Lymphocytes Stimulate Dehydroepiandrosterone Production through ...

0021-972X/99/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 1999 by The Endocrine Society

Vol. 84, No. 11 Printed in U.S.A.

Lymphocytes Stimulate Dehydroepiandrosterone Production through Direct Cellular Contact with Adrenal Zona Reticularis Cells: A Novel Mechanism of Immune-Endocrine Interaction* ¨ RFER, TOBIAS LOHMANN, CHRISTIAN MARX, GERNOT W. WOLKERSDO ¨ SABINE SCHRODER, ROBERT PFEIFFER, HANS-DETLEF STAHL, WERNER A. SCHERBAUM, GEORGE P. CHROUSOS, AND STEFAN R. BORNSTEIN Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health (G.W.W., G.P.C., S.R.B.), Bethesda, Maryland 20892; Diabetes Research Institute, University of Dusseldorf (W.A.S.), Dusseldorf 40001, Germany; and the Department of Internal Medicine, University of Leipzig (T.L., S.S., R.P., H.-D.S., S.R.B.), Leipzig 04103, Germany ABSTRACT Adrenal androgen production was reduced by 80% in patients receiving T lymphocyte-suppressive medications compared to that in age-matched controls. In vitro, however, neither tacrolimus nor cyclosporin A reduced dehydroepiandrosterone (DHEA) release by adrenocortical cells. Therefore, we examined the potential role of lymphocytes in adrenal androgen production, using cocultures of human T lymphocytes and adrenocortical primary or transformed cells. Cocultures led to a 4-fold elevation of DHEA levels (490.4 6 94.8% over basal), which was greater than the increase observed after the addition of maximal concentrations of ACTH (117.4 6 14.8%). Separation of cells by semipermeable membranes abolished this effect, and transfer of leukocyte-conditioned medium had little androgen-stim-

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EHYDROEPIANDROSTERONE (DHEA) and its sulfate are quantitatively the most abundant adrenal androgens; these molecules are produced in the inner zone of the adrenal cortex, the zona reticularis. The main known regulator of adrenal androgen production is pituitary ACTH; indeed, in the absence of pituitary ACTH, adrenal androgen levels are low. Exogenous ACTH replacement in hypophysectomized great apes maintains normal cortisol, but not DHEA, secretion (1). This suggests that factors in addition to ACTH participate in the regulation of adrenal androgen secretion. In chronic autoimmune diseases, such as rheumatoid arthritis, there is a decline in circulating adrenal androgen levels, whereas cortisol concentrations remain within the normal range (2–10). DHEA was proposed to have immunoregulatory effects in vitro (11–13) and in small animals (14 –17), and therapeutic effects of DHEA were observed in patients with systemic lupus erythematosus in placebo-conReceived May 13, 1999. Revision received July 20, 1999. Accepted July 26, 1999. Address all correspondence and requests for reprints to: Dr. G. W. Wolkersdo¨rfer, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892. E-mail: WolkersdoerferG@ netscape.net. * This work was supported by a grant from Studienstiftung des Deutschen Volkes and BASF Aktiengesellschaft (to G.W.W.) and a Heisenberg grant (to S.R.B.).

ulating effect. These data suggested that the observed stimulation of androgen secretion required cell contact rather than soluble paracrine factor(s). Furthermore, we examined human adrenal glands for the presence of T lymphocytes and contact between these cells and steroid-secreting cells of the zona reticularis. Indeed, T lymphocytes expressing CD4 and CD8 antigens were present within human adrenal zona reticularis by immunohistochemical subtyping. Electron microscopic analyses demonstrated direct cell-cell contact between T lymphocytes and adrenocortical cells in situ. This study provides evidence for a novel mechanism of immune-endocrine interactions of direct T lymphocyte-adrenocortical cell contact-mediated stimulation of adrenal androgen secretion. (J Clin Endocrinol Metab 84: 4220 – 4227, 1999)

