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Clinical Studies |
(TNF) Production in Humans: High Sensitivity of TNF and Resistance of IL-6
Clinical Neuroendocrinology Branch, National Institute of Mental Health, National Institutes of Health (R.D., D.M., B.K., E.S., P.G.), Bethesda, Maryland 20892; the Departments of Physiology and Military and Emergency Medicine, Uniformed Services of the University of the Health Sciences,(J.P., E.G., P.D.), Bethesda, Maryland 20814; and CytImmune Sciences Inc. (G.P.), College Park, Maryland 20740
Address all correspondence and requests for reprints to: Dr. R. H. DeRijk, Leiden/Amsterdam Center for Drug Research, Center for Bio-Pharmaceutical Sciences, Sylvius Laboratories, Wassenaarseweg 72, 2300 RA Leiden, The Netherlands. E-mail: r.rijk{at}lacdr.leidenuniv.nl
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Abstract |
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(TNF) production in the whole
blood of healthy volunteers studied under conditions chosen to
approximate either physiological or pharmacological glucocorticoid
levels.
Administration of a pharmacological dose of hydrocortisone suppressed
the production of all three cytokines, whereas administration of a
physiological dose of hydrocortisone suppressed only TNF production.
Stress-induced levels of glucocorticoids, achieved during exercise at
100% maximal oxygen utilization, suppressed IL-1ß and TNF
production, but were without effect on IL-6 production. In addition,
circadian variations of cortisol were associated with decreased TNF
production, but were without effect on IL-1ß or IL-6 production.
These studies challenge the generally accepted idea that
glucocorticoids consistently suppress cytokine production and indicate
a hierarchy of sensitivity, with TNF having the greatest
sensitivity, IL-1ß having intermediate sensitivity, and IL-6 being
resistant. The resistance of IL-6 production to glucocorticoid
suppression is compatible with data suggesting an antiinflammatory as
well as a proinflammatory action for this cytokine.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In addition to stimulation of the hypothalamic-pituitary-adrenal (HPA) axis and glucocorticoids by stressful stimuli, it is also now known that a negative feedback loop exists between peripheral inflammatory cytokines and the HPA axis, in which cytokines promote hypothalamic CRH release and subsequent activation of the pituitary-adrenal axis (2). The resultant adrenocorticosteroid response is thought to protect against overstimulation of the immune system by peripheral inflammatory mediators. We have previously demonstrated the functional relevance of this immune system-HPA axis feedback loop in experimental animals by showing that inflammatory susceptibility in genetically inflammatory susceptible Lewis (LEW/N) rats is related to their severely blunted HPA axis responsiveness, whereas inflammatory resistance in F344/N rats is related to their robust HPA axis response (3). Furthermore, interruption of this feedback loop surgically or pharmacologically results in highly increased severity and susceptibility to the lethal effects of proinflammatory stimuli. Conversely, reconstitution of the HPA axis by hypothalamic transplantation from F344/N into LEW/N rats virtually eradicates the LEW/N peripheral inflammatory response to carrageenan (4).
Case reports in humans suggest that even basal nonstress levels of glucocorticoids influence immune function (5). Patients with Addison’s disease show increased immunoreactivity, and adrenalectomy or chemical inhibition of cortisol release in humans exacerbates rheumatoid arthritis (5, 6, 7). However, the effects of basal nonstress concentrations of endogenous corticosteroids on specific aspects of immune function are largely unknown. Furthermore, the effects of physiological fluctuations in corticosteroid levels have not been systematically examined in humans. Thus, although animal studies indicate an important regulatory role of the HPA axis on inflammatory susceptibility, definitive evidence for the precise regulation of immune functions by the HPA axis in humans does not exist.
Stress and nonstress levels of glucocorticoids are regulated through two different types of glucocorticoid receptors. The type 1 high affinity, or mineralocorticoid receptor (MR), mediates nonstress circadian fluctuations in glucocorticoids, whereas the type 2 low affinity, or glucocorticoid receptor (GR), mediates stress levels of glucocorticoids (8). Thus, if specific immune responses are differentially affected by low or high levels of corticosteroids, specific type 1 or type 2 glucocorticoid receptor mediation may be expected to play a role in various aspects of immune regulation.
