenalectomized and adrenalectomized
tions. The basic pattern of stress-induced changes in the
intact and sham adrenalectomized groups was similar to
those observed in the experiments described above. Im-
portantly, the only significant effect of stress that was ob-
served in adrenalectomized animals was a large increase
(25%) in relative percentages of B cells.
stress no stress
Corticosterone administration to adrenalectomized
180
240 300
Time (min)
animals replicates stress-induced changes in
leukocyte distribution observed in intact animals
Figure 9A depicts a rapid and significant increase in plasma
corticosterone after a single, S.C. injection of corticosterone (5
mgkg) in adrenalectomized animals. Figure 9B shows cor-
responding changes in numbers of WBC, lymphocytes, and
neutrophils. Plasma corticosterone and leukocyte numbers
were measured at 0 min (baseline, measured immediately
before injection) and at 30, 60, 120, and 240 min after injec-
tion. The changes in leukocyte numbers were characterized
by a rapid and significant decrease in WBC and lymphocyte
numbers, which were accompanied by a rapid and significant
increase in neutrophil numbers. The percentage change in
leukocyte numbers at 2 h and 4 h after the administration of
corticosterone (relative to 0 h baseline) is also indicated (Fig.
9B). Two hours after corticosterone administration, WBC
and lymphocyte numbers were reduced by 36% and 62%
respectively, whereas neutrophil numbers increased by 25%.
Four hours after corticosterone administration, no fur-
ther decrease in WBC or lymphocyte numbers was ob-
served. Although WBC and lymphocyte numbers were
stillreduced (37% and 54% respectively),neutrophil
numbers had returned back to baseline levels 4 h after
corticosterone administration.
Plasma lactate dehydrogenase (L DH) levels
Table V shows plasma LDH levels during 2 h of stress,
and during recovery after the cessation of stress. An in-
crease in plasma LDH, an intracellular enzyme, would be
indicative of cell death or tissue damage caused by the
stressor (15-17). There was no increase in plasma LDH
The Journal of Immunology 551 9

DARK /ACTIVE PERIOD levels either during or after the stress session. This sug-
gests that thestress-inducedreductionsin immune cell
numbers were not the result of leukocyte destruction.
50 - "Corticosterone
Discussion
Our results indicate that a single session of relatively mild
40 -
stress induces rapid, selective, and large-magnitude changes
in numbers and percentages of leukocyte subpopulations in
the peripheral blood. Although the basic phenomenon of
30 - glucocorticoid- (18-21) and stress (2, 6, 22-25)-induced
reductions in blood lymphocyte numbers has been previ-
ously recognized, the present study is the first character-
20 - ization of the kinetics and leukocyte subpopulation spec-
ificity of stress-induced changes in blood
leukocyte
distribution.Moreover,thisstudyclearlyillustrates that
10- theeffects of acute stressare reversible, that they are
largely adrenal-dependent, and that the profile of stress-

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induced changes in leukocyte numbers is very similar to
that induced by acute injection of corticosterone.
0 60240
180
120 300 It isimportant to recognize that althoughperipheral
time (min) blood immune cellsrepresent a small portion of total body
leukocytes, theblood is animportant compartment serving
-
-
20 "WBC as a conduit by which leukocytes travel between different
18- Lymphocytes immune tissues. Furthermore, in humans, blood is theonly
Neutrophils easily accessible immune compartment, and provides the
16- substrate for numerous experimental and diagnostic mea-
sures which often involve monitoring changes in immune
14-
cell numbers. The changes in blood leukocytenumbers
12- described in our animal model may provide important in-
formation about the kinetics and hormone dependency of
10-
leukocyte redistribution in thebody.Moreover,these
8- changes in leukocyte distribution may havesignificant
consequences for the ability of the immune system to re-
6- spond to challenge.
4-
Dynamics and magnitude of stress-induced changes
2-

