Healthy ageing is associated with reduced lymphocyte sensitivity to glucocorticoids

Instituto de Pesquisas Biomédicas, Instituto de Geriatria e Gerontologia and Faculdade de Biociências, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil.

Corresponding author: Dr Moisés E. Bauer, Laboratory of Cellular and Molecular Immu-nology, Instituto de Pesquisas Biomédicas, Hospital São Lucas - PUCRS, Av. Ipiranga 6690, 2º andar – PO Box 1429. 90.610-000 Porto Alegre, RS. Brazil.
Phone: +55 51 3320 3000/x2725, Fax: +55 51 33203312.
Email: mebauer@pucrs.br

Abstract

Ageing has been associated with immunological changes that resemble those observed fol-lowing chronic stress or glucocorticoid (GC) treatment. However, it remains controversial whether these changes cause or are caused by underlying disease in humans. In this study we evaluated both neuroendocrine and immune functions of healthy elders by measuring dehy-droepiandrosterone (DHEA) levels as well as T-cell function and sensitivity to steroids in vitro. Forty-six elderly subjects (72.0 ± 8.5 yrs) and 33 young adults (27.4 ± 6.7 yrs) were recruited accordingly the SENIEUR protocol. Salivary and serum DHEA levels were meas-ured by radioimmunoassays. Mitogen-induced T cell proliferation was evaluated as an index of non-specific cell-mediated immunity. Peripheral sensitivity to steroids was assessed in vi-tro by dexamethasone, cortisol or DHEA inhibition of T-cell proliferation. Healthy elders had 46% lower dehydroepiandrosterone levels than young adults (p < 0.01). In addition, elders displayed a blunted T-cell proliferation ( 44%, p < 0.001) and reduced sensitivity to dexa-methasone (-19%, p < 0.001). These data indicate that healthy ageing is associated with blunted salivary DHEA production and impaired neuroendocrine-immunoregulation at the level of the lymphocyte.

Key words: ageing, psychoneuroimmunology, glucocorticoids, cortisol, lymphocyte prolif-eration

1. Introduction

Ageing is associated with several physiological alterations including changes in the immune system (immunosenescence). However, it remains controversial whether these changes cause or are caused by underlying disease in humans. Strenuous efforts have been made to circumvent this problem by separating “disease” from “ageing”, as exemplified by the application of the SENIEUR protocol (Ligthart et al., 1984) that defines rigorous criteria for selecting healthy individuals in immunogerontological studies. When diseased subjects are excluded, immunosenescence involves thymic involution, impaired humoral responses and blunted T-cell proliferation (Pawelec et al., 2002). The latter is one of the most documented age-related change observed during ageing (Murasko et al., 1987; Liu et al., 1997) and seems to be due to both numerical and functional cellular changes. There is considerable amount of evidence suggesting that ageing impairs the signal transduction events that follow mitogen-induced T-cell activation and interleukin (IL)-2 secretion (Candore et al., 1992; Pawelec et al., 2002). For instance, it has been shown that the frequency of T cells responding to phytohe-magglutinin (PHA) by secreting IL-2 decreased with age in American SENIEUR donors (Jackola et al., 1994). The clinical consequences of immunosenescence include increased sus-ceptibility to infectious diseases, neoplasias and autoimmune diseases (Castle, 2000). How-ever, this altered morbidity is not evenly distributed and should be influenced by other im-mune-modulating factors. These data led us to consider that other factors may potentially con-tribute to the heterogeneity of these changes, including neuroendocrine pathways. Therefore, the understanding of the interplay between the immune, endocrine and nervous systems in the elderly is of paramount importance.