The purpose of the present report is to further explore in human
subjects the effects of endogenous and exogenous glucocorticoids on a
variety of inflammatory cytokines that are known to participate in the
immune response and inflammatory disease. We wished to determine
whether several of the principal peripheral inflammatory cytokines,
including interleukin-1ß (IL-1ß), tumor necrosis factor-
(TNF), and IL-6, are affected by physiological or stress-induced
endogenous glucocorticoid secretion in a similar or dissimilar
fashion.
To explore the effects of physiological variations in plasma
glucocorticoid concentrations on cytokine production, we measured the
production of IL-1ß, IL-6, and TNF and employed three different
approaches to determine changes in plasma cortisol concentrations.
First, we administered hydrocortisone (HC) in doses that produce plasma
glucocorticoid levels comparable to those seen during ordinarily
stressful situations, such as exercise. For comparison, we also
administered HC in pharmacological doses. Next, the impact of elevated
plasma glucocorticoid levels associated with a naturalistic stressor
was studied by subjecting volunteers to graded treadmill exercise at
100% oxygen consumption (VO2 max). In addition, we
measured IL-1ß, IL-6, and TNF production in the morning and
evening, as plasma glucocorticoid levels are typically 3 times higher
in the morning than in the evening.
Demonstration of differences in cytokine responses to lipopolysaccharide (LPS) to such subtle naturalistic fluctuations in plasma cortisol levels would indicate that endogenous cytokine release is sensitive to even slight changes in HPA axis function. Moreover, such changes could help explain circadian differences in immunologic function in human subjects with and without inflammatory disease. Finally, we also explored whether differences in corticosteroid sensitivity between cytokines could be related to specific MR or GR actions. Such differences could underlie a differentially mediated modulation of inflammatory cytokines in humans by various levels of cortisol.
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Subjects and Methods |
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Volunteers were recruited by advertising in the local community. All volunteers were medication free. Medical and psychiatric histories were obtained, and subjects underwent physical examination by a board-certified psychiatrist (D.M.) as well as screening laboratory examination, including routine chemistry and complete blood counts. All subjects gave informed consent before participation. The study was approved by the NIMH institutional review board. In the case of the HC-loading experiments, the mean age (\SD) was 41.1 \ 8.2 yr, and the group consisted of three men and four women. In the experiment involving blood collection in the morning and the evening, the subjects were six male volunteers, with an average age of 36.2 \ 5 yr.
Strenuous exercise
Five healthy men and one woman free of medication and endocrine disorders, 25–37 yr of age (mean, 32 yr), participated in the exercise study, using a protocol that has been previously validated under a variety of conditions by Kyle et al. (9). Briefly, the subjects reported to the laboratory after having refrained from caffeine, alcohol, and strenuous exercise 24 h before testing and from food for 6 h before exercise. During this visit, a medical history, a physical examination, and a resting 12-lead electrocardiogram were obtained, and each subject underwent a progressive maximal treadmill test to exhaustion. The test was conducted on a motorized treadmill and began with a 10-min warm-up walk at 3.5 mph up a 5% grade. Treadmill speed then increased to 7 mph on a 0% grade for 2 min, after which the treadmill grade increased by 2.5% increments every 2 min. Exercise continued to volitional exhaustion. Verification that each subject actually reached 100% VO2 max at the end of the maximal oxygen uptake test consisted of the following criteria: 1) achieving predicted maximal heart rate, 2) a Borg’s perceived exertion scale rating of 17 or higher, 3) a respiratory exchange ratio of 1.10 or more, 4) an increase in oxygen uptake of 150 mL or less for an increase in workload, and 5) a lactate concentration of 10.0 mmol/L or higher. Oxygen uptake and CO2 production during all exercise tests were determined with a Metabolic Measurement Cart 2900c (SensorMedics, Yorba Linda, CA). Electrocardiograms and heart rates were monitored continuously throughout the exercise protocol. After arriving at the laboratory, an iv catheter for blood sampling was placed in one forearm vein 50 min before testing. Blood was drawn 10 min before the start of the exercise for baseline measurements, at the end of exercise, and 20 min after the end of exercise. Blood collections were used for in vitro incubations, leukocyte counts, and determination of plasma cortisol. Heart rate was also monitored before each blood drawing.
