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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0067
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 11 5978-5984
Copyright © 2005 by The Endocrine Society

Glucocorticoid Sensitivity in Young Healthy Individuals: in Vitro and in Vivo Studies

Rosangela Soares Chriguer, Lucila Leico Kagohara Elias, Ivan Moreira da Silva, Jr., Jose Gilberto Henriques Vieira, Ayrton Custodio Moreira and Margaret de Castro

Departments of Internal Medicine (R.S.C., I.M.d.S., J.G.H.V., A.C.M., M.d.C.) and Physiology (L.L.K.E.), School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil

Address all correspondence and requests for reprints to: Dr. Margaret de Castro, School of Medicine of Ribeirao Preto, University of Sao Paulo, Avenida dos Bandeirantes 3900, 14049-900, Ribeirao Preto, Sao Paulo, Brazil. E-mail: castrom{at}fmrp.usp.br.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Interindividual variation and tissue specificity of glucocorticoid (GC) sensitivity may occur in healthy subjects.

Objective and Participants: The objective of this study was to evaluate the GC sensitivity in 40 healthy young subjects (21 women and 19 men; 22–42 yr old).

Design: We measured salivary and plasma cortisol levels before and after the administration of 0.25, 0.5, and 1 mg dexamethasone (DEX), given at 2300 h. We also evaluated the pattern of DEX-mediated inhibition of concanavalin A-stimulated peripheral blood mononuclear cell proliferation using different DEX doses, the number of binding sites, and the affinity of the GC receptor (Kd).

Results: The increasing DEX doses resulted in a dose-dependent decrease in cortisol levels. The majority of the subjects (70%) suppressed cortisol with DEX doses lower than 0.5 mg, and two did not suppress even with 1 mg DEX. The binding capacity was 4.1 ± 0.3 fmol/mg protein, and the Kd was 8.1 ± 1.3 nM. Four individuals presented with elevated Kd. Peripheral blood mononuclear cell proliferation was inhibited by DEX in a dose-dependent pattern. The median IC50 value was 7.1 x 10–7 mol/liter. We found 77.5% (31 of 40) concordance among all three tests; 29 subjects showed all parameters between the 10th and 90th percentiles (P10-P90), one above P90, and one below P10. These two subjects could be classified as more GC resistant or sensitive, respectively. No concordance between in vivo and in vitro tests in two subjects suggested a tissue-specific sensitivity.

Conclusions: This is the first report that, taking advantage of three bioassays performed on the same subject, demonstrated a considerable interindividual variability and tissue-specific GC sensitivity in a young healthy population.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOIDS (GCs) AFFECT a variety of important functions throughout the body, such as glucose and fat metabolism, mediate the stress response, influence immune and central nervous system activities, and have numerous effects on development and cell differentiation. The regulation of serum GC concentrations is under the influence of the hypothalamus-pituitary-adrenal (HPA) axis, and circulating GCs themselves exert a negative feedback on hypothalamic and pituitary levels. GCs act via the cytoplasmic GC receptor (GR), which is a member of the nuclear receptor family (1, 2). GR precursor mRNA is processed into at least two alternative splice products, human (h) GR{alpha} and a non-ligand-binding isoform, hGRß, which acts as a dominant negative inhibitor of hGR{alpha} on glucocorticoid-responsive promoters (3, 4).

Mutations in the hormone-binding domain of the GR gene are responsible for the generalized familial GC resistance syndrome (5, 6, 7, 8, 9). In contrast, Iida et al. (10) described a patient who presented with signs and symptoms of Cushing’s syndrome despite persistent hypocortisolemia. The molecular basis of this condition has not been established. In addition to this evidence, variability in GC sensitivity can be observed in several other conditions. GCs are routinely used in the pharmacological treatment of patients with chronic inflammatory or autoimmune diseases. However, some patients are resistant to the GC antiinflammatory effects in the presence of several side effects known to reflect normal sensitivity to GCs, including suppression of the HPA axis. Moreover, the negative feedback action of GCs on the HPA axis varies in normal elderly individuals (11), and it can be associated with polymorphisms in the GR gene (12). However, there are few studies of GC sensitivity in a young healthy population (13, 14).

Controversy about whether the GC sensitivity of one target tissue reflects the sensitivity of other tissues in healthy subjects may be due to several methods used to study GC sensitivity in different target tissues. Among these methods, dexamethasone (DEX)-mediated inhibition of mitogen-stimulated peripheral blood mononuclear cell (PBMC) proliferation has been used to assess the GC sensitivity of the immune system, one of the most important GC target tissues. The other bioassay that can be used is the GC binding assay to determine the number and affinity of GR. To test the feedback integrity of the HPA axis in vivo, DEX suppression test has been used for several decades (15).

