Eur. J. Irnmunol. 1990. 20: 35-40
Magnus Von Knebel Doeberitz", Susanne KochO, Heiko DrzonekO and Harald Zur Hauseno Institut fur Virusforschung/ATVO and Institut fiir Immunologieo, Deutsches Krebsforschungszentrum, Heidelberg
Glucocorticoids reduce h4HC class I expression
35
Glucocorticoid hormones reduce the expression of major histocompatibility class I antigens on human epithelial cells Expression of a critical level of major histocompatibility complex (MHC) class I antigens on epithelial cells is a prerequisite for the action of specific cytolytic immune response cells. Glucocorticoid hormones have strong immunosuppressive effects. Therefore, we investigated the influence of the synthetic glucocorticoid dexamethasone on the expression level of MHC class I antigens on human epithelial cell lines. Long-term treatment with dexamethasone leads to reduced MHC class I surface antigen expression and to decreased total membrane-bound MHC class I protein. The steady-state mRNA level is significantly decreased and the transcription rate of MHC class I genes is reduced.
1 Introduction MHC class I antigens are composed of highly polymorphic transmembranous glycoproteins that are noncovalently associated with P2-microglobulin (P2m) [l]. They play a central role in the regulation of immune functions, e.g., as parts of the target structures recognized by CTL, and mediate cytotoxic responses to virally infected cells [2]. They also represent the target antigens for cytolytic responses in graft injection reactions after tissue heterotransplantation [3]. MHC class I antigens are expressed on almost all nucleated mammalian cells with the exception of neurons and trophoblasts [4, 51. The expression level of these antigens is regulated by various exogenous and endogenous immunomodulating factors. IFN and TNF for example increase the level of MHC class I mRNA [6-111, while certain oncogenic viruses can reduce the MHC class I expression level [12, 131. The expression of a critical threshold level of MHC class I antigens on keratinocytes is necessary for the class I-mediated immune functions, e.g., the lytic attack of CTL [14]. Glucocorticoid hormones are potent anti-inflammatory and immunosuppressive agents. They are part of almost all therapeutic protocols to suppress rejection reactions in organ transplant recipients. They have an anti-proliferative effect on in vim-stimulated lymphocytes [15-17] and interfere with the synthesis of various mediators of inflammation, e.g., prostaglandins or IL [18-201. The exact mechanisms of their immunosuppressive action, however, is still not well understood. Glucocorticoid hormones activate the transcription of many genes by binding as a ligand-receptor complex to certain DNA target sequences (glucocorticoid responsive element; GRE) in regulatory elements of the respective genes [21]. They can also reduce the expression of various genes [22-261. Among immunoregulatory proteins the
[I 79451 Correspondence: Magnus Von Knebel Doeberitz, Institut fiir Virusforschung/AW, Deutsches Krebsforschungszentrm, Irn Neuenheirner Feld 506, D-6900 Heidelberg, FRG Abbreviation: DXM: Dexarnethasone 0 VCH Verlagsgesellschaft rnbH, D-6940 Weinheirn, 1990
expression of the IL 1P is inhibited by glucocorticoid hormones both by reduced transcription and stability of the mRNA [20]. In murine macrophages and B cells glucocorticoids inhibit Ia antigen expression at the level of mRNA transcription [27, 281. Immunofluorescence assays and RIA revealed that glucocorticoid hormones decrease the level of MHC class I antigens on human PBL by approximately 15% [29]. Since a reduction of the MHC class I antigen expression on epithelial cells by glucocorticoid hormones might have important implications for their immunosuppressive effects, we investigated the action of dexamethasone (DXM) on the MHC class I expression on various human epithelial cell lines. Experiments described here reveal that DXM significantly decreases the expression of MHC class I antigens on human epithelial cell lines. Decreased expression is accompanied by reduction of the mRNA in the cytoplasm; however, its stability is not affected at any detectable extent. Nuclear "run-on" experiments revealed that DXM leads to a decreased transcription rate of MHC class I genes.
2 Materials and methods 2.1 Cell l i e s and cell culture C4-I [30], HT-3 [31], SiHa [32], Me180 [33], Caski [34], A431 [35] and SW756 cell lines [36] were derived from human genital squamous carcinomas. The HaCaT cells represent a nontumorigenic epithelial cell line with a capacity to differentiate in vivo [37]. All cells were cultured in DMEM (Gibco, Eggenstein, FRG) supplemented with 10% FCS, 40 IU of penicillin and 50 pg of streptomycidml at 37"C, 5% C02. Where indicated, DXM (Sigma, Miinchen, FRG) was used at a final concentration of 1 PM. Cycloheximide (Boehringer Mannheim, Mannheim, FRG) and actinomycin D (Sigma) were used at a final concentration of 50 pg/ml and 10 pg/ml, respectively. 2.2 Immunoprecipitation assay Immunoprecipitation was performed as described [38]. C4-I cells were grown for 1week either in the presence or 0014-2980/90/0 101-0035$02.50/0
36
M.Von Knebel Doeberitz, S. Koch, H. Drzonek and H. Zur Hausen
absence of DXM. Cells were washed twice with methionine-free DMEM and incubated with this medium for 30 min at 37°C. Cells were labeled with 0.1 mCi/ml= 3.7 MBq/ml [35S]-methioninefor 90 min at 37 "C and then harvested with a rubber policeman and resuspended in Tris-buffered saline (TBS) containing 1% NP40, 1mM PMSF and 1pg/ml aprotinin. The total radioactivity of the labeled cell lysates was determined and equal amounts of labeled proteins were subjected to immunoprecipitation. Cell lysate was preabsorbed with protein A-coated Sepharose beads (Pharmacia, Uppsala, Sweden) for 2 h at 4°C. The beads were removed and the SN was incubated with the respective antibody and fresh protein A-Sepharose at 4 "C overnight. The beads were then washed twice in TBS, 0.25% NP40 and subsequently resuspended in SDS sample buffer. Precipitated material was analyzed on a polyacrylamide gel (7.5% -15% acrylamide). After electrophoresis the gel was treated with DMSO (Roth, Karslruhe, FRG), 20% (w/v) diphenyloxazole (Roth) and water as described [39], dried and exposed to X-ray films.