Stimulation of whole blood
Venous blood was collected in heparinized tubes (15 IU/mL blood, sodium heparin, 8-mL tubes, no. 6541, Becton and Dickinson, Rutherford, NJ) and, when several tubes were collected, pooled in a 50-mL Falcon tube (Becton & Dickinson, Lincoln Park, NJ). Previous results showed that using blood diluted with 10% (vol/vol) culture medium (no. 31053, Life Technologies, Gaithersburg, MD) gave more consistent results than undiluted blood, and this was, therefore, employed throughout the study. The blood was incubated with LPS (Difco 055:B5, Westphal, Difco Laboratories, Detroit, MI) and/or dexamethasone-21-phosphate (Dex; no. D1159, Sigma Chemical Co., St. Louis, MO), both dissolved in pyrogen-free saline (no. 314, Biofluids, Rockville, MD). To equalize the amount of blood incubated in each well in the various experiments, 400 µL blood were always added to 50 µL LPS or saline and to 50 µL Dex or saline in a 48-well plate (no. 3548, Costar, Cambridge, MA). After 6 h of incubation in a humidified atmosphere at 37 C in 5% CO2, the plate was centrifuged for 10 min at 2000 x g at 4 C, and plasma was collected by pipetting and stored at -80 C until assayed. A final dose of 30 ng/mL LPS was used in all experiments in which cytokine production was inhibited with Dex. To stimulate T cells, staphylococcal enterotoxin B (SEB; no. S-4881, Sigma Chemical Co.) was dissolved in saline and incubated with whole blood, as was done with LPS.
Assays
The plasma IL-1ß, IL-6, and TNF present in stimulated blood
were measured using commercial kits (CytImmune Science, College Park,
MD). This assay is a competitive binding immunoassay based on
competition between the cytokine and biotinylated cytokine for a rabbit
antibody raised against the recombinant human cytokine, as previously
described (10) with some minor modifications. Briefly, 96-well plates
were coated with a goat antirabbit antibody, and 50 µL plasma sample,
50 µL assay diluant, and 25 µL antibody against the cytokine were
incubated for 3 h, followed by the addition of 25 µL
biotinylated cytokine. After an additional 30 min, the plates were
washed, and a conjugate of streptavidin and alkaline phosphatase was
added for 30 min. The enzyme was washed out, and color development was
achieved by adding the substrate (NADPH) 20 min later followed by an
enhancer (formazan). Optical density was measured at 495 nm, whereas
cytokine concentrations were calculated using Microplate Manager
(Bio-Rad Laboratories, Richmond, CA). The detection limit was 200
pg/mL, intraassay variability was 8–9%, and interassay variability
was 11–12% for all assays.
Plasma cortisol was measured using a commercial RIA kit (Diagnostic Systems Laboratory, Webster, TX).
Lymphocyte counts
Before the addition of medium to the pooled blood, a 2-mL sample was taken, and total cell blood counts (10,000 cells counted) and differential counts were made using a Cell-Dyn 3500-SL (Abbott Diagnostics, Santa Clara, CA) as routinely performed by the hematology laboratory of the Clinical Center at the NIH (Bethesda, MD).
Calculations and statistics
As a measure of corticosteroid sensitivity, we determined the dose of Dex that produced 50% inhibition (ID50) of the LPS-induced cytokine response. This was determined from the graph for every individual separately. Total cytokine production was determined by conducting the net integrated response using the trapezoidal approximation method.
Data are represented as the mean \ SEM. Statistical testing for the LPS dose-response curves involved repeated measures using SPSSPC. Differences among cell counts or plasma cortisol concentrations were determined using ANOVA followed by Scheffe’s F test and/or paired t tests. In the absence of normal distribution, nonparametric testing (Wilcoxon matched pairs) was used [e.g. areas under the curve (AUCs)].
