Cellular immune-endocrine interaction in adrenocortical tissues
C. Marx*, S. R. Bornstein*+ and G. W. Wolkersdörfer*+
*Department of Internal Medicine III, University of Leipzig, Germany; +National Institute of Child Health and Human Development, NIH Clinical Center, Bethesda, MD, USA
| Abstract | Background The detection of important immunocompetence-like features on endocrine steroid cells raises questions about direct intercellular communication between the adrenal and immune systems. This article summarizes our recent work and new data on immune-adrenal interactions. |
| Materials and methods RT-PCR and immunohistochemistry were performed to exam- ine MHC class II (HLA-DR) expression in adrenocortical tumours. Coculture systems of NCI-H295 adrenocortical carcinoma cells and HLA-matched lymphocytes were used to examine effects on steroid production and survival of lymphocytes .. Results HLA-DR m-RNA is found in both benign and malignant adrenals, except the NCI-H295 cell line. Under direct coculture conditions with NCI-H295 cell line, spon- taneous apoptosis of immune cells was reduced. Synthesis of cortisol and especially of dehydroepiandrosterone production of tumour cells was markedly increased. Differences by separating CD4+ and CD8+ T cells were not detected. Conclusions Direct cellular contact between lymphocytes and adrenocortical cells seems to be involved in the peripheral regulation of androgen synthesis in the adrenal. The molecular basis of this interaction is no tknown. With regard to normal adrenals, ligation of MHC class II antigens could be a potential mechanism for a peripheral regulation of androgen secretion. Keywords adrenal gland, androgen production, immune-endocrine interaction. Eur J Clin Invest 2000; 30 (Suppl. 3): 1-5 |
The adrenal gland, as the main effector organ of the hypo- thalamic-pituitary-adrenal (HPA)-axis, shows unique morphologic and physiologic alterations during life, which depend on the state of activation of the HPA-axis. In the past decade it has become evident that the function of the adrenal cortex has to be seen in close morphological and functional relation to the sympathoadrenal system and the immune system [1]. The physiological function of each of these systems has been extensively examined. Never- theless, it is still a long way toward a more connecting and integrative concept that should have great importance for the pathophysiology in several diseases.
Department of Internal Medicine III, University of Leipzig, Germany (C. Marx, G. W. Wolkersdörfer); National Institute of Child Health and Human Development, NIH Clinical Center, Bethesda, MD, USA (S. R. Bornstein).
Correspondence to: Stefan R. Bornstein, PhD, National Institute of Child Health and Human Development, Building 10, Room 10N262, Bethesda MD 20814, USA.
Most of our knowledge about the interaction between immune systems and the HPA-axis is based on studies exploring the effects of mediator-like substances such as cytokines (reviewed in [2]) deriving from the immune sys- tem, and steroid hormones from the endocrine system that exert cross-talk effects on the endocrine and immune system, respectively.
The detection of important immunocompetence-like features on endocrine steroid cells such as major histo- compatibility complex (MHC/HLA) class II expression on androgen-producing cells [3,4] or the expression of Fas and Fas-ligand (Fas-L) on steroidogenic cells [5], patterns originally thought to be a characteristic of cellular immunity, raises questions about a direct inter- cellular communication between both systems. Here we summarize and discuss recent morphological and func- tional studies that allow us to speculate on the physio- logical importance of a cross-talk on the cellular level between immune cells and adrenal steroid cells.
The expression of MHC class II molecules and Fas antigen, which were originally thought to be restricted to
the immune system, in endocrine active organs such as normal adrenal cortex (Fig. 1) has been extensively exam- ined [3-5]. In normal human adrenals, MHC class II molecules are expressed on steroidogenic cells of the innermost cortical zona reticularis, where androgen pre- cursors such as dehydroepiandrosterone-sulfate (DHEAS) and dehyrdoepiandrosterone (DHEA) are synthesized. A correlation between MHC class II expression and the pro- duction of adrenocortical androgens has been suggested, as the onset of the MHC class II expression at approxi- mately 4 years of age (Fig. 2) coincides with the increase of androgen production during childhood.
