The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright © 1999 by The Endocrine Society Dehydroepiandrosterone Sulfate Enhances Natural Killer Cell Cytotoxicity in Humans Via Locally Generated Immunoreactive Insulin-Like Growth Fact

Experimental and clinical investigations suggest the hypothesis that dehydroepiandrosterone sulfate (DHEAS) can positively influence natural killer (NK) immunity via locally produced insulin-like growth factor I (IGF-I) from NK cells. In the present study, the NK cell cytotoxicity (NKCC) and IGF-I levels in the supernatant of NK cells were studied at baseline and after exposure to various molar concentrations of DHEAS (from 10(-5)-10(-8) mol/L x mL/7.75 x 10(6) NK cells) in healthy subjects of young and old age. DHEAS-induced NKCC was also determined after DHEAS coincubation with somatostatin-14 (10-6) mol/L x mL/7.75 x 10(6) NK cells) and with interleukin-2 (IL-2; 100 IU/mL x 7.75 x 10(6) NK cells). NK cells were previously isolated by Ficoll-Hypaque density gradient and then by immunomagnetic procedure; the purity obtained was 97 +/- 1%. NKCC was determined against K562 tumoral targets. We observed that the increase in NKCC after DHEAS exposure was dose dependent and was correlated with the amount of IGF-I released in the supernatant of cultured NK cells. NKCC and IGF-I generation from NK cells were more elevated in healthy elder subjects than in healthy young subjects. The coincubation of DHEAS with somatostatin-14 significantly suppressed NKCC and IGF-I release from NK in both groups, whereas higher NKCC was found after DHEAS plus IL-2 exposure than after incubation with DHEAS alone. Taken together, this study suggests a role for NK-generated IGF-I in the modulation of NKCC by DHEAS in humans. Although DHEAS may contribute to the IL-2-mediated NKCC, its activity on NK cytolytic function can be dependent on a autocrine mechanism (IGF-I-mediated), probably independent of cytokine activation. The higher NKCC response to DHEAS found in old subjects than in younger might counterbalance the age-dependent decline in circulating DHEAS, thus contributing to maintain the pattern of NK immunity during aging.

NK larger granular cells are a distinct subpopulation of lymphocytes involved in nonmajor histocompatibility complex-restricted cytotoxic activity against tumoral and viral targets.The cytotoxic function of NK cells is independent of prior sensitization and Ig action, and is mainly regulated by an autocrine mechanism related to cytokines [e.g.interleukin-2 (IL-2), interferon-␥, and interferon-␤] and by a protein kinase C (PKC)-dependent mobilization of proteolytic granules.
The functional mechanism that may link DHEAS with NK cytotoxic function is still not defined.However, it has been suggested that DHEAS might promote NK cytolytic function by increasing the synthesis and secretion of bioactive immunoreactive insulin-like growth factor I (IGF-I) from NK cells (24,25) and enhancing the bioavailability of IGF-I (26).In fact, IGF-I can positively regulate spontaneous and cytokine-stimulated NK cytotoxicity (27), whereas type I IGF receptors may be functional on this cell type (28).
To gain insight into the role of DHEAS in IGF-I-mediated regulation of NK cell cytotoxicity (NKCC), the release of IGF-I from NK cells was studied after incubation with various molar concentrations of DHEAS (from 10 Ϫ5 -10 Ϫ8 mol/ L⅐mL/7.75 ϫ 10 6 NK cells) in healthy subjects of young and old age.DHEAS-induced NKCC was also measured after coincubation of DHEAS with somatostatin-14 (SST; 10 Ϫ6 mol/L⅐mL/7.75 ϫ 10 6 NK cells) and after coincubation of SST with IL-2 (100 IU/mL⅐7.75 ϫ 10 6 NK cells).
In this report we show that the enhancement of NKCC is dose dependent after DHEAS exposure and is correlated with the amount of IGF-I found in the supernatant of cultured NK cells.DHEAS-induced release of IGF-I from NK is also more pronounced in healthy old compared to healthy young subjects and is associated with an increased response of NKCC to DHEAS.The coincubation of DHEAS with SST inhibits NKCC and suppresses IGF-I release from NK cells in both groups, whereas higher NKCC was demonstrated after coincubation of DHEAS with IL-2.These findings suggest that the immune regulatory function of NK during DHEAS exposure involves the generation and the release of IGF-I from these cells.Although DHEAS may potentiate IL-2-mediated activation of NKCC, the molecular mechanism related to DHEAS/IGF-I modulation of NKCC might be independent of cytokine activation.

