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Blood First Edition Paper, prepublished online March 17, 2009; DOI 10.1182/blood-2008-11-190769
Cortisol and epinephrine control opposing circadian rhythms in T-cell subsets
Stoyan Dimitrov,1 Christian Benedict,1 Dennis Heutling,1 Jürgen Westermann,2 Jan Born1 and
Tanja Lange1
1
Department of Neuroendocrinology, University of Lübeck, Germany
2
Department of Anatomy, University of Lübeck, Germany
Running head: Hormones and T-cell subpopulations
Address correspondence to: Tanja Lange, Department of Neuroendocrinology, University
of Lübeck, Ratzeburger Allee 160, Haus 23a, 23538 Lübeck, Germany. Phone: ++49-451500-4603; Fax: ++49-451-500-3640; E-mail: [email protected]
Present adresses of S.D. and D.H. : Stoyan Dimitrov, Cousins Center for Psychoneuroimmunology, University of California Los Angeles, USA; Dennis Heutling, Department of
Nephrology and Hypertension, University of Magdeburg, Germany.
1
Copyright © 2009 American Society of Hematology
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Abstract
Pronounced circadian rhythms in numbers of circulating T-cells reflect a systemic control of
adaptive immunity whose mechanisms are obscure. Here, we show that circadian variations in
T-cell subpopulations in human blood are differentially regulated via release of cortisol and
catecholamines. Within the CD4+ and CD8+ T-cell subsets, naive cells show pronounced
circadian rhythms with a daytime nadir, whereas (terminally differentiated) effector CD8+ Tcell counts peak during daytime. Naive T-cells were negatively correlated with cortisol
rhythms, decreased after low-dose cortisol infusion, and showed highest expression of
CXCR4 which was upregulated by cortisol. Effector CD8+ T-cells were positively correlated
with epinephrine rhythms, increased after low-dose epinephrine infusion, and showed highest
expression of β-adrenergic and fractalkine receptors (CX3CR1). Daytime increases in cortisol
via CXCR4 likely acts to redistribute naive T-cells to bone marrow whereas daytime
increases in catecholamines via β-adrenoceptors and, possibly, a suppression of fractalkine
signaling promote mobilization of effector CD8+ T-cells from the marginal pool. Thus,
activation of the major stress hormones during daytime favor immediate effector defense but
diminish capabilities for initiating adaptive immune responses.
2
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Introduction
Whereas recent research has provided some valuable insights into the complex processes of
systems regulation underlying the organism's immunity, circadian rhythms have been barely
investigated in this context, although many immune functions exhibit striking circadian
rhythms1-3. T-cells are the major mediators of adaptive immunity. In humans, the number of
circulating T-cells during the circadian nadir (14:00h) is decreased by about 40% compared to
T-cell counts during the circadian peak (2:00h)1,4,5.
The two major stress-systems, i.e., the sympathetic nervous system and the hypothalamo-pituitary-adrenal (HPA) system play a key role in conveying circadian rhythms to the
immune system controlling also the redistribution of T-cells2,6. Plasma concentrations of
cortisol and catecholamines exhibit most distinct circadian variations. In humans, levels are
minimal at night and rise to a maximum in the beginning of the day, with this rise considered
to prepare the organism for the demands of the active period4,7,8. Circadian variations in the
global population of circulating T-cells show a quite robust negative correlation with 24-h
changes in plasma cortisol5,9 and administration of corticosteroids to humans and animals
reduces blood lymphocytes counts, by redistributing these cells to the bone marrow6,10,11.
Opposite to corticosteroids, catecholamines generally increase cell numbers in blood. Infusion
of catecholamines induces rapid and transient increases in CD8+ (cytotoxic) T-cells and
natural killer (NK) cells12,13, and both populations are decreased when endogenous catecholamines are suppressed by stellate ganglion block14.
CD4+ and CD8+ T-cells are, in itself, heterogeneous populations. Each can be divided into at least four subsets with different function and recirculating patterns: (i) CD45RA+
naive and (ii) CD45RA– central memory T-cells which express CD62L and CCR7 molecules
and therefore recirculate through secondary lymphoid organs, and (iii) CD45RA– memory
effector and (iv) CD45RA+ (terminally differentiated) effector T-cells which lack CD62L and
CCR7 expression and thus are excluded from lymph nodes, but expected to migrate to
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peripheral nonlymphoid tissue15-17. To what extent the redistribution of these subsets is
differentially regulated by circadian release of corticosteroids and catecholamines is
obscure18,19.
Here we demonstrate specific circadian rhythms for these CD4+ and CD8+ T-cell
subsets that differ in phase, due to their primary regulation via glucorticoid and catecholamine
signaling, respectively. Naive CD4+ and CD8+ T-cells displayed clear circadian rhythms that
peaked during nighttime and were conveyed primarily via cortisol and CXCR4 signaling,
whereas effector CD8+ T-cells peaked during daytime and were modulated via catecholamine
and fractalkine receptor (CX3CR1) signaling.
Material and Methods
Subjects
Fourteen healthy men participated in the main experiment to assess circadian rhythms of Tcell subpopulations (mean age 25yrs, range 21-30yrs). All individuals were non-smokers
presenting with a normal nocturnal sleep pattern and did not take any medication at the time
of the experiments. Acute and chronic illness was excluded by medical history, physical
examination and routine laboratory investigation.
The men were synchronized by daily activities and nocturnal rest. They had a regular
sleep-wake rhythm for at least six weeks before the experiments and no signs of sleep
disturbances including apnea and nocturnal myoclonus. All subjects were adjusted to the
experimental setting by spending at least one adaptation night in the laboratory. The study
was approved by the Ethics Committee of the University of Lübeck. All men gave written
informed consent in accordance with the Declaration of Helsinki.
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Experimental design
Each participant was examined during two 24-h periods starting each at 20:00h, one including
a regular sleep-wake cycle whereas during the other period the subject remained awake
continuously (24-h continuous wakefulness). Both conditions for a subject were separated by
at least four weeks, and the order of conditions was balanced across subjects.