trolled studies (18). Although DHEA and its sulfate may provide small amounts of androgen and estrogen activity after peripheral conversion, via the androgen and estrogen receptors, respectively, suggestions have been made that it may also have distinct effects through membrane g-aminobutyric acid type A and excitatory amino acid and/or nuclear peroxisome proliferator-activated receptors (19 –22). Adrenal androgen-producing cells of the zona reticularis are the only adrenocortical cells that constitutively express major histocompatibility complex (MHC) class II molecules (23). Expression of these molecules is related to the maturation of the gland during adrenarche and correlates with the age-dependent gain of androgen secretory capacity, reaching peak activity in the third decade of life (23, 24). Although the size and the secretory function of this androgen-producing zone start declining in parallel with decreasing adrenal androgen levels after the third decade of life (25–28), the numbers of resident T lymphocytes within it increase reciprocally, in an infiltration-like manner (29). Resident monocytes/macrophages are present within the zona reticularis at all stages (30, 31). To analyze the role of the immune system in adrenal androgen secretion we assessed circulating adrenal androgen levels in patients receiving immunosuppressive antilymphocytic therapy and compared these concentrations with those in an age-matched normal control group. Then, we evaluated

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the capacity of such agents to directly influence the hormone secretion of adrenocortical cells in culture. To define the mechanisms of immune-endocrine interactions at the level of the androgen-producing cell, we analyzed the in vitro capacity of primary and transformed adrenocortical cells to secrete androgens when cocultured with immune cells, including CD41 and CD81 T lymphocytes. Furthermore, a coculture system allowing or preventing cellular contact was employed to determine whether the effects observed were mediated by soluble factors or required direct cell to cell contact. We employed polyclonal stimulation by phytohemagglutinin and specific T cell activation by OKT-3 antibodies to examine the influence of T cell activation on adrenal androgen production. Finally, we characterized the localization and distribution of immune cell subtypes within the human adrenal cortex in situ by immunohistochemistry and electron microscopy. Subjects and Methods Subjects Blood samples were drawn from 24 patients (11 men and 13 women; mean age, 48.2 6 2.8 yr) treated with T lymphocyte-suppressive agents (either tacrolimus or cyclosporin A) according to standard protocols. The treatment indications were uveitis (n 5 5) or kidney transplantation (n 5 19); 16 of the 19 kidney-transplanted patients received prednisolone (7.5 mg/day). All patients had normal serum electrolytes and glucose parameters and were studied at least 9 months after transplantation. Patients with overt metabolic disease were excluded. None had Cushing’s syndrome or Addison’s disease. Normal function of the hypothalamicpituitary-adrenal axis was assessed by determination of normal morning and evening plasma ACTH levels, normal morning cortisol levels (.5 mg/dL), and/or normal 24-h urinary free cortisol excretion. Patients with suppression of plasma ACTH or loss of diurnal variation of ACTH and/or cortisol were excluded. Blood samples were drawn 30 min after insertion of an iv cannula and bed rest at 0800 and 2200 h. Age- and sex-matched volunteers (n 5 50) without any reported endocrine or immune disease or receiving immunosuppressive therapy served as controls (mean age, 44.9 6 3.7 yr). The study was approved by the ethical committee of the University of Leipzig and post-hoc by the Office of Human Subjects Research, NIH.

Hormone measurement Hormone concentrations in serum/plasma and/or incubation medium were measured by enzyme immunoassay and RIA using the following kits: DYNOtest ACTH (Brahms Diagnostica, Berlin, Germany; sensitivity, 0.44 pmol/L), Cortisol-RIA (Biermann, Bad Neuheim, Germany; sensitivity, 5.5 nmol/L; cross-reactivity with cortisol, 100%; with prednisolone, 76%; with 11-deoxycortisol, 11.4%; with prednisone, 2.3%; with other steroids, ,1%; intra- and interassay variations, 5.1% and 6.4%, respectively), DHEA-RIA (Diagnostics Systems Laboratories, Inc., Sinsheim, Germany; sensitivity, 0.02 ng/mL; cross-reactivity with DHEA, 100%; with other steroids, ,0.88%; intra- and interassay variations, 10.6% and 10.2%, respectively), and Immulite DHEA-SO4 (Diagnostic Products, Los Angeles, CA; sensitivity, 0.07 mg/dL; crossreactivity with DHEA-SO4, 100%; with DHEA, 0.049%; with DHEAglucuronide, 0.054%; with androstenedione, 0.147%; with androsteroneSO4, 0.231%; with estrone-3-SO4, 0.459%; with other steroids, ,0.04%; intra- and interassay variations, 9.5% and 15.0%, respectively).