Subjects and Methods Subjects
Nineteen healthy young subjects (10 women and 9 men; mean Ϯ sd age, 33 Ϯ 5.3 yr) and 16 healthy elderly subjects (8 women and 8 men; 75.3 Ϯ 4.4 yr) were included in the study.Healthy young subjects were matched for body weight with healthy old subjects (body mass index, 22.3 Ϯ 1.4 kg/m 2 in healthy young subjects and 21.9 Ϯ 1.3 kg/m 2 in healthy elderly subjects).Healthy elderly subjects were carefully selected on the basis of the SENIEUR protocol (29) to avoid the presence of clinical and immunological alterations.Cognitive disorders in old people were excluded by using the criteria of the Diagnostic and Statistical Manual of Mental Disorders (30), and the healthy status of old subjects was supported by physical and neurological examinations and laboratory evaluations (including a complete biochemical assessment of nutritional status).All subjects were free of alcohol and drug abuse known to affect immunological function.
The study was conducted in accordance with the Declaration of Helsinki and was approved by the ethical committee of the Department of Internal Medicine of the School of Medicine of the University of Pavia.Written informed consent was obtained from all subjects.
Biochemical and metabolic parameters and DHEAS serum concentrations were determined at 0800 h after an overnight fast.DHEAS was also evaluated at 2000 and 0400 h.

Biochemical and hormonal study
DHEAS was determined in serum by a specific RIA (Coat-A-Count DHEA-SO 4 , Diagnostic Products, Los Angeles, CA); the results were expressed as micromoles per L. The intra-and interassay coefficients of variation were always below 8%, and the lowest detectable dose of DHEAS was 0.029 mol/L.Metabolic parameters (blood glucose, cholesterol, and triglycerides) and blood count (hematocrit, hemoglobin, and total lymphocytes) were evaluated by automatic procedures (Dasit Ise-Autoanalyzer and Sysmex Toa F800 Microcell Counter, Dasit, Bareggio, Italy).Circulating serum proteins (prealbumin, albumin, transferrin, and retinol-binding protein) were measured by immunonephelometry (BNA, Nephelometer, Behringwerke AG, Marburg, Germany).