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Results |
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LPS- and SEB-induced cytokine responses in whole blood.
To
determine the selectivity for induction of specific cytokines, whole
blood was incubated for a period of 18 h with increasing doses of
LPS, a typical stimulus for cells of the macrophage lineage, or SEB, a
superantigen specifically reactive with the T cell receptor. LPS
(3–3000 ng/mL) induced a dose-dependent increase in IL-1ß, IL-6, and
TNF (typically 10–30 times above saline incubation), but not of
IL-2 (data not shown). In contrast, addition of increasing doses of SEB
(10–1000 ng/mL) induced dose-dependent increases in IL-2, up to 10
ng/mL (saline incubation resulted in IL-2 levels of 0.8 ng/mL), with
minor effects (2–3 times above saline incubation) on IL-1ß, IL-6,
and TNF only at the highest dose of SEB.
To determine the optimal incubation time to measure IL-1ß, IL-6, and
TNF, whole blood was incubated with LPS to a final concentration of
30 ng/mL or with saline. Significant increases were measured after
3 h for TNF and IL-6 and after 4 h for IL-1ß. IL-6
concentrations reached a plateau at 4 h, with levels around 7
ng/mL, whereas IL-1ß levels increased up to 14 ng/mL. TNF,
however, showed the highest level of 12 ng/mL at 6 h, whereas the
concentration declined to 2.5 ng/mL at 24 h. As all three
cytokines could be measured after 6 h of LPS stimulation, we used
this time point throughout the study.
Effect of HC administration on LPS-induced IL-1ß, IL-6, and
TNF production
To test whether cortisol administration suppresses ex vivo LPS-induced cytokine production, seven healthy subjects were given oral HC (cortisol). A dose of 20 mg HC has been reported to increase plasma cortisol for several hours to levels within the physiological range, such as those seen during exercise. A dose of 80 mg HC induces longer lasting increases and is considered a pharmacological dose (11).
Plasma cortisol levels after oral HC administration.
In this
double blind study, a placebo or 20 or 80 mg HC was orally administered
in the morning (0800–0830 h), and blood was collected in the afternoon
(1600–1730 h). At the time of blood collection in the afternoon, the
cortisol level in the placebo group was 165.5 \ 19.3 nmol/L, the
plasma cortisol level in the 20 mg HC group was 157.2 \ 22
nmol/L, whereas 689.5 \ 72 nmol/L cortisol was measured in the 80
mg HC group (Fig. 1
, left panel). The
cortisol levels in the placebo group and those in the 20 mg HC group
were not different from each other, whereas the high cortisol levels in
the 80 mg HC group were significantly different from those in the other
two groups.
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, left panel). Lymphocytes showed a minor, but
statistically significant, decrease in cell number after administration
of 20 mg HC.
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, IL-1ß, and IL-6
production, whereas low dose HC administration decreases LPS-induced
TNF, but not IL-1ß or IL-6, production.
Administration of 80
mg HC significantly suppressed LPS-induced IL-1ß, IL-6, and TNF
production compared to the effect of placebo or 20 mg HC
(P < 0.001, by repeated measure for all three
cytokines; Fig. 2). Twenty milligrams of HC did not
significantly inhibit ex vivo IL-1ß (F = 0.24;
P = 0.6) or IL-6 (F = 0.3; P =
0.6) production. However, TNF was significantly decreased after
treatment with 20 mg HC compared to the level after placebo
administration (F = 7; P = 0.012). These data were
confirmed by assessing the AUCs as a measure of total cytokine
production; all three TNF groups (placebo, 20 mg HC, and 80 mg HC)
were significantly different from each other (P <
0.05). Eighty milligrams of HC, but not 20 mg HC, significantly
inhibited IL-1ß and IL-6 (P < 0.001).
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production, as expected. In
contrast, 20 mg HC only suppressed TNF production, without affecting
IL-1ß or IL-6 production or monocyte numbers.