On experimental days, subjects arrived at the laboratory at 18:00h for preparing blood
sampling and, in the regular sleep-wake cycle condition, for standard polysomnographic
recordings. Sleep was allowed between 23:00h (lights off) and 7:00h in the morning. In the
condition of 24-h wakefulness subjects stayed awake in bed during this period in a half-supine
position, watching TV, reading, listening to music and talking to the experimenter at normal
room light (about 300lx). During daytime between 7:00 and 20:00h subjects stayed in the
laboratory in both conditions. Standardized meals were provided at appropriate times for
breakfast (8:00h), lunch (12:00h), and dinner (18:00h).
Blood was sampled first at 20:00h, then every 1.5 hours between 23:00 and 8:00h, and
every three hours between 8:00 and 20:00h the next day. During sleep (23:00 – 7:00h), blood
was sampled via an intravenous forearm catheter which was connected to a long thin tube and
enabled blood collection from an adjacent room without the subject's awareness (comparison
of blood collected at 23.00h in the absence and presence of the additional long tube used for
sampling during sleep, did not provide any hint at a biasing effect of the tube on the blood
measures of interest). To prevent clotting, about 700ml of saline solution were infused
throughout the 24-h experimental period. The total volume of blood sampled during a 24-h
period was 250ml. Blood samples were always processed immediately after sampling.
Standard polysomnographical recordings assured normal nocturnal sleep in the sleep
condition.
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T-cell subpopulations
Absolute counts of CD3+ total T-cells, CD4+ T-helper cells and CD8+ cytotoxic T-cells were
determined by a ‘lyse no-wash’ flow cytometry procedure. Briefly, 50µl of an undiluted blood
sample were immunostained with anti-CD3/FITC, anti-CD8/PE, anti-CD45/PerCP and antiCD4/APC in Trucount tubes (BD Biosciences, San Jose, CA). After 15min incubation at
room temperature, 0.45ml of FACS lysing solution (BD Biosciences) was added followed by
incubation for 15min. Finally, samples were mixed gently and at least 10,000 CD3+ cells were
acquired on a FACSCalibur using CellQuest Software (BD Biosciences).
For detection of naive, central memory, effector memory and effector T-cells, whole
blood was incubated with anti-CD4/FITC and anti-CD8/FITC (Diatec, Oslo, Norway), antiCD62L/PE and anti-CD3/PerCP (BD Biosciences) and anti-CD45RA/APC (Invitrogen, Carlsbad, CA). Cells were lysed with FACS lysing solution (BD Biosciences), washed, resuspended and at least 10,000 CD4+ or CD8+ T-cells were acquired on the FACSCalibur using
CellQuest Software (BD Biosciences). Absolute counts of T-cell subsets were calculated
based on the proportion of the respective CD4+ and CD8+ T-cell subpopulation and on
absolute counts obtained by the ‘lyse no-wash’ procedure.
Hormone assays
Samples for measuring hormone concentrations were kept frozen at –70°C until assay.
Cortisol was measured in serum using a commercial assay (Immulite, DPC-Biermann GmbH,
Bad Nauheim, Germany). Epinephrine and norepinephrine were measured in plasma by
standard high-performance liquid chromatography. Sensitivity and intra- and interassay
coefficients of variation were as follows: cortisol 0.2µg/dl, < 10%; epinephrine 2.0pg/ml, <
5.6%; norepinephrine 5.0pg/ml, < 6.1%.
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Administration of cortisol and epinephrine
Supplementary experiments were performed in ten healthy men who were intravenously
infused for 30min with sodium chloride (placebo), cortisol (8µg/kg/min, Hydrocortison 100®,
Rotexmedica, Trittau, Germany), or epinephrine (0.005µg/kg/min, Suprarenin®, SanofiAventis, Frankfurt, Germany) on three different occasions, according to a double-blind
within-subject cross-over design. The doses were chosen to induce changes in blood
concentration comparable with the nadir-to-peak difference in the circadian variation of the
respective hormone. Infusions started always in the evening (21:00h) when levels of
endogenous cortisol are close to the circadian nadir. Subject characteristics were the same as
in the main experiment. Subjects were prepared for blood sampling one hour before infusions
started and remained in a supine position throughout the experimental session. Blood samples
to determine hormone concentrations and immunologic parameters were drawn via a second
catheter (inserted into the vein of the other arm) before (baseline), during (15, 30min) and
after (60 to 300min) the start of the infusion. Heart rate and an electrocardiogram were
continuously monitored to exclude any adverse effects. Hormones and T-cell subpopulations
were determined and analysed in the same way as in the main experiments, except that cells
were acquired on a FACSCanto II using FACSDiva Software (BD Biosciences).
Determination of glucocorticoid and adrenergic receptors
Whole blood was used for simultaneous immunophenotyping and intracellular glucocorticoid
receptor (GR) determination in T-cell subpopulations20. The following panel of monoclonal
antibodies was employed: anti-GR/FITC and anti-mouse isotope control (AbD Serotec,
Raleigh, NY); anti-CD62L/PE, anti-CD8/PerCP, anti-CD3/PE-Cy7, anti-CD45RA/APC and
anti-CD4/APC-Cy7 (all from BD Biosciences). The staining was performed by incubating
100µl of undiluted blood sampled in the morning with the surface antibodies for 15min. Cells
were then fixed, permeabilized and stained against intracellular GR according to the
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manufacturer’s instructions using reagents from BD Biosciences. At least 1,000 naive, central
memory, effector memory and effector CD4+ or CD8+ T-cells were acquired and subsequently
analyzed for expression of GR by a FACSCanto II using FACSDiva Software (BD
Biosciences). The relative quantity of GR (mean GR fluorescence) expressed as mean
fluorescence intensity (MFI), was calculated as the difference between mean values of GR
and isotype control labeled samples.