Immunological procedures
Immunological study was performed in a fully sterile manner using a biological safety cabinet class II (Microflow 51426, MDH Ltd., Andover, UK).Complete medium containing RPMI 1640 medium (Hy-Clone Laboratories, Inc., Logan, UT) enriched with 10% inactivated FBS (HyClone Laboratories, Inc.), 1% glutamine (HyClone Laboratories, Inc.), and 50 g/mL/gentamicin (Irvine Scientific, Santa Ana, CA) was used routinely for all cultures and cytotoxicity assays.Inactivation of FBS was performed by treatment with dithiothreitol using a procedure that eliminates all detectable IGF-I and IGF-II (31).
The human myeloid cell line K562 was the source of sensitive targets for measurements of NK cytotoxicity (32,33).The cell line K562 was maintained in our laboratory in suspension culture flasks at 37 C in 5% CO 2 incubator (BB 6220, Heraeus, Hanau, Germany).All target cells used were more than 90% viable, as measured by trypan blue dye exclusion (0.4% trypan blue solution; Sigma Chimica, Milan, Italy).
Peripheral blood mononuclear cells (PBMC) were obtained from heparinized venous blood samples (Vacutainer, lithium heparin, Becton Dickinson and Co., Meylan, France) derived from elderly controls and patients with SDAT while fasting for 12 h before venipuncture.PBMC cells were immediately separated by Ficoll-Hypaque density centrifugation (34) (Lympholyte-H, Cedarlane Laboratories Ltd., Hornby, Canada).Plastic-adherent cells were removed by incubation at 37 C in petri dishes for 1 h.The remaining nonadherent cell population was passed through nylon wool columns preincubated for 1 h with RPMI 1640 supplemented with 10% heat-inactivated autologous serum at 37 C (5% CO 2 in air).T/NK cells were obtained by rinsing the columns with tissue culture medium, which leaves B cells and remaining monocytes attached to the nylon wool (35).The enriched fraction of PBMC containing T/NK cells was used for the separations in the magnetic field.The procedure of separating NK cells was carried out under sterile conditions.For the immunomagnetic separation we used the magnetic cell separation (MACS) system and the NK cells isolation kit for negative enrichment (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany).Washed PBMC were resuspended in 80 L buffer/10 7 total cells containing phosphatebuffered saline and supplemented with 0.5% BSA.PBMC were first incubated for 15 min at 6 C with 20 L reagent consisting of modified CD3, CD4, CD19, and CD33 antibodies of mouse IgG1 isotype to label non-NK cells.Thereafter, the cells were washed once in phosphatebuffered saline and incubated for 15 min at 6 C with 20 L colloidal superparamagnetic MACS microbeads recognizing non-NK cells.Labeled and unlabeled cells were separated in a high gradient magnetic field generated in a steel wool matrix inserted into the field of a permanent magnet (36,37).The columns were sterilized in an autoclave at 120 C shortly before use.The negative unlabeled cells, representing the enriched nonmagnetic NK cell fraction, were eluted from the separation column outside the magnetic field in a laminar flow to ensure appropriate asepsis.The efficiency of separation was evaluated by flow cytometry, using a FACScan (Becton Dickinson and Co., Mountain View, CA).The sample obtained from the negative fraction was stained with fluorescein isothiocyanate-conjugated NK cells antibodies (CD56 ϩ , CD16 ϩ ) and counted for total NK cell number.Anti-Leu 11b (anti-CD16) and anti-Leu 19 (anti-CD56) were purchased from Becton Dickinson and Co..The MACS procedure allowed us to separate the negative NK cell population in approximately 2 h with yields of more than 95% and a purity of 97 Ϯ 1% for CD16 ϩ CD56 ϩ NK cells.The viability of the NK subpopulation was determined by trypan blue uptake (0.4% trypan blue solution; Sigma Chimica) before the cytotoxicity assay against K562 cells.All NK cells used were more than 95% viable in healthy subjects of young and old age, without differences between the two groups.
After the magnetic separation, NK cells were washed three times (with 0.9% saline and complete RPMI medium) and finally resuspended to a measured (Sysmex Toa F800 Microcell Counter, Dasit) density of 7.75 ϫ 10 6 cells/mL complete medium.NK effector cells were incubated for 20 h (38, 39) at 37 C in a humidified atmosphere of 95% air and 5% CO 2 with DHEAS, DHEAS plus SST, SST plus IL-2, SST, and IL-2 and without the use of modulators (to measure the spontaneous basal cytotoxicity).DHEAS (Sigma Chimica) was diluted in complete fresh medium (in a 0.1-mL final volume) and used at final concentrations of 10 Ϫ8 , 10 Ϫ7 , 10 Ϫ6 , and 10 Ϫ5 mol/L⅐mL/7.75 ϫ 10 6 NK cells.DHEAS (at all molar concentrations) was also coincubated with SST (Sigma Chemical Co., St. Louis, MO) at concentration of 10 Ϫ6 mol/L⅐mL/7.75 ϫ 10 6 NK cells and with IL-2 (recombinant human IL-2, Proleukin, Chiron Corp., Emeryville, CA) at a concentration of 100 IU/mL⅐7.75 ϫ 10 6 NK cells.Finally, SST (10 Ϫ6 mol/L⅐mL/7.75 ϫ 10 6 NK cells) was coincubated with IL-2 (100 IU/mL⅐7.75 ϫ 10 6 NK cells).To verify the possibility that SST may be toxic for NK cells, a survivability test for these cells was also performed after SST exposure.The survivability of NK cells was more than 95% after incubation with 10 Ϫ6 mol/L⅐mL SST; the viability of NK cells also remained more than 95% after 20-h exposure to SST at 10 Ϫ7 and 10 Ϫ5 mol/L⅐mL/7.75 ϫ 10 6 cells.
After incubation, the NK cells were washed twice with 0.9% saline and then once with complete medium containing modified medium 199 and 5% fraction V bovine albumin (Sigma Chemical Co.).
After washing three times with 9% saline and complete medium (medium 199 and 5% albumin, fraction V), 3 ϫ 10 4 target cells in 0.1 mL complete medium were mixed in triplicate with various concentrations of NK effector cells in the wells of a round-bottom, 96-hole, standard microtiter plate (TPP, Celbio, Pero-Milan, Italy), at a final total volume of 0.2 mL.These mixtures gave final effector/target cell ratios of 25:1, 12.5:1, 6.25:1, and 3.125:1.After a second incubation for 4 h at 37 C in a 5% CO 2 atmosphere, the microtiter plate was centrifuged, and a fixed aliquot (0.1 mL) of supernatant was extracted from each well and transferred to the corresponding wells of a flat-bottom microtiter plate.The cytotoxicity assay of NK cells was based on kinetic measurement by a computer-assisted (Milenia Kinetic Analyzer, Diagnostic Products) microtiter plate reader of the amount of the lactate dehydrogenase released in the supernatant of target cells, according to the calculation of Korzeniewski and Callewaert (40).Subsequently, 0.1 mL lactic acid dehydrogenase substrate mixture (41) was added to each well at intervals of 3 s.
Data for NK activity of effector cells incubated with modifiers were expressed as lytic units (LU) per 10 7 cells (41) and as the percent increase and decrease in specific lysis.The reproducibility of the cytotoxicity assay was evaluated in triplicate measurements and was always below 2%.