Because there are few studies on interindividual variation and tissue specificity of GC sensitivity in normal subjects, in the present study we addressed the question of whether young normal subjects would have variability in GC sensitivity, as observed in pathological conditions. For this purpose, we took advantage of three different bioassays performed on the same subject to evaluate in vivo the negative feedback action of GCs on the HPA axis and two in vitro tests to evaluate the number and affinity of GR and DEX-mediated inhibition of PBMC proliferation in young healthy individuals.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

This prospective study was approved by the institutional review board for human research of University Hospital of School of Medicine of Ribeirao Preto, University of Sao Paulo, and informed consent was obtained from all subjects. We studied 40 healthy subjects (19 men and 21 women) ranging in age from 22–42 yr. Subjects with acute, psychiatric, or endocrine diseases were excluded from the study. None of the individuals was treated with any medication, including estrogen. At the time of the study, the body weights and heights of the volunteers were determined, and the body mass index (mean ± SE) was 26.2 ± 0.5 kg/m2 in men and 20.8 ± 0.4 kg/m2 in women.

Plasma and salivary cortisol measurements after overnight 0.25, 0.5, and 1.0 mg DEX suppression tests

Blood and saliva samples were obtained from healthy individuals between 0800 and 0900 h before each dose of DEX (basal levels). At 2300 h, randomized doses of DEX (0.25, 0.5, and 1.0 mg) were administered orally at an interval of at least 1 wk. The next morning, between 0800 and 0900 h, post-DEX samples were taken for plasma and salivary cortisol measurements. Plasma and salivary cortisol concentrations were measured by RIA, as previously described (16). DEX levels were measured by RIA using a polyclonal antiserum raised in rabbits immunized with DEX 21-acetate (Sigma-Aldrich Corp., St. Louis, MO) and [3H]DEX (Amersham Biosciences, Little Chalfont, UK). Intra- and interassay variations and sensitivity were 5.1%, 9.3%, and 0.04 nmol/liter, respectively. All samples obtained from each individual were analyzed in duplicate in the same assay.

Cell preparation

PBMC were isolated by density gradient centrifugation using Ficoll-Hypaque (Histopaque, Sigma-Aldrich Corp.) as we previously described (17). Briefly, cells were washed three times in Hanks’ buffered saline solution and resuspended in RPMI 1640 (Invitrogen Life Technologies, Gaithersburg, MD) containing 2 mmol/liter HEPES buffer (Sigma-Aldrich Corp.), 10% fetal calf serum, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 10 mg/ml gentamicin.

Binding assay

The PBMC DEX-binding assay was performed as previously described (17, 18). Cells were suspended in RPMI medium, adjusted to 2 x 106 cells/tube in duplicate, and incubated with six concentrations (1.56–50 nmol/liter) of DEX ([1,2,4,6,7-3H]DEX, Amersham Biosciences) at 37 C in the presence or absence of a 1000-fold molar excess of unlabeled DEX (Sigma-Aldrich Corp.) for 1 h. After incubation, the cells were washed three times to separate bound from free steroid with 1.5 ml cold PBS and centrifuged at 400 x g for 10 min. After the third wash, the pellets were suspended in 100 µl RPMI and transferred to vials, and radioactivity was counted in a ß-counter. Specific binding was calculated by subtracting nonspecific binding from total binding. Receptor assay data were analyzed by the method of Scatchard using computerized linear regression analysis. The binding capacity (Bmax) was expressed as femtomoles of DEX bound per milligram of protein, and the dissociation constant (Kd), inversely proportional to ligand affinity, was expressed as nanomoles per liter. Protein levels were determined by bicinchoninic acid protein assay reagent (Pierce Chemical Co., Rockford, IL), and BSA was used as a standard. The binding assay was performed twice in all individuals who showed abnormal results.

Proliferation and in vitro corticosteroid sensitivity assay

To perform the in vitro steroid sensitivity assay, we measured the inhibitory effect of DEX on concanavalin A (Con A)-stimulated PBMC proliferation. PBMC (2 x 106 cells/well) were plated onto 96-well, flat-bottomed plates (Nunc, Copenhagen, Denmark) in triplicate and cultured at 37 C in the presence of 5% CO2. Con A at a dose of 50 µg/ml was used to stimulate cells in the absence or presence of different doses (10–10, 10–8, 10–6, and 10–4 mol/liter) of DEX. After 48 h of culture, the cells were pulsed with 1 µCi/well tritiated thymidine ([3H]thymidine; Amersham Biosciences) for 18 h. The cells were harvested with a multiple automated sample harvester, and radioactivity was counted in a liquid scintillation ß-counter (Beckman Coulter, Fullerton, CA).

Data analysis and statistics

Suppression of the HPA axis after treatment with different doses of DEX (0.25, 0.5, and 1.0 mg) was assumed when the concentration of plasma cortisol was less than 50 nmol/liter or 1.8 µg/dl (19). Because there was no criterion for salivary cortisol suppression after different doses of DEX, we assumed a cutoff point of 2.6 nmol/liter (92 ng/dl), after 1.0 mg DEX based on the highest level observed in normal population, as we previously published (16). For the analysis of DEX-mediated inhibition of Con A-stimulated PBMC proliferation, the IC50 was defined as the concentration of DEX that caused 50% inhibition of cell proliferation. Subjects with an IC50 values lower than or equal to percentile 10 (P10) and equal to or higher than P90 were considered more sensitive or more resistant to glucocorticoid, respectively, in a spectrum of intervariability of GC sensitivity.