2.3 Immunoslot blot analysis A quantitative immunoslot blot assay has been described recently [ a ] . Briefly, 10 pg of membrane protein was loaded onto nitrocellulose filters and incubated with the W6/32 mAb. Subsequently the filters were washed, incubated with lzI-labeled protein A and exposed to X-ray films. The signal obtained was quantified by densitometry in an LKB scanner (LKB-Pharmacia, Freiburg, FRG). 2.4 RNA analysis Cytoplasmic RNA was extracted as described [41]. For Northern blot and RNA slot blot analysis, 10 pg of cytoplasmic RNA was either separated in non-denaturing 1% agarose gels in MOPS buffer (20 mM MOPS, 5 m~ sodium acetate, 1m~ EDTA) and transferred onto Gene Screen Plus filters (NEN, Boston, MA) or loaded directly onto nitrocellulose filters (Schleicher and Schiill, Dassel, FRG) using the minifold slot blot device (Schleicher and Schull). The filters were hybridized with a 32P-labeled cDNA probe [42] for the MHC class1 antigen HLA-B8 (kindlyprovided by Dr. E.Weiss, Munich, FRG). RNA slot blot filters were also hybridized with a probe for ribosomal RNA [43] to assure that indeed comparable amounts of cytoplasmic RNA were bound to the filter. To evaluate the relative amount of MHC class I mRNA autoradiograms of RNA slot blot filters were evaluated by densitometry in an LKB scanner. 2.5 Nuclear run-on analysis
Cells were grown for 1week either with or without DXM. They were then washed twice with ice-cold PBS and harvested by scraping with a rubber policeman. They were lysed in 1 O m ~Tris-HC1, pH8.0, 10 mM NaCl, 3 mM MgC12, 0.5% NP40, 100 pM PMSF on ice and the nuclei were pelleted by centrifugation at 4000 x g for 5 min at 4 "C. Nuclei were washed twice in storage buffer (50 mM TrisHC1, pH 8.0, 40% glycerol, 5 m~ MgC12, 100 p~ EDTA, 100 p~ PMSF) and then stored at a concentration of 2 x lo7
Eur. J. Immunol. 1990. 20: 35-40
nuclei1100 p1 storage buffer in liquid nitrogen. The nuclear run-on assay was performed with modifications as described [44]. Briefly, 2 x lo7nuclei were incubated in 5 mM Tris-HC1,pH 8.0, 2.5 mM MgC12 ,. 0.15 mM KCl, 0.25 mM ATE GTF', CTP and 250 pCi 32P-labeledUTP (10 mCi/ml, 400 Cilmmol, Amersham, Braunschweig, FRG) for 60 min at 28°C. Cellular DNA was digested with DNase I (Worthington Diagnostics, Freehold, NJ) at a final concentration of 20 pg/ml for 5 min at room temperature. To terminate the reaction 200 p1 of 1% SDS, 10 mM EDTA and 20 mM Tris-HC1, pH 7.4 was added. Proteins were digested .in this mixture for 1 h with proteinase K (100 pg/ml) at 37°C and then extracted with phenolkhloroform. The radiolabeled RNA was precipitated in 2.5 M ammonium acetate and isopropanol. After dissolving it in 10mM Tris-HC1, pH 7 . 4 , l mM EDTA the RNA was purified on a Sephadex G-50 fine (Pharmacia, Freiburg, FRG) column from remaining free nucleotides. The radioactivity of the eluted RNA fraction was determined and comparable amounts of labeled RNA (106cpm/ml) were used as hybridization probe [45] for nitrocellulose filters with 10 pg of the respective plasmid DNA and 2 pg of total cellular DNA. Following hybridization for 2 days the filters were washed three times for 30 min at 70°C in 2 x SSC, 0.1% SDS and then exposed to X-ray films.
3 Results 3.1 Reduction of the MHC class I antigens on epithelial cells
To investigate the influence of glucocorticoid hormones on the expression level of the MHC class1 antigens on epithelial cells immunofluorescence studies were performed on C4-I cervical squamous carcinoma cells with W6132 mAb [46]. DXM-treated cells showed an up to 40% reduction of the cell surface expression of MHC class I antigens as determined by immunofluorescence analysis (Table 1). The reduced expression of the MHC class I antigens on C4-I cells is not due to a generalized inhibition of protein synthesis in DXM-treated cells, since expression of other membrane antigens, e.g., the receptor for the epidermal growth factor (EGF), is even enhanced [40] while still other antigens (e.g., the HEA125 marker) are not significantly affected by the hormone. Furthermore, DXM has strong growth-stimulating effects on C4-I cells 1471. Fluorescence microscopy of C4-I cells stained with the W6132 mAb and a fluorescence-labeled anti-mouse lg antibody revealed that most of the MHC class I antigens remain in the intracellular compartment. This is in line with reports that cell surface expression of MHC antigens is significantly disturbed in malignant cell lines [48]. Therefore, surface immunofluorescence studies on living cells might significantly underestimate the degree of reduction. Immunoprecipitation assays for the MHC class I antigens with the W6132 mAb confirmed the specific reduction of the 43-kDa MHC class I antigen in DXM-treated cells (Fig. 1). The antigen recognized by the HEA125 mAb [49] is not significantly influenced by DXM treatment.