IGF-I assay
The supernatant fluids of NK cells (final measured density of 7.75 ϫ 10 6 cells/mL) were lyophilized and then resuspended in 300 L sterile water.Concentrated fluids were analyzed in triplicate for their IGF-I content (RIA procedure) using a commercial kit available from Nichols Institute Diagnostics (San Juan Capistrano, CA).The RIA kit includes solvents and buffer solution for acid-ethanol extraction.This technique involves the separation of soluble IGF-I from binding proteins that are precipitated with 87.5% ethanol and 12.5% 2 n HCl; after incubation (at room temperature for 30 min), the supernatant was neutralized with Tris-base solution (42).The recovery of IGF-I in the RIA after extraction was greater than 90%.The antiserum for IGF-I has less than 0.5% crossreactivity with IGF-II, human GH, TSH, insulin, proinsulin, transforming growth factor, and fibroblast-derived growth factor.The sensitivity of the method was 0.03 ng/mL.The intraassay variation was below 3%, and the interassay variation was between 5-7%.

Statistical analysis
Parametric unpaired Student's t test was employed to evaluate differences in DHEAS concentrations between healthy subjects and patients with SDAT.One-way ANOVA (F test) was employed to measure differences in clinical, metabolic, hematological, and nutritional parameters among healthy young and healthy old subjects.Parametric paired and unpaired Student's t test and ANOVA were used to evaluate differences in cytotoxicity recorded under basal conditions (spontaneous NKCC) and after incubation with DHEAS, DHEAS plus SST-14, SST-14 plus IL-2, IL-2 plus DHEAS, and IL-2.These differences were also evaluated between healthy young and old subjects.Parametric Pearson's correlation coefficient was employed to analyze correlations between the amount of IGF-I released in the supernatants of NK cells (after DHEAS) and NKCC.P Ͻ 0.05 was considered significant.All analyses were run using SPSS, Inc./PCϩ version 3.0 statistical package (SPSS, Inc., Chicago, IL).

Biochemical and hormonal parameters (Table 1)
Table 1 summarizes the clinical and laboratory features of healthy young and old subjects.Hematological, metabolic, and nutritional parameters were similar in the two groups of subjects.On the contrary, lower circulating DHEAS levels (at 0800, 2000, and 0400 h) were found in healthy elderly than in healthy young subjects (P Ͻ 0.001).