mark), using the avidin-biotin staining method, as described previously (32). In brief, sections were deparaffinized in xylene and hydrated in a descending ethanol row. Endogenous peroxidase was quenched by 1.5% H2O2-10% methanol in phosphate-buffered saline (PBS) for 10 min, followed by a Triton X-100 incubation with 0.5% in PBS for 5 min. A blocking preincubation for 30 min in 10% normal serum of the secondary antibody species (normal rabbit serum; Dakopatts) in PBS was followed by overnight exposure to the specific antiserum at 4 C at a 1:50 dilution. After washing in PBS, the color reaction was carried out using the avidin-biotin staining method (CSA system, DAKO Corp.) with 3-amino-9-ethylcarbazole chromogen (Immunotech, Hamburg, Germany). In controls, the specific antiserum was replaced by an isotype-immune serum (mouse IgG1, PharMingen, rabbit immune serum, DAKO Corp.) and showed no nonspecific staining.

Electron microscopy Small tissue pieces of adrenal gland were fixed in 4% paraformaldehyde-1% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.3, for 3 h, postfixed in 2% OsO4 in 0.1 mol/L cacodylate, pH 7.3, dehydrated in ethanol, and embedded in epoxy resin. Seventy-nanometer sections were stained with uranyl acetate and lead citrate and examined at 80 kV under a Phillips electron microscope 301 (Phillips, Rahway, NJ).

Leukocyte separation and separation of CD41 and CD81 cells Peripheral blood mononuclear cells (PBMC) were obtained from whole blood after Ficoll gradient separation. Aliquots of PBMC suspension were used to separate CD41 or CD81 cells by antibody-linked magnetic beads, according to the supplier’s protocol (Dynal). Stimulation of lymphocytes was achieved by incubation with phytohemagglutinin (PHA) at 10 mg/mL or with OKT3 antibody-containing medium at 10 mg/mL for 24 h. Before adding lymphocytes into the coculture dish, lymphocytes have been washed three times in RPMI, harvested by centrifugation, and resuspended in coculture medium. Indomethacin supplementation was used to suppress inflammatory mediator actions at 10 and 100 mmol/L during the coculture experiments.

Cell culture and coculture procedure Normal adrenal glands for primary culture were obtained from two subjects undergoing nephrectomy due to nonpapillous carcinoma of kidney, trimmed free of adipose tissue, and transported in ice-cold PBS. The medulla was removed, and the cortex was scraped off the capsule. Small pieces were washed three times in washing medium [DMEMHam’s F-12 (Life Technologies, Inc., Egenstein, Germany) containing 200 U/mL penicillin, 200 mg/mL streptomycin, and 50 mg/mL gentamicin]. Cells were dispersed by digestion with collagenase (0.1%, wt/vol) and deoxyribonuclease I (0.01%, wt/vol) and mechanical disaggregation. Viability was checked by trypan blue exclusion test. Cell preparation was cleared from erythrocytes by erythrocyte lysis with 0.15 mol/L NH4Cl, 0.1 mmol/L Na2 ethylenediamine tetraacetate and 12 mmol/L NaHCO3 at 37 C for 2 min, and lysis was stopped by adding ice-cold PBS. Preparations were cultured in DMEM-Ham’s 12 containing 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% FCS (wt/vol) at 37 C under 5% CO2 and seeded in 24-well plates at a density of 150,000 cells/well. In addition, adrenocortical cells were cocultured either with PBMC directly or with PBMC separated by inserts with 0.2-mm anopore membrane (Nunc, Roskilde, Denmark) at equivalent density. After 3 days, hormone release was measured. TABLE 1. Plasma ACTH and serum cortisol in 24 patients receiving immunosuppressive therapy