Eur. J. Immunol. 1990. 20: 35-40
Glucocorticoids reduce MHC class I expression
Table 1. Reduction of MHC class I antigen expression on C4-I cellsa)
MI(CCII
37
Pmc
C4-I
Hours of DXM treatment
Mean fluorescence (channel numbers) W6/32 antibody
dex. U-1
(% )
dox.
c
.-0
4-
5
C4-1
0 18
48 62 120
1126 1008 859
100 89
738
65 59
660
76
dex. C4-1
n
dox. C4-1
dox.
a) C4-I cells were cultured for the time periods indicated in the presence of 1 p~ DXM.The cells were harvested and stained for 60 min with the W6132 mAb on ice. After three washes with PBS 0.1% BSA, bound mouse Ig was detected by staining for 60 min with a FITC-conjugated goat anti-mouse antiserum (Dianova, Hamburg, FRG). After three washes with PBS0.1% BSA mean fluorescence of cells was determined using a Becton Dickinson (Mountain View, CA) FACS III.
Immunoprecipitation assays, however, do not allow precise quantitation of specific proteins. Therefore, a quantitative immunoslot blot assay was used to determine the rate of reduction of the total cellular amount of MHC class I antigen in C4-I cells (Fig. 2). DXM treatment led to an about tenfold reduction of the total MHC class I antigen content in C4-I cells. A comparable reduction of the MHC class I antigen expression was observed in other epithelial cell lines (e.g., HT-3, HaCaT, SW756, SiHa, ME180; data not shown). Using the same assay, the HEA125 marker was not influenced by hormone treatment,while other antigens, e.g. the receptor for the EGF, were expressed at significantly increased rates [40].
dax.
Figure 2. Amount of MHC class I antigen on membranes of DXM-treated or untreated cells. Membrane proteins (10 pg) of C4-I cervical cancer cells either treated (1 p ~ B) , or not (0)with DXM for 1 week in five independent experiments was loaded on a nitrocellulose filter and incubated with the W6/32 mAb directed against the MHC class I antigens and subsequently with Iz5Ilabeled protein A. Densitometric evaluation of an autoradiogram of this filter revealed that upon DXM treatment about tenfold less radioactivity was bound to the membrane preparations in this assay. The average value and the standard deviation of five independent experiments are given. As positive control about 10 pg of membrane protein of A431 cells either treated or not with 1 p~ DXM was loaded on the same filter. No decrease of the MHC C1-I content upon DXM treatment was observed for this cell line. A second filter (Prot.) with identical amounts of the respective protein preparations was stained with Coomassie blue. It indicates that comparable amounts of protein were loaded in each slot.
3.2 DXM reduces the steady-state level of MHC class I mRNA
To investigate the influence of DXM on the MHC class I mRNA steady-state level, cytoplasmic RNA was extracted from treated and untreated cells and subjected to quantitative RNA slot blot analysis [50] using a 32P-labeledcDNA of the HL,A-B8 antigens as probe. DXM treatment for 1week led to decreased steady-state levels of MHC class I transcripts in C4-I, HT-3, HaCaT, SW 756, SiHa and ME180 cells. However, no reduction of MHC class I transcripts was observed in Caski and A431 cells (Fig. 3). Since other DXM-regulated proteins (e.g., the E GF receptor) are also not influenced in these cells, this is most likely due to the lack of glucocorticoid receptors in these cells.
Figure 1. Immunoprecipitation of MHC class I antigens in hormone-treated and untreated cells. C4-I cells were cultured for 1 week in the absence or presence of 1 p~ DXM. After labeling of cellular proteins with [35S]methioninefor 90 min, the MHC class I antigens were immunoprecipitated as described in the method section with the W6132 mAb or the HEA125 mAb and analyzed on a polyacrylamide gel. In DXM-treated cells (dex.) significantly less labeled 43-kDa protein was precipitated with the W6132 mAb compared t o untreated controls. The P2m is linked to the MHC class I antigens and, therefore, coprecipitated with the MHC class I molecules.The same quantitative relation is, thus, observed for this 12-kDa protein. With the HEA125 rnAb about the same amount of antigen was precipitated in treated or untreated cells.
Down-regulation of the MHC class1 mFWA is dose dependent. Already at DXM concentrations of M there is a reduction of the mRNA detectable. At concentrations of M DXM in the culture medium reduction of MHC class I expression reaches its maximum extent. The new reduced equilibrium state state of MHC class I mRNA levels is reached approximately 36-48 h after onset of treatment. Twenty-four hours after hormone application the steady-state level was reduced by 50% (data not shown). Inhibition of transcription of some genes of DXM was shown to be mediated directly by cis-acting elements [51-551. However, inhibition of Ia antigen expression on murine B lymphocytes by DXM was reported to be mediated by a short-lived transacting factor [20].
Eur. J. Immunol. 1990.20: 35-40
M. Von Knebel Doeberitz, S. Koch, H. Drzonek and H. Zur Hausen
38 1
0
C4-1
HT-3
HaCaT
SW 756
SiHa
ME 180
Cask1 A431
Figure 3. Steady-state level of MHC class I mRNA in cells either ) or not ( 0 )with DXM. Cytoplasmic RNA of epithelial cell lines cultured with or without DXM (1 pM) for 1week was loaded on nitrocellulose filters and hybridized with a 3*P-labeled probe for the MHC class I HLA-B8 cDNA. A parallel filter of the same RNA was hybridized with a probe for ribosomal RNA [43]. The relative amount of MHC class I mRNA in DXM-treated cells was calculated by dividing the signal intensity of the hormone-treated cells by the untreated controls after standardization against ribosomal RNA.The relative reduction of the MHC class I transcripts in the hormone-treated cells is shown.