In addition to immunosenescence, the endocrine system also undergoes important changes during ageing. From both human and animal studies, it has been demonstrated a de-cline in growth hormone, sex hormones and dehydroepiandrosterone (DHEA) with ageing (Roshan et al., 1999). DHEA is the major secretory product of the human adrenal. The hor-mone is uniquely sulfated (DHEAS) before entering the plasma, and this prohormone is con-verted to DHEA and its metabolites in various peripheral tissues (Canning et al., 2000). Se-rum DHEA levels decrease by the second decade of life reaching about 5% of the original level in the elderly (Migeon et al., 1957). However, it remains to be established whether this hormonal change can also be observed when stress-free collection procedures are employed (e.g. salivary samples). DHEA and its sulfated form have been reported to have immuno-modulatory properties, including increased mitogen-stimulated IL-2 production (Daynes et al., 1990; Suzuki et al., 1991), diminished TNF-? or IL-6 production (Di Santo et al., 1996; Straub et al., 1998), inhibition of natural killer cell differentiation (Risdon et al., 1991) or ro-dent mitogen-induced lymphocyte proliferation (Padgett and Loria, 1994). Furthermore, DHEA has been proposed as exerting restoring effects on immunosenescence, including im-portant adjuvant effect on the immunization of aged mice with recombinant hepatitis B sur-face antigen (Araneo et al., 1993) or influenza (Danenberg et al., 1995). However, DHEA treatment did not produce any beneficial to the immune response to influenza vaccination in elderly subjects (Danenberg et al., 1997). In addition, the immunological effects of DHEA treatment on healthy human aging have poorly been explored so far.

Glucocorticoid immunoregulation is orchestrated by specific binding of GCs on two distinct cytoplasmic receptors. Type I adrenal receptor is primary mineralocorticoid binding but also has a high binding affinity for GCs. The type II adrenal receptor binds to dexa-methasone (DEX), a synthetic GC, with a greater affinity than natural GCs. Although the type I receptors have higher affinity for circulating GCs than the type II, most (if not all) effects on the immune system are mediated via type II adrenal receptors (McEwen et al., 1997). The presence of these receptors indicates that the immune system is prepared for hypothalamic-pituitary-adrenal (HPA) axis activation and the subsequent elevation in endogenous GCs. However, it has been suggested that chronically elevated cortisol levels may produce a state of steroid resistance enabling lymphocytes to respond with less intensity to GCs. However, this has been poorly explored in healthy ageing so far.

We have recently demonstrated that strictly healthy ageing was associated with sig-nificant psychological distress and increased salivary cortisol levels in parallel with changes in T-cell subsets (Collaziol et al., 2004) and unaltered pro-inflammatory cytokine production (Luz et al., 2003). In this study we further describe this elderly cohort investigating if sali-vary/serum DHEA levels were related to changes in mitogen-induced T cell proliferation. In addition, we investigated whether lymphocytes differed in sensitivity to steroids and so exam-ined whether ageing was associated with alterations in neuroendocrine-immune regulation. This was achieved by investigating in vitro lymphocyte sensitivity to DEX, cortisol and DHEA.

2. Materials and methods

2.1. Subjects

Forty six non-institutionalized healthy elderly (31 females), aged from 60 to 91 years (mean ± SD, 72.00 ± 8.51 yrs), were recruited from an existing database of 1,118 community-dwelling elderly subjects who had previously participated in research at the Institute of Geri-atrics and Gerontology (PUCRS). All subjects were recruited from local community centres and registered at the Office for Social Care in Gravataí (RS). This elderly population corre-sponded both ethically and socio-economically to the general population of our State (RS). All subjects took part in the GENESIS Program for the study on the genetic-environmental interactions on human ageing. Thirty three healthy young adults, (18 females), aged from 20 to 40 years (mean ± SD, 27.40 ± 6.70 yrs), also took part in this study and were all students or employees from the PUCRS.

All subjects were recruited accordingly to the SENIEUR protocol (Ligthart et al., 1984) that defines rigorous criteria for selecting healthy individuals in immunogerontological studies. The health conditions were checked accordingly to accurate clinical investigations and to haematological and biochemical parameters. The exclusion criteria included: infec-tions, acute or chronic inflammation, autoimmune diseases, heart disease, under nourishment, anaemia, leucopoenia, mood disorders, caregiving, neurodegenerative disease, neoplasias and use of hormones (steroids) and drugs (alcohol, antidepressants, immunosuppressants, antico-agulants).