Exercise-related inhibition of LPS-induced IL-1ß, IL-6, and
TNF production
The previous section showed that exogenous HC, even in the physiological range, has an effect on LPS-induced cytokine production. This series of experiments addressed the effect of exercise-induced elevations of endogenous cortisol on cytokine production by comparing cytokine production in blood obtained before and after exercise.
Plasma cortisol levels and cell numbers after exercise.
Plasma
cortisol levels increased from 535 \ 66.2 nmol/L before exercise
to 943 \ 127 nmol/L, as measured 20 min after ending exercise
(Fig. 1
, middle panel). Increases in plasma cortisol have
been associated with changes in peripheral blood leukocyte numbers
(12). Upon completing exercise, significant increases in white blood
cells (WBC), granulocytes, lymphocytes, and monocytes (increasing up to
200%) were noted (data not shown). However, at the points when
ex vivo cytokine responses were induced, before exercise and
20 min after ending exercise, no differences in total WBC,
granulocytes, lymphocytes, or monocytes were seen (Fig. 3, middle panel).
LPS-induced IL-1ß, IL-6, and TNF production after
exercise.
Blood obtained 20 min after ending exercise showed
significantly lower IL-1ß (F = 13; P < 0.001)
and TNF (F = 35; P < 0.001) concentrations
than blood collected before exercise (Fig. 4). For
example, at a concentration of 300 ng/mL LPS, IL-1ß decreased from
19.6 \ 3.1 to 9.2 \ 1.3 ng/mL, whereas TNF decreased
from 16.3 \ 2.1 to 6.7 \ 1.5 ng/mL. In contrast, no
significant effect was seen on IL-6 production (F = 0.4;
P = 0.54). This suggests that the increase in plasma
cortisol seen after exercise is associated with inhibition of
LPS-induced IL-1ß and TNF, whereas IL-6 is relatively
resistant. In light of the lack of changes in cell numbers, it is
unlikely that the changes in LPS-induced IL-1ß and TNF production
observed before and 20 min after exercise result from changes in the
number of monocytes, which are the major cellular source of these
proinflammatory cytokines.
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, IL-1ß, or IL-6
productionIf the large increases in endogenous cortisol, as seen during exercise, affect LPS-induced cytokine production, the next question is whether the naturally occurring lower levels of circadian variation in cortisol can also influence cytokine production. To determine the effect of circadian cortisol variation on cytokine production, blood was collected from six healthy individuals in the morning (0800–0830 h) and on the subsequent evening (2000–2030 h).
Plasma cortisol and leukocyte numbers during circadian rhythm.
The plasma cortisol level was 375 \ 30 nmol/L in the morning and
decreased to 135 \ 12.4 nmol/L in the evening (Fig. 1, right panel).
Differences in blood cell numbers during circadian rhythm.
Changes in numbers of circulating cells have been described during the
circadian rhythm; therefore, changes in LPS-induced cytokine production
could be a direct result of blood collection at different times of the
day. Circulating cells were counted and expressed as total cell numbers
(Fig. 3, right panel). No significant change was seen in
monocytes, granulocytes, or total WBC. Only lymphocytes showed a
moderate increase \[1595 \ 462/µL (morning) vs.
2370 \ 628/µL (evening); P < 0.001\].
Circadian rhythm differentially affects TNF, but not IL-1ß and
IL-6, production.
LPS-induced cytokine production, as shown in
Fig. 5, did not show a significant difference for
IL-1ß when morning and evening values were compared (repeated
measure: F = 0.16; P = 0.7). In addition, plasma
concentrations of IL-6 were almost identical in the morning and
evening, and the difference did not reach significance (F = 0.01;
P = 0.9). In contrast, LPS-induced TNF was
significantly less in the morning than in the evening (F = 6.5;
P = 0.013). By calculation, the AUCs (as a measure of
cytokine production) for IL-1ß, IL-6, and TNF, showed the same
pattern; TNF production showed a significant decrease in the morning
compared to the evening (P < 0.03), whereas no such
difference was seen for IL-1ß (P = 0.12) or IL-6
(P = 0.46, by nonparametric testing). In light of the
lack of circadian change in monocyte numbers, the differences in
LPS-induced cytokine production in relation to circadian rhythm are
unlikely to be due to changes in monocytes and are more likely
secondary to circadian fluctuations in plasma cortisol.