To obtain more estimated individual values of the DEX dose needed to attain the cutoff values of plasma cortisol of 50 nmol/liter and salivary cortisol of 2.6 nmol/liter and the individual values of IC50 for the DEX-mediated inhibition of Con A-stimulated PBMC proliferation, we performed a nonlinear sigmoidal dose-response regression (GraphPad Prism version 3.0). Additionally, the in vivo and the in vitro data were adjusted to a nonlinear mixed logistic growth model with fixed and random effects (20) based on the equation yij = {b1 + µi1/1 + exp[–(dij – b2)/b3]} + {epsilon}ij, where yij is the percent inhibition of PBMC in a j dose of DEX in an ih observation, dij represents the doses of DEX, b1 represents the asymptotic regression, b2 is the point of inflection, b3 is the parameter form, µi1 represents individual residual effects, and {epsilon}ij represents residual errors. For analysis and data simulation, we used the PROC NL MIXED software SAS version 8.02 (SAS Institute, Cary, NC).

All results are expressed as the median, mean ± SEM, and percentiles when appropriate. Basal plasma and salivary cortisol concentrations in men and women were compared using the two-sample Wilcoxon rank-sum test. The Wilcoxon matched pairs signed-rank-sum test was used to compare serum or salivary cortisol concentrations before and after DEX administration. Spearman’s rank correlation coefficient was used to assess the association between salivary and plasma cortisol before and after the administration of 0.25, 0.5, and 1.0 mg DEX. We also used Spearman correlation to compare the individual IC50 values obtained from the PMBC proliferation test with the dose of DEX that suppressed plasma cortisol levels to less than 50 nmol/liter from the same subject. Significance was assumed at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Plasma and salivary cortisol measurements after overnight DEX suppression tests

There was no difference among the three basal levels of plasma and salivary cortisol for each subject (Fig. 1Go); therefore, for all additional statistical analyses, the baseline concentration was considered the mean of the three samples obtained before each DEX suppression test. Figure 2Go shows individual plasma (upper panel) and salivary (bottom panel) cortisol concentrations after various doses of DEX in 40 healthy individuals. The administration of increasing doses of DEX (0.25, 0.5, and 1.0 mg) resulted in a dose-dependent increase in mean circulating DEX levels (1.4 ± 0.1, 2.6 ± 0.2, and 5.8 ± 0.4 nmol/liter, respectively) and a dose-dependent decrease in cortisol levels (plasma: 198.5 ± 19.4, 57.1 ± 9.0, and 37.9 ± 2.6 nmol/liter; salivary: 26.2 ± 3.3, 4.7 ± 1.4, and 2.17 ± 0.3 nmol/liter) compared with the basal cortisol levels (plasma, 381.6 ± 22.3 nmol/liter; salivary, 46.5 ± 3.0 nmol/liter; Fig. 3Go). Except under basal conditions, none of the individuals had an undetectable circulating DEX level, indicating that all participants indeed ingested the DEX tablets.



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  		FIG. 1. Basal plasma (upper panel) and salivary (bottom panel) cortisol concentrations (nanomoles per liter) of 40 healthy subjects obtained at 0800 h on three different occasions.

 


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  		FIG. 2. Plasma and salivary cortisol concentrations (nanomoles per liter) under baseline conditions and after DEX suppression test (0.25, 0.5, and 1.0 mg DEX) in 40 healthy individuals. Dotted lines represent the suppression of the HPA axis, assumed when the concentrations of plasma and salivary cortisol were less than 50 and less than 2.6 nmol/liter, respectively. •, Plasma and salivary cortisol levels of two subjects more resistant to GC (subjects 15 and 19).

 


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  		FIG. 3. Individual plasma DEX (nanomoles per liter) and cortisol (upper panel) concentrations and salivary cortisol concentrations (bottom panel; nanomoles per liter) after each dose of DEX. •, Subjects 15 and 19, who showed no suppression with 1.0 mg DEX.

 
There was a considerable individual variability in plasma and salivary cortisol suppression in response to a given DEX concentration. Thirty-eight of 40 healthy individuals (95%) showed plasma and salivary cortisol concentrations, after the administration of 1 mg DEX that were less than 50 or 2.6 nmol/liter, respectively. There was no difference between the DEX doses required to suppress plasma and salivary cortisol to these levels. Therefore, in Table 1Go, we show only the individual DEX doses needed to suppress plasma cortisol. We observed that the majority of young healthy individuals (70%) suppressed salivary and plasma cortisol with DEX doses lower than 0.5 mg. Seven individuals (17.5%) needed slightly higher doses (0.55–0.66 mg), three individuals (7.5%) needed 0.79 and 0.83 mg, and two (5%) did not suppress even with 1.0 mg DEX. As a group, the median values of DEX that suppressed plasma and salivary cortisol to the cutoff levels were 0.51 and 0.50 mg, respectively (range, 0.25–1.0 mg). The P10 and P90 values for both plasma and salivary cortisol were 0.25 and 0.83 mg, respectively. According to these cutoff values, subjects 12, 17, 18, 24, 33, and 39 were considered more sensitive to GC, and subjects 2, 15, and 19 were considered more resistant to GC.