2.2. Procedures

Subjects reported to the laboratory between 7–8 h and were promptly examined by a geriatrician. After the clinical examination, subjects were asked to collect the fist saliva sam-ple (9 h) and blood was immediately drawn for the immune measures. Before leaving the laboratory, subjects were asked to collect the second saliva sample (12 h) and were instructed to collect the third sample (20 h) at home. The latter was kept in the fridge and returned to lab within a week. The study protocol was approved by both scientific and ethics committees (Pontifical Catholic University of Rio Grande do Sul, PUCRS, Porto Alegre, Brazil) and writ-ten informed consent was obtained from all subjects.

2.3. Nutritional analyses

Nutritional status was assessed in this investigation because it is known to influence immune function (Krause et al., 2000). The assessments consisted of both body mass index (BMI: weight / height2) and serum proteins (total serum proteins, albumin, vitamin B12, folic acid and ferritin). These parameters have been used previously as markers of nutritional status in gerontological studies (Nikolaus et al., 1995), as well as in previous work exploring the effects of stress on the immune system (Bauer et al., 2000). Albumin was also measure here as a major serum transport protein for DHEA (85% is bound to albumin) (Ganong, 1991) and alterations ascribed to this carrier may thus change the active free levels occurred in the tis-sue. Serum vitamin B12 and folic acid were measured by electroquimioluminescence kits (Elecsys 2010, Roche). Ferritin was assessed by quimioluminescence kits (Immulite I, Diag-nostic Products Corporation Medlab, São Paulo, Brazil). Albumin was measured by the stan-dard enzymatic method of Biuret (540 nm) in combination with staining procedures (kit GT-Labs., Buenos Aires, Argentina). The measurement of these variables allowed us to examine the extent to which any observed immune impairment could be explained by these factors.

2.5. Collection of salivary samples and measurements of DHEA

The assessment of steroids in saliva has proven to be a valid and reliable reflection of the unbound hormone in the blood (Walker et al., 1978), with salivary cortisol and DHEA concentrations reflecting 5-10% of the levels present in serum (Kahn et al., 1988; Bauer et al., 2003). Participants were asked to collect three saliva samples with the help of cotton rolls over the course of the experimental day at 9, 12 and 20 h, always before meals and venepunc-ture. Sampling was performed across the day to assess some aspects of circadian pattern. Upon arrival in the laboratory, the samples were centrifuged and frozen at -20?C. Salivary DHEA was analyzed by a modified RIA (DPC Medlab) accordingly to previous work (Granger et al., 1999). The sensitivity of this assay was estimated in 0.031 nmol/L. The intra- and inter-assay coefficients of variation were less than 10%. Results from each of the sam-pling times were expressed in nmol/L. In addition, integrated hormonal levels over the sam-pling time frame (i.e. 9am to 10pm) were estimated using the trapezoidal rule to calculate the area under the curve (AUC) and were expressed as nmol per liter per hour.

2.6. Serum DHEA

Aliquots of peripheral blood were also collected without anticoagulant in order to measuring serum DHEA. Hormone levels were assessed by a radioimmunoassay kit (Diag-nostic Systems Laboratories, Webster, TX, USA). The sensitivity of these assays was esti-mated in 0.031 nmol/L. The intra- and inter-assay coefficients of variation were less than 10%. Results from each of the sampling times were expressed in pg/ml.

2.7. Collection of peripheral blood and isolation of mononuclear cells

Twenty millilitres of peripheral blood was collected by venepuncture in the morning (between 9–10 h) and samples stored into lithium-heparin tubes prior to analyses. Samples were always collected at the same time of day to minimize circadian variations. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over a Ficoll-Hypaque (Sigma) gradient (900 g, 30 min). Cells were counted by means of microscopy (100 x) and viability always exceeded 95%, as judged from their ability to exclude trypan blue (Sigma).