NKCC after DHEAS (Figs. 1a and 2a and Table 2)
The mean variations in NKCC, expressed as the percent increase in cytotoxicity and as the lytic response (LU ϫ 10 7 NK cells) after DHEAS incubation (from 10 Ϫ8 -10 Ϫ5 mol/ L⅐mL/7.75 ϫ 10 6 NK cells) are shown in Figs.1a and 2a and Table 2.The mean levels of NKCC after DHEAS treatment were significantly increased (P Ͻ 0.001, by ANOVA) at all molar concentrations of DHEAS (from 10 Ϫ8 -10 Ϫ5 mol/L⅐mL/ 7.75 ϫ 10 6 NK cells) compared to spontaneous NKCC (no addition of DHEAS; Fig. 2a).The mean increase in NKCC ranged from 39 -149% in healthy young subjects and from 58 -174% in healthy elder subjects (Fig. 1a).Significant differences of NKCC (from P Ͻ 0.05 to P Ͻ 0.01), evaluated as the lytic response (Fig. 2a) and as the percent increase in NKCC (Fig. 1a), were also demonstrated between healthy young and healthy old subjects.

IGF-I after DHEAS (Fig. 3a and Table 2)
The supernatant fluids from NK cells were analyzed by RIA for immunoreactive IGF-I (irIGF-I).The measurements of irIGF-I were performed in spontaneous conditions (no addition of DHEAS) and after 24 h of DHEAS exposure (from 10 Ϫ8 -10 Ϫ5 mol/L/mL⅐7.75 ϫ 10 6 NK cells; Fig. 3a and Table 2).The supernatant fluids contained at baseline 1.3 ng/L irIGF-I ϫ 7.75 ϫ 10 6 NK cells.This concentration is approximately similar to the amount of IGF-I produced by leukocytes (43) and is 60 times less than the IGF-I produced by hepatocytes (44).No differences in irIGF-I derived from NK cells at baseline were found between healthy young and old subjects.A significant increase in irIGF-I concentrations from cultured NK cells was demonstrated after exposure to DHEAS (P Ͻ 0.001, by ANOVA).The mean increase in irIGF-I ranged from 2.3-16.7 ng/L⅐7.75ϫ 10 6 NK cells in healthy young subjects and from 4.5-21.4ng/L⅐7.75ϫ 10 6 NK cells in healthy old subjects.Significant differences in irIGF-I levels after DHEAS (P Ͻ 0.05) were also found between these two groups.

NKCC and IGF-I after DHEAS plus SST (Figs. 1b, 2b, and 3b and Table 2)
Changes in NKCC, measured as the percent increase in cytotoxicity and as the lytic response (LU ϫ 10 7 NK cells), after DHEAS coincubation (from 10 Ϫ8 -10 Ϫ5 mol/L⅐mL/  and 2a).The mean increase in NKCC after DHEAS plus SST ranged from 12-39% in healthy young subjects and from 15-44% in healthy old subjects, without differences between the two groups (Fig. 1b).The loss of NKCC response during DHEAS plus SST was approximately 3-4 times less than that of NKCC response during DHEAS alone (Figs. 1a  and 2a).No variations in NKCC from baseline were found after exposure to SST without DHEAS (Fig. 4).The supernatant fluids from NK cells were analyzed for irIGF-I in spontaneous conditions (no addition of DHEAS plus SST) and after 24-h incubation with SST (10 Ϫ6 mol/L⅐mL) and with DHEAS plus SST (Fig. 3b).The exposure of NK cells to SST and SST plus DHEAS significantly suppressed irIGF-I production by NK cells (P Ͻ 0.001 from baseline); irIGF-I release from NK was, therefore, completely suppressed compared with the irIGF-I concentrations found in the supernatant fluids of NK cells after incubation with DHEAS alone (Fig. 3a).