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  		TABLE 1. Clinical findings and in vivo and in vitro GC sensitivity evaluation in 40 normal individuals

 
Binding assay

The linearity of the Scatchard plots (r2 = 0.91 ± 0.01) indicates a single class of binding site affinity. As a group, the mean ± SEM number of binding sites of GR (Bmax) in PBMC and the dissociation constant (Kd) were 4.1 ± 0.3 fmol/mg protein and 8.1 ± 1.3 nmol/liter, respectively. Considering the values above P95 (Bmax, >8.1 fmol/mg protein; Kd, >24.9 nmol/liter) of the studied group, four of 40 individuals showed an elevated Kd (10%), suggesting a decrease in GR affinity. Only one of 40 individuals (2.5%) showed an elevated Bmax. Figure 4Go shows a representative saturation curve and Scatchard plot of one individual with a normal Kd value and one individual with an elevated Kd value.



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  		FIG. 4. Representative saturation curves of [3H]DEX binding to PBMC and the Scatchard plot (inset) of GR binding studies of one individual with a normal Kd ({square}) and one individual with an elevated Kd ({diamondsuit}).

 
Proliferation assay

Basal lymphocyte proliferation was stimulated by Con A (761 ± 61 vs. 36,866 ± 2,656 cpm). Increasing concentrations of DEX (10–10, 10–8, 10–6, and 10–4 mol/liter) inhibited lymphocyte proliferation in a dose-dependent manner (34,488 ± 2,467, 29,693 ± 2,295, 15,946 ± 1,590, and 13,263 ± 1,457 cpm). Figure 5Go shows the individual percent inhibition of lymphocyte proliferation after incubation in RPMI with Con A plus different doses of DEX.



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  		FIG. 5. Percent inhibition of lymphocyte proliferation after incubation in RPMI with Con A plus different doses of DEX (10–10, 10–8, 10–6, and 10–4 mol/liter), in 40 healthy individuals. Boxes indicate the 95% confidence interval in the individuals studied.

 
The individual IC50 values estimated by the nonlinear sigmoid dose-response curve are shown in Table 1Go. As a group, the median IC50 value was 7.1 x 10–7 mol/liter (range, 2.5 x 10–9 to 1 x 10–4 mol/liter). The P10 and P90 IC50 values were 2.7 x 10–8 and 1.1 x 10–5 mol/liter, respectively. According to these cutoff values, subjects 1, 4, 8, 9, and 35 were considered more sensitive to GC, and subjects 15, 19, 27, 32, and 36 were considered more resistant to GC.

Using the PROC NL MIXED software for in vitro GC sensitivity analysis, three individuals did not fit the logistic model, indicating that the DEX dose necessary to suppress 50% of PBMC proliferation in these individuals was higher than 10–4 mol/liter. For the purpose of statistical analysis, in these three individuals we considered the highest dose used in the experiment (1 x 10–4 mol/liter). The GC sensitivity profile estimated by the nonlinear sigmoidal dose response was similar for all individuals except two, compared with that obtained by the PROC NL MIXED software analysis.

Comparison between in vivo and in vitro assays

In 31 of 40 subjects (77.5%), we found concordance among all three tests. Twenty-nine subjects showed suppression of plasma and salivary cortisol with a DEX dose between 0.25–0.83 mg (P10–90), an IC50 between 1.7 x 10–8 and 1.1 x 10–5 nmol/liter (P10–90) in the in vitro corticosteroid sensitivity assay, and a normal Kd. Also, concordance was observed in subject 19, who showed no suppression of plasma and salivary cortisol with 1.0 mg DEX, an IC50 of 2 x 10–5 nmol/liter (higher than P90), and an elevated Kd. Therefore, this young healthy subject could be classified as more resistant to GC. Subject 39 suppressed plasma and salivary cortisol with 0.25 mg DEX, showed an IC50 of 1 x 10–8 mol/liter (below P10), and a normal Kd. Therefore, this young individual could be classified as more sensitive to GC.

Although concordance was observed in the majority of the individuals, there were some exceptions. We observed no concordance between in vivo (HPA axis) and in vitro (immune system) tests in subjects 27 and 32. Both showed IC50 values equal to or higher than 1.1 x 10–5 mol/liter (above P90) and elevated Kd.; however, both suppressed plasma and salivary cortisol levels with 0.55 mg DEX between P10–90, suggesting a tissue-specific sensitivity to GC in these two individuals, with less sensitivity in the immune system.

In the present study we observed no concordance among the three tests in nine individuals (22.5%). No concordance between the two in vitro tests was observed in subjects 2 and 15. Subject 2 showed IC50 between P10–90 in the in vitro GC sensitivity assay, but showed an elevated Kd. In contrast, subject 15 showed IC50 higher than P90 and had a normal Kd. In the in vivo test, subject 2 required 0.83 mg (P90), and subject 15 did not suppress plasma and salivary cortisol at any dose of DEX. Therefore, in both cases, one of the in vitro tests was in accordance with the in vivo study, indicating that subjects 2 and 15 could have a GC-resistant profile.

No concordance between the two in vitro tests was also observed in subjects 1, 4, 8, 35, and 36 who showed normal Kd and required doses of DEX between P10–90. However, these subjects showed IC50 below P10 (subjects 1, 4, 8, 35, and 39) or above P90 (subject 36). Because two tests were considered in the normal range of this population, these subjects could be considered as having a normal GC sensitivity pattern.