2.8. Steroid sensitivity assays

PBMCs were cultured in a final concentration of 1.5x105 cells/well in complete cul-ture medium (RPMI-1640, supplemented with gentamicin 0.5%, glutamine 1%, hepes 1%, and fetal calf serum 10%; all from Sigma) for 96 h at 37?C in 5% CO2 atmosphere. Stimula-tion by PHA (Gibco) was performed in triplicate (100 ?l/well) to yield the following final concentrations: 2, 1 and 0.5%. To assess in vitro GC sensitivity, 10-9 to 10-4 M of dexa-methasone (selective type II adrenal receptor agonist) and cortisol (which binds to both types of adrenal receptors) were added in duplicates (50 ?l/well; both water-soluble substances pur-chased from Sigma) to mitogen-stimulated lymphocyte cultures. Glucocorticoid concentra-tions were used in a range that free GCs would reach during resting state (10 8 M), stress (10 6 M), or under pharmacological treatment (10 5 M) in vivo (Kirschbaum et al., 1995; Wilckens and Rijk, 1997). Only two optimal DHEA concentrations (10-6 and 10-5 M) were used because of unresponsiveness of lower concentrations and toxicity of higher doses (i.e. > 10-5 M). One optimum PHA concentration (1%) was used for all steroid cultures. Fifty micro-litres of culture medium were added to all stimulated cultures. In spontaneous (without lectin) cultures, mitogen was substituted by 150 ?l of culture medium. Data are presented as percent-age of basal proliferation (0; PHA 1% without steroids).

2.9. Cell proliferation/viability assay

The proliferative responses were determined by a modified colorimetric assay (Mosmann, 1983). In the last 4 h of culture, 100 ?l of the supernatant was gently discarded and 30 ?l of freshly prepared MTT (3-(4,5-diamethyl 2-thiazolyl) 2,5 diphenyl-2H-tetrazolium, Sigma) solution (5 mg/ml in RPMI-1640) was added to each well. The dehydro-genase enzymes in metabolically active cells convert this substrate to formazan, producing a dark blue precipitate. The cell cultures were incubated for 4 h at 37?C in 5% CO2 atmosphere. After completely removal of the supernatant, 100 ?l of dimethyl sulfoxide (Sigma) was added to each well. The optical density (OD) was determined using Biorad ELISA plate reader at a wavelength of 570 and 630 nm. Proliferation/viability was expressed as OD of stimulated – OD of nonstimulated cultures.

2.10. Statistical analysis

All variables were tested for normality of distribution by means of the Kolmogorov-Smirnov test. The salivary DHEA levels were log-transformed to correct for skewed distribu-tions. Proliferation and salivary DHEA data were analyzed by repeated measures ANOVA that included one between-subjects variable (elderly versus young) and one within-subjects variable (DHEA, mitogen or steroid levels). Multiple comparisons among levels were checked with Tukey post hoc test. Differences between demographic and nutritional variables were assessed by Student’s t test and differences in proportions between groups were tested by means of ?2 test. Data are expressed as mean ? SE in all figures and tables. A statistical software (SPSS 11.0, USA) was used for the statistical analyses.

3. Results

3.1. Demographic data and nutritional analyses

Independent community-living elderly and young adults took part in this study. All subjects were recruited accordingly to the SENIEUR protocol (Ligthart et al., 1984) that de-fines rigorous criteria for selecting healthy individuals. Both demographic and nutritional data are presented in Table 1. Most elderly (95.6%) and young subjects (81.0%) were Caucasian and the female/male ratio did not differ between groups, ?2 = 1.35, p = 0.25. In addition, there were no differences regarding smoking habits between groups, ?2 = 5.51, p = 0.14.

In order to ensure that only strictly healthy individuals were selected, the nutritional status was also investigated in this study. The elderly subjects had an elevated body mass in-dex (27.18 ? 0.84) compared to young adults (22.89 ? 0.63), p = 0.0001. No statistically sig-nificant differences were noted for the total serum proteins, vitamin 12 or albumin levels be-tween elders and young individuals (Table 1). In contrast, elders had significant higher folic acid and ferritin levels compared to young adults. Nevertheless, both variables were found within the normal reference range (folic acid: 3-17 ng/ml and ferritin: 9-370 ng/ml).