To investigate whether a short-lived transacting factor might also be involved in glucocorticoid-mediated reduction of MHC class I antigen expression, cells were treated with DXM and cycloheximide, a known protein-synthesis inhibitor acting at the level of translation. Cells were either cultured with DXM and cycloheximide for 6 h or treated for lweek with DXM and then for 6 h with DXM and cycloheximide. RNA was extracted from the respective cells and the amount of MHC class I transcripts was estimated from Northern blots (Fig. 4). Long-term treatment for 1 week with DXM reduces the amount of MHC class I transcripts. Short-term treatment for only 6 h has no significant influence on the MHC class I mRNA steady-state level. Cycloheximide treatment for 6 h leads to an increased MHC class I mRNA steady-state level in C4-I cells. In cells pretreated with DXM for 1 week application of cycloheximide for 6 h also led to an increase
xI xI 0
c (0
d 0
+
J
J
0
P
0 P
c
c
c
(D
P)
0
(0
0
c (0
+ J
9)
0 Y P)
t
of the cytoplasmic mRNA level compared to cells treated for 1 week with DXM. However, it did not reach the level of untreated cells. Cycloheximide is highly toxic if cells are treated for longer then 6 h. Therefore, cycloheximide experiments could not be extended and it is not possible to differentiate between direct or indirect effects of DXM on MHC class I mRNA levels mediated by proteins with a longer half-life time. The increased steady-state levels of MHC class I mRNA upon cycloheximide treatment of either hormone-treated or untreated cells suggests that expression of MHC class I genes might possibly be regulated by labile transacting factors in these cells. Evidence for negative regulating factors for MHC class I gene expression has been given by other experimental systems [56]. The steady-state level of other transcripts in C4-I cells is not affected by cycloheximide ([57] and Von Knebel Doeberitz, unpublished results).Thus, there is no evidence that cycloheximideleads to increased levels of class I transcripts by an unspecific stabilization of mRNA as has been described in other experimental systems [58]. 3.3 Stability of MHC class I transcripts in C 4 I cells in the presence or absence of DXM
To exclude an increased degradation of the MHC class I mRNA in the cytoplasm under DXM treatment, the half-life time of the mature MHC class I transcripts in the cytoplasm of C4-I cells was analyzed by inhibiting the “de novo” synthesis of mRNA with actinomycin D.The amount of MHC class I mRNA was then analyzed at various time intervals after application of actinomycin D. Cells were seeded in 10-cm culture dishes and cultured either in the presence or absence of the hormone for 1week. Actinomycin D was then added and RNA was extracted 90, 180, 320 and 420 min after actinomycin D treamtent. After > 420 min following application of the RNA synthesis inhibitor toxic effects on the cells became detectable, thus no further extension of actinomycin D treatment was possible. Northern blots were prepared and hybridized with the MHC class I probe. To control for the actinomycin D effect, the same blots were washed and rehybridized with a 32P-labeledprobe for the third exon of the c-myc gene [59] since the c-myc mRNA has a relatively short half-life time [60]. No significant reduction of the
r
CHX 50 pp/ml
dex. 1 pY a ea
180 320 420
a
90 180
320 420
0
80 180 320 420 mln. acllnomycln D
MHC CI I
pmyc exm 111
MHC CI I
Figure 5. Stability of MHC class I transcripts in hormone-treated and untreated cells. RNA was extracted from C4-I cells either Figure 4. Effect of cycloheximide (CHX.) on DXM (dex.)-treated treated with DXM (dex.) (1 p ~ or ) cycloheximide (CHX., and untreated cells. Cytoplasmic RNA of C4-I cells treated with 50 pg/ml) or not at 0, 90, 180, 320, 420 min after application of cycloheximide (50 pg) for 6 h, DXM (1 p ~for ) 6 h, DXM (1 p ~ ) actinomycin D (10 pglml). Northern blots were prepared as deand cycloheximide (50 pg/ml) for 6 h, DXM for 1 week as well with scribed in Sect. 2.4 and hybridized first with the MHC class1 DXM (1 PM) for 1week and cycloheximide (50 pg) for 6 h was probe. After autoradiography, the probe was completely washed extracted and anlyzed on a Northern blot after electrophoresis in a off at the melting temperature of DNA (Tm -0°C) as proposed by 1% nondenaturing MOPS agarose gel. The filter was hybridized the manufacturer, and the filters were rehybridized with a probe for with a probe for the MHC class I transcripts. the third exon of the c-myc gene.
Eur. J. Immunol. 1990. 20: 35-40
Glucocorticoids reduce MHC class I expression
MHC class I transcripts could be observed within 420 min (Fig. 5). DXM or cycloheximide treatment did not alter the half-life time of the MHC class I mRNA to the detectable extent. However, in DXM-treated cells the MHC class I probe gave a weaker signal compared to cycloheximide or untreated controls. Hybridization of the same filters with the c-myc probe revealed as expected that no c-myc transcripts could be detected after 90 min of actinomycin D treatment in DXM or untreated cells. In cycloheximidetreated cells, however, the half-life time of the c-myc gene was prolonged to about 120 min leading to increased levels of the steady-state level of c-myc mRNA.This is consistant with similar observations made in other cell systems
WI. These findings indicate that the MHC class I transcripts in the cytoplasm are very stable.The DXM- or cycloheximideinduced alterations of the MHC class I mRNA steady-state level is, thus, not due to significant alterations of the half-life time as far as it can be judged within the time range of 420 min. Alterations of the half-life time beyond 420 min cannot be excluded with this assay. 3.4 DXM treatment leads to decreased transcription of
the MHC class I genes To investigate the influence of DXM on the transcription rate of the MHC class I genes nuclear run-on experiments were performed. C4-I cells were cultured for 1week either in the presence or absence of DXM. Nuclei were prepared and nuclear run-on experiments were performed. 32P-labeledand in vitro synthesized RNA of nuclei of C4-I cells which were either treated or not with the hormone for 1week was hybridized to nitrocellulose filters with 10 pg of the respective plasmid DNA. Autoradiograms of these filters were scanned. In three independent experiments DXM treatment reduced transcription of the MHC class I gene by about 50% compared to untreated controls (Fig. 5). Despite the high steady-state level of MHC class I transcripts in the cytoplasm, the transcription rate as determined with nuclear run-on experiments in our hands appeared to be relatively low. 200,
dex.