To determine numbers of β-adrenergic receptors (β-AR) in T-cell subpopulations,
cell purification was first performed. PBMC were isolated from the buffy coat of 300ml
morning blood of healthy donors by Ficoll-Paque gradient centrifugation in Leucosep tubes
(Greiner Bio-One, Frickenhausen, Germany) according to the manufacturer’s instruction.
Purified CD4+ and CD8+ T-cells were separated from PBMC by negative isolation using the
magnetic-activated cell sorting (MACS) technology (Miltenyi Biotec, Bergisch Gladbach,
Germany). CD62L positive and negative CD4+ and CD8+ cells were then sorted using antiCD62L/FITC antibody and anti-FITC MultiSort Kit (Miltenyi Biotec). Finally, the remaining
microbeads of the positive populations were removed by releasing reagent. Effector memory
and effector CD8+ T-cells were additionally isolated from the CD62L– fraction by MoFlo Hi
Speed Cell Sorter using anti-CD45RA antibody (Dako, Carpinteria, CA). The purity of the
isolated T-cell subpopulations was evaluated by flow cytometry and exceeded 90% in all
experiments performed.
The number of β-AR on the purified T-cell subpopulations was determined as described before21. Briefly, aliquots of 3 x 105 cells were incubated for 60min at 37°C with five
different concentrations of
125
I-CYP (I-cyanopindolol; PerkinElmer, Waltham, MA), ranging
from 12.5 to 200pM 125I-CYP. Radioactivity was determined in an LKB 1282 CompuGamma
CS gamma counter (LKB Wallac, Turku, Finland). Unspecific binding of 125I-CYP was determined in parallel by incubating the T-cell subpopulations in the presence of 1µM propranolol,
a competitive antagonist of
125
I-CYP. Specific binding of
125
I-CYP was determined by sub8
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tracting unspecific binding from the total binding capacity. The maximum number of
125
I-
CYP binding sites (Bmax), representing the number of β-AR was calculated according to the
method of Scatchard.
Detection of adhesion molecules and chemokine receptors
Whole blood from six healthy subjects sampled at 9:00 and 21:00h (on the same day) was
immediately labeled with anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, anti-CD62L, antiCD11a, anti-CD11b, anti-CD49d, anti-CCR5, anti-CCR7, anti-CXCR1, anti-CXCR3, antiCXCR4 (all from BD Biosciences) and anti-CX3CR1 (MBL International, Woburn, MA).
Samples were then lysed, acquired and analyzed for the expression of adhesion molecules and
chemokine receptors on T-cell subsets using the FACSCanto II with FACSDiva Software
(BD Biosciences).
To test the influence of cortisol on the expression of CXCR4 on T-cell subsets, blood
samples taken at 21:00h were additionally incubated at 37°C in the absence or presence of
20µg/dl cortisol for up to six hours. CXCR4 expression measured as MFI was analyzed
before and every hour after the start of incubation.
Statistical analysis
Data are generally presented as means ± s.e.m. To identify circadian rhythms, cosinor analysis
was performed separately for the conditions of a regular sleep-wake cycle and of 24-h
continuous wakefulness as well as collapsed across both conditions using Chronolab22
(Cosinor analysis identifies circadian rhythm based on a least square fit of a cosine wave to
the time series data). To assess temporal relationships between circadian rhythms in T-cell
subpopulations of interest and diurnal variations in hormonal concentrations, cross-correlation
functions were calculated. For these analyses individual data were normalized (i.e, values
were ln-transformed and the individual 24-h mean was subtracted). Then product-moment
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correlation coefficients and stepwise linear regression coefficients were calculated across all
time points and subjects. Bonferroni correction was applied on resulting coefficients yielding
a significance level of P=0.002. Analyses of effects of intravenous cortisol and epinephrine
infusions on T-cell subsets were based on analysis of variance (ANOVA) including a
substance factor (“Cort/Plac” and “Epi/Plac”), a “Population” factor representing the different T-cell subsets, and a “Time” factor reflecting the different time points of measurement.
Considering the different kinetics of hormone actions six time points (60 to 300min after start
of infusion) were chosen for cortisol and two time points (15 and 30min after start of
infusion) were chosen for epinephrine. In case of significant substance effects, differences
between effects of placebo and, respectively, cortisol and epinephrine were analyzed at single
time points using paired t-test. ANOVA and subsequent t-tests were also used to analyze
differences in GR, β-AR and adhesion molecule/chemokine receptor expression among T-cell
subsets, including the factors “Population” and “Time” (for daytime differences in molecule
expression and to confirm CXCR4 upregulation after in vitro incubation with cortisol).
Degrees of freedom were adjusted according to the Greenhouse-Geisser procedure where
appropriate. A P<0.05 was considered significant.
Results
Undifferentiated total T-cell populations show strong circadian rhythms
Total numbers of CD3+, CD4+, and CD8+ T-cells showed the expected pronounced circadian
rhythm, with maximum values (acrophase) at ~2:00h at night and minimum values (nadir) at
~14:00h (P<0.001, derived from cosinor analysis, Table 1). Circadian rhythms of these cell
populations were remarkably similar during regular sleep-wake conditions and 24-h
continuous wakefulness, except for a slight but consistent decrease in cell counts when
subjects slept during night-time (P<0.05, for a comparison of night-time levels4,5). Because
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overall the effects of sleep remained small and did not essentially alter circadian rhythm
characteristics in the cell counts of interest, we focused our further analyses on data collapsed
across both the regular sleep-wake condition and the condition of 24-h continuous
wakefulness.
Opposing rhythms for T-cell subpopulations
We classified CD4+ and CD8+ T-cells into four distinct subpopulations: naive, central memory, effector memory, and effector T-cells. Cosinor analysis revealed distinct and significant
(P<0.05) circadian rhythms in absolute cell counts for all subpopulations of interest, except
for effector CD4+ T-cells (summarized in Figure 1, Table 1). The identified rhythms differed
remarkably with respect to their peak times and relative amplitudes. Acrophases during the
night were evident for naive, central memory and effector memory CD4+ and CD8+ T-cells
(between 01:31 and 02:41h). In contrast, effector CD8+ T-cell counts peaked during daytime,
in the afternoon hours (15:34h). Rhythms were most pronounced for naive CD4+ and CD8+ Tcells. With reference to peak counts, cell counts during nadir times were reduced,
respectively, by 41% and 46%, in these two populations (Table 2).