The IC50 obtained from the PMBC proliferation test (in vitro study) correlated positively (r = 0.37; P < 0.01) with the dose of DEX that suppressed plasma cortisol levels to below 50 nmol/liter in the same subject (in vivo study).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In humans, large individual variations have been demonstrated in both baseline and stimulated cortisol levels in healthy elderly individuals (11). In a young healthy population, we observed no difference among the three basal levels of plasma and salivary cortisol for each subject, indicating a marked individual stability of baseline cortisol concentrations. Over the years, the effects of gender and age on the function of the HPA axis have been investigated. We observed no difference in baseline serum and salivary cortisol concentrations between young men and women, as previously observed in normal and elderly subjects (11, 21), in contrast with another study (22) that showed a gender difference.

We also evaluated the interindividual variability of the feedback sensitivity of the HPA axis in this young population. There is a controversy in the literature concerning the influence of age on the DEX suppression test; in some studies (13, 23, 24) there was a significant correlation between age and post-DEX cortisol concentrations, whereas in others no such correlation could be demonstrated (11, 25). We found young healthy individuals who were less sensitive to the inhibitory effects of GCs on HPA axis. Therefore, a decreased sensitivity to GC feedback may be prevalent in very elderly (>80 yr) individuals (13, 24), but this phenomenon can also occur in a young population.

In the present study we also compared the performance of plasma cortisol and that of salivary cortisol to gain more insight into the interindividual variability in the feedback sensitivity of the HPA axis in a normal population. Salivary cortisol showed more profound suppression than plasma cortisol, with a correlation between both measurements basally and after different DEX doses. Previously, we demonstrated that salivary cortisol also presented more profound suppression than plasma cortisol or ACTH in a dose-response pattern after different doses of DEX in patients with Cushing’s syndrome (26). Therefore, based on the accuracy, simplicity, and cost-effectiveness, salivary cortisol, besides its use as a main method to screen for Cushing’s syndrome (16, 27, 28, 29), may also be useful to evaluate GC sensitivity in normal subjects.

This study estimated the dose of DEX needed to suppress plasma and salivary cortisol in a normal young population. The increasing DEX doses resulted in a dose-dependent increase in circulating DEX levels and a dose-dependent decrease in cortisol levels. We observed clearly that the majority of the young healthy individuals (70%) suppressed salivary and plasma cortisol with lower DEX doses (0.25–0.5 mg DEX), Seven individuals (17.5%) needed a slightly higher doses (0.55–0.66 mg), three individuals needed 0.79 and 0.83 mg, and two did not suppress even with 1.0 mg DEX. Therefore, after analysis of the present data, we could state that 1.0 mg DEX is too high a suppressive dose to detect individual differences in the sensitivity of the HPA axis to GCs within a normal population, as suggested in previous reports in the elderly (11). These data show, for the first time, a spectrum of GC sensitivity in normal individuals. We found that 5% of subjects (subjects 15 and 19) had no suppression of plasma and salivary cortisol after 1 mg DEX even in the presence of a plasma DEX concentration in the normal range (6.38 and 10.19 nmol/liter, respectively), excluding altered metabolism of DEX in these subjects. This finding is in accordance with previous studies that showed no cortisol suppression in 2.3–9.1% of the studied subjects (11, 13). It is important to point out that subjects 15 and 19 have no clinical findings of Cushing’s syndrome, and they suppressed plasma and salivary cortisol after a DEX dose of 2 mg/d for 2 d. However, considering the high incidence of adrenal incidentaloma in the general population and subclinical Cushing’s syndrome, these subjects should be submitted to periodical clinical, radiological, and hormonal evaluations.

We also evaluated the number of binding sites and the affinity of GRs in young healthy controls and demonstrated no gender difference. Previously, data from our laboratory also demonstrated no difference in binding sites and affinity of GRs in normal children and young healthy controls (17). The mean Kd observed in this study (8.1 ± 1.3 nmol/liter) is in accordance with data (9.3 ± 0.5 nmol/liter) recently published (30). Considering the values above P95 of the studied group, four of 40 individuals (10%) showed an elevated Kd, suggesting a decrease in GR affinity. Two of these subjects did not suppress or needed a high DEX dose to suppress the HPA axis, suggesting a GC resistance profile. Decreased ligand affinity, rather than GC receptor down-regulation, has been demonstrated in patients with endogenous Cushing’s syndrome (30). These researchers postulated that a diminished GR affinity of as yet unknown origin might partially protect cells from the high cortisol levels. Thus, we could speculate that this putative mechanism could also be present as an adaptive physiological phenomenon in normal individuals who are more resistant to GC. Taken together, these data suggest that GRs might mediate the GC resistance observed in some individuals from a normal population. It is also important to point out that binding assays evaluate GR{alpha}, which is the classic ligand-binding protein for GC. Therefore, we cannot rule out an overexpression of GRß, a dominant negative inhibitor of GR{alpha}, inducing steroid insensitivity in some normal individuals, as well established in asthmatic patients (3, 31).

