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THE MERCY OF ADRENOCORTICAL TUMOR CELLS ON LYMPHOCYTES
Gernot W. Wolkersdörfer, Christian Marx, Tobias Lohmann, Silke Brauer, Sabine Schröder1, John Brown2, Nicolas Mitsiades4, George P. Chrousos3, Stefan R. Bornstein1
Department of Internal Medicine, University of Leipzig, Germany.
1 Institute of Clinical Immunology, University of Leipzig, Germany.
2Division of Applied Marine Physics, University of Miami, USA.
3National Institute of Child Health and Human Development, Bethesda, USA.
4 National Cancer Institute, Bethesda, USA.
ABSTRACT
Immunologic escape includes the loss of Fas-receptor and the gain of Fas-ligand expression. Normal adrenal glands express the Fas-receptor and MHC class II molecules in inner cortical zones. A distinctive feature of adrenocortical tumors is the loss of MHC class II expression.
Here we demonstrate loss of Fas and gain of Fas-ligand expression in the adrenocortical carcinoma cell line NCI-H295 by immunohistochemistry and RT-PCR. In a co-culture system of tumor cells and HLA-matched leukocytes, CD 8-positive or CD 4-positive lymphocytes, we examined the immunologic escape and the ability to induce apoptosis in the immune cells. The direct co-culture with either leukocytes, CD 8-positive or CD 4-positive lymphocytes reduced spontaneous apoptosis in immune cells from 49.9% to 13.0%, 8.6% and 15.3%, respectively, as determined by FACS analysis of Annexin V binding and LDH release in the medium. In co-culture, cortisol secretion increased up to 200%.
Cellular communication does not induce apoptosis in immune cells, but promotes their survival. This may be due to partial HLA class I mismatches contributing to immunologic activity. The viability of the tumor cells was not affected, and these cells were stimulated to secrete cortisol. In summary, immune escape of adrenocortical carcinomas may occur because of altered Fas/Fas-L system expression and loss of MHC class H expression.
INTRODUCTION
Binding of Fas-ligand to Fas-antigen, belonging to the superfamily of tumor necrosis factor receptor, induces apoptosis (1). This phenomenon contributes to
immune homeostasis. Blunting of immunologic activity by the Fas/Fas-L mediated apoptosis is enhanced by MHC class II antigens (2).
Recent data on Fas-L expression by various cancers (3-6) has suggested that Fas- L-mediated cytotoxic effect on immune cells is utilized by these tumors. Immune evasion and lack of immunologic response to neoplastic degeneration might be at least in part due to this cytotoxic activity of the tumors. MHC class II molecules, expressed within the zona reticularis of normal adrenal glands (7), allow intercellular communication between adrenocortical and immune cells. Also Fas is expressed within the zona reticularis (8). Interestingly, a feature of adrenocortical carcinomas consists in the loss of MHC class II expression (9) and a decreased rate of apoptotic cells compared to normal adrenals (10).
To study the role of immunoadrenal interaction in carcinomas we assessed the expression of Fas and Fas-L in an adrenocortical cell line. In addition, we performed co-culture experiments of this cell line with HLA-matched immune cells to investigate its influence on immune cell and adrenocortical cell function and survival.
MATERIALS AND METHODS
Cell culture: Adrenocortical carcinoma cell line, NCI-H295, was cultured in RPMI 1640 containing penicillin (100 U/ml), streptomycin (0.1% wt/vol.), and 2% FCS at 37℃ under 5% CO2.
Immunohistochemistry: Specimens were immunostained with anti-CD 95 (mouse anti human, clone DX2, Dianova) and anti-Fas-L ((C-20), rabbit anti-human, polyclonal, Santa Cruz Biotechnology) as described in (8).
RNA isolation was done according to supplier’s protocol of RNeasy total RNA kit (Quiagen), treated with 10 U 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 forward primer 5’- TATCACCACTATTGCTGGAGTC-3’, and complementary reverse primer 5’- AACATCCTTTGAGGCAGAATC-3’ for Fas, and forward primer 5’- AGCCCTTCAATTACCCATATCC-3’, and complementary reverse primer 5’- AGTTCTGCCAGCTCCTTCTGTA-3’for Fas-L. cDNA was amplified as described previously (11) using 0.5 U Pfu polymerase (Stratagene) and an annealing temperature of 65.5℃, 35 cycles. Samples were electrophoresed through 1.5% agarose gel and amplified bands were identified by ethidium bromide staining. Leukocyte separation and separation of CD 4- and CD 8-positive cells: Peripheral blood mononuclear cells (PBMC) were collected from whole blood after ficoll gradient separation. Aliquots of PBMC suspension were used to separate CD 4- or CD 8-positive cells by antibody-linked magnetic beads (DYNAL).