Circadian rhythms of cortisol and catecholamines
Plasma concentrations of both cortisol and catecholamines were characterized by distinct
circadian rhythms which were equally observed during conditions of regular sleep and 24-h
wakefulness (P<0.01 for collapsed data, Table 1). Cortisol concentrations showed a minimum
~0:30h and a maximum around the time of morning awakening ~8:00h (Figure 2A).
Concentration of both epinephrine and norepinephrine were decreased during nighttime hours
(with a slightly more pronounced decrease for norepinephrine when subject slept; P<0.05),
and reached maximum levels in the morning between 8:00 and 11:00h, with epinephrine
showing a second increase in the evening hours (Figure 2B,C).
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Temporal associations between T-cell subset counts and hormone concentrations
There was a marked negative correlation between the rhythm of cortisol and those of total
CD4+ and total CD8+ T-cell counts. Correlations were maximal when CD4+ and CD8+ T-cell
counts were measured with a delay of three hours with reference to cortisol concentrations
(r=–0.65 and –0.64, respectively, P<0.001). Although less strong, epinephrine and norepinephrine concentrations also correlated negatively with total numbers of CD4+ and CD8+ Tcells, maximally at a zero time lag (r =–0.27 to –0.40, P<0.001).
T-cell subset counts were also correlated to hormone concentrations (summarized in
Table 2). Stepwise linear regression analyses indicated marked negative correlations of naive,
central memory and effector memory CD4+ and CD8+ T-cells to cortisol concentrations. In
contrast, effector CD8+ T-cells counts were correlated positively with epinephrine levels. As
for the total CD4+ and CD8+ T-cell counts, correlations with cortisol were maximal for cell
subset counts measured with a delay of three hours.
Influence of cortisol and epinephrine on the redistribution of T-cell subpopulations
During infusion of cortisol and epinephrine plasma concentrations of the respective hormone
increased, reaching peak values after 30min. Thereafter, concentrations declined, with a more
gradual decrease for cortisol than epinephrine concentrations, reflecting the different halflives of the hormones. Cortisol administration did not affect plasma epinephrine concentrations and, vice versa, epinephrine administration did not affect cortisol concentrations
(P>0.5, for all comparisons).
As expected, infusion of cortisol compared with placebo induced a clear decrease in
both total CD4+ and CD8+ T-cell counts, which started 90min after infusion onset and reached
minimum levels after 180 min (F(1,9)=41.4 and 60.1, respectively, P<0.001, for Cort/Plac).
However, there were also systematic differences in the size of the suppression among the
subpopulations of interest (F(7,63)=24.5, P<0.001, for Population x Cort/Plac). The
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suppression was strongest for naive T-cells, intermediate for central memory T-cells, weaker
for effector memory T-cells, and entirely absent for effector T-cells (see Figure 4I,J and Table
2 for percent changes in comparison to placebo 180min after starting cortisol infusion). Of
note, effector CD4+ and CD8+ T-cells were the only subsets not influenced by cortisol (Figure
4D,H).
In striking contrast to the effects of cortisol, infusion of epinephrine induced a
selective increase in numbers of effector CD8+ T-cells only (F(1,9)=7.0, P<0.05, for
Epi/Plac). The increase was short-lived and concentrated around the end of the infusion
period when epinephrine levels were maximal (Figure 4H). All other T-cell subsets remained
unchanged by epinephrine infusion (P>0.35). Comparable effects were revealed when subpopulations were categorized by CCR7 and CD45RA expression (data not shown).
Expression of glucocorticoid and adrenergic receptors on T-cell subpopulations
Differences in GR expression between subpopulations were only subtle although highly
significant, with generally higher levels in CD8+ than CD4+ cells (F(7,49)=37.1, P<0.001; see
Table 2 for pairwise comparisons between subpopulations). Surprisingly, however, cells (like
CD4+ naive T-cells) that were highly sensitive to the reducing effect of cortisol, showed on
average even slightly lower rather than higher GR expression.
When we analyzed expression of β-AR on MACS-sorted T-cell subsets, we found
striking differences between different subpopulations in the expected direction (F(3,12)=9.3,
P<0.05, see Table 2 for pairwise comparisons between subpopulations)23. β-AR levels were
higher on CD8+ than CD4+ T-cells and within these cell populations higher on CD62L–
(effector memory and effector T-cells) than on CD62L+ T-cells (naive and central memory
cells; Figure 5). However, the specific binding of
125
I-CYP was comparable in effector
memory and effector CD8+ T-cells (P>0.6).
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Expression of adhesion molecules and chemokine receptors on T-cell subpopulations
We revealed quite distinct expression patterns of adhesion molecules and chemokine
receptors in the different T-cell subpopulations (Figure 6). CXCR4 expression was highest in
naive, lower in central memory and lowest in effector memory and effector CD4+ and CD8+
T-cells (F(7,35)=94.2, P<0.001, see Figure 6E,F for pairwise comparison between subpopulations). By contrast, CX3CR1 was preferentially expressed on effector CD8+ T-cells
(F(7,35)=50.2, respectively, P<0.001, Figure 6G,H). Effector CD8+ T-cells were also
charactarized by highest expression of CXCR1, CD11a and CD11b, whereas CD49d and
CCR5 density was lowest on naive CD4+ and CD8+ T-cells. CXCR3 expression was not
selective for any subset (data not shown). Of note, the ratio of CXCR4/CX3CR1 expression
on T-cell subsets strongly correlated with percent changes of the respective subpopulation
from night to daytime (Figure 6I).
We assessed these markers additionally in blood samples collected in the evening
(21:00h) when cortisol concentrations are low in comparison with morning levels.