o! *
-
,
0
1
stress
1

60240
180
I

120
, .
recovery
# . , I 1

300
.
Time (min)
FIGURE 6. Stress-induced changes in peripheral blood
leukocyte distribution observed during the dark phase of the
diurnal cycle. The plasma corticosterone response during 2-h
restraint stress and subsequent return of hormone levels to
baseline after cessation of stress is shown ( A ) . The time
course of stress-induced changes in WBC, lymphocyte and
neutrophil numbers, and recovery after cessation of stress is
also shown ( B ) . Total WBC countswereobtained on a
Coulter counter, and lymphocyte and neutrophil numbers
were obtained from smears of peripheral blood (modified
Wright-Giemsa stain). The percentage change in leukocyte
numbers after 120 min stress relative to baseline (0 min), is
shown in ( B ) . Data are expressed as means 5 SEM. Statisti-
cally significant differences are indicated: * p < 0.05; ** p <
0.005, significantly different from 0-h baseline (paired t-test).
5 p < 0.05, significantly differentfrom the 2-h stress time
point (paired t-test).
in leukocyte distribution
The experiments described here illustrate how readily the
numbers and proportions of immune cells in the circula-
tion are regulated by acute stress. An examination of num-
bers as well as percentages of leukocyte subpopulations
enabled us to obtain a complete picture of the dynamic
changes intherelativeproportions of bloodleukocytes
induced by stress. The effects of stress on the distribution
of peripheral blood leukocytes were apparent within 30
min after applying the stressor. In all experiments, there
was a large decrease (45-50%) in WBC number. This de-
creasewas accompanied by adecreasein lymphocyte
number (50-60% decrease) and percentage (20-40% de-
crease), and an increaseinneutrophilnumber (10-30%
increase)andpercentage(60-130%increase).Cessation
of stress resulted in a rapid reversal of the stress-induced
reductionsin numbers and percentages of all leukocyte
subpopulations except neutrophils. Recovery from stress-
induced changes was apparent within 1 h after the cessa-
tion of stress. Furthermore,leukocyte numbers and per-
5520 EFFECTS OF STRESS O N IMMUNE CELL DISTRIBUTION

DARK / ACTIVE PERIOD

5 "
Th
'
'"1
" "
7A "-A"- B cells NK cells
CTL/Ts 2.5 "A"
T
Monocytes
3.5
T
4
4 2.0
T

8 3 p 1.5
3, e.
d d
8 2 8
c9 1.0
H
I I
-65%
1 0.5
-55%

- stress recovery - stress recovery

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0 0.0 I . , . , . , . , . , .

0 60
120
180
240 300 0 60
120
180
240 300 0 60
120
180
240 300
(min) Time (min)Time (min) Time
FIGURE 7. Subpopulation specificity of stress-induced changes in peripheral blood leukocyte distribution and recovery after
cessation of stress, during the dark phase of the diurnal cycle. Changes in numbers of T cells ( A ) , B cells ( B ) and NK cells and
monocytes ( C ) during 2 h of stress and recovery after cessation of stress are shown. The percentage change in leukocyte
numbers after 120 min stress relative to baseline (0 min) is indicated for each subpopulation in each panel. Leukocyte sub-
populations were measured using two-color flow cytometry with the following mAbs: Cytotoxic (CTL)/suppressor (Ts) T cells
(OX1 9+,W3/25Y), Th cells (OX19+, W3/25+), B cells (OX33+), NK cells (OX1 9-, OX8+),and monocytes (OX19-, W3/25+).
Data are expressed as means 2 SEM. Statistically significant differences are indicated: * p < 0.05; ** p < 0.005, significantly
different from 0-h baseline (paired t-test). 4 p < 0.05, significantly different from 2-h stress timepoint (paired t-test).

2.8 -
8A
2.6 -

d"l
*
2.4 - 2.4
T
2.2 - 2.2 -
m 00
6
t6 2.0:
1.8-
2
8
2.0-

1.8-

1.6- 1.6-
1
1.4-

1.2 - LIGHT PERIOD

1.4
1.24
1 LIGHT PERIOD
1.44

DARK PERIOD
stress stress recovery
0
-1
. 1.01 .I
. . . .
. 1.o (. I I I

0 30 60 90 120 0 60
120
180
240 300 0 60 120
180
240 300
Time (min) Time (min) Time (min)
FIGURE 8. Stress-induced changes in CD4:CD8 ratio in peripheral blood. Changes in CD4:CD8 ratio during 2 h of stress
(light period) ( A ) , during recovery after cessation of stress (light period) ( B ) , and during 2-h stress and recovery (dark period)
(C) are shown. T cell subpopulations were measured using two-color flow cytometry with the following mAbs: CD4 cells
(OX19+, W3/25+), CD8 cells (OX19+, W3/25-). Data are expressed as means t SEM. Statistically significant differences are
indicated: * p < 0.05, significantly different from 0-h baseline (paired t-test). 4 p < 0.05, significantly different from 2-h stress
timepoint (paired t-test).
 The Journal of Immunology 5521