In an attempt to identify the response to GC in the immune system, we evaluated the pattern of DEX-mediated inhibition of Con A-stimulated PBMC proliferation. Increasing concentrations of DEX inhibited lymphocyte proliferation in a dose-dependent manner. Using this method, based on the individual IC50 values estimated by the nonlinear sigmoid dose-response curve, we also observed a spectrum of GC sensitivity in the immune system in a normal young population.

We found a high concordance (77.5%) among all three tests based on the P10–90 of each test. It is important to point out that these tests performed in the same individual allowed us to determine the normal ranges for each test and also to discriminate one subject more resistant (subject 19) and one subject more sensitive (subject 39) to GC. Although concordance was observed in the majority of individuals, there were some exceptions. We observed no concordance between the two in vitro tests in subjects 2 and 15. In both cases, one of the in vitro tests was in accordance with the in vivo study, showing a GC-resistant profile. The dissociation between the two in vitro tests can be ascribed to the variability in the biological assays. As expected for any biological assay, there is no 100% sensitivity and specificity.

We observed no concordance between in vivo (HPA axis) and in vitro (immune system) tests in subjects 27 and 32, suggesting a tissue-specific sensitivity to GC, with less sensitivity in the immune system. Therefore, our data demonstrated that there is a variability of GC sensitivity in specific target tissues in healthy subjects. These data are in accordance with a few studies reporting no association between DEX suppression and skin blanching in hypertensive and normotensive subjects (32) or DEX suppression and sensitivity in peripheral blood mononuclear leukocytes in elderly women with osteoporosis (33). More recently, GC sensitivity was measured in 36 healthy subjects in three target tissues (the immune system, the cardiovascular system, and the HPA axis), and there was no correspondence among the GC sensitivity parameters (14). However, controversy concerning the correspondence of GC sensitivity across tissues has been found in the literature. Few studies reported significant association between GC sensitivity measured by clinical response to GC and their actions on skin blanching in asthma (34) and DEX suppression of the HPA axis and lymphocyte mitogenesis in depressed and nondepressed subjects (35).

The discordance of GC sensitivity in different target tissues might be influenced by a large number of tissue-specific factors, such as GR expression levels, intracellular GC availability, hormone binding affinity, heat shock protein complexes, and modulation of gene transcription. In addition, an important role for 11ß-hydroxysteroid dehydrogenase type 1 activity in normal physiology, with the relative contributions of dehydrogenase and reductase activities being important in controlling the overall equilibrium of local GC levels, can be considered to modulate GC sensitivity in a tissue-specific manner (36). Several polymorphisms in the locus of the GR gene have been described; however, it is still unclear to what extent the observed response variability can be due to GR gene polymorphisms (37, 38). We observed a clear interindividual variation in GC sensitivity as a possible individual trait. We speculate that the long-term effects of increased sensitivity or resistance to GC in normal subjects might influence the outcome of development of early and/or serious side effects during therapy with GCs or may be associated with an increased risk to develop insulin resistance, obesity, and osteoporosis (39, 40, 41).

In conclusion, this is the first report that, taking advantage of three bioassays performed on the same subject, evaluated GC sensitivity in a normal young population. This strategy allowed us to define normal ranges for in vivo and in vitro tests and also to discriminate subjects more sensitive or more resistant to GC, indicating considerable interindividual variability and a spectrum of GC sensitivity in normal subjects. In addition, we demonstrated tissue-specific GC sensitivity in a young normal population. These findings are important because they can contribute to clarifying the role of GC sensitivity in a normal population and might help to predict which individuals are at greater risk for side effects associated with GC therapy and which individuals could have a genetic predisposition to diseases related to GC sensitivity.


   Acknowledgments
 
We thank Mr. Jose Roberto da Silva and Mrs. Maria Aparecida Nunes Ferreira for technical assistance. We also thank Prof. Dr. Edson Zangiacomi Martinez for all suggestions and the discussion of the statistical analysis.


   Footnotes
 
This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo Grants 01/04013-2 and 01/07670-4.

First Published Online August 9, 2005

Abbreviations: Bmax, Binding capacity; Con A, concanavalin A; DEX, dexamethasone; GC, glucocorticoid; GR, GC receptor; h, human; HPA, hypothalamus-pituitary-adrenal; P, percentile; PBMC, peripheral blood mononuclear cell.

Received January 12, 2005.