Immunohistochemistry Normal adrenal glands were obtained from subjects undergoing nephrectomy due to nonpapillous carcinoma of the kidney. Donors’ ages ranged from 34 –58 yr. Formaldehyde-fixed and paraffin-embedded tissue specimens and 1.5% glutaraldehyde-fixed cryostat specimens were separately immunostained for CD4 (Novocastra Laboratories Ltd., Newcastle, UK), CD8, CD45, and CD22 (DAKO Corp., Copenhagen, Den-

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0800 h 1000 h

ACTH (pmol/L)

Cortisol (nmol/L)

5.99 6 1.0 (2.2–13.2)a 2.84 6 0.8 (1.3– 6.6)

275.7 6 38.5 (137.9 – 689.7) 87.38 6 18.4 (55.2–331.1)

Values are the mean 6 SE. a Normal ranges are in parentheses.

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FIG. 1. Serum DHEA levels in patients receiving immunosuppressive therapy and in untreated normal age-matched controls. Blood samples were drawn 30 min after insertion of an iv cannula and bed rest at 0800 h. Mean DHEA levels, measured by specific RIA, were 1.5 6 0.25 ng/mL (mean 6 SE) in immunosuppressant-treated individuals vs. 9.5 6 0.43 ng/mL in age-matched controls (P , 0.0001).

FIG. 2. DHEA and cortisol secretion of primary adrenocortical cell cultures under basal conditions, direct coculture, or coculture with semipermeable membrane separation. Cells were seeded in 24-well plates at a density of 150,000 cells/well and cocultured either with PBMC directly or with PBMC, separated by inserts with 0.2-mm anopore membrane at equivalent density. Cortisol secretion increased 2.7-fold over baseline concentration, whereas DHEA secretion increased 3.7-fold.

Adrenocortical carcinoma cell line (NCI-H295) was routinely cultured in RPMI 1640 containing penicillin (100 U/mL), streptomycin (0.1%, wt/vol), and 2% FCS and supplemented with apotransferrin (100 mg/mL; Sigma Chemical Co., St. Louis, MO), insulin (5 mg/mL; Sigma Chemical Co.), sodium selenite (0.03 mmol/L; Sigma Chemcial Co.), and b-estradiol (0.01 mmol/L; Sigma Chemical Co.) at 37 C under 5% CO2. Cells were placed in 24-well dishes. Culture in medium containing immunosuppressive agents was carried out with 2,000,000 adrenocortical cells/wellz4 mL medium. The medium was supplemented with tacrolimus (1:250 in ethanol containing 33% castor oil) or cyclosporin A (1:500 in ethanol containing 33% castor oil). Coculture was carried out either with 500,000 adrenocortical cells and 500,000 lymphocytes in 1 well or in 1 well separated by semipermeable membranes (0.02-mm anopore membrane, Nunc), allowing passage of soluble factors. Culture medium was collected for determining hormone concentrations after 3 days or as conditioned medium for further incubation of adrenocortical cells. HLA-matched lymphocytes were used in all coculture experiments. Each experiment was carried out in triplicate at least four times. Statistical analysis were made using Prism2 software and the MannWhitney U test.

FIG. 3. A, Steroid secretion of transformed adrenocortical cells cocultured directly with CD41 lymphocytes compared to secretion in 72-h coculture-primed medium. Coculture was carried out with 500,000 adrenocortical cells and 500,000 lymphocytes in 1 well. Culture medium was collected for determining hormone concentration after 3 days or collected as conditioned medium for further incubation of adrenocortical cells. The graph shows the significant stimulatory effect of direct coculture on steroid hormone secretion (data are a percentage of baseline activity), whereas primed medium led to a significant decrease. B, Effect of tacrolimus on DHEA secretion in vitro. Two million transformed adrenocortical cells per well/4 mL medium were cultured in medium supplemented with tacrolimus (c, 1:250 in ethanol containing 33% castor oil). DHEA secretion of transformed adrenocortical cells cultured in tacrolimus-containing medium is dose and time dependent.