Table 11. Efect of adrenalectomy on stress-induced changes in peripheral bloodleukocyte number during the light period
of the diurnal cycle

Leukocyte Number (lOOO/pl)

Stress
adx
Condition adx
Intact Sham
~

WBC 0 h baseline 15.42

13.30 i 1.25 ? 0.43 12.72 t 0.48
2 h stress 7.55 t 1.1 1 ** 6.92 2 0.67** 10.28 t 0.70*+
Lymphocyte 0 h baseline 1 4 . 1 7 2 1.19 11.26 t- 0.43 10.31 t 0.43
2 h stress 6.12 2 0.81 ** 4.88 5 0.51 *+ 7.29 t 0.34**
Neutrophil baseline
0h 1.07 t 0.1 2 1.68 5 0.1 9 1.98 t 0.28*
2 h stress 1.51 t 0.12 1.98 2 0.21 2.61 2 0.20
cell Th baseline
0h 5.42 i 0.52 4.23 2 0.22 3.77 t 0.1 25
2 h stress 3.03 2 0.34** 2.46 5 0.25** 2.66 t 0.09**
CTL/Ts 0 h baseline 3.40 2 0.41 2.74 2 0.18 2.44 t 0.26
2 h stress 1.50 ? 0.19** 1.17 5 0.11** 1.65 t 0.20**
B cell 0 h baseline 1.80 ? 0.20 1.46 ? 0.14 1.75 t 0.14
2 h stress 0.70 t 0.14** 0.58 2 0.09** 1.39 t 0.19
NK cell baseline
0h 2.01 2 0.27 1.50 -t 0.1 2 1.35 t 0.10
2 h stress 0.65 2 0.1 5** 0.48 2 0.08** 1 .oo t 0.1 3
Monocyte 0 h baseline 1.27 t 0.23 1.08 2 0.1 6 1.16 t 0.19

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2 h stress 0.28 ? 0.10 0.28 4 0.06** 0.66 5 0.10*+
a Absolute numbers of blood leukocyte subpopulations in intact, sham adrenalectomized (sham adx),and adrenalectomized (adx) animals. Baseline (0 h) as well
as post-stress (2 h) numbers (lOOO/pl) are shown for each leukocyte subpopulation in each treatment group. The stress test wasconducted during the light (inactive)
period of the animals diurnal cycle. Data are expressed as means 2 SEM. Statistically significant differences are indicated: * p < 0.05; * * p < 0.005, significantly
different from corresponding 0 h baseline (dependent t-test). p< 0.05, significantly different from corresponding intact level (by Tukey test). tp < 0.05, significantly
different from corresponding intact and sham adx level (by Tukey test).

Table 111. fffectof adrenalectomy on stress-induced changes in peripheral blood leukocyte number during the dark period
of the diurnaf cycle

Leukocyte Number (lOOO/pl)