WC class I cell. DNA
dex.
Figure 6. Nuclear transcription rate of the MHC class I genes in DXM (dex.)-treated or untreated cells. Nuclear run-on assays were performed as outlined in Sect. 2.5. 32P-labeledin vitro synthesized nuclear RNA was hybridized to nitrocellulose filters with 10 pg of plasmid DNA of the MHC class I B8 cDNA and 2 pg of total cellular DNA (HeLa).The signal intensity of the respective slots was scanned. To calculate the reduction or increase of the respective signals, the intensity of the signal obtained with total cellular DNA served as reference. The signal for class I transcription in untreated cells was set as 100 and the degree of downregulation in hormone-treated cells was calculated.The SD of three independent experiments is given.
39
4 Discussion The experiments described here reveal that DXM reduced MHC class I gene expression in various human epithelial cell lines. The phenomenon was observed in all lines tested which express functional glucocorticoid receptors. Downregulation of MHC class I antigens by DXM was accompanied by a reduced steady-state level of the mRNA in the cytoplasm. After application of the hormone it took a rather long-time interval of about 48 h until the final reduced steady-state level was reached. Cycloheximide treatment led to an about twofold increase of the MHC class I mRNA steady-state level suggesting that labile protein factors either repressing transcription or enhancing degradation might possibly be involved in the regulation of MHC class I antigen expression. Inhibition of the synthesis of such a factor by cycloheximide might lead to increased mRNA levels. In cells treated for 1week with DXM addition of cycloheximide for 6 h also led to an about twofold increase of the MHC class I mRNA. This might be due to inhibition of synthesis of a DXM-induced transacting repressor acting either on the transcription or degradation of MHC class1 mRNA or to a DXM-independent mechanisms as observed in hormone-untreated cells. The half-life time of the MHC class I mRNA in DXMtreated cells and untreated controls is rather long, since no significant reduction was observed even more than 6 h after application of actinomycin D. DXM or cycloheximide had no detectable influence on the stability of the MHC class I mRNA within this time range. These data suggested that alterations of the MHC class I mRNA steady-state level might rather be due to variations of the transcription initiation or elongation rates than to a significantly decreased stability of the mRNA in the cytoplasm upon DXM treatment. Repeated nuclear run-on experiments clearly indicated a reproducible reduction of about 50% of the transcription rate of the MHC class I genes in DXM-treated C4-I cells. The transcription rate determined by nuclear run-on assays appeared to be relatively low compared to other genes.The high steady-state level of the mRNA in the cytoplasm can, therefore, be attributed to a slow degradation rate of mature transcripts. This is in good agreement with the longevity of the MHC class I mRNA. Small alterations of the transcription rate may, thus, lead to significant changes of the steady-state level of mRNA after a prolonged period of time. Similarly, small variations of the degradation rate might also lead to significantly reduced steady-state levels. Such changes might not be detectable with the actinomycin D assay and can, therefore, not be excluded on the basis of these experiments. Isolation and detailed characterization of the glucocorticoid-responsive regulatory squences of MHC class I genes is necessary to analyze the exact mechanism of DXM-mediated down-regulation of MHC class I antigen expression. The data presented here may explain some aspect of the immunosuppressive actions of glucocorticoid hormones, since expression of a critical threshold level of MHC class I antigens on epithelial target cells is an obligatory prerequisite for MHC class I-mediated immune functions [14]. Even if there is no complete down-regulation of the MHC class I
40
M.Von Knebel Doeberitz, S. Koch, H. Drzonek and H. Zur Hausen
expression, these findings clearly demonstrate t h e potential role of glucocorticoid hormones t o modulate t h e expression level of MHC antigens on epithelial cells, thereby maybe interfering with MHC class I-independent reactions, e.g., t h e attack of specific CTL. For critical reading of the manuscript and helpful suggestions we wish to thank E. Schwarz, J. Wolf and H . Jakobsen.
Received September 8, 1989.