Results DHEA serum levels in immunosuppressed patients and healthy control subjects

DHEA serum levels were measured in immunosuppressed patients (n 5 24) with preserved diurnal rhythm of ACTH and cortisol, indicating a functionally, albeit grossly, intact hypothalamic-pituitary-zona fasciculata axis (Table 1). Data were compared to DHEA serum levels of age-matched controls (n 5 50) who did not receive immunosuppressive therapy (Fig. 1). The mean DHEA levels in patients receiving immunosuppressive therapy were 1.5 6 0.25 ng/mL (mean 6 se) compared to 9.5 6 0.43 ng/mL in controls (P , 0.0001). There was no difference in ACTH, cortisol, or DHEA concentrations between subjects receiving or not receiving


prednisolone at any time (ACTH: 0800 h, P 5 0.53; 2200 h, P 5 0.18; cortisol: 0800 h, P 5 0.93; 2200 h, P 5 0.07; DHEA: 0800 h, P 5 0.54; 2200 h, P 5 0.25). Coculture results

The functional relevance of cellular contact between T lymphocytes and adrenocortical cells was assessed in coculture experiments. Primary adrenocortical cells in coculture with purified T lymphocytes responded with a 3.7-fold increase in DHEA (369.3 6 28.7%) and a 2.7-fold increase in cortisol (273.3 6 17.3), whereas separation with semipermeable membranes allowing free medium exchange, decreased DHEA secretion to 203.0 6 2.0% and cortisol secretion to 182.4 6 7.9% (Fig. 2). Incubation of naive transformed adrenocortical cells with conditioned medium from lymphocyte primary cultures or from 72-h direct cocultures did not increase steroid secretion (Fig. 3a). To the contrary, the cortisol secretion decreased to 82.52 6 12.2% (P , 0.05) and DHEA to 51.71 6 3.8% (P , 0.0001). Basal DHEA secretion from adrenocortical cells cultured in tacrolimus-containing medium did not decrease. In contrast, hormone secretion showed a slight time and dosedependent increase (Fig. 3b). Comparable data were ob-

FIG. 4. A, Secretion of steroid hormones under basal conditions, ACTH challenge and direct coculture with either CD41 or CD81 lymphocytes. Hormone secretion in cocultures of 500,000 transformed adrenocortical cells and 500,000 CD41 or CD81 lymphocytes in 1 well is compared to half-maximal ACTH stimulation (1028 mol/L; data are given as a percentage of basal activity; P values refer to comparison of stimulated vs. basal activities). B, Influence of the prostanoid synthesis inhibitor indomethacin on lymphocytestimulated DHEA release. The addition of indomethacin at a concentration of 100 mmol/L to the culture medium did not influence lymphocyte-mediated DHEA release.

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tained in cultures with cyclosporin A-supplemented medium (data not shown). Basal androgen secretion by primary adrenocortical cells in direct cocultures with lymphocytes was higher than ACTH-stimulated secretion (Fig. 4a). ACTH stimulation resulted in 170.9 6 7.3% (mean 6 sem) cortisol secretion over basal concentration. ACTH-stimulated DHEA secretion was weak (117.4 6 14.75%). Direct coculture with CD41 lymphocytes resulted in a 179.0 6 4.3% increase in cortisol secretion (P , 0.0001; in relation to basal hormone secretion) and a 449.8 6 158.8% stimulation of DHEA (P , 0.005) compared to 183.7 6 8.0% and 490.4 6 94.8%, respectively, when cocultured with CD81 cells. The prostanoid synthesis inhibitor indomethacin at 10 or 100 mmol/L did not abolish the direct contact-induced hormone secretion as shown in Fig. 4b. Pretreatment of CD41 and CD81 lymphocytes with phytohemagglutinin- or antiOKT3-stimulated DHEA secretion when these cells were subsequently cocultured with adrenocortical cells. Activation of CD41 or CD81 T cells with phytohemagglutinin resulted in 132.7 6 6.91% and 141.2 6 10.26% increases in DHEA release, respectively, whereas anti-OKT3 treatment induced a 162.5 6 39.0% and 181.2 6 12.99% increase during


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FIG. 5. Effect of lymphocyte activation on DHEA secretion by transformed adrenocortical cells. Lymphocytes were activated by incubation with PHA at 10 mg/mL or with OKT3 antibody-containing medium at 10 mg/mL for 24 h before coculture experiments. T lymphocyte-specific stimulation was superior to pan-lymphocyte stimulation for both CD4- and CD8-positive cells.