Stress
adx
Condition adx
Intact Sham

WBC 0 h baseline 13.87 2 1.45 12.25 i- 1.06 13.95 i 1.42

2 h stress 9.35 t 0.80* 5.25 t 0.53** 11.91 i 0.915
Lymphocyte 0 h baseline 11.16 t 1.17 9.1 5 t 0.52 10.87 ? 0.88
2 h stress 6.29 t 0.36** 4.00 f 0.55*** 9.03 ? 0.47+
Neutrophil 0 h baseline 2.31 t 2.66 2.49 t 0.48 2.27 ? 0.38
2 h stress 3.31 i 0.45* 0.98 t 0.10* 2.18 t 0.39
Th cell 0 h baseline 3.98 i 0.38 3.60 i- 0.33 3.89 2 0.38
2 h stress 2.54 2 0.1 7* 2.01 5 0.30* 3.15 ? 0.28
CTL/Ts 0 h baseline 2.40 5 0.44 1.96 t 0.23 2.23 i 0.16
2 h stress 1.30 2 0.14 0.89 t 0.1 8** 1.68 i 0.14*
B cell 0 h baseline 2.1 7 t 0.22 1.84 t 0.23 2.37 t 0.20
2 h stress 1.13? 0.11** 0.59 t 0.10** 2.45 2 0.16
NK cell 0 h baseline 1.19 t 0.09 0.79 i: 0.1 1 1.08 i 0.06
2 h stress 0.71 t 0.05** 0.33 t 0.04* 0.85 t 0.06
Monocyte 0 h baseline 0.84 t 0.10 0.62 f 0.08 0.74 t 0.05
2 h stress 0.44 2 0.06* 0.21 ? 0.02** 0.78 2 0.09+
a Absolute numbers of blood leukocyte subpopulations in intact, sham adrenalectomized (sham adx),and adrenalectomized (adx) animals. Baseline (0 h) as well
as post-stress (2 h) numbers (lOOO/pI) are shown for each leukocyte subpopulation in each treatment group. The stress test wasconducted during the dark (active)
period of the animals diurnal cycle. Data are expressed as means 2 SEM. Statistically significant differences are indicated: * p < 0.05; * * p < 0.005, significantly
different from corresponding 0 h baseline (dependent t-test). p < 0.05, significantly different from corresponding intact level (by Tukey test). tp < 0.05, significantly
different from corresponding intact and sham adx level (by Tukey test).

centages returned to pre-stress baseline levels within 3 h stress. B cells, NK cells, and monocytesexhibiteda
after cessation of stress, with the exception of some sub- greater stress-induced decrease in numbers and percent-
populations, which showedincomplete recovery during ages than did T cells. Peripheral blood was relatively en-
the active (dark phase) of the diurnal cycle. riched in Th cells during the stress session, and Th cells
were the only nongranulocyte subpopulation of immune
specificity Of stress-induced changes cells to show a stress-induced increase intheir relative
in leukocyte distribution
proportion in the blood. Interestingly, Beer and Center
All leukocyte subpopulations showed a large decrease (ex- (26) have also reported that rat splenic B cells are more
cept neutrophils which increased) in cell numbers during sensitive to inhibition of migration by glucocorticoids than
5522 EFFECTS OF STRESS ON IMMUNE CELL DISTRIBUTION

Table IV. The effect of adrenalectomy on stress-induced changes in relative percentages of peripheral blood leukocytes during the dark
period of the diurnal cycle"

Leukocyte %
Stress
Intact Condition Sham adx adx
~ ~

Lymphocyte 0 h baseline 80.50 t 2.09 75.67 ? 2.65 78.67 t 2.50

2 h stress 68.50 t 4.40* 74.67 2 3.47 76.50 f 2.31
Neutrophil 0 h baseline 16.67 2 2.08 19.50 f 2.1 3 16.00 f 1.77
2 h stress 27.1 7 t 4.57* 19.83 f 2.77 18.1 7 t 1.89
Th cell 0 h baseline 35.90 t 1.41 39.22 t 2.06 35.73 f 1.41
2 h stress 40.48 t 1.45* 50.53 f 2.90*' 34.72 2 1.90
CTLfls 0 h baseline 20.75 ? 1.63 21.63 t 2.70 20.68 f 0.87
2 h stress 20.67 t 1.57 22.05 -+ 2.34 18.58 t 1.1 1
B cell 0 h baseline 19.62 -+ 1.31 20.02 t 2.21 21.86 t 0.84
2 h stress 17.92 t 0.90 14.67 t 1.56* 27.22 + 1.32**'
NK cell 0 h baseline 10.82 t 0.56 8.40 f 0.799 10.02 f 0.43
2 h stress 11.28 f 0.56 8.78 2 0.95 9.45 f 0.44
Monocvte 0 h baseline 7.52 ? 0.47 6.68 % 0.55 6.95 f 0.52
2 h stress 6.88 t 0.84 5.62 t 0.52 8.68 t 0.99
Relative percentages of blood leukocyte suhpopulations in intact, sham adrenalectomized (sham adx), and adrenalectomized (adx) animals. Baseline (0 h) as