Accepted August 2, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889–892[Abstract/Free Full Text]
  2. Bamberger CM, Schulte HM, Chrousos GP 1996 Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids. Endocr Rev 17:245–261[Abstract]
  3. Castro M, Elliot S, Kino T, Bamberger C, Karl M, Webster E, Chrousos GP 1996 The non-ligand binding ß-isoform of the human glucocorticoid receptor (hGRß): Tissue levels, mechanism of action, and potential physiologic role. Mol Med 2:597–607[Medline]
  4. Yudt MR, Cidlowski JA 2002 The glucocorticoid receptor: coding a diversity of proteins and responses through a single gene. J Mol Endocrinol 16:1719–1726
  5. Hurley DM, Accili D, Stratakis CA, Karl M, Vamvakopoulos N, Rorer E 1991 Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance. J Clin Invest 87:680–686
  6. Karl M, Lamberts SWJ, Detera-Wadleigh SD, Enico IJ, Stratakis CA, Hurley DM 1993 Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene. J Clin Endocrinol Metab 76:683–689[Abstract]
  7. Malchoff DM, Brufsky A, Reardon G, McDermott P, Javier EC, Bergh CH 1993 A mutation of the glucocorticoid receptor in primary cortisol resistance. J Clin Invest 91:1918–1925
  8. Vottero A, Kino T, Combe H, Lecomte P, Chrousos GP 2002 A novel, C-terminal dominant negative mutation of the GR causes familial glucocorticoid resistance through abnormal interactions with p160 steroid receptor coactivators. J Clin Endocrinol Metab 87:2658–2667[Abstract/Free Full Text]
  9. Mendonca BB, Leite MV, de Castro M, Kino T, Elias LL, Bachega TA, Arnhold IJ, Chrousos GP, Latronico AC 2002 Female pseudohermaphroditism caused by a novel homozygous missense mutation of the GR gene. J Clin Endocrinol Metab 87:1805–1809[Abstract/Free Full Text]
  10. Iida S, Nakamura Y, Fujii H, Nishimura J, Tsugawa M, Gomi M, Fukata J, Tarui S, Moriwaki K, Kitani T 1990 A patient with hypocortisolism and Cushing’s syndrome-like manifestations: cortisol hyperreactive syndrome. J Clin Endocrinol Metab 70:729–737[Abstract]
  11. Huizenga NA, Koper JW, de Lange P, Pols HA, Stolk RP, Grobbee DE, de Jong FH, Lamberts SW 1998 Interperson variability but intraperson stability of baseline plasma cortisol concentrations, and its relation to feedback sensitivity of the hypothalamo-pituitary-adrenal axis to a low dose of dexamethasone in elderly individuals. J Clin Endocrinol Metab 83:47–54[Abstract/Free Full Text]
  12. Huizenga NA, Koper JW, de Lange P, Pols HA, Stolk RP, Burger H, Grobbee DE, Brinkmann AO, De Jong FH, Lamberts SW 1998 A polymorphism in the glucocorticoid receptor gene may be associated with an increased sensitivity to glucocorticoids in vivo. J Clin Endocrinol Metab 83:144–151[Abstract/Free Full Text]
  13. Georgotas A, McCue RE, Kim OM, Hapworth WE, Reisberg B, Stoll PM, Sinaiko E, Fanelli C, Stokes PE 1986 Dexamethasone suppression in dementia, depression, and normal aging. Am J Psychiatry 143:452–456[Abstract/Free Full Text]
  14. Ebrecht M, Buske-Kirschbaum A, Hellhammer D, Kern S, Rohleder N, Walker B, Kirschbaum C 2000 Tissue specificity of glucocorticoid sensitivity in healthy adults. J Clin Endocrinol Metab 85:3733–3739[Abstract/Free Full Text]
  15. Liddle GW 1960 Tests of pituitary-adrenal suppressibility in the diagnosis of Cushing’s syndrome. J Clin Endocrinol Metab 20:1539–1560
  16. Castro M, Elias PCL, Quidute ARP, Halah FPB, Moreira AC 1999 Outpatient screening for Cushing’s syndrome: the sensitivity of the combination of circadian rhythm and overnight dexamethasone suppression salivary cortisol tests. J Clin Endocrinol Metab 84:878–882[Abstract/Free Full Text]
  17. Carlotti AP, Franco PB, Elias LL, Facincani I, Costa EL, Foss N, Moreira AC, de Castro M 2004 Glucocorticoid receptors, in vitro steroid sensitivity, and cytokine secretion in idiopathic nephritic syndrome. Kidney Int 65:403–408[CrossRef][Medline]
  18. De Antonio, Blotta HM, Mamoni RL, Louzada P, Bertolo MB, Foss NT, Moreira AC, Castro M 2002 Effects of dexamethasone on lymphocyte proliferation and cytokine production in rheumatoid arthritis. J Rheumatol 29:46–51[Medline]
  19. Newell-Price J, Trainer P, Besser M, Grossman A 1998 The diagnosis and differential diagnosis of Cushing’s syndrome and pseudo-Cushing’s states. Endocr Rev 19:647–672[Abstract/Free Full Text]
  20. Lindstrom, MJ, Bates, DM 1990 Nonlinear mixed effects models for repeated measures data. Biometrics 46:673–687[CrossRef][Medline]
  21. Raff H, Raff JL, Duthie EH, Wilson CR, Sasse EA, Rudman I, Mattson D 1999 Elevated salivary cortisol in the evening in healthy elderly men and women: correlation with bone mineral density. J Gerontol A Biol Sci Med Sci 54:M479–M483