5 References 1 Ploegh, H. L., Orr, H.T. and Strominger, J. L., Cell 1981.24: 287. 2 Doherty, I? C., Blanden, R. V and Zinkernagel, R. B., Transplant. Rev. 1976. 29: 89. 3 Zinkernagel, R. M. and Doherty, P. C., Adv. Immunol. 1979. 27: 51. 4 Faulk,W. D., Sanderson, A. R. and Temple, A., Transplant. Proc. 1977. 9: 1379. 5 Lampson, L. A., Fisher, C. A. and Whelan, J. I?, J. Immunol. 1983. 130: 2471. 6 Lindahl, P., Leary, I? and Gresser, I., Eur. J. Imrnunol. 1974.4: 779. 7 Imai, K., Pellegrino, M. A., Ng, A. K. andFerron, S., Scand. J. Immunol. 1981. 14: 529. 8 Burrone, 0. H. and Milstein, C., EMBO J. 1982. I : 345. 9 Satz, M. and Singer, D. S., J. Immunol. 1984. 132: 496. 10 Friedman, R. L. and Stark, G. R., Nature 1985. 314: 637. 11 Collins, T., Lapierre, L. A., Fiers, W., Strominger, J. L. and Pober, J. S., Proc. Natl. Acad. Sci. USA 1986. 83: 446. 12 Meruelo, D., Kornreich, R., Rossomondo, A., Pampeno, C., Mellor, A. L., Weis, E. H., Flavell, R. A. and Pellicier, A., Proc. Natl. Acad. Sci. USA 1984. 81: 1804. 13 Miyazaki, J., Appella, E. and Ozato, K., Proc. Natl. Acad. Sci. USA 1986. 83: 9537. 14 Niederwieser, D., Aubock, J., Troppmair, J., Herold, M., Schuler, G., Boeck, G., Lotz, J., Fritsch, I? and Huber, C., J. Irnmunol. 1988.140: 2556. 15 White, A. and Makman, M. H., Adv. Enzyme Res. 1966. 5: 317. 16 Ono, T., Terayama, H., Takaku, F. and Nakao, K., Biochim. Biophys. Acta 1968. 161: 361. 17 Heilman, D. H. and Lechner, J. I?, in Schwarz, M. R. (Ed.), Proceedings of the 6th Leucocyte Culture Conference, Academic Press, London, New York 1972, p. 581. 18 Flower, R. J. and Blackwell, G. J., Nature 1979. 278: 456. 19 Hirata, F., Schiffman, E.,Venhatasubramanian, K., Salomon, D. and Axelrod, J., Proc. Natl. Acad. Sci. USA 1980. 77: 2533. 20 Lee, S.W. ,Tsou, A. P., Chan, H. ,Thomas, J., Petrie, K., Eugui, E. M. and Allsison, A. C., Proc. Natl. Acad. Sci. USA 1988.85: 1204. 21 Beato, M., Biochim. Biophys. Acta 1987. 910: 95. 22 Eberweine, J. H. and Roberts, J. L., J. Biol. Chem. 1984.259: 2166. 23 Turcotte, B., Gueqtin, M., Chevrette, M. and Belanger, L., Nucleic Acids Res. 1985. 13: 2387. 24 Camper, S. A.,Yao,Y. A, S. and Rothman, F. M., J. Biol. Chem. 1985. 260: 12246. 25 Nechustan, H., Benvenisty, N., Brandeis, R. and Reshef, L., Nucleic Acids Res. 1987. 15: 6405. 26 Adler, S. ,Waterman, M. L., He, X. and Rosenfeld, M. G., Cell 1987. 52: 685. 27 Fertsch-Ruggio, D., Schoenberg, D. R. and Vogel, S. N., J. Immunol. 1988. 141: 1582. 28 McMillan,V M., Dennis, G. J., Glimcher, L. H., Finkelman, E D. and Mond, J. J., J. Immunol. 1988. 140: 2549. 29 Hokland, M., Larsen, B.. Heron, I. and Plesner,T., Clin. Exp. Immunol. 1981. 44: 239.
Eur. J. Immunol. 1990. 20: 35-40
30 Auersperg, N . and Hawryluk, A. I?, J. Natl. Cancer Inst. 1962. 28: 605. 31 Fough, J. andTrempe, G., inFogh, J. (Ed.), Humanturnorcells in vitro, Plenum Press, New York, London 1975, p. 115. 32 Friedl, F., Kimura, I., Osato, T. and Ito,Y., Proc. SOC. Enp. Biol. Med. 1970. 135: 543. 33 Sykes, J. A., Whiteseaver, J., Jernstrom, I?, Nolan, F. J. and Byatt, P., J. Natl. Cancer Inst. 1970. 45: 107. 34 Patillo, R. A., Hussa, R. O., Sory, M. T., Ruckert, A. C. F., Shalaby, M. R. and Mattingly, R. E., Science 1977. 196: 1456. 35 Giard, D. J., Aaronson, S. A., Todaro, G. J., Amstein, I?, Kersey, J. H., Dosik, H. and Parks, W. I?, J. Natl. Cancer Inst. 1973. 51: 1417. 36 Freedman, R. S., Bowen, J. M., Leibovitz, A., Pathak, S., Siciliano, M. J., Gallagher, H. S. and Giovanella, B. C., In Vitro1982. 18: 719. 37 Boukamp, I?, Petrussevska, R.T., Breitkreuz, D., Hornung, J., Markham, A. and Fusenig, N., J. Cell Biol.. 1988. 106: 761. 38 Koch, S., Schultz, A. andKoch, N., J. Immunol. Methods 1987. 103: 211. 39 Bonner, W. M. and Laskey, R. A., Eur. J. Biochern. 1974.46: 83. 40 Von Knebel Doeberitz, M., Drzonek, H., Koch, S. and Becker, C. M., J. Immunol. Methods 1989. 122: 259. 41 De Villiers, J. and Schaffner, W., in Flavell, R. A. (Ed.), Techniques in the Life Sciences, B5, Nucleic acid biochemistry, Elsevier-Ireland, Dublin 1983, p. 1. 42 Feinberg, A. and Vogelstein, B., Anal. Biochem. 1984. 137: 266. 43 Rungger, D., Ackerman, H. and Crippa, M., Proc. Natl. Acad. Sci. USA 1979. 76: 3957. 44 Linial, M., Gunderson, N. and Groudine, M., Science 1985. 230: 1126. 45 Melton, D. A., Krieg, I? A,, Rebagliati, M. R., Maniatis, T., Zinn, K. and Green, M. R., Nucleic Acids Res. 1984. 12: 7035. 46 Barnstable, C. J., Bodmer, W. F., Brown, G., Galfrk, G., Milstein, C., Williams, A. F. and Ziegler, A., Cell 1978. 14: 9. 47 Von Knebel Doeberitz, M., Oltersdorf, T., Schwarz, E. and Gissmann, L., Cancer Res. 1988. 48: 3780. 48 Durrant, L. G., Ballantyne, K. C., Armitage, N. C., Robins, R. A., Marksman, R., Hardcastle, J. D. and Baldwin, R. W., Br. J. Cancer 1987. 56: 425. 49 Moldenhauer, G., Momburg, E, Moller, €!, Schwa-, R. and Hammerling, G., Br. J. Cancer 1987. 56: 714. 50 Gasser, C. S., Simonsen, C. C., Schilling, J. W. and Schimke, R. T., Proc. Natl. Acad. Sci. USA 1982. 79: 6522. 51 Israel, A. and Cohen, S. N., Mol. Cell. Biol. 1985. 5: 2443. 52 Charron, J. and Dourin, J., Proc. Natl. Acad. Sci. USA 1986. 83: 8903. 53 Weiner, F. R., Czaja, M. J., Jefferson, D. M, Giambrone, M. A., Kaspa, R. T., Reid, L. M. and Zern, M. A., J. Biol. Chem. 1987. 262: 6955. 54 Guertin, M., LaRue, H., Bernier, D., Wrange, O., Chevrette, M., Gingras, M. C. and Belanger, L., Mol. Cell. Biol. 1988.8: 1398. 55 Akerblom, I. E., Slater, E. I?, Beato, M., Baxter, J. D. and Mellon, I? L., Science 1988. 241: 350. 56 Ehrlich, R., Maguire, J. E. and Singer, D. S., Mol. Cell. Biol. 1988. 8: 695. 57 Kleiner, E., Dietrich, W. and Pfister, H., EMBO J. 1986. 5: 1945. 58 Stiles, C. D., Lee, K. L. and Kenney, F. T., Proc. Natl. Acad. Sci. USA 1976. 73: 2634. 59 Battey, J., Moulding, C., Taub, R., Murphy, W., Stewart, T., Potter, H., Lenoir, G. and Leder, I?, Cell 1983. 34: 779. 60 Dani, C., Blanchard, J., Piechaczyk, M., El Sabouty, S., Marty, L. and Jeanteur, P., Proc. Natl. Acad. Sci. USA 1984. 81: 7046.