1a
1b
Fas-L
Fas
1000
800
700
600
500
400
300
200
334bp
100
492bp
Co-culture was carried out either with 500,000 adrenocortical cells and 500,000 immune cells in one well or in one well separated by inserts with a 0.02-um anopore membrane (Nunc). Culture medium was collected for determining hormone concentration after three 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 a Cortisol-RIA (Biermann). LDH activity in culture medium was assessed photometrically after pyruvate reduction.
RESULTS
Immunohistochemistry demonstrates reduced ability to express Fas-receptor but expression of Fas-ligand in adrenocortical tumor cells (Figure la). RT-PCR shows both Fas and Fas-L expression (Figure lb). FACS analysis (Fig. 2) revealed reduction of cell death upon direct co-culture conditions from 49.9% to 13.0% in leukocytes, 8.6% in CD 8- and 15.3% in CD 4-positive cells, while in insert-separated co-culture experiments, leukocyte or lymphocyte death was comparable to controls without cancer cells. LDH release was in general low in co-cultures (1.87±0.149 umol/L*s; meantSEM) compared to basal cancer cells release (4.26±1.11).
After three days, cortisol secretion increased to 190.9 % in leukocyte/cancer cell co-culture, to 197.6 % in CD8- positive/cancer cell co-culture and to 171.0 % in CD4
Annexin V-binding in % of total cells
C
S
50-
75
Immune cells (IC)
Irm Tumor cells
Tumor cells (TC)
IC + TC
IC + TC. sep.
-
CD8 + TO
( D)8 + TC. sep.
Immune cells
CD4 + TO
(D)4 + TC. sep.
LDH in pmol/L*s
0.0
2.5-
5.0-
7.5-
Immune cells (IC)
Tumor cells (TC)
IC + TC
4
IC + TC. sep.
CD8 + TC
CD8 + TC. sep.
CD4 + TC
CD4 + TC. sep.
Cortisol in nmol/L
250
500-
750
0
Immune cells (KC)
Tumor cells (T(*)
IC + TC
4
1C + TC. sep.
1
CD8 + TC
4
CD8 + TC. sep.
H
CD4 + TC
CD4 + TC, sep.
positive/cancer cell co-culture compared to basal secretion. In insert-separated co- cultures, cortisol secretion was only 70-80% of basal secretion (Fig. 2).
DISCUSSION
Malignant adrenocortical tumors are rare and have a poor prognosis. Hence, tumor cells are well endowed with opportunities to evade immunologic mechanisms. Later, their capacity to undergo apoptosis is diminished (10). The expression of Fas-L by cancer cells could help them to kill lymphocytes in a Fas-dependent way (12), and thus allow for an escape from immunologic mechanisms. In this study, we show expression of Fas-L and abrogation of Fas-receptor in adrenocortical tumor cells
potentially conferring them with immune resistance. However, immune cells did not undergo apoptotic cell death in the presence of Fas-L producing tumor cells. In contrast, direct cellular interaction led to increased immune cell survival. This immune cell survival might be due to partial HLA-mismatches within MHC class I antigens between tumor cells and homologous PBMC contributing to immunologic activation. This activation, in turn, may facilitate cytokine production and hence increase adrenocortical hormone secretion. The cortisol increase did not affect immune cell’s survival nor could a cell death promoting effect by MHC class I activated lymphocytes on tumor cells be detected. The phenomenon of immune escape in adrenocortical tumors may be unique and differ from other tumors as follows, (i) the natural interaction between adrenocortical cells and immune system via MHC class II molecules is disrupted (9), (ii) adrenocortical tumor cell line expresses immunolytic agents, cortisol and Fas-L, without utilising cytotoxic effects on immune cells.
This work was supported by grants from BASS to G.W.W., BMBF IZKF B I and a Heisenberg grant to S.R.B.
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