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Original Studies |
Center for Psychobiological and Psychosomatic Research, University of Trier (M.E., A.B.-K., D.H., S.K., N.R., C.K.), D-54286 Trier, Germany; Department of Medical Sciences, University of Edinburgh (B.W.), EH4 2XU Edinburgh, United Kingdom; and Department of Psychology, University of Düsseldorf (C.K.), D-40225 Düsseldorf, Germany
Address all correspondence and requests for reprints to: Prof. Dr. Clemens Kirschbaum, Department of Psychology, Heinrich Heine Universität Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany. E-mail: ck{at}uni-duesseldorf.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production in peripheral leukocytes,
beclomethasone dipropionate-induced skin blanching, and suppression of
cortisol levels after low-dose (0.5 mg) dexamethasone suppression test
were determined in each subject.
The results showed the expected glucocorticoid-induced suppression of
interleukin-6 and tumor necrosis factor- production (both
P < 0.001), dose-dependent skin blanching
(P < 0.001), and suppression of salivary cortisol
response to awakening (P < 0.001). However,
neither simple correlations nor cluster analysis revealed a significant
association among the three bioassays for GC sensitivity. In contrast
to the idea that interindividual variation in GC sensitivity is an
intrinsic trait affecting all tissues, these results suggest that this
variability is target tissue specific in healthy subjects.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mutations causing generalized pathological GC resistance and GC hypersensitivity have been described (7), in which resistance includes hypothalamic and pituitary tissues and is caused by point mutations or microdeletions in the GC receptor (GR) gene lowering the affinity of the GR for its cognate hormone (8). Acquired generalized GC resistance also exists in a subgroup of patients suffering from acquired immune deficiency syndrome even though the exact molecular mechanism of this effect is yet unclear (9). The rare full-scale generalized glucocorticoid hypersensitivity is believed to be mediated either by point mutations leading to enhanced hormone binding of GR and/or defects in dominant negative inhibitors of glucocorticoid action, for example the ß-isoform of the human GR (10).
In contrast to these extreme generalized disorders of GR signaling, it is still unknown whether in healthy individuals the GC sensitivity of one target tissue reflects the GC sensitivity of other organs. There are several methods that allow us to study GC sensitivity in healthy subjects. One important target tissue for GC actions is blood vessels. Since GCs and mineralocorticoids interact with vascular receptors, they indirectly influence vascular tone by increasing vascular sensitivity to noradrenaline, as seen in Cushing’s syndrome (11). Topically applied GCs cause vasoconstriction of subdermal blood vessels, resulting in temporary blanching of the treated skin area (12). The intensity of dermal blanching by GCs is a quantifiable marker that reflects the GC sensitivity of the blood vessels (13).
Another prominent target tissue for GCs is the immune system, where GCs
inhibit the release of the proinflammatory cytokines interleukin-6
(IL-6) and tumor necrosis factor- (TNF) from monocytes and
macrophages. The recent development of an in vitro bioassay
in which a dose-dependent dexamethasone (DEX)-induced inhibition of
cytokine production is measured provides an estimate of GC sensitivity
of circulating leukocytes (14, 15).
Moreover, GCs exert a strong feedback signal at different levels of the HPA axis. For several decades, the DEX suppression test has been used to test the feedback integrity of the axis (16, 17). Therefore, the degree of DEX-induced suppression of cortisol levels can be viewed as yet another index of GC sensitivity.
Taking advantage of these three bioassays (blood vessels, monocytes, and HPA axis), the present study compared the GC sensitivities of these different target tissues in a sample of healthy adults.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nineteen male and 22 female healthy subjects (mean ± SD age, 30.56 ± 9.52 yr) were recruited through local newspaper and e-mail advertisements at the University of Trier. Exclusion criteria were cigarette smoking, intake of glucocorticoid medication, hormonal and endocrine dysfunctions, allergic diseases, and depressive illness. Female subjects using oral contraceptives were also excluded from participation.
Subjects reported to the laboratory on 3 consecutive days. On the first day a blood sample was drawn between 1000–1100 h to assess GC sensitivity in peripheral leukocytes (see below). Thereafter, skin blanching was induced as described below, and subjects received salivasampling devices (Salivette, Sarstedt, Rommelsdorf, Germany) for saliva collection at home. They were instructed to collect saliva to assess the early morning cortisol response to awakening and the circadian cortisol profile (see below). On the second day they returned their saliva samples to the laboratory, and the degree of skin blanching was rated. Subjects received another set of Salivettes and a tablet containing 0.5 mg oral DEX (Fortecortin, Merck & Co., Germany), which they had to take at 2300 h on the same day. On the third day subjects again reported to the laboratory to deliver their saliva samples. The study protocol was approved by a local ethics committee, and written informed consent was obtained from each subject.