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, IL-1ß,
or IL-6 production
The unique sensitivity of TNF to cortisol during the circadian
rhythm under conditions of very low GR occupancy suggests predominantly
MR control of this cytokine. In contrast, IL-1ß was only inhibited
after stress levels of cortisol, suggesting GR control. To test the
possibility of preferential GR control of IL-1ß, we incubated whole
blood with LPS and Dex, a GR agonist. Figure 6 shows
dose-dependent inhibition by Dex of LPS-induced IL-1ß, IL-6, and
TNF. IL-6 is 10–20 times less sensitive to Dex compared to IL-1ß
or TNF (P < 0.001), as determined by comparing
IC50 values. Comparing IL-1ß and TNF, expressed as the
-log of the IC50 values, in blood obtained from both
morning or evening, IL-1ß was significantly more sensitive to
inhibition by Dex than TNF [morning: TNF, 7.15 \ 0.08;
IL-1ß, 7.5 \ 0.08 (P = 0.005); evening: TNF,
7.0 \ 0.08; IL-1ß, 7.4 \ 0.08 (P =
0.02)]. No significant differences were detected when comparing
evening vs. morning values for all three cytokines.
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and the different responses seen at
different times of the day suggest differential regulation of these
cytokines by various corticosteroid receptors. |
Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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is very sensitive to glucocorticoids. These studies indicate
that physiological variations in glucocorticoids may play an important
role in the regulation of specific cytokine production. Furthermore,
together with in vitro studies showing differential cytokine
sensitivity to Dex suppression, these studies suggest that cytokines
may be differentially regulated by the type I or type II GR.
The exercise paradigm employed here has been extensively validated and
has been shown to produce a dose-dependent plasma cortisol response
based on percent oxygen utilization. Moreover, cortisol responses to a
given level of oxygen utilization in this context are similar
regardless of the individual’s level of physical conditioning and are
thus independent of prior exposure to this naturalistic stressor (13).
We have previously reported that exercise at 90% oxygen consumption
produced glucocorticoid responses similar to those presented here
without causing an increase in plasma IL-6 levels (14). However, at
that time we did not measure other cytokines, such as IL-1ß or
TNF.
Our finding that pharmacological levels of glucocorticoids effectively suppress all cytokines measured is consistent with the dogma that glucocorticoids globally suppress cytokines and inflammation (15). However, the pharmacological state may not be representative of varying physiological conditions.
When we attempted to mimic a physiological state with HC administration
by producing glucocorticoid levels ordinarily seen during a
naturalistic stressor such as exercise, we saw suppression of TNF
and IL-1ß, but not of IL-6. The differential responsiveness of IL-6
to glucocorticoid administration was also mirrored in the context of
the application of a naturalistic physiological stressor, exercise.
Here we saw once again suppression of TNF and IL-1ß production,
with escape of IL-6 production. TNF was the most sensitive to even
subtle changes in endogenous glucocorticoid levels, such as circadian
variations, showing higher production when plasma cortisol levels were
low in the evening, whereas IL-1ß and IL-6 were unaffected.
TNF is a major regulator of early immune responses and is highly
proinflammatory, as can be deduced from its crucial role during septic
shock (16). Our finding that TNF is the most sensitive to
suppression by cortisol, including nonstress circadian variation
levels, underscores the tight regulatory control of the production of
this cytokine. Furthermore, as TNF is a major factor in inflammation
associated with autoimmune diseases, such as rheumatoid arthritis (17),
the circadian variations in cortisol-regulated TNF production could
explain circadian variations in the symptoms of such illnesses
(18).
IL-1ß production is also sensitive to cortisol suppression, although
at higher concentrations of cortisol than TNF. At higher stress
levels of plasma cortisol, both LPS-induced IL-1ß and TNF
production were suppressed.