Magnus Von Knebel Doeberitz", Susanne KochO, Heiko DrzonekO and Harald Zur Hauseno Institut fur Virusforschung/ATVO and Institut fiir Immunologieo, Deutsches Krebsforschungszentrum, Heidelberg
Glucocorticoids reduce h4HC class I expression
35
Glucocorticoid hormones reduce the expression of major histocompatibility class I antigens on human epithelial cells Expression of a critical level of major histocompatibility complex (MHC) class I antigens on epithelial cells is a prerequisite for the action of specific cytolytic immune response cells. Glucocorticoid hormones have strong immunosuppressive effects. Therefore, we investigated the influence of the synthetic glucocorticoid dexamethasone on the expression level of MHC class I antigens on human epithelial cell lines. Long-term treatment with dexamethasone leads to reduced MHC class I surface antigen expression and to decreased total membrane-bound MHC class I protein. The steady-state mRNA level is significantly decreased and the transcription rate of MHC class I genes is reduced.
1 Introduction MHC class I antigens are composed of highly polymorphic transmembranous glycoproteins that are noncovalently associated with P2-microglobulin (P2m) [l]. They play a central role in the regulation of immune functions, e.g., as parts of the target structures recognized by CTL, and mediate cytotoxic responses to virally infected cells [2]. They also represent the target antigens for cytolytic responses in graft injection reactions after tissue heterotransplantation [3]. MHC class I antigens are expressed on almost all nucleated mammalian cells with the exception of neurons and trophoblasts [4, 51. The expression level of these antigens is regulated by various exogenous and endogenous immunomodulating factors. IFN and TNF for example increase the level of MHC class I mRNA [6-111, while certain oncogenic viruses can reduce the MHC class I expression level [12, 131. The expression of a critical threshold level of MHC class I antigens on keratinocytes is necessary for the class I-mediated immune functions, e.g., the lytic attack of CTL [14]. Glucocorticoid hormones are potent anti-inflammatory and immunosuppressive agents. They are part of almost all therapeutic protocols to suppress rejection reactions in organ transplant recipients. They have an anti-proliferative effect on in vim-stimulated lymphocytes [15-17] and interfere with the synthesis of various mediators of inflammation, e.g., prostaglandins or IL [18-201. The exact mechanisms of their immunosuppressive action, however, is still not well understood. Glucocorticoid hormones activate the transcription of many genes by binding as a ligand-receptor complex to certain DNA target sequences (glucocorticoid responsive element; GRE) in regulatory elements of the respective genes [21]. They can also reduce the expression of various genes [22-261. Among immunoregulatory proteins the
[I 79451 Correspondence: Magnus Von Knebel Doeberitz, Institut fiir Virusforschung/AW, Deutsches Krebsforschungszentrm, Irn Neuenheirner Feld 506, D-6900 Heidelberg, FRG Abbreviation: DXM: Dexarnethasone 0 VCH Verlagsgesellschaft rnbH, D-6940 Weinheirn, 1990
expression of the IL 1P is inhibited by glucocorticoid hormones both by reduced transcription and stability of the mRNA [20]. In murine macrophages and B cells glucocorticoids inhibit Ia antigen expression at the level of mRNA transcription [27, 281. Immunofluorescence assays and RIA revealed that glucocorticoid hormones decrease the level of MHC class I antigens on human PBL by approximately 15% [29]. Since a reduction of the MHC class I antigen expression on epithelial cells by glucocorticoid hormones might have important implications for their immunosuppressive effects, we investigated the action of dexamethasone (DXM) on the MHC class I expression on various human epithelial cell lines. Experiments described here reveal that DXM significantly decreases the expression of MHC class I antigens on human epithelial cell lines. Decreased expression is accompanied by reduction of the mRNA in the cytoplasm; however, its stability is not affected at any detectable extent. Nuclear "run-on" experiments revealed that DXM leads to a decreased transcription rate of MHC class I genes.