DEX suppression of IL-6 and TNF production in peripheral
leukocytes
On the first day an iv catheter (Braun, Melsungen, Germany) was
inserted, and a venous blood sample was collected in heparinized tubes
(Braun) 35 min later. Before analysis the blood sample was diluted 10:1
with saline (Braun) and subsequently incubated with lipopolysaccharide
(LPS; derived from Escherichia coli 055:B5, Difco, Augsburg,
Germany) and different concentrations of DEX (Sigma,
Deisenhofen, Germany). Diluted whole blood (400 µL) was added to 50
µL LPS and 50 µL of different DEX concentrations with final
concentrations of 30 ng/mL (LPS) and 0,
10-10,
10-9,
10-8, and
10-7 mol/L (DEX),
respectively. After 6 h of incubation at 37 C in 5%
CO2 the plates were centrifuged for 10 min at
2000 x g at 4 C. The supernatant was collected and
stored at -80 C until assayed. To account for interindividual
variations in monocytes producing IL-6 and TNF, a differential blood
cell count was performed with an SE-9000 cell counter (Sysmex,
Norderstedt, Germany). This assay was adapted from a previously
reported protocol (14).
Skin vasoconstriction assay
Solutions of beclomethasone dipropionate (Sigma) were prepared in ethanol/water (95:5, vol/vol) at concentrations of 0, 0.2, 1, 5, 10, and 20 µg/mL. Six circles with a 20-mm diameter were outlined on the volar aspect of the subject’s forearm. The surrounding skin area was covered with a hard rubber stencil. Between 1200–1300 h 50 µL of each solution were applied to a corresponding circle. The order of application was randomized, with each circle receiving a different concentration of the glucocorticoid. After evaporation of the ethanol, the forearm was covered with polyethylene vacuum foil (dm-Drogerie Markt, Karlsruhe, Germany). The occlusive dressing was removed the following morning between 1000–1100 h. About 1 h later the intensity of the skin blanching for each circle was rated. The test areas were examined under standardized light conditions by two trained, double blinded raters. Scores on a standardized rating scale ranged from 0 (no blanching), 1 (faint blanching), 2 (obvious blanching not extending the circle), to 3 (intense blanching extending over the margin of the circle). Interobserver agreement showed a satisfactory reliability of r = 0.74. The blanching score has been validated against objective recordings with reflectance spectrophotometry (13). Each subject’s response to beclomethasone was computed as the total of the six blanching scores. Pilot studies in this laboratory revealed neither order effects of topically applied beclomethasone dipropionate on the cortisol response to awakening nor effects on the DEX suppression test the following day (unpublished observations). This method was previously used in different studies (18, 19).
Low-dose DEX suppression test
Free cortisol levels after awakening have been reported to reliably reflect the individual’s adrenocortical activity (20). Subjects were instructed to sample saliva for cortisol assessment on 2 days. The first day served as a baseline for comparison with the following day after DEX suppression. Saliva samples were obtained by the subjects using Salivette sampling devices. The first sample on each day was collected immediately after awakening. Four additional samples followed 10, 20, 30, and 60 min later. Also on each day a 1400 h and a 2000 h sample were obtained to monitor early escape from suppression of cortisol levels. Subjects took 0.5 mg oral DEX (Fortecortin, Merck & Co.) at 2300 h on the control day. The low-dose DEX test (0.5 mg) has been shown to have a greater sensitivity in differentiating between clinical subgroups than when using higher doses (i.e. 1.0 mg) (16, 17). Saliva samples were stored at -20 C until assay. Nine subjects of the total sample did not complete the DEX test.
Biochemical analysis
After thawing, saliva samples were centrifuged at 3000 rpm for 5 min, which resulted in a clear supernatant of low viscosity. Fifty microliters of saliva were used for duplicate analysis. Cortisol levels were determined by a time-resolved immunoassay with fluorometric end-point detection (DELFIA, Wallac, Gaithersburg, MD) with an intraassay coefficient of variance below 10% as described in detail previously (21). The assay has a lower detection limit of 0.78 nmol/L. To reduce error variance caused by intraassay imprecisions, all samples from one subject were analyzed in the same run.