Downloaded from http://www.jimmunol.org/ by guest on July 1, 2017

well as post-stress (2 h) percentages are shown for each leukocyte subpopulation in each treatment group. The stress test was conducted during the dark (active)
period of the animal's diurnal cycle. Data are expressed as means 5 SEM. Statistically significant differences are Indicated: ' p < 0.05; *"p 10.005, signiflcantly
different from corresponding0 h baseline (dependent t-test). 4p< 0.05, significantly different from corresponding intact level (by
Tukey test). ' p < 0.05, significantly
different from corresponding Intact and sham adx level (by Tukey test).

are rat splenic T cells. We have previously reported similar
changes in the distribution of peripheral blood leukocytes
which vary according to the diurnal corticosterone rhythm
(2), and after long-term treatment with selective adrenal
steroid agonists (8).
Hormone dependency of stress-induced changes in
leukocyte distribution
Our results suggest that hormones released by the adrenal
gland in response to stress are the major mediators of the
observedeffects on leukocyte numbers andpercentages.
Compared with adrenal intact animals, adrenalectomized
animals showeda significantly lower magnitude of change
in numbers and percentages of leukocyte subpopulations
after stress. Moreover, the attenuating effect of adrenalec-
tomy was most pronounced on those leukocyte subpopu-
lations (B cells, NK cells, and monocytes) which showed
the greatestresponsivity to the effects of stress.Impor-
tantly, in adrenalectomizedanimals,thestress-induced
change in B cell percentage was opposite in direction (in-
crease rather than decrease), compared with the change in
B cell percentage observed in intact and sham adrenalec-
tomized animals.
Even though these changes in immune cell distribution
seem to beregulated by adrenalsteroids, we observed
small but significant stress-induced changes in some leu-
kocyte subpopulations in adrenalectomized animals. Sev-
eral reasons might account for this: First, adrenalectomy
results in severe disruption in the release of neuroendo-
crine hormones (corticotropin-releasing hormone, adreno-
corticotropin, and the endorphins). In the absence of in-
hibitory feedback (by corticosterone),adrenalectomized
animals exhibit significantly higher basal and stress levels
of these hormones than do intact animals. High levels of
these hormones released during stress in adrenalectomized
animals may mediate the observed effects on leukocytes.
Furthermore, T cells may be more sensitive to the effects
of these (and other) hormones than are the other leukocyte
subpopulations. Second, there is alsoevidence of local
corticosteroid production in immune organs (27). Cortico-
steroids produced in certain compartments during stress in
adrenalectomized animals may mediate the residual stress-
induced changes in leukocyte numbers observed in adre-
nalectomizedanimals.Third,the residual response to
stress in adrenalectomized animals also raises the possi-
bility that stress-induced changes in leukocyte distribution
may be partially mediated by nonadrenal mediators such
as endogenous opioids,neural catecholamines, gonadal
steroids,andprolactin. However, it is important to note
that catecholamines and endogenousopioids have been
shown to induce changes in lymphocyte distribution that
are opposite in direction to those described in this study
(22, 28-31). Finally, it is also possible that adrenal hor-
mones and some nonadrenal mediators act synergistically
to produce the stress-induced changes in leukcoyte distri-
bution. In adrenalectomizedanimals,nonadrenal factors
may mediate partial changes in leukocyte distribution even
in the absence of the major adrenal stress hormones.
To further examine the role played by the adrenal ste-
roid corticosterone in inducing changes in leukocyte num-
bersduringstress, it was important to examine whether
acute administration of corticosterone to unstressed adre-
nalectomized animals resulted in changes in leukocyte dis-
tribution which were similar to those observed after stress-
ingadrenalintactanimals. The experimentdescribed in
Figure 9 shows that acute corticosterone treatment of adre-
nalectomized animals closelyreplicatedthe changes in
The Journal of Immunology 5523

corticosterone is an importantmediator of thestress-in-

9A 140-
duced changes in blood leukocyte distribution.
-E
h

120-
8 Diurnal effects on stress-induced changes in
5
v
100- leukocyte distribution

80 - In addition to being released during stress, corticosterone

exhibits a significant diurnal rhythm. In rodents, cortico-
60 - sterone levels reach a diurnal trough at the beginning of

i \
the inactive (light) period, and a diurnal peak at the be-
ginning of the active (dark) period. In a previous study, we