BMI = body mass index. TSP = total serum proteins. NS = non-significant. Data expressed as mean ± SE.

3.2. Endocrine assessments

In this study we assessed HPA axis function by means of reliably collection of multi-ple salivary samples. Salivary (free) DHEA levels differed significantly across the day (Fig. 1A), F(2,128) = 3.68, p < 0.05. Elders had significantly lower salivary DHEA levels com-pared to young adults, F(1,64) = 31.91, p < 0.0001. Accordingly, we observed that elders pre-sented significantly lower AUC DHEA levels compared to young adults (3.75 ± 0.40 nmol/L/h vs. 8.15 ? 0.86 nmol/L/h, respectively), t = 5.28, df = 67, p < 0.0001. In addition, there was a significant interaction between DHEA vs. group, F(2,128) = 12.02, p < 0.0001. Post hoc analyses revealed that only the young adults displayed a regular circadian pattern, with peak DHEA levels in the morning and lower levels in the evening (p < 0.05). In contrast, the elderly subjects presented a flat circadian pattern. In addition, morning serum (total) DHEA levels were found significantly reduced in the elderly compared to young subjects (Fig. 1B), t = 5.63, df = 70, p < 0.0001. Although young men presented significantly higher serum DHEA levels than young women (25.01 ± 2.04 vs. 16.71 ± 2.58 nmol/L, p < 0.05), there were no gender-related differences in the elderly group.
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Fig. 1. Human ageing is associated with reduced DHEA levels. Both salivary (A) or serum (B) DHEA levels were detected by RIAs. The mean (± SE) levels of DHEA at each time point are shown. Serum DHEA was only measured at 9am. Statistically significant differences are indicated: *** p < 0.001.

3.3. Lymphocyte proliferation and sensitivity to steroids

Mitogen-induced T cell proliferation was evaluated as an index of non-specific cell-mediated immunity. Basal proliferation/viability was found slightly increased in elderly (OD = 0.20 ± 0.007) compared to young subjects (OD = 0.17 ± 0.10), t = 2.28, df = 50.41, p = 0.03. PHA-induced proliferative responses differed significantly across the three mitogen concentrations, F(2,134) = 7.57, p = 0.001. Repeated measures ANOVA revealed that elderly had significantly lower PHA proliferation than young subjects, F(1,67) = 25.14, p < 0.0001 (see Fig. 2). There was not a statistically significant interaction between group (elderly vs. young) and mitogen concentration, F(2,134) = 0.18, p = 0.83.

Fig. 2. Healthy ageing is associated with a reduced T-cell proliferation. PBMCs were stimu-lated with phytohemagglutinin (PHA) for 96h and proliferation estimated by colorimetric as-say. Results are shown as OD stimulated – OD nonstimulated cultures. Statistical significance differences are indicated: *** p < 0.001.

In view of evidence that, during chronic GC exposure, lymphocytes appear to become resistant to the immunosuppressive effects of steroids (Rupprecht et al., 1991; Wodarz et al., 1991; Bauer et al., 2000) we examined lymphocyte sensitivity to steroids in both elderly and young adults. This was explored by incubating PHA-stimulated peripheral lymphocytes with GCs. Both DEX and cortisol produced a significant dose-dependent suppression of T-cell proliferation, F(5,360) = 127.43, p < 0.0001 and F(5,330) = 56.56, p < 0.0001, respectively. These data highlight the efficacy of these steroids in suppressing in vitro proliferation. Inter-estingly, we observed that lymphocytes from elders were less sensitive to in vitro GC treat-ment compared with young subjects (see Fig. 3A). In particular, it was observed that cells from elders required higher DEX concentrations to suppress PHA proliferation to the same extent as cells from young subjects, F(1,72) = 8.19, p = 0.006. Although cortisol treatment produced a similar effect (Fig. 3B), it did not reach statistical significance, F(1,66) = 1.64, p = 0.20.