Therefore, the development of the complete physiologi- cal activity of the adrenal seems to be related to the maturation of T-cell immunity, which occurs in parallel [6] and might be linked to expression of MHC class II antigens by steroid cells, thus making cellular communi- cation between T helper cells and steroid cells possible. Previously, we have extensively pointed out the potential pathophysiological relevance of this interaction [7]. It is known that adrenocortical cells derive from cells of the outer cortical layers and are believed to migrate toward
the inner cortical layers, thereby switching from cortisol toward androgen synthesis. We hypothesized that CD4+ T helper cells might be able to interact via T cell receptor with MHC class II expressing androgenic steroid cells. Since these T cells may express Fas-L, they could induce apoptotic cell death of centripetally streaming adreno- cortical steroid cells that are able to express Fas antigen, thus becoming receptive for immune-induced apoptotic cell death.
Since regulation of androgen synthesis in the adrenal cortex is still insufficiently understood and differs from the regulation of glucocorticoid synthesis during various situations such as critical disease, stress and adrenocorti- cal insufficiency, this mechanism could contribute to the regulation of androgen synthesis. Vice versa, adrenal androgens and glucocorticoids have been suggested to influence the differentiation shift of T-helper cells toward the Th1 cytokine pattern or toward Th2, respectively [8]. In summary, a peripheral cellular interaction between adrenal steroid cells and T helper cells could be an impor- tant mechanism to coordinate and integrate immune system and endocrine-adrenal function during immune response and stress.
In a similar way, cellular interaction could have impli- cations for adrenocortical tumour pathophysiology, as has already been shown for other tumours. The phenomenon of immunologic escape includes loss of Fas-receptor by tumour cells and gain of Fas-L expression. Binding of Fas-L to Fas antigen has been shown to induce apoptosis. This phenomenon blunts activation and overactivation, contributing to immune homeostasis. Data on Fas-L expression by various cancers led to the discovery of a Fas-L mediated cytotoxic effect on immune cells utilized by these tumours. Immune evasion and the lack of immunologic response against neoplastic degeneration might at least in part be due to this cytotoxic activity of the tumours.
Blunting of immunologic activity by the Fas/Fas-L mediated apoptosis has been found to be enhanced by MHC class II antigens [9].
Recently, we could demonstrate loss of Fas-receptor and the ability to express Fas-L in the adrenocortical carcinoma cell line NCI-H295 by immunohistochemistry [10]. RT-PCR showed expression of both Fas and Fas-L.
In a coculture system of NCI-H295 adrenocortical carcinoma cells and HLA-matched CD4+ or CD8+ lymphocytes [10, 11], we examined the immunologic escape and the ability to induce apoptosis in the immune cells (Fig. 3). Flow-cytometric analysis of Annexin V bind- ing revealed a 50% reduction of spontaneous apoptotic cell death upon direct coculture conditions, while in insert separated coculture experiments lymphocyte death was comparable with controls without cancer cells. LDH release, indicating unspecific cytolytic cell death, did not significantly differ between insert separated cocultures and cocultures with direct contact of immune cells and cancer cells (not shown). After 3 days, cortisol secretion was increased to 190% in lymphocyte/cancer cell co- culture, compared with basal secretion. In insert
Immune cells
75
Tumour cells
T
50
25
0
1
2
3
4
5
6
7
8
separated coculture experiments, cortisol secretion did not significantly differ from basal secretion. Most im- portantly, androgen release was stimulated to a much higher extent than cortisol release (Fig. 4).
Cellular communication in this model does not induce apoptosis in immune cells, but promotes their survival. This may also be due to partial HLA class I mismatches contributing to immunologic activity. Therefore, HLA- identical lymphocytes are necessary to examine an im- mune escape mechanism in this tumour. However, adrenocortical tumour cells were not affected in viability, but appeared to release androgens and cortisol due to direct cellular contact.
This reduction of immune cell death and stimulation of hormone secretion were proven by incubation of cancer cells along with media previously obtained from coculture without inserts, where immune cells and cancer cells had been in direct cellular contact (Fig. 5).
In this way, we were not able to detect any difference by separating CD4+ and CD8+ T cells and performing co- incubation with NCI H295 cells. This might be due to a
700
Secretion ( in % activity)
Cortisol
p < 0.005
DHEA
600
p < 0.005
500
400
p < 0.0001
p < 0.0001
p < 0.0001
300
200
100
0
basal
ACTH
CD 4
CD 8
basal
ACTH
CD 4
CD 8
700
Cortisol
DHEA
p < 0.005
600
500
400
300
p< 0.0001
200
100
p< 0.05
p<0.0001
0
basal
co-culture medium transfer
basal co-culture medium transfer
lack of MHC class II antigen expression in the NCI-H295 cell line, as determined by flow cytometry (not shown) and RT-PCR for HLA-DR alpha chain (Fig. 6). Whereas HLA-DR m-RNA is also found in benign (not shown) and malignant adrenocortical tumours (Fig. 6), the majority of these carcinomas do not express HLA class II DR, DP and DQ antigens on the protein level [12].