::j
0
i reported diurnal changes in peripheral blood immune cells,
which coincided with the diurnal increase in plasma cor-
ticosterone and which were similar in subpopulation spec-
ificity to thestress-induced changes describedhere(2).
Therefore, it was important to determine whether the di-
1 2 3 4 5

$ time (hour) urnal rhythm of the animal influences the stress-induced

changes in leukocytedistribution in theblood. For this

Downloaded from http://www.jimmunol.org/ by guest on July 1, 2017

corticosterone
administration reason, the experiments described in this manuscript (with

20

18-
- - WBC
- - - 0 - - - Lymphocytes
Neutrophils
the exception of the corticosterone administration study)
were conducted during theactive(dark)as well as the
inactive (light) phase of the rats diurnal cycle. Our results
indicate that the dynamics, magnitude, leukocyte subpopu-
16-
lation specificity, and hormone dependency of the stress-
induced changes in leukocyte distribution are not signifi-
14- cantly affected by the diurnal rhythm. We did not observe
12-
a diurnal difference in baseline leukocyte numbers in the
kinetic studies described here. However, we did observe a
10- diurnal difference in baseline leukocyte numbers of intact
8- and sham adrenalectomized animals in the experiments
described in Tables II to IV. To reliablydetectdiurnal
6-
differences in basal numbers of leukocytes, it may be nec-
4- essary to obtain blood samples overa 24-h period, asshifts
- 62 % -5 % in the timing of the diurnal corticosterone peak may cause
2-
shifts in diurnal changes in peripheral blood leukocytes. It
o! , , I , I . I 1 I . I may also be necessary to conduct diurnal studies on the