In addition to GCs, we also evaluated the lymphocyte sensitivity to DHEA (Fig. 3C). DHEA produced a significant dose-dependent suppression of T-cell proliferation, F(1,72) = 90.78, p < 0.0001. However, lymphocytes from young adults and elders presented similar sensitivities to DHEA treatment in vitro, F(1,72) = 0.11, p = 0.74.

Fig. 3. Ageing is associated with reduced lymphocyte sensitivity to glucocorticoids in vitro. Glucocorticoid sensitivity was assessed by incubating peripheral blood mononuclear cells with phytohemagglutinin (1%) and increasing concentrations of dexamethasone, cortisol and DHEA. After 96-h of incubation, proliferation was estimated by colorimetric assay. Data pre-sented as percentage of basal proliferation (0 = PHA 1% without steroids). ** p < 0.01 and * p < 0.05 (Tukey).


4. Discussion

Ageing is associated with several immune-related diseases including neoplasias, auto-immune and infectious diseases. However, this altered morbidity is not evenly distributed and should be thus influenced by other immune-modulating factors. It has repeatedly been shown that there is a bidirectional communication between the nervous system, endocrine and im-mune system (Blalock, 1989). In this study we explored the role of endocrine factors in regu-lating cell-mediated immunity in the elderly. To control for age-related diseases that would interfere with our immunological analyses, strictly healthy individuals were recruited by means of the SENIEUR protocol (Ligthart et al., 1984). However, we observed that SENIEUR elderly subjects had elevated BMI compared to young adults. Changes in BMI could be related to both obesity and lack of regular exercise that have been implicated to im-munological changes (Pedersen and Toft, 2000; Lamas et al., 2002). However, our elderly cohort did not seem to have nutritional changes as they reported no changes in serum vitamin B12 levels and significantly higher ferritin and folic acid levels compared to young adults.

We observed that healthy elderly subjects had reduced serum and salivary DHEA lev-els compared to young adults. These data are in agreement with previous studies (Migeon et al., 1957; Candore et al., 1992; Deuschle et al., 1997; Luz et al., 2003). Most studies have previously undertaken misleading one time point evaluation of peripheral hormones. In this study, multiple daily salivary sampling lead us to describe that elders presented a flat cir-cadian pattern for salivary DHEA production. Healthy elderly subjects had also increased cortisol/DHEA ratios compared to young adults. Indeed, we have recently observed that strictly healthy ageing was associated with significant psychological distress in parallel with increased salivary cortisol production (Luz et al., 2003). Taken together these data suggest that healthy ageing is associated with significant neuroendocrine changes. The cortisol/DHEA ratio may be more informative than isolated values of cortisol and DHEA and it may indicate that high cortisol and low DHEA levels in ageing are contributory to a toxic action in the brain. DHEA is an ACTH-regulated steroid that possesses anti-glucocorticoid properties (Daynes et al., 1990; Canning et al., 2000). The antagonist action of DHEA to cortisol in the brain suggests that measurement of cortisol alone may provide an incomplete estimate of hy-percortisolemia.