TNF and IL-6 were determined using commercial enzyme-linked
immunosorbent assay (ELISA) kits (PharMingen, San Diego,
CA). This sandwich ELISA uses antihuman TNF monoclonal antibody as
capture antibody and biotinylated antihuman TNF monoclonal antibody
as detection antibody. A 96-well plate was coated with the capture
antibody and incubated overnight. Then the plate was blocked with 200
µL assay diluent and washed. One hundred microliters of standard or
plasma sample (diluted 1:600 for IL-6 and 1:50 for TNF) were added
to each well and incubated for 2 h. After another wash step, 100
µL detection antibody were added, and the plate was incubated for
1 h and washed again. Then, 100 µL substrate solution
(tetramethylbenzidine and hydrogen peroxide) were added and incubated
for 30 min followed by the addition of 50 µL stop solution (2
N
H2SO4). The plate was read
in an ELISA reader (Dynatech Corp., Denkendorf, Germany)
at 450 nm vs. 630 nm.
Statistical analysis
ANOVAs for repeated measures were computed to detect differences
in salivary free cortisol levels, cytokine production, and skin
blanching scores, with Greenhouse-Geisser corrections where
appropriate. Cumulative measures for the overall GC sensitivity in each
target tissue were established as follows. The area under the curve
(AUC) for saliva cortisol samples 1–5 on the day after DEX suppression
was subtracted from the same individual’s AUC on the control day. This
variable (DEXAUC) was used as an index of
suppression of basal HPA axis activity. IC50
values were calculated for IL-6 and TNF, representing the
concentration of DEX used for 50% inhibition of LPS-induced cytokine
production. IC50 values were determined by
plotting a curve of the respective cytokine production using an
exponential fit with r2 > 0.90. For the total
skin blanching score (SUMSKIN), the sum of the
ratings for all six skin areas treated with beclomethasone solutions
was computed. Rank order correlations and cluster analysis of the
glucocorticoid sensitivity markers were performed. Data are presented
as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). ANOVA results support the
effectiveness of DEX reduction of the waking response (F = 263.94;
P < .001). The mean (±SD) value
for suppression of cortisol levels by DEX
(AUCDEX) was 95.91 ± 25.29 (minimu, 34.05;
maximum, 151.09).
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shows the production of IL-6 and TNF
standardized for 106 peripheral monocytes. The
highest levels for both cytokines were obtained after stimulation with
LPS alone (IL-6, 80.77 ng/mL; TNF, 15.78 ng/mL). Rising
concentrations of DEX
(10-10,
10-9,
10-8, and
10-7 mol/L) resulted in
declining amounts of cytokine production. ANOVA confirmed the
dose-dependent reduction of cytokine production for IL-6 (F =
91.03; P < .001) and TNF (F = 103.67;
P < .001). The mean concentration of DEX required for
50% inhibition of LPS-induced cytokine production
(IC50) was 5.91 x
10-8 mol/L
(±SD, 2.41; minimum, 1.36; maximum, 15.12) for
IL-6 and 4.46 x 10-8
mol/L for TNF (±SD, 1.39; minimum, 1.27;
maximum, 8.24).
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). ANOVA
for repeated measures revealed a significant effect of the applied dose
on the total variation in skin blanching (F = 27.31;
P < 0.001). The mean (±SD) sum
of blanching scores (SUMSKIN) was 9.19 ±
5.04 (minimum, 0.0; maximum, 16.50).
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(rs = 0.68; P < 0.001), no
significant correlations were found among IC50
values, DEX suppression of cortisol levels, or the total blanching
scores, respectively (see Table 1 and
Fig. 4).
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between two clusters formed on
the basis of IC50 for IL-6 (F = 17.55;
P < 0.001). Vice versa, two clusters based
on IC50 for TNF discriminated between high and
low IC50 for IL-6 (F = 18.11;
P < 0.001). Neither cluster analyses based on
DEX-suppressed cytokine production (IC50 for
IL-6/TNF) or DEX-suppressed morning cortisol
(DEXAUC) nor skin blanching scores
(SUMSKIN) yielded any significant difference
between clusters for the other variables (data not shown), supporting
the pattern of results obtained by correlations. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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However, other studies support the present results. No association was reported between skin blanching and DEX suppression in a sample of patients with Alzheimer’s disease or polymyalgia rheumatica and healthy controls (24). In elderly women suffering from hip fractures, DEX nonsuppression occurred together with normal sensitivity in peripheral blood mononuclear leukocytes (25). In a sample consisting of hypertensive and normotensive subjects, DEX suppression of plasma cortisol levels was not correlated with beclomethasone-induced skin blanching scores (6). Also, normotensive subjects with a GR polymorphism showed no intraindividual correlation between DEX inhibition of lysozyme release by mononuclear leukocytes and budesonide-induced skin blanching (26).
GC sensitivity is influenced by a large number of tissue-specific factors that may account for these observations. These include GR expression levels, intracellular glucocorticoid availability, hormone binding affinity, heat shock protein (HSP) complexes, and modulation of gene transcription (10).