1
corticosterone
administration
1 2
time (hour)
3 4
FIGURE 9. Corticosterone-induced changes in peripheral
blood leukocyte distribution. A time course of changes in
plasma corticosterone ( A ) , and WBC, lymphocyte, and neu-
tropihl numbers ( B ) is shown. Corticosterone (5 mg/kg) was
administered S.C. to previously adrenalectomized animals.
WBC countswereobtained on a Coulter counter. Lym-
phoycte and neutrophil numbers were obtained from smears
of peripheral blood (modified Wright-Giemsa stain). Data are
expressed as means 2 SEM. Statistically significant differ-
ences are indicated: * p < 0.05, ** p < 0.005, significantly
different from pre-injection baseline (0 h) (paired t-test).
WBC, lymphocyte, and neutrophil numbers that were in-
duced by stress in intact animals. Vehicleinjection of
adrenalectomized animalsdid not result in similar changes
(data not shown). These results support the
conclusion that
5
samegroup of animals, or simultaneously on diurnal
phase-matched groups, as comparisons across studies con-
ducted at different times may not yield significant results
because of between-study variability.
Stress-induced changes in feukocyte numbers are
mediated by leukocyte redistribution and not by
leukocyte destruction
The observed changes in leukocyte numbers and percent-
ages could be the result of stress-induced changes in leu-
kocyte turnover (production/destruction), or of stress-in-
ducedchanges in leukocyte distribution. It has been
previously suggested that glucocorticoid-induced changes
in bloodleukocytenumbersrepresent changes in leuko-
cyte redistribution in steroid-resistant species (humans
and guinea pig), and leukocyte lysis in steroid-sensitive
species (mouse, rat, and rabbit) (32, 33). However, a large
body of evidence indicates that even in species previously
labeled
steroid
sensitive,
pharmacologically
induced
changes in adrenal steroids produce changes in leukocyte
5524
Table V. Plasma lactate dehydrogenase (LDH) levels during 2 h stress and after cessation of stressa
~ ~~
Oh
Time (stress)
(baseline)
LDH activity ( U N 336.2 5 305.4
24.7
Plasma lactate dehvdroaenase
. I levels durinR 2 h stress and following recoveryafter the cessation of stress. LDH activity is expressed as U/L. Data are expressed
as means C SEM.
distribution rather than an increase in leukocyte destruc-
tion (19, 20, 34-38).
We have previously hypothesized that stress-induced
changes in leukocyte redistribution, rather than changes in
leukocyte turnover, may account for the observed effects
(2). The following reasons support this hypothesis: First,
in the present series of experiments, we have demonstrated
that although acute stresscaused significant changes in
numbers and percentages of peripheral blood leukocytes,
cessation of stress resulted in a rapid return of leukocyte
numbers and percentages to baseline levels (Figs. 3
through 8). It is unlikely that over 40 to 70% of the cir-
culating leukocyte pool would be destroyed within 2 h of
exposure to mild stress, and completely replaced within 1
to 3 h after the cessation of the stress. Second, measure-
ments of plasma LDH levels during and after the stress
manipulation showed no significant increase in plasma
LDH either during or after the cessation of stress (Table
V). An increase in plasma LDH concentration is an indi-
cator of cell or tissue damage (15-17). Therefore, these
findings suggest that the observed reductions in immune
cell numbers in the blood were not the result of leukocyte
destruction. Finally, a significant amount of evidence in-
dicates that pharmacologic treatment with glucocorticoids
induces changes in leukocytedistribution (19, 20, 34-38).
These changes are similar to those observed with stress,
and are not the result of leukocyte destruction. It has been
shown in human and nonhuman species that glucocorti-
coids induce a neutrophilia in the blood by increasing the
release of neutrophilsfromthe marginated granulocyte
pool (39, 40). It has also been demonstrated that infusion
of the synthetic glucocorticoid (prednisolone) into rats re-
sults in a decrease in lymphocyte numbers in the blood
accompanied by a retention of recirculating lymphocytes
within the bone marrow, spleen, and lymph nodes (20).
Thus, we suggest that leukocyte redistribution (rather than
leukocyte lysis) is the primary mechanism underlying the
observed stress-induced changes in peripheral blood leu-
kocyte numbers and percentages.
Stress-induced changes in leukocyte distribution-
potential mechanisms
It is likely that the observed changes in leukocyte redis-
tribution are mediated by stress-induced changes in either
the expression or affinity of surface adhesion molecules on
leukocytes and/or endothelial cells. During stress, specific
leukocyte subpopulations (being transported by blood and
EFFECTS OF STRESS ON IMMUNE CELL DISTRIBUTION
~~ ~~ ~ ~ ~~
l h 2 h 3h 5h
(stress) (recovery) (recovery)
5298.1
29.7 281.5
5 25 2 279.4
27.7 2 23.9
lymph through different body compartments) may be se-
lectively retained in those compartments in which they
encounter a glucocorticoid-induced adhesion match on
the surface of a tissue endothelial cell. As a result, the
proportion of these leukocyte subpopulations would de-
creaseinthe blood. Alternatively,other leukocyte sub-
populations may be released from certain immune com-
partments, and as a result, their proportion would increase
Downloaded from http://www.jimmunol.org/ by guest on July 1, 2017
in the peripheral blood. Pharmacologic studiessupport this
hypothesis: Prednisolone has been shown to induce the
retention of circulating lymphocytes within the bone mar-
row, spleen, and some lymph nodes, thus resulting in a
decrease in lymphocyte numbers in the thoracic duct and
a concomitant decrease in numbers in the peripheral blood
(20, 34). Dexamethasone has been shown to inhibit adhe-
sion of T cells to endothelial cells and synovial cells under
specific conditions (41). Importantly, glucocorticoidsmay
also indirectly influence leukocyte-endothelial cell adhe-
sion interactions by affecting the production of cytokines
(42-44), and lipocortins (45), which in turn can affect the
surface adhesion properties of leukocytes and endothelial