In contrast to that of both TNF and IL-1ß, we found that IL-6
production is resistant to suppression by cortisol levels within the
physiological range. Even stress-elevated cortisol levels, exceeding
the binding capacity of circulating cortisol-binding proteins and,
therefore, resulting in high free levels of active cortisol, were
virtually unable to induce a change in LPS-induced IL-6. These findings
differ from the generally accepted idea that glucocorticoids
consistently inhibit the production of all proinflammatory cytokines,
including IL-6 (15). On the other hand, our data are consistent with an
emerging series of recent studies in patients with endocrine disorders
or using the pharmacological administration of glucocorticoids to
healthy volunteers, which suggests that glucocorticoids may not simply
profoundly inhibit IL-6 secretion. Yamada et al. (19) showed
that prednisolone given to patients with thyroiditis actually promoted
an increase in plasma IL-6 levels, whereas Papanicolaou et
al. (20) reported circulating plasma IL-6 levels in patients with
Cushing’s disease. Berber et al. (21) showed that although
high dose iv hydrocortisone administration to healthy volunteers
completely suppressed plasma TNF responses to bolus LPS
administration, IL-6 levels were only slightly inhibited. Studies in
the rat by Persoons et al. (22) showed that although
stress-associated increases in corticosteroids decreased in
vitro LPS-induced IL-1ß and TNF production, the production of
IL-6 was unaffected.
Although IL-6 is considered proinflammatory, a synergism exists between
IL-6 and glucocorticoids in regulating the development of B cells (23)
and the production of acute phase proteins by the liver (24). These
include several proteins with antiinflammatory properties that could
function as a protective mechanism during sepsis (25). Glucocorticoids
also up-regulate the expression of the IL-6 receptor, which could
further contribute to the synergistic effects of IL-6 and
corticosteroids under some circumstances (24, 26). Moreover, IL-6
inhibits IL-1ß and TNF production not only by directly suppressing
their production and release (27), but also by stimulating the
induction of the IL-1ß receptor antagonist and the soluble TNF
receptor (p55) (28). Our data showing that IL-6 is cortisol resistant
are thus compatible with these studies, suggesting that under special
circumstances IL-6, rather than acting solely as a proinflammatory
cytokine, could have an antiinflammatory action in synergism with
cortisol.
One mechanism by which differential corticosteroid levels regulate
different effects is by preferential binding to the two glucocorticoid
receptor subtypes, the type I, or MR, and the type II, or GR. Under
nonstress corticosteroid levels, the MR is almost fully occupied by
cortisol, whereas the GR is largely unoccupied due to the difference in
affinity of both receptors for cortisol (8). After elevations in plasma
cortisol to stress levels, the GR also becomes increasingly occupied
and activated (8). As both receptors have been detected in immune cells
(29, 30), the differential sensitivity of TNF to low levels of
circulating cortisol suggest that TNF production may be more
sensitive to the actions of MR, whereas IL-1ß is preferentially
regulated by the GR. The in vitro data showing differential
sensitivity to dexamethasone suppression, with IL-1ß more sensitive
than TNF, also supports this hypothesis.
Monocytes are the principal source of TNF and IL-1ß (31, 32). In
contrast, IL-6 released during inflammation is derived not only from
monocytes, but also from noncirculating sources, such as endothelial
cells and fibroblasts (33). However, when using whole blood stimulated
by LPS, monocytes are likely to be the principal source of IL-6. It has
been well documented that fluctuations in plasma cortisol influence the
relative numbers of different circulating WBC (12, 34), most of which
have the ability to produce and release cytokines. The lack of IL-2
production that we observed after LPS stimulation supports the
selective activation in our system of monocytes rather than T cells. T
cells are reactive under these whole blood-stimulated conditions
however, as SEB, a T cell superantigen that directly interacts with the
T cell receptor, stimulated a profound IL-2 induction but did not
induce an IL-1ß or TNF response and only induced a minor IL-6
response at the highest dose used. Granulocytes can produce fairly
large amounts of cytokines, including IL-1ß and TNF (35). However,
monocytes are almost 1000-fold more sensitive to LPS than granulocytes,
whereas on a per cell basis, monocytes produce approximately 10 times
more IL-1ß and TNF than granulocytes. Moreover, granulocytes have
not been observed to produce detectable amounts of IL-1ß, IL-6, or
TNF at the doses of LPS used in our study (35). Furthermore, when
cells of the macrophage lineage were depleted in vivo in
rats, the LPS-induced increase in plasma IL-1ß was completely
abrogated, whereas normal amounts of granulocytes and lymphocytes were
present (31). Importantly, the dichlormethylene diphosponate liposomes
used to deplete cells of the macrophage lineage were without effect on
T cells or granulocytes (36). Finally, the positive correlation between
monocytes and IL-1ß and TNF production (IL-1ß-AUCs and
TNF-AUCs), which was not seen with other cells, suggests that in our
system monocytes are the major producers of IL-1ß and TNF. As in
our studies the numbers of monocytes did not change significantly after
placebo treatment, after 20 mg HC administration, during exercise at
the time points tested, or during fluctuations in the circadian rhythm,
it is unlikely that changes in cytokine production are caused by
changes in monocyte number.