Interestingly, expression was higher in the morning than evening for CD62L, CD49d,
CXCR1, CXCR4 and CX3CR1 (all P<0.05), whereas CCR7 expression did not differ
between the time points. Daytime differences in CD62L and CXCR4 expression were most
pronounced for naive and central memory CD4+ and CD8+ T-cells, whereas daytime
differences in CX3CR1 expression were most pronounced in effector memory and effector Tcells (for CXCR4 and CX3CR1 see Figure 6E-H).
We incubated T-cells drawn at 21:00h in the evening (when endogenous cortisol is
low, <5µg/dl) with cortisol at concentrations mimicking those in the morning (20µg/dl).
Cortisol induced a distinct linear increase in CXCR4 expression in both naive and central
memory CD4+ and CD8+ T-cells (P<0.01, for naive T-cells see Figure 6J,K). The increase
was detectable as early as one hour after the start of incubation and continued throughout the
6-h monitoring period. It was comparable in all subpopulations of interest.
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Discussion
We show here in humans a fine-tuned systems regulation of circadian rhythms in circulating
T-cell subpopulations that is controlled by the brain's major stress-hormone systems, i.e., by
release of glucocorticoids via the HPA system and release of catecholamines via the
sympathetic nervous system. Distinct rhythms with nocturnal peaks were evident for naive
and, less pronounced, for central memory and effector memory CD4+ and CD8+ T-cells. In
contrast, the rhythm of (terminally differentiated) effector CD8+ T-cells peaked during
daytime. The rhythms were not substantially affected by sleep (compared with a condition of
24-h continuous wakefulness), and showed robust temporal associations to the circadian
variations in plasma cortisol and epinephrine. T-cell subsets with nocturnal peak rhythms
were negatively correlated with cortisol whereas effector CD8+ T-cells were positively
correlated with epinephrine. Administration of cortisol and epinephrine, at low doses
mimicking in size endogenous increases in blood concentrations of these hormones, produced
strikingly different effects on the T-cell subpopulations. Cortisol with a delay of three hours
decreased to the greatest extent naive T-cells and to a lesser extent central memory and
effector memory T-cells, but left effector T-cells unaffected. In contrast, epinephrine induced
an immediate and highly selective increase in effector CD8+ T-cells, but did not affect any
other subpopulation of interest. Greater sensitivity to suppressing influences of cortisol on Tcell subset counts was associated with increased expression of CXCR4, a candidate molecule
mediating effects of cortisol on T-cell migration. Epinephrine induced increases in effector
CD8+ T-cells on the other hand are likely conveyed by an increased expression of β-AR and
CX3CR1 on these cells.
The pronounced global circadian variation in total numbers of circulating T-cells,
peaking during nighttime (at ~2:00h) is well-documented4,5,9. However, since T-cells constitute a functionally highly heterogeneous population, assessment of total T-cell counts
masks the specific rhythms characterizing the various CD4+ and CD8+ T-cell subpopulations.
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Rhythms of naive, central memory and effector memory T-cells were critically controlled by
cortisol, i.e., the greater the sensitivity of the respective subset to the reducing influence of
cortisol the higher was the rhythm amplitude in this subpopulation. Cortisol infusion at
concentrations mimicking morning increases in cortisol under natural conditions reduced
naive T-cell counts by ~40%, i.e., an effect size remarkably similar to the peak-to-nadir
difference in the circadian rhythm of this subpopulation. Previous studies showed that
glucocorticoid administration and exercise induced endogenous cortisol release suppress
CD45RA+ and CD62L+ T-cells to a greater extent than their CD45RA– and CD62L–
counterparts18,24. Thus, co-expression of both surface markers on naive T-cells identifies this
subpopulation as the one most sensitive to the reducing effects of cortisol. In combination, the
data strongly support the notion that the marked circadian rhythm in naive T-cells peaking
during night-time when cortisol reaches nadir levels, is controlled by the brain via HPA
glucocorticoid release2. Surprisingly, however, graded sensitivity to cortisol among the T-cell
subpopulations did not relate to differences in glucocorticoid receptor expression, although
cortisol is known to influence T-cell migration through this receptor25.
In contrast to naive T-cells, effector CD8+ T-cells increased during daytime. The
increase in cell numbers of this subset synchronized with the daytime increase in epinephrine,
reflects a particular sensitivity of these cells that are CD62L– to the influence of catecholamines26. Accordingly, infusion of epinephrine at concentrations that mimicked normal
day values produced an increase only in circulating effector CD8+ T-cells, but had no
appreciable effect on the other T-cell subsets. This finding concurs with previous studies in
which exercise, mental stress and administration of sympathomimetics selectively mobilized
CD62L– and CCR7– T-cells via β2-AR19,24,27,28. As a rule, the mobilization of these CD62L–
or CCR7– subsets is much more pronounced for CD45RA+ effector than CD45RA– effector
memory T-cells27 and also for CD8+ than CD4+ cells19,24. In our experiments, β-AR density
partly reflected the differences in catecholamine sensitivity with higher receptor numbers in
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CD8+ than CD4+ T-cells and higher numbers in CD62L– than CD62L+ T-cells. However,
expression of β-AR in effector CD8+ T-cells was comparable to that in effector memory CD8+
T-cells which were not affected by epinephrine injection. Therefore, the effect of epinephrine
selectively increasing numbers of effector CD8+ T-cells, is not sufficiently explained by β-AR
expression alone.
Fast recovery of T-cell numbers in blood after concentrations of cortisol or
epinephrine had normalized (following infusion of the hormones), suggests that the changes
induced by these hormones represent a redistribution, rather than effects on production rate or
apoptosis of the respective T-cell subpopulation10,12,29,30. Therefore, and because neither
expression of GR nor of β-AR could fully account for the differential responses to cortisol
and epinephrine in the T-cell subsets of interest, we concentrated on an analysis of adhesion
molecule and chemokine receptor profiles regulating migratory behavior of these cells.