At the level of the HPA axis, the expression of GR numbers is a crucial
factor distinguishing between pituitary tissue and other potential GC
target tissues. Whereas in peripheral immune tissue and brain, GRs are
down-regulated after high doses of corticosterone, pituitary GRs are
relatively insensitive to this effect (27). Likewise, no significant
correlation between GR numbers and in vitro GC sensitivity
could be observed in white blood cells from healthy and leukemic
patients (28) or in human precursor mononuclear phagocytes (29).
Tissue-specific concentrations of cytokines and different states of
immune activation are other potential sources of dissociation. GC
sensitivity is increased in sepsis, and numerous cytokines, including
IL-2, IL-4, IL-6, and TNF, can lead to regional reduction of GC
sensitivity (30, 31). Moreover, intracellular GC availability is
modulated by the 11ß-hydroxysteroid dehydrogenase (11ßHSD) enzymes.
In tissues expressing 11ßHSD type 2, such as distal nephron, sweat
glands, and colon, GC are inactivated before reaching receptors. In the
many tissues expressing 11ßHSD type 1, inactive metabolites such as
cortisone may be reactivated. 11ßHSDs are not expressed in peripheral
blood leukocytes, but 11ßHSD1 may be important in pituitary and
supra-pituitary sites where negative feedback occurs, and both
isozymes may be expressed in blood vessels.
There have been conflicting findings on the influence of GR polymorphisms on the tissue specificity of GC action. The N363S polymorphism of the GR gene seems to be associated with a greater sensitivity to exogenously administered GC. Individuals carrying this polymorphism showed significantly enhanced cortisol suppression by DEX and a trend toward stronger reduction of lymphocyte proliferation by DEX compared with healthy controls (32). In contrast, the BclI restriction fragment length polymorphism of the GR gene seems to influence in vivo and in vitro GC sensitivity in opposite ways (26). Subjects homozygous for the large allele of the GR gene were significantly more sensitive to budesonide-induced skin blanching, but showed a trend toward lower sensitivity for DEX inhibition of lysozyme release by mononuclear cells compared to subjects homozygous for the small allele. It has been proposed that this polymorphism affects the GR gene promoter in a tissue-specific manner, thus resulting in tissue-specific GR expression levels (26).
It appears tempting to speculate that certain more extreme forms, such as primary hereditary GC resistance, could uniformly up- or down-regulate GC sensitivity in different tissues. However, those cases are rare (4), and therefore are unlikely to account for the positive findings on intraindividual correlations of GC sensitivity reported in asthma and depression patients and healthy subjects described above.
Finally, the inactive form of the GR in the cytoplasm is bound to HSPs
of the 90K (HSP90 and HSP90ß) and the 70K (HSP70) families.
Particularly HSP90, which is predominantly expressed in the
periphery, is a potential modulator of differential GC sensitivity in
these tissues (33).
In the present study only a single dose of DEX (0.5 mg) was administered in the DEX suppression test; thus, the conclusions that can be drawn from these data are somewhat limited. The main reason for using only the 0.5-mg dose was that in healthy younger adults, larger doses (1 mg or higher) would result in the suppression of salivary cortisol levels to less than 2 nmol/L in 70–90% of the subjects. This would provide only small variation between subjects, thus reducing the chance of significant covariation with the two other measures of GC sensitivity employed here. Furthermore, we used two different synthetic GCs (DEX and beclomethasone dipropionate); therefore, ligand-specific effects at different sites of action cannot be excluded. In an animal model (Bianchi-Milan hypertensive rats) lymphocyte GR sensitivity to corticosterone, but not to DEX, was altered (34). Moreover, 11ßHSDs modulate DEX availability (35), but there is evidence that beclomethasone dipropionate is unaffected by this enzyme (36). Finally, factors such as polymorphisms in HSPs or 11ßHSDs could increase the noise in the data (37) or even have effects that offset those of GR polymorphisms.
Nevertheless, we conclude that in healthy subjects there is little evidence for a generalized pattern of GC sensitivity in different body tissues, given that more extreme polymorphisms that could clearly alter GC sensitivity are not evident. Although an overall heightened or lowered response to GCs may appear as a consequence of such rare mutations, in health there is no correspondence of GC sensitivity markers across tissues.
Received February 21, 2000.
Revised June 26, 2000.
Accepted June 29, 2000.
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References |
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(TNF) production in humans: high sensitivity of
TNF and resistance of IL-6. J Clin Endocrinol Metab. 82:2182–2191.
synthesis. J Immunol. 161:7007–7014.This article has been cited by other articles:
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) and 30 ng/mL
LPS plus 10-10, 10-9,
10-8, and 10-7 mol/L
DEX.