In accordance to previous studies (Murasko et al., 1987; Blauer et al., 1991; Liu et al., 1997; Castle et al., 1999; Douziech et al., 2002; Schindowski et al., 2002), we observed that healthy elders had significant lower (-53.6%) T-cell proliferation. Blunted lymphocyte prolif-eration is indeed one of the most documented age-related change observed across different species and may thus possible contribute to a poor in vivo immune response against new anti-gens. This specific change may be related to several underlying mechanisms, including: thymic involution, cytokine changes (i.e. lower IL-2 and higher IL-10 production), alterations in cell trafficking, decreased replicative senescence and accessory cell function (Pawelec et al., 2002). In addition, elders had a minor but statistically significant increase of unstimulated PBMC viability/proliferation. This change could be attributable to monocytes as these cells remain viable in culture. However, no changes in peripheral counts of monocytes or lympho-cytes were noted in this cohort (Collaziol et al., 2004).
In this study, we also investigated the lymphocyte sensitivity to both synthetic (DEX) and natural occurring steroids (cortisol and DHEA) and so examined whether ageing was as-sociated with alterations in neuroendocrine-immunoregulation. We reported that strictly healthy (SENIEUR) elders had a reduced in vitro lymphocyte sensitivity to DEX (but not cor-tisol or DHEA) when compared to young adults. These data indicate that during ageing, lym-phocytes appear to be less sensitive to DEX. Furthermore, this age-related change in steroid sensitivity seems to be type II adrenal receptor-specific since cells from elders and young adults showed a comparable lymphocyte sensitivity to cortisol (which binds to both type I and II receptors). There is considerable evidence for a shift in lymphocyte sensitivity to GCs dur-ing the ontogeny. For instance, peripheral T-cells of infants younger than 12 months have been reported to be highly sensitive to DEX treatment in vitro (Kavelaars et al., 1996). After this period the lymphocyte sensitivity to steroids is gradually decreasing, reaching the adult levels at one year of life. A reduced sensitivity to GCs can also be observed at the central level during ageing. Indeed, higher cortisol levels in old than in young subjects have been described during some pharmacological challenges, such as the DEX suppression test, the stimulation by human or ovine corticotrophin-releasing hormone or by physostigmine (Raskind et al., 1994; Ferrari et al., 2001). Finally, it has been recently shown that ageing is associated with changes in GC sensitivity of pro-inflammatory cytokine (TNF-? and IL-6) production following psychosocial stress (Rohleder et al., 2002). In particular, monocytes of healthy (but non-SENIEUR) elderly men had a higher sensitivity to DEX treatment in vitro at baseline and showed a reduced sensitivity to this steroid following acute stress exposure (speech coupled to mental arithmetic task). These data suggest that psychological factors may be implicated in regulating peripheral GC sensitivity during healthy ageing.

We have previously reported that chronic stress renders lymphocytes of healthy eld-erly more resistant to GC treatment in vitro compared to non-stressed subjects (Bauer et al., 2000). These data support the notion that chronic stress would thus worsen a trait phenome-non (marker) of the healthy ageing. The mechanisms underlying acquired steroid resistance are poorly understood. Based on our previous observations (Luz et al., 2003) we suggest that higher cortisol levels would render lymphocytes to be less sensitive to the effects of GCs in vitro. Indeed, there is some evidence in the literature suggesting that changes in GC sensitiv-ity could be the result of chronic GC treatment (Kloet, 1991; Chiappelli et al., 1994). Reduced intracellular GC receptors may account for the putative underlying mechanisms of age-related GC resistance (Risdon et al., 1991; Zovato et al., 1996; Grasso et al., 1997) but changes in GC receptor affinity cannot be ruled out. Glucocorticoid-induced acquired resistance may have an important physiological significance of protecting cells from the dangerous effects of pro-longed GC-related immunosuppression. Additionally, altered steroid immunoregulation may have important therapeutic implications in clinical situations where GCs are administered, including treatment of autoimmune diseases, organ transplantation, and allergies.

Overall, our data suggest that ageing is associated with changes in neuroendocrine-immunoregulation. We particularly demonstrated that blunted lymphocyte responses were associated with altered lymphocyte sensitivity to a synthetic GC. However, the underlying mechanisms of acquired steroid resistance require further investigation. In addition, we do not know yet whether this altered GC sensitivity is associated with resistance to other medications and we are currently investigating this possibility.

Acknowledgements
The authors would like to acknowledge the excellent technical assistance of Ingrid Manfredi (Office for Social Care, Gravataí). We are grateful to the city hall of Gravataí for setting up special facilities for the recruitment of the elderly subjects. We thank Dr Sidia Marques (Department of Genetics, UFRGS, Porto Alegre, Brazil) and Dr Luiz Glock (PUCRS) for statistical assistance. This study was supported by grants from FAPERGS (00/0168.9, M.E.B.) and CNPq (551180/01-3, M.E.B.).

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