Correlations between NKCC after DHEAS and irIGF-I
The increase in NKCC after DHEAS exposure was significantly correlated with irIGF-I concentrations measured in the supernatant fluids of NK cells.In fact, several positive correlations between the percent increase in NKCC after DHEAS exposure and irIGF-I levels were found in healthy young subjects (r ϭ 0.51; P Ͻ 0.05 at 10 Ϫ8 mol/L; r ϭ 0.59; P Ͻ 0.01 at 10 Ϫ7 mol/L; r ϭ 0.61; P Ͻ 0.01 at 10 Ϫ6 mol/L; r ϭ 0.64; P Ͻ 0.01 at 10 Ϫ5 mol/L) as well as in healthy elderly subjects (r ϭ 0.55; P Ͻ 0.05 at 10 Ϫ8 mol/L; r ϭ 0.57; P Ͻ 0.05 at 10 Ϫ7 mol/L; r ϭ 0.63; P Ͻ 0.01 at 10 Ϫ6 mol/L; r ϭ 0.65; P Ͻ 0.01 at 10 Ϫ5 mol/L).These correlations fail to be significant when DHEAS was coincubated with SST (data not reported).

Discussion
In the present study we have demonstrated that DHEAS can positively modulate the immune function of NK cells by increasing, in a dose-dependent manner, the NKCC against tumoral target K562.This effect was observed in healthy young and old subjects even if, in agreement with our preliminary study (19), the NKCC response to DHEAS was higher in healthy old than in healthy young subjects.This evidence may be of a certain interest, because, as first re-ported by Orentreich et al. (45), circulating DHEAS levels were significantly reduced during aging.In fact, although the age-dependent decline of DHEAS is a common feature of aging, the demonstration of a more pronounced NKCC after DHEAS exposure might contribute to maintain a physiological pattern of cytotoxicity in our elderly subjects, even in the presence of low circulating DHEAS levels.The increased NKCC response to DHEAS found in healthy elderly subjects may therefore counterbalance the loss of DHEAS in these subjects.
Nevertheless, the mechanism by which DHEAS can stimulate NKCC is still unknown.The first consideration concerns the hypothesis that DHEAS might directly induce NKCC by means of its own activity on IL-2 release from immune cells (9 -14).The increase in IL-2 production from lymphocytes and the enhanced availability of IL-2 during DHEAS exposure may be two main factors related to the physiological activation of NKCC (22,23).On the other hand, a further interesting aspect of the interaction between DHEAS and NK immunity may regard the effects of DHEAS on IGF-I generation and release from immune cells, in particular from NK (24 -26).
Experimental data have already demonstrated that IGF-I is produced by immune cells (43, 46 -48) and that these cells express functional IGF-I receptors (24,28,49).Moreover, the immune-derived IGF-I might act by inducing an autocrine/ paracrine mechanism responsible for the control of immune cell replication and/or function (28,43,50).
Taken together, these findings might suggest a physiological role of DHEAS in IGF-I release from NK cells and in the control of NKCC.Within this context, our study is the first experimental investigation that demonstrates the possibility of IGF-I generation and release from a pure population of NK cells (CD16 ϩ , CD56 ϩ ), obtained by immunomagnetic separation, and that de novo synthesis of IGF-I from NK and NKCC might be regulated by the amount of DHEAS in the medium of cultured NK cells.In fact, DHEAS has de- termined a prompt release of IGF-I from NK cells in healthy subjects of young and old age, and this release has been associated with DHEAS concentrations (from 10 Ϫ6 -10 Ϫ5 mol/L) and with the increase in NKCC.Thus, our data have clearly indicated that NK cells can function as a source of IGF-I and that DHEAS might contribute to the synthesis of IGF-I from these cells.
Our hypothesis concerning the role of DHEAS on local generation of IGF-I from NK cells as regulatory mechanism of NKCC may be sustained by the near-total blockade of IGF-I release and NKCC after incubation of NK with SST.In fact, SST (10 Ϫ6 mol/L⅐mL) totally suppressed IGF-I release from NK cells when incubated alone or when coincubated with different molar concentrations of DHEAS.The disappearance of IGF-I in the supernatant of NK cells demonstrates that NK are responsive to the inhibitory effect of SST and that SST receptors might be present and function normally in NK immune effectors.Previous studies have already indicated that distinct subsets of SST receptors are present on cultured human lymphocytes (51) and that SST have inhibitory effects on NKCC against tumoral target K562 at both high and low molar concentrations (52).