cells (46-49). Further investigation of the effects of nat-
ural glucocorticoids (administered in physiologicdoses
and kinetic conditions) on changes in expressionlactivity
of cell surface adhesion molecules and on leukocyte-en-
dothelial cell adhesion is necessary.
The effects of adrenal steroids on cellular function have
traditionally been thought to be mediated by changes in
gene expression induced by activated intracellular adrenal
steroid receptors (50). The presence of intracellular adre-
nal steroid receptors has been demonstrated in isolated leu-
kocytes (51, 52), and immune tissues (53-55). Ithas also
been hypothesizedthat adrenal steroids can exert non-
genomic effects throughmembrane-bound receptors (56,57).
Nongenomic actions of adrenal steroids are expected to be
more rapid than genomic actions. The rapidity of the stress-
and corticosterone-inducedchanges described in our studies
raises the possibility thatsome of the observed changes may
be mediated by nongenomic actions of adrenal steroids.
It is clear that the observed reductions in lymphocyte
subsets are not caused by stress-induced changes in sub-
population-specific surface marker expression. This is be-
cause we observed a decrease in absolute numbers of pe-
ripheral blood leukocytes. These measures of lymphocyte
number were independent of surface markers becausethey
were made using standard hematologic cell enumerating
The Journal of Immunology
techniques. Similarly, measurements of neutrophil num-
bers and percentages (which increased) were also indepen-
dent of surface markers. Furthermore, 85 to 90% of the
lymphocyte pool was accounted for (in terms of positive
staining for flow cytometry) at all time points and in all skin, mucosa, spleen, and other tissues). It is possible that
treatment groups. Decreases in relative proportions of B
cells, NK cells, and monocytes werematched by increases
in relative proportions of T cells, thus implying changesin
relative subpopulation percentages rather than alterations
in surface marker expression.
Stress-induced changes in leukocyte distribution-
functional implications
The large magnitude, selectivity, and reproducibility of the
observed effects, and the relatively mild stress which in-
duces these effects, suggest that stress-induced changes in
leukocyte numbers and proportions may have significant
functional implications. Results from some studies suggest challenge that mightbeinitiated
that stress-induced changes in leukocyte distribution may
mediate the observed effects of stress on immune function.
For example, it has been suggested that stress-induced
suppression of splenic and peripheral blood NK activity is
related to stress-induced migration of NK cellsout of these
compartments (5), and that a decrease in peripheral blood
NK activity afterexaminationstressis mediated by a
stress-induced decrease in blood NK cell number (6). Our
results indicate that the timing of the administration of the stress may serve to enhance immune preparedness for po-
immune challenge (relative to the timing and duration of
stress) andthe route of administration of challenge may be
crucial in determining the overall effect of stress on the
ability of the immune system to respond to the challenge.
i n addition to being released during stress, corticoste-
rone is also released according to a circadian rhythm (2,
53, 58). Furthermore, corticosterone secretion is induced
by antigenic challenge (59), viral infection (60, 61), tissue
damage (62), and by the administration of cytokines (63-
65). We hypothesize that corticosteronereleased under
these different physiologic conditions may induce a redis-
tribution of leukocytesbetween different immunecom-
partments. Further studies need to be conducted to deter-
minethepattern of redistribution of leukocytesamong
various tissues, and to examine the effects that this redis-
tribution may have on the ability of the immune system to
respond to challenge.
It is important to note that the stressor used in our ex-
periments is relatively mild and acute. Humans as well as
animals undergo a similar amount of stress in conjunction
with many standard laboratory manipulations. Unintended
stressors may cause a significant redistribution of leuko-
cytes between different immune compartments, and as a
result affect the immune parameter(s) being measured.
Thus, it may be important to minimize these changes in
leukocyte distribution or toaccount for these changes
when conducting certain diagnostic tests and immunologic
experiments.
5525
In conclusion, we suggestthat neural and endocrine fac-
tors released during stresssignal specific leukocytes to exit
theperipheralbloodandenter otherimmunecompart-
ments, (lymph nodes, Peyers patches, bone marrow, lung,
some leukocytes migrate to certain compartments to be
protected from potential deleterious effects of stress. Al-
ternatively, other leukocytesmay migrate to immune com-
partments which serve as battle stations where leuko-
cytesare likely to encounter Ags,pathogens, or other
activated immune cells. In either case, stress-induced re-
distribution of immune cells may have significant conse-
quences for the ability of the immune system to perform
its surveillance and effector functions.
We hypothesize that an important function of endocrine
mediators released under conditions of acute stress may be
toensure thatappropriate leukocytes arepresent in the
Downloaded from http://www.jimmunol.org/ by guest on July 1, 2017
right place and at the right time to respond to an immune
by thestress-inducing
agent (e.g., attack by a predator, invasion by a pathogen,
etc.). The modulation of immune cell distribution by acute
stress may be an adaptive response designed to enhance
immune vigilance andincrease the capacityof the immune
system to respond to challenge in immune compartments
(such as the skin and epithelia of lung, gastrointestinal and
urogenital tracts), which serve as major defense barriers
for the body. Thus, endocrine mediators released during
tential (or ongoing) immune challenge.
Acknowledgments
We thank Dr. Doina Ciurea and Libang Zhang for their assistance with
the hematologic measures described in these studies.
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