Cortisol and epinephrine responsive T-cell subpopulations greatly differ in their recirculating
pattern and function with two extremes: Whereas naive T-cells are critically involved in
initiating adaptive immune responses in secondary lymphatic tissues, effector CD8+ T-cells
exert immediate, mainly cytotoxic effector functions in the periphery16,17,31. Cortisol depletes
T-cells from peripheral blood and redistributes them to the bone marrow11, whereas epinephrine mobilizes T-cells from the so called marginal pool – cells that roll along the
endothelial layer – by demargination12,13,32. Both facets of cell migration essentially depend
on different selectins, integrins, chemokines and respective receptors, i.e., molecules regulating the attraction and adhesion of the cells to the vascular endothelium33,34.
Bone marrow homing of T-cells is mainly regulated by the expression of CD62L,
CD49d and CXCR435. Of these molecules, expression of CXCR4 showed the strongest
association with the cells' sensitivity to the redistributing effects of cortisol with highest
CXCR4 expression in naive T-cells, intermediate in central memory and low expression in
effector memory and effector T-cells36. In functional studies, high CXCR4 expression on
17
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naive and central memory CD8+ T-cells was revealed to be critical for the facilitated homing
of these cells to bone marrow, in comparison with effector subpopulations35. Glucocorticoids
enhance CXCR4 expression and signaling in T-cells in vitro37,38. Here we extend this
observation by showing increased CXCR4 expression in the different T-cell subsets of
interest after incubation with low, physiological concentrations of cortisol. This analysis
together with comparisons of blood sampled in the morning versus evening points to a most
pronounced cortisol-induced up-regulation of CXCR4 in naive and central memory CD4+ and
CD8+ T-cell subsets. Interestingly, not only CXCR4 but also bone marrow production of
CXCL12, i.e., the ligand of CXCR4, follows a strong circadian rhythm with a peak during the
active period, although influences of cortisol on this rhythm have as yet not been elucidated39.
Overall data provide good evidence that up-regulation of CXCR4 substantially contributes to
the cortisol dependent control of circadian rhythms especially in circulating naive and central
memory T-cells, leading to enhanced redistribution of the cells to bone marrow in the
morning hours.
Catecholamines, in contrast to cortisol, act by recruiting immune cells with high
expression of β 2-AR from the marginal pool to the circulation, by reducing the adhesive
properties of the cells12. This process is fast occuring within minutes12,40 probably through
conformational changes of adhesion molecules. Of the CD8+ T-cells with high β-AR density,
effector CD8+ T-cells were hallmarked by a profile comprising high expression of CD11a,
CD11b, CXCR1 and especially of CX3CR1. Stress, exercise and catecholamine injections are
known to increase CD11a+, CD11b+19,24,41, CXCR1+ and CX3CR1+ cells (S.D., J.B., and T.L.,
unpublished, March 7, 2009). One likely candidate contributing to margination of effector Tcells and their demargination by catecholamines appears to be CX3CR1: This molecule replaces function of selectin as adhesion molecule on CD62L– cells31,34,42, it regulates conformational changes of integrins like CD11a33,42 and, like β-AR, it is a G protein coupled receptor,
18
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enabling immediate suppressing influences of catecholamines on CX3CR1 signaling, e.g., via
cAMP43,44.
In sum, our results point to a coordinate system regulation of circadian rhythms in
circulating T-cell subpopulations (Figure 7). This regulation presumably originates from the
brain's major circadian oscillators (in the nucleus suprachiasmaticus and associated hypothalamic structures) and synchronizes immune functions via two separate pathways, the HPA
axis and the sympathetic nervous system, leading eventually to rhythms differing in phase.
Naive, central memory and, less pronounced, effector memory T-cells show clear circadian
rhythms peaking during nighttime. The higher the rhythm's amplitude the stronger in these
subsets is the expression of CXCR4, making these cells sensitive to the daily rhythm in
cortisol. In contrast, effector CD8+ T-cells show a rhythm peaking during daytime. These
cells with a CD62L– phenotype show high expression of β-AR, CD11a and CD11b and, unlike the other T-cell subsets, of CXCR1 and CX3CR1, making these cells particularly sensitive to the immediate demarginating influences of catecholamines. Given that circadian blood
T-cell counts run in parallel with content of the cells in lymph nodes45,46, the release of naive
T-cells from bone marrow during nighttime might allow a facilitated homing of the cells to
secondary lymphatic tissues thus supporting the initiation of adaptive immune responses. On
the other hand, the increase in circulating effector CD8+ T-cells during daytime represents a
circadian feature that is shared with other cytotoxic cell populations sensitive to catecholamines, like NK-cells5,26. It presumably acts to strengthen effector immune defense against
tissue damage and infection encountered during the active phase12,47,48. The temporally
disparate activation and distribution of T-cell subsets and associated immune functions by
glucocorticoids and catecholamines may form part of a more basic mechanism optimizing the
organism's immunological adaptation not only to the changing demands of the circadian restactivity cycle but also to stress in general.
19
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Acknowledgments
We are grateful to C. Otten, A. Otterbein, T. Kriesen and E. Böschen for technical assistance.
Author Contributions
SD, CB, DH, JB, and TL designed the study; SD, CB, DH and TL performed experiments and
collected data; SD, JB, and TL analyzed and interpreted data and performed statistical analyses; SD, CB, JW, JB and TL wrote the manuscript.
`Funding
The study was funded by a grant from the Deutsche Forschungsgemeinschaft (SFB 654:
Plasticity and Sleep). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interest
The authors have declared that no competing interests exist.