Nevertheless it is interesting that direct cellular contact between T cells and adrenocortical cells is able to stimu- late androgen synthesis to a higher degree than cortisol release. This supports our hypothesis of a peripheral regu- lation of androgen synthesis within the adrenal gland, which may occur in addition to the effects of ACTH, which stimulates mainly glucocorticoid- but also andro- gen synthesis. However, in our model the exact mechanism of the observed effects is not known. Assuming a regulation circuit between MHC class II and T-helper cells in the normal adrenal, our results show that this interaction would not be applicable for malignant adrenocortical tissues, which are devoid of MHC class II expression and clinically also often reveal dysregulations in androgen synthesis. It should, therefore, be of high interest to elucidate further the molecular basis of this direct cellular interaction with relevance for the interplay of the immune and endocrine systems.
Materials and methods
Cell culture Adrenocortical carcinoma cell line (NCI- H295) was cultured in RPMI 1640 containing penicillin (100 U mL-1), streptomycin (0.1% w/v) and 2% fetal calf serum at 37℃ under 5% CO2.
Immunohistochemistry After fixation in 1.5% para- formaldehyde, specimens were immunostained with anti-CD 95 (mouse anti-human, clone DX2, Dianova) and anti-Fas-L [(C-20), rabbit anti-human, polyclonal,
1
2
3
4
5
1 2 3 4 5
Santa Cruz Biotechnology] at dilutions of 1:50 for 12 h at 4℃. Furthermore, antibodies for mouse anti-human HLA-DR, DP, DQ (Dako) were used to stain paraffin- and acetone-fixed sections from normal adrenal tissues using avidin-biotin staining methods (LSAB and CSA system, Dako). In controls, the specific antiserum was replaced by an isotype-immune serum (Mouse IgG1, Pharmingen; rabbit immune serum, Dako) and showed no unspecific staining. RNA isolation was done according to the suppliers protocol of RNeasy total RNA kit (Quiagen). The resulting RNA was resolved in DEPC water, treated with 10 U of DNase I, RNase free (Boehringer) and reversely transcribed (Ready-to-go T-primed first-strand kit, Pharmacia Biotech). PCR amplification of cDNA was carried out using the forward primer 5’-TATCACCACTATTGCTGGAGTC-3’, and the complementary reverse primer 5’-AACATCCTTT- GAGGCAGAATC-3’ for Fas, and the forward primer 5’-AGCCCTTCAATTACCCATATCC-3’, and the com- plementary reverse primer 5’-AGTTCTGCCAGCT- CCTTCTGTA-3’ for Fas-L. For detection of HLA-DR we used 5’-TACTCCGATCACCAATGTACCT-3’ and the reverse primer 5’-CAAACTCCCAGTGCTTGA- GAAG-3’.
cDNA was amplified using 0.5 U Pfu polymerase (Stratagene) and an annealing temperature of 65.5℃ for Fas and Fas-L, 60℃ for HLA-DR for 35 cycles. Samples were electrophoresed through a 1.5% agarose gel and amplified bands were identified by ethidium bromide staining.
Leukocyte separation and separation of CD4+ and CD8+ cells Peripheral blood mononuclear cells (PBMC) of (NCI- H295 cell line) HLA-matched donors were collected from whole blood after ficoll gradient separation. Aliquots of the PBMC suspension were used to separate CD4+ or CD8+ cells by antibody linked magnetic beads (DYNAL). Coculture was carried out either with 500 000 adrenocor- tical cells and 500 000 immune cells in one well or in one well separated by inserts with a 0.02 um anopore mem- brane (Nunc). Culture medium was collected for determining hormone concentration after 3 days. Annexin V-labelling of FITC-labelled annexin V (Bender Med Systems) was used to assess the percentage of apoptotic cells by FACS analysis. Hormone measurements were carried out using cortisol and DHEA radioimmunoassay (Biermann). LDH activity in culture medium was assessed photometrically after pyruvate reduction.
Acknowledgements
This work was supported by grants from BASF to G.W.W., BMBF IZKF B1 and a Heisenberg grant to S.R.B. We thank S. Brauer for excellent technical assist- ance.
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