In addition to cortisol, several other hormones are elevated during
exercise, including PRL, GH, and catecholamines (13). Although PRL and
GH have been shown to be permissive factors for the development and the
tonic activity of the immune system, neither has been shown to acutely
influence the secretion of cytokines or the immune response (37). In
contrast, catecholamines have been shown to exert a rapid acute
influence on cytokine production in whole blood (38, 39). It should be
noted, however, that we evaluated ex vivo cytokine release
in blood obtained at the peak of the cortisol response to exercise,
well after catecholamine responses had peaked and returned to baseline.
On the other hand, in blood drawn at the peak of the catecholamine
response and before a substantial rise in ACTH-induced cortisol
release, we did not observe a significant change in LPS-induced IL-1ß
or IL-6 production when corrected for the increased amount of monocytes
(data not shown). At this time point, mean TNF production, which was
shown to be highly sensitive to catecholamines, was decreased, although
this did not reach significance (P = 0.077, by repeated
measurement), compared to preexercise production. These data suggest
that the inhibitory effect of exercise on IL-1ß and TNF production
at the time points used in this study are mediated predominantly by
cortisol rather than catecholamines.
In conclusion, this study provides evidence that the naturalistic
activation of the HPA axis in humans is involved in physiological
restraint of one aspect of the immune responses, that of differential
cytokine production. The specificity, sensitivity, and consistency of
suppression of TNF production in response to nonstress
concentrations of plasma cortisol, as seen during the circadian rhythm,
further supports this physiological glucocorticoid restraint. The
resistance of IL-6 production to corticosteroid feedback and the
numerous synergistic effects of IL-6 and cortisol suggest a
physiological role of IL-6 in the suppression of inflammation. Taken
together, we propose that a more complex physiological regulation of
immune responses by corticosteroids exists, as opposed to the simple
inhibition seen with pharmacological doses. Thus, patterns of cytokine
production may be determined by both differential cytokine-specific
corticosteroid sensitivity coupled with differential physiological
plasma cortisol concentrations. In this schema, each cytokine pattern
would have its own characteristics. As therapeutic intervention of
inflammatory disease is almost entirely predicated on the use of high
pharmacological doses of glucocorticoids, more detailed definition of
the effects of physiological corticosteroid concentrations on patterns
of cytokine production, with their resultant differential effects on
regulation of inflammation, could have implications for more precise
therapeutic approaches to the treatment of inflammatory disease.
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Acknowledgments |
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Received November 6, 1996.
Revised March 14, 1997.
Accepted March 20, 1997.
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References |
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), 20 mg HC (•), or 80 mg HC (
). LPS
concentrations varied from 0 (sal. = saline) to 300 ng/mL. Blood was
collected in the afternoon (1600–1630 h) from seven healthy
individuals (three men and four women) after they took HC/placebo in
the morning (0800–0830 h). Using repeated measurements, 80 mg HC
administration decreased the levels of all three cytokines
significantly (P < 0.05). Twenty milligrams of HC
decreased ex vivo TNF