2 Materials and methods 2.1 Cell l i e s and cell culture C4-I [30], HT-3 [31], SiHa [32], Me180 [33], Caski [34], A431 [35] and SW756 cell lines [36] were derived from human genital squamous carcinomas. The HaCaT cells represent a nontumorigenic epithelial cell line with a capacity to differentiate in vivo [37]. All cells were cultured in DMEM (Gibco, Eggenstein, FRG) supplemented with 10% FCS, 40 IU of penicillin and 50 pg of streptomycidml at 37"C, 5% C02. Where indicated, DXM (Sigma, Miinchen, FRG) was used at a final concentration of 1 PM. Cycloheximide (Boehringer Mannheim, Mannheim, FRG) and actinomycin D (Sigma) were used at a final concentration of 50 pg/ml and 10 pg/ml, respectively. 2.2 Immunoprecipitation assay Immunoprecipitation was performed as described [38]. C4-I cells were grown for 1week either in the presence or 0014-2980/90/0 101-0035$02.50/0
36
M.Von Knebel Doeberitz, S. Koch, H. Drzonek and H. Zur Hausen
absence of DXM. Cells were washed twice with methionine-free DMEM and incubated with this medium for 30 min at 37°C. Cells were labeled with 0.1 mCi/ml= 3.7 MBq/ml [35S]-methioninefor 90 min at 37 "C and then harvested with a rubber policeman and resuspended in Tris-buffered saline (TBS) containing 1% NP40, 1mM PMSF and 1pg/ml aprotinin. The total radioactivity of the labeled cell lysates was determined and equal amounts of labeled proteins were subjected to immunoprecipitation. Cell lysate was preabsorbed with protein A-coated Sepharose beads (Pharmacia, Uppsala, Sweden) for 2 h at 4°C. The beads were removed and the SN was incubated with the respective antibody and fresh protein A-Sepharose at 4 "C overnight. The beads were then washed twice in TBS, 0.25% NP40 and subsequently resuspended in SDS sample buffer. Precipitated material was analyzed on a polyacrylamide gel (7.5% -15% acrylamide). After electrophoresis the gel was treated with DMSO (Roth, Karslruhe, FRG), 20% (w/v) diphenyloxazole (Roth) and water as described [39], dried and exposed to X-ray films.
2.3 Immunoslot blot analysis A quantitative immunoslot blot assay has been described recently [ a ] . Briefly, 10 pg of membrane protein was loaded onto nitrocellulose filters and incubated with the W6/32 mAb. Subsequently the filters were washed, incubated with lzI-labeled protein A and exposed to X-ray films. The signal obtained was quantified by densitometry in an LKB scanner (LKB-Pharmacia, Freiburg, FRG). 2.4 RNA analysis Cytoplasmic RNA was extracted as described [41]. For Northern blot and RNA slot blot analysis, 10 pg of cytoplasmic RNA was either separated in non-denaturing 1% agarose gels in MOPS buffer (20 mM MOPS, 5 m~ sodium acetate, 1m~ EDTA) and transferred onto Gene Screen Plus filters (NEN, Boston, MA) or loaded directly onto nitrocellulose filters (Schleicher and Schiill, Dassel, FRG) using the minifold slot blot device (Schleicher and Schull). The filters were hybridized with a 32P-labeled cDNA probe [42] for the MHC class1 antigen HLA-B8 (kindlyprovided by Dr. E.Weiss, Munich, FRG). RNA slot blot filters were also hybridized with a probe for ribosomal RNA [43] to assure that indeed comparable amounts of cytoplasmic RNA were bound to the filter. To evaluate the relative amount of MHC class I mRNA autoradiograms of RNA slot blot filters were evaluated by densitometry in an LKB scanner. 2.5 Nuclear run-on analysis
Cells were grown for 1week either with or without DXM. They were then washed twice with ice-cold PBS and harvested by scraping with a rubber policeman. They were lysed in 1 O m ~Tris-HC1, pH8.0, 10 mM NaCl, 3 mM MgC12, 0.5% NP40, 100 pM PMSF on ice and the nuclei were pelleted by centrifugation at 4000 x g for 5 min at 4 "C. Nuclei were washed twice in storage buffer (50 mM TrisHC1, pH 8.0, 40% glycerol, 5 m~ MgC12, 100 p~ EDTA, 100 p~ PMSF) and then stored at a concentration of 2 x lo7
Eur. J. Immunol. 1990. 20: 35-40
nuclei1100 p1 storage buffer in liquid nitrogen. The nuclear run-on assay was performed with modifications as described [44]. Briefly, 2 x lo7nuclei were incubated in 5 mM Tris-HC1,pH 8.0, 2.5 mM MgC12 ,. 0.15 mM KCl, 0.25 mM ATE GTF', CTP and 250 pCi 32P-labeledUTP (10 mCi/ml, 400 Cilmmol, Amersham, Braunschweig, FRG) for 60 min at 28°C. Cellular DNA was digested with DNase I (Worthington Diagnostics, Freehold, NJ) at a final concentration of 20 pg/ml for 5 min at room temperature. To terminate the reaction 200 p1 of 1% SDS, 10 mM EDTA and 20 mM Tris-HC1, pH 7.4 was added. Proteins were digested .in this mixture for 1 h with proteinase K (100 pg/ml) at 37°C and then extracted with phenolkhloroform. The radiolabeled RNA was precipitated in 2.5 M ammonium acetate and isopropanol. After dissolving it in 10mM Tris-HC1, pH 7 . 4 , l mM EDTA the RNA was purified on a Sephadex G-50 fine (Pharmacia, Freiburg, FRG) column from remaining free nucleotides. The radioactivity of the eluted RNA fraction was determined and comparable amounts of labeled RNA (106cpm/ml) were used as hybridization probe [45] for nitrocellulose filters with 10 pg of the respective plasmid DNA and 2 pg of total cellular DNA. Following hybridization for 2 days the filters were washed three times for 30 min at 70°C in 2 x SSC, 0.1% SDS and then exposed to X-ray films.
3 Results 3.1 Reduction of the MHC class I antigens on epithelial cells
To investigate the influence of glucocorticoid hormones on the expression level of the MHC class1 antigens on epithelial cells immunofluorescence studies were performed on C4-I cervical squamous carcinoma cells with W6132 mAb [46]. DXM-treated cells showed an up to 40% reduction of the cell surface expression of MHC class I antigens