Abbreviations
ANOVA
analysis of variance
β-AR
β-adrenergic receptor
GR
glucocorticoid receptor
HPA
hypothalamo-pituitary-adrenal
I-CYP
I-cyanopindolol
MACS
magnetic-activated cell sorting
MFI
mean fluorescence intensity
NK
natural killer
20
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PBMC
peripheral blood mononuclear cells
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Table 1. Cosinor data for circulating T-cell subsets and hormone concentrations
Mesor
Amplitude
Acrophase
Significance
CD3+ T-cells /μl
1902.08 ± 85.00
387.91 ± 38.10
02:10 h ± 20min
<0.01
CD4+ T-helper cells /μl
1120.42 ± 70.29
243.99 ± 25.4
02:17 h ± 22min
<0.01
Naive /μl
510.20 ± 43.13
131.65 ± 16.00
02:41 h ± 23min
<0.01
Central memory /μl
349.87 ± 18.15
74.58 ± 6.60
02:02 h ± 20min
<0.01
Effector memory /μl
221.36 ± 15.40
42.31 ± 5.20
01:31 h ± 23min
<0.01
Effector /μl
-
-
-
ns
CD8+ cytotoxic T-cells /μl
658.71 ± 30.08
140.01 ± 13.80
02:03 h ± 19min
<0.01
Naive /μl
326.49 ± 22.27
96.22 ± 9.90
02:09 h ± 20min
<0.01
Central memory /μl
86.52 ± 6.76
15.33 ± 2.00
02:16 h ± 26min
<0.01
Effector memory /μl
188.87 ± 12.37
24.72 ± 4.50
01:56 h ± 29min
<0.01
Effector /μl
66.69 ± 11.64
6.96 ± 3.4
15:34 h ± 116min
<0.05
Cortisol μg/dl
7.68 ± 0.22
4.64 ± 0.20
10:40 h ± 19min
<0.01
Epinephrine pg/ml
25.55 ± 3.03
6.69 ± 1.70
16:10 h ± 54min
<0.01
Norepinephrine pg/ml
239.28 ± 17.18
71.41 ± 10.80
14:05 h ± 16min
<0.01
T-cell counts and hormone concentrations were collapsed across conditions of a regular sleepwake cycle and 24-h continuous wakefulness and analysed by the cosinor method. Mesor:
average value during 24-h period. Amplitude: difference between estimated maximum/minium and Mesor; peak and nadir values can be calculated by adding and substracting
the amplitude to/from Mesor values, respectively. Acrophase: time of day of the maximum,
s.e.m. is presented in min. Significance: P-value of the zero amplitude test.
24
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Table 2. T-cell subset characteristics
+
+
CD4 T-helper cells
CD8 cytotoxic T-cells
Naive
Central
memory
Effector
memory
Effector
Naive
Central
memory
Effector
memory
Effector
CD62L
+
+
-
-
+
+
-
-
CD45RA
+
-
-
+
+
-
-
+
Changes from
night to daya
-41%
-35%
-32%
ns
-46%
-30%
-23%
+23%
Correlation with
cortisolb
-0.65*
-0.62*
-0.54*
ns
-0.68*
-0.54*
-0.42*
ns
Changes after
cortisolc
-42%
-38%
-35%
ns
-39%
-27%
-21%
ns
GR expressiond
1063
±31*
1098
±34***
1167
±35
1181
±36
1168
±33
1228
±50*
1280
±51**
1367
±55
Correlation with
epinephrineb
ns
ns
ns
ns
ns
ns
ns
+0.26*
Changes after
epinephrinec
ns
ns
ns
ns
ns
ns
ns
+30%
e
ß-AR expression
Naive and
Central memory
Effector memory
and Effector
Naive and
Central memory
Effector
memory
Effector
197
±42*
827
±239
727
±181**
2045
±579
2112
±602
Columns show data of naive (CD45RA+CD62L+), central memory (CD45RA–CD62L+),
effector memory (CD45RA–CD62L–), and effector (CD45RA+CD62L–) CD4+ and CD8+ Tcells. aPercent changes comparing the two extremes of the circadian rhythm during nighttime
and daytime. bCorrelations assessed by stepwise linear regression analyses between circadian
variation in cortisol/epinephrine and respective T-cell subpopulation, measured with a delay
(time lag) of three and zero hours with reference to cortisol and epinephrine, respectively; *
P<0.001. cPercent change in cell numbers 180 and 30min after starting cortisol and
epinephrine infusions, respectively, compared to placebo numbers (see also Figure 4I,J),
n=10. dMorning levels of GR expression in MFI, mean±s.e.m., n=8; * P<0.05, ** P<0.01 and
*** P<0.001 for pairwise comparisons between naive vs. central memory, central memory vs.
effector memory and effector memory vs. effector T-cells. eMorning levels of β-AR expres25
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sion measured by specific binding of
125
I-CYP in isolated T-cells, data of saturation curves
(Figure 5) were analyzed using nonlinear regression plot and the maximum number of
125
I-
CYP binding sites (Bmax) is presented as mean±s.e.m.; n=6, * P<0.05 and ** P<0.01 for
pairwise comparisons between CD62+ vs. CD62L– T-cells and effector memory vs. effector
CD8+ T-cells.
ns = non significant
26
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Figure Legends
FIGURE 1. Rhythms of circulating T-cell subsets. Mean (±s.e.m.) of (A, E) naive
(CD45RA+CD62L+), (B, F) central memory (CD45RA–CD62L+), (C, G) effector memory
(CD45RA–CD62L–) and (D, H) effector (CD45RA+CD62L–) CD4+ (left) and CD8+ (right) Tcells; n=14. Rhythms with nocuturnal peaks are more pronounced for naive T-cells than for
central memory and effector memory T-cells. In contrast, effector CD8+ T-cells peak during
the day. Cosinor analysis of circadian rhythms was performed on data collapsed across
conditions of a regular sleep-wake cycle and 24-h continuous wakefulness (see also Table 1
for rhythm characteristics). Adapted cosine curves are indicated for confirmed circadian
rhythm. Shaded area indicates bed time.
FIGURE 2. Circadian variations in stress hormone concentrations. Mean (±s.e.m.)
plasma concentrations of (A) cortisol, (B) epinephrine and (C) norepinephrine during the 24-h
period collapsed across conditions of a regular sleep-wake cycle and 24-h continuous wakefulness; n=14. Shaded area indicates bed time.