  22. Laudat MH, Cerdas S, Fournier C, Guiban D, Guilhaume B, Luton JP 1988 Salivary cortisol measurement: a practical approach to assess pituitary-adrenal function. J Clin Endocrinol Metab 66:343–348[Abstract]
  23. Weiner MF, Davis BM, Mohs RC, Davis KL 1987 Influence of age and relative weight on cortisol suppression in normal subjects. Am J Psychiatry 144:646–649[Abstract/Free Full Text]
  24. Wilkinson CW, Peskind ER, Raskind MA 1997 Decreased hypothalamic-pituitary-adrenal axis sensitivity to cortisol feedback inhibition in human aging. Neuroendocrinology 65:79–90[Medline]
  25. Tourigny-Rivard MF, Raskind M, Rivard D 1981 The dexamethasone suppression test in an elderly population. Biol Psychiatry 16:1177–1184[Medline]
  26. Castro M, Elias LL, Elias PC, Moreira AC 2003 A dose-response study of salivary cortisol after dexamethasone suppression test in Cushing’s disease and its potential use in the differential diagnosis of Cushing’s syndrome. Clin Endocrinol (Oxf) 59:800–805[CrossRef][Medline]
  27. Raff H, Raff JL, Findling JW 1998 Late-night salivary cortisol as a screening test for Cushing’s syndrome. J Clin Endocrinol Metab 83:2681–2686[Abstract/Free Full Text]
  28. Papanicolaou DA, Mullen N, Kyrou I, Nieman LK 2002 Nighttime salivary cortisol: a useful test for the diagnosis of Cushing’s syndrome. J Clin Endocrinol Metab 87:4515–4521[Abstract/Free Full Text]
  29. Putignano P, Toja P, Dubini A, Giraldi FP, Corsello SM, Cavagnini F 2003 Midnight salivary cortisol versus urinary free and midnight serum cortisol as screening tests for Cushing’s syndrome. J Clin Endocrinol Metab 88:4153–4157[Abstract/Free Full Text]
  30. Huizenga NA, De Herder WW, Koper JW, de Lange P, v D Lely AJ, Brinkmann AO, de Jong FH, Lamberts SW 2000 Decreased ligand affinity rather than glucocorticoid receptor down-regulation in patients with endogenous Cushing’s syndrome. Eur J Endocrinol 142:472–476[Abstract]
  31. Sousa AR, Lane SJ, Cidlowski JA, Staynov DZ, Lee TH 2000 Glucocorticoid resistance in asthma is associated with elevated in vivo expression of the glucocorticoid receptor ß-isoform. J Allergy Clin Immunol 105:943–950[CrossRef][Medline]
  32. Walker BR, Best R, Shackleton CH, Padfield PL, Edwards CR 1996 Increased vasoconstrictor sensitivity to glucocorticoids in essential hypertension. Hypertension 27:190–196[Abstract/Free Full Text]
  33. vanRijen E, Harvey RA, Barton RN, Rose JG, Horan MA 1998 Sensitivity of mononuclear leucocytes to glucocorticoids in elderly hip-fracture patients resistant to suppression of plasma cortisol by dexamethasone. Eur J Endocrinol 138:659–666[Abstract]
  34. Brown PH, Teelucksingh S, Matusiewicz SP, Greening AP, Crompton GK, Edwards CR 1991 Cutaneous vasoconstrictor response to glucocorticoids in asthma. Lancet 337:576–580[CrossRef][Medline]
  35. Lowy MT, Reder AT, Gormley GJ, Meltzer HY 1988 Comparison of in vivo and in vitro glucocorticoid sensitivity in depression: relationship to the dexamethasone suppression test. Biol Psychiatry 24:619–630[CrossRef][Medline]
  36. Tomlinson JW, Walker EA, Bujalska IJ, Draper N, Lavery GG, Cooper MS, Hewison M, Stewart PM 2004 11ß-Hydroxysteroid dehydrogenase type 1: a tissue-specific regulator of glucocorticoid response. Endocr Rev 25:831–866[Abstract/Free Full Text]
  37. van Rossum EF, Feelders RA, van den Beld AW, Uitterlinden AG, Janssen JA, Ester W, Brinkmann AO, Grobbee DE, de Jong FH, Pols HA, Koper JW, Lamberts SW 2004 Polymorphisms in the glucocorticoid receptor gene and their associations with metabolic parameters and body composition. Recent Prog Horm Res 59:333–357[Abstract/Free Full Text]
  38. Stevens A, Ray DW, Zeggini E, John S, Richards HL, Griffiths CE, Donn R 2004 Glucocorticoid sensitivity is determined by a specific glucocorticoid receptor haplotype. J Clin Endocrinol Metab 89:892–897[Abstract/Free Full Text]
  39. Weaver JU, Hitman GA, Kopelman PG 1992 An association between a Bc1I restriction fragment length polymorphism of the glucocorticoid receptor locus and hyperinsulinaemia in obese women. J Mol Endocrinol 9:295–300[Abstract]
  40. Panarelli M, Holloway CD, Fraser R, Connell JM, Ingram MC, Anderson NH, Kenyon CJ 1998 Glucocorticoid receptor polymorphism, skin vasoconstriction, and other metabolic intermediate phenotypes in normal human subjects. J Clin Endocrinol Metab 83:1846–1852[Abstract/Free Full Text]
  41. Lin RC, Wang XL, Dalziel B, Caterson ID, Morris BJ 2003 Association of obesity, but not diabetes or hypertension, with glucocorticoid receptor N363S variant. Obes Res 11:802–808[Medline]



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