coculture (Fig. 5). Lymphocyte activation did not alter cortisol secretion (data not shown). Immunohistochemistry

Normal adrenal glands obtained from subjects undergoing nephrectomy due to renal carcinoma were used in all histological investigations. The tissue was stained with antisera against CD45, CD4, CD8, and CD22 to determine immune cell distribution. CD451 leukocytes were present throughout the entire gland, while CD41 and CD81 T cells were preferentially located in the inner cortical zones (Fig. 6). Their immunohistochemical localization suggests a close contact to epithelial cells of the adrenal cortex. CD221 B cells were seen rarely within the entire cortex. Electron microscopy

At the ultrastructural level, lymphocytes were seen in direct contact with adrenocortical cells in the zona reticularis (Fig. 7). Adrenocortical cells were characterized by their typical tubulovesicular mitochondria and ample smooth endoplasmic reticulum (SER). Lymphocytes presented with characteristic large nuclei and sparse undifferentiated cytoplasm with few large rod-shaped mitochondria, some SER, and polyribosomes. Adrenocortical cells were extending filopodia toward the lymphocytes. Lymphocytes connected to adrenocortical cells by cell junctions could be detected. Adrenocortical cells in direct contact with lymphocytes showed signs of stimulation with large vesicular mitochondria and dilated SER (Fig. 7). Discussion

Here we provide evidence that intact T cell function is required for normal adrenal androgen production. Suppression of T cell function in humans with immunosuppressants, such as tacrolimus or cyclosporin A, led to a marked reduction in adrenal androgen secretion while at the same time basal adrenal glucocorticoid production was maintained within the normal range. Incubation of human adrenocorti-

FIG. 6. Human adrenal gland. Immunostaining against CD41 lymphocytes within the adrenal cortex. CD41 lymphocytes appear to be more frequent within the zona reticularis than in the two other zonae (arrowheads). ZF, Zona fasciculata; ZR zona reticularis; ZM, zona medullaris.

cal cells with cyclosporin A or tacrolimus showed a small dose- and time-dependent increase in steroid hormone secretion, excluding a direct inhibitory effect of these substances on DHEA secretion. As ACTH is a major regulator of adrenal androgen production, suppression of the entire hypothalamic-pituitary-adrenal axis by glucocorticoid therapy could also have led to a decrease in plasma androgen levels. However, normal ACTH and cortisol levels exclude this possibility. Furthermore, we noted a similar decline in adrenal androgen production in uveitis and kidney transplantation patients receiving immunosuppressive treatment without prednisolone as in those receiving the glucocorticoid. Therefore, other indirect mechanisms may have caused the dissociation of adrenal androgen and cortisol secretion in these patients. There are many physiological and pathophysiological conditions in which dissociation between adrenal androgen and cortisol secretion has been observed, and ACTH alone seems to be unable to maintain a normal cortisol to androgen ratio (1, 33–36). At times of chronic or severe illness, steroid synthesis may be diverted from adrenal androgen to glucocor-


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FIG. 7. Electron microscopy of human adrenal cortex. The figure shows a lymphocyte with typical nucleus (NUC) in direct contact with an adrenocortical cell in the zona reticularis. The adrenocortical cells exhibit typical round mitochondria with tubulo-vesicular internal membranes (MIT) and abundant SER. The adrenal cell extends filopodia toward the lymphocyte (small arrowheads). The cells are connected by cellular junctions (large arrowheads). The lymphocyte has a small rim of clear cytoplasm (bar, 0.2 mm).