FIGURE 3. Low-dose cortisol and epinephrine infusions to mimick nadir-to-peak
concentration difference in circadian rhythm. Mean (±s.e.m) plasma concentrations of (A)
cortisol, (B) epinephrine, and (C) norepinephrine in healthy men before (21:00h), during (15,
30min), and after (60, 90, 120, 180, 240, 300min) 30-min infusions (horizontal grey bar) of
placebo (sodium chloride, open circles), cortisol (8µg/kg/min, black circles) and epinephrine
(0.005µg/kg/min, grey circles); n=10. * P<0.05, ** P<0.01, *** P<0.001, for pairwise comparisons between cortisol/placebo (A), and epinephrine/placebo conditions (B) at single time
points.
27
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FIGURE 4. Cortisol and epinephrine differentially influence T-cell subpopulations.
Mean (±s.e.m) numbers of (A, E) naive (CD45RA+CD62L+), (B, F) central memory
(CD45RA–CD62L+), (C, G) effector memory (CD45RA–CD62L–) and (D, H) effector
(CD45RA+CD62L–) CD4+ (left) and CD8+ (right) T-cells following a 30-min intravenous infusion (horizontal grey bar) of placebo (sodium chloride, open circles), cortisol (filled circles)
and epinephrine (grey circles). n=10, * P<0.05, ** P<0.01, *** P<0.001 for pairwise comparison between cortisol/placebo conditions (A, B, C, E, F, G), and epinephrine/placebo
conditions (H) at single time points. Bar charts show percent changes in (I) CD4+ and (J)
CD8+ T-cell subsets 180min after cortisol (black bars) and 30min after epinephrine infusion
(grey bar), respectively, compared to placebo (see also Table 2); * P<0.05, ** P<0.01, ***
P<0.001 for ANOVA main effect (Cort/Plac, Epi/Plac) and for pairwise comparison of
cortisol effects between naive vs. central memory, central memory vs. effector memory and
effector memory vs. effector T-cells.
FIGURE 5. Saturation curves indicating β-AR expression in T-cell subsets. Mean
(±s.e.m.) specific binding of β-AR agonist 125I-cyanopindolol (125I-CYP) per 100,000 cells at
different 125I-CYP concentrations; n=6. β-AR expression is greater in CD62L– (filled circles)
than CD62L+ (open circles) T-cells and in CD8+ (B) than CD4+ T-cells (A). Values of specific
binding were obtained by subtracting nonspecific binding from total binding. For Bmax see
Table 2.
FIGURE 6. CXCR4 and CX3CR1 expression on T-cell subsets. Dotplots from a representative subject of CXCR4+ (red, A, C) and CX3CR1+ (blue, B, D) naive (N,
CD45RA+CD62L+), central memory (CM, CD45RA–CD62L+), effector memory (EM,
CD45RA–CD62L–) and effector (E, CD45RA+CD62L–) CD4+ (left) and CD8+ (right) T-cells.
Chemokine receptor postitive cells are shown as coloured dots and mean percentages indicate
28
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respective subpopulation with the highest proportion of chemokine receptor positive cells,
n=6. Bar charts show mean±s.e.m. of (E, F) CXCR4 and (G, H) CX3CR1 expression indicated as MFI, n=6; * P<0.05, ** P<0.01 and *** P<0.001 for pairwise comparison between
morning (9:00h, red and blue bars) and evening (21:00h, dark red and dark blue bars) levels
and pairwise comparisons between naive vs. central memory, central memory vs. effector memory and effector memory vs. effector T-cell counts (collapsed across morning and evening
samples). (I) Correlation of CXCR4/CX3CR1 ratio (ln(CXCR4 MFI/CX3CR1 MFI), mean of
n=6) and percent changes from night to day (see also Table 2) for naive (N), central memory
(CM), effector memory (EM) and effector (E) CD4+ (open circles) and CD8+ (filled circles)
T-cells, * P<0.05. A high ratio predicts a strong daytime decrease, while a low ratio predicts a
daytime increase. In an additional experiment, whole blood was sampled at 21:00h and
cultured in the presence or absence of cortisol at normal daytime concentrations (20µg/dl).
CXCR4 expression in naive (CD45RA+CD62L+) (J) CD4+ and (K) CD8+ T-cells is presented
as fluorescence intensity. Cells were stained with control immunoglobulin (filled histograms)
or anti-CXCR4 antibody (empty histograms). FACS profiles are shown (from a representative
subject) for gated T-cells before (left empty histogram, thin line) and after three hours of
culture without (middle empty histogram, grey line) and with cortisol (right empty histogram,
red line).
FIGURE 7. Synopsis. Numbers of circulating T-cell subpopulations show opposing rhythms
with a nighttime peak (represented by naive CD4+ T-cells – red cosine curve) or daytime peak
(effector CD8+ T-cells – blue cosine curve). Circadian rhythms peaking at night are controlled
through the release of cortisol which strongly increases during the early hours of daytime and
via GR activation (with a delay of three hours) redistributes T-cells from the circulation to
bone marrow. Rhythms peaking during daytime are controlled by release of catecholamines
which is increased during daytime and via β-AR activation leads to an immediate
29
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demargination of the cells from the vascular endothelium. Cortisol sensitive T-cells are
characterized by high CXCR4 expression, whereas epinephrine sensitive cells show highest
CX3CR1 expression. The two chemokine receptors mark two distinct T-cell populations with
only few double positive cells (see conture plots for CXCR4 and CX3CR1 expression on
CD4+ (top) and CD8+ T-cells (bottom)). CXCR4 mediates homing of T-cells to bone marrow
with cortisol supporting this traffic by up-regulating CXCR4. Available data on CX3CR1
tempts to speculate that this molecule facilitates margination of T-cells to vascular endothelium, with epinephrine inducing demargination by suppressing adhesive fractalkine
signaling.
30
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Prepublished online March 17, 2009;
doi:10.1182/blood-2008-11-190769
Cortisol and epinephrine control opposing circadian rhythms in T-cell
subsets
Stoyan Dimitrov, Christian Benedict, Dennis Heutling, Jurgen Westermann, Jan Born and Tanja Lange
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