Although an association between NKCC and NK-derived IGF-I was demonstrated during DHEAS incubation, spontaneous NKCC activity (i.e.natural cytotoxicity without modulators) would seem not to be regulated by IGF-I generation from NK.In fact, the spontaneous NKCC was iologically expressed during SST incubation without DHEAS, suggesting that this activity may be controlled by cytokines (20 -23).
As NKCC was not completely suppressed, as demonstrated for IGF-I release, after SST coincubation with DHEAS (20% of residual NKCC activity at DHEAS: 10 Ϫ5 mol/L), other factors may be involved in the control of NKCC during DHEAS exposure.IGF-I may be an important factor in the regulation of NKCC after DHEAS, and DHEAS-mediated NKCC might be related to a strengthening effect of DHEAS on IL-2 action and release (9 -14).Our data have indicated that the percent increase in NKCC was higher after coincubation of DHEAS with IL-2 than after exposure to IL-2 alone; the further increase in NK cytotoxicity after DHEAS plus IL-2 ranged from 40% (10 Ϫ8 mol/L DHEAS) to 150% (10 Ϫ5 mol/L DHEAS) compared with NKCC after incubation of IL-2 without DHEAS, and this effect was evident in both group of subjects.Therefore, the immune-enhancing action of DHEAS on NKCC may be linked to a regulatory mechanism involving IL-2 or other cytokines.In this context, the NK-dependent release of IGF-I during DHEAS exposure might increase the effectiveness of IL-2 signaling on cytotoxic mechanism activation (22,23).molecular mechanism similar to that of IL-2 (57).The slight reduction (30% decrease) of NKCC after IL-2 coincubation with SST would seem to confirm the autocrine role of IGF-I in IL-2-mediated NKCC.
However, the physiological mechanism by which DHEAS may activate NKCC remains to be elucidated.The immunoendocrine effect of DHEAS on NKCC might concern the intracellular signaling pathway involving PKC.PKC is a serine-threonine phosphorylating enzyme that stimulates granule exocytosis in NK cells.PKC also induces an increase in cytotoxicity and determines several positive effects on cytokine gene transcription and cell surface receptor expression (23,58).DHEAS might induce NKCC by increasing PKC system activation (i.e.PKC-␤II isoform activity) in NK cells, and by this mechanism IGF-I might exert a key role.In fact, IGF-I has been found to directly stimulate diacylglycerol generation and PKC function in mouse myocytes (59) and to increase polymorphonuclear degranulation in response to phorbol ester activation of PKC in healthy humans (50).
The present investigation raises two important questions: can the effects of DHEAS be blocked with an androgenic receptor blocker, and will other androgens (e.g.testosterone or dihydrotestosterone) have the same effects as DHEAS on NKCC and IGF-I?As androgens may have negative effects on immunity (60), our hypothesis is that the NK immune activation related to DHEAS could be independent of its androgenic potential.
In conclusion, an important regulatory effect of DHEAS on NKCC was demonstrated in healthy subjects, in particular in those of more advanced age.The mechanism by which DHEAS can influence NKCC may involve the generation and release of IGF-I from NK cells and is dependent on the amount of DHEAS molar concentrations in the medium.DHEAS can also regulate NKCC during IL-2 exposure, and this effect may determine an expansion of NK functional activity during cytokine modulation.All of these effects may be antagonized by SST.In fact, SST completely suppresses IGF-I production from NK and NKCC responses to DHEAS, also reducing IL-2-dependent NKCC.Further studies of the molecular mechanisms related to DHEAS effects on NKCC will be useful to define the immunoendocrine regulation of NK compartment.Anyhow, as suggested by Khrorram et al. (53,61), the immune-stimulating effects of DHEAS on NK autocrine regulation may have relevant benefits in an attempt to improve natural immunity against viral and tumoral targets in immunodeficiency-related diseases, even if the results obtained in peripheral cells cannot completely reflect those expected in the whole immune system (62).

TABLE 1 .
Clinical and laboratory parameters of healthy young and elderly subjectsData are presented as the mean Ϯ SD.BMI, Body mass index.

TABLE 2 .
Variations in NKCC and IGF-I release from NK cells in healthy young and old subjects in spontaneous conditions and after modulation with DHEAS, DHEAS plus SST, and DHEAS plus IL-2