ticoid production, providing maintenance of high glucocorticoid levels, which may be crucial for coping with the illness (37– 40). A differential regulation of 17,20-desmolase expression, which governs the bioynthesis of D5-adrenal androgens through a specific factor, has, however, not been defined, and such a factor has not been isolated as yet. Adrenal androgens are produced within the zona reticularis of the inner adrenal cortex; a distinct immunological feature of the zona reticularis is the expression of MHC class II surface molecules, which facilitate cellular interactions of these cells with lymphocytes and other immune cells (41), suggesting that these cells are predestined for direct interaction with the immune system (42). Coculture of lymphocytes with human adrenocortical cells stimulated adrenal androgen synthesis 4-fold, whereas incubation with high doses of ACTH only led to a 2-fold stimulation. In a transformed adrenocortical cell line that did not contain other blood cells or cells of the adrenal medulla, this effect was shown to be strongly selective for adrenal androgens. In line with these findings, immune reconstitution of athymic Swiss

nude mice by injecting lymphocyte-enriched splenocyte fractions increased adrenocortical steroid levels (43), whereas a decrease in CD41 T lymphocytes in stage IV acquired immunodeficiency syndrome patients was accompanied by a decrease in adrenal DHEA secretion (44 – 46). What are the mechanisms of this lymphocyte-mediated regulation of adrenal DHEA production? Soluble factors, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-a (TNFa), are known to stimulate adrenal steroid production (47–53), and hence, cytokines released from lymphocytes could explain the findings. However, IL-1, IL-6, and TNFa have been shown to primarily regulate adrenal cortisol production (54 –56), and this does not explain the predominant effect of lymphocytes on adrenal androgen secretion. Furthermore, and even more importantly, incubation of adrenocortical cells with lymphocyte-conditioned medium did not increase DHEA secretion. Rather, medium, conditioned by 3 days of direct coculture led to a significant decrease in steroid hormone secretion, suggesting that soluble factors, possibly TNFa and/or TGFb, known to exert an inhibitory


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role in steroidogenesis, are produced by activated leukocytes (57– 62). Thus, in this setting, the immune-endocrine interaction is not mediated by cytokines, but requires direct cellcell interaction between lymphocytes and the adrenal androgen-producing cells. Does this concept relate to the in vivo situation? By specific immunostaining, CD41 and CD81 T lymphocytes were identified in normal human adrenal glands; most of the cells were located in the inner cortical zones. Ultrastructural analysis demonstrated lymphocytes in direct cellular contact with adrenocortical cells of the zona reticularis. Adrenocortical cells extended filopodia toward lymphocytes, and cell junctions were depicted between these cells. Therefore, the presence of MHC class II molecules selectively on androgenproducing adrenal cells and the cell-cell contacts with local lymphocytes may trigger signaling pathways that activate steroidogenesis. The human adrenal gland, as the main stress organ in the human body, is extremely well vascularized and receives 10 times the amount of circulating blood for its weight as the average supply of other organs. During stress or inflammation, there is further adrenal vessel dilatation and/or hypervascularization that may provide an increased supply of lymphocytes to the adrenal gland. Prestimulation of lymphocytes with either phytohemagglutinin or OKT3, both of which activate lymphocytes, as does inflammation in vivo, and incubation of these activated lymphocytes with adrenocortical cells further augmented the release of adrenal androgens. Our findings of activated lymphocyte-mediated stimulation of adrenal androgen secretion are in apparent contradiction with the fact that certain autoimmune diseases, such as rheumatoid arthritis, have been associated with low adrenal androgen secretion (2, 4 –7, 10, 63– 66). We have two possible explanations for this paradox. First, in some autoimmune diseases, the activation of lymphocytes could be altered to allow attack of certain tissues, but be deficient in stimulating adrenal androgen secretion. Second, excessively or chronically activated lymphocytes, in the context of some autoimmune diseases, could accelerate the apoptosis of zona reticularis cells, resulting in low androgen secretion. These are testable hypotheses. In summary, T cells within the adrenal gland have direct cell-cell contact to epithelial cells of the adrenal zona reticularis; this provides a mechanism for immune system-mediated stimulation of androgen secretion in vitro and helps explain how impaired T cell function results in decreased androgen levels in vivo. Also, it provides evidence for a non-ACTH-mediated mechanism of adrenocortical androgen regulation. These findings may provide an evidencebased strategy for DHEA treatment in some disorders of the immune system. Patients with systemic lupus erythematosus, rheumatoid arthritis, and AIDS have low DHEA levels and might benefit from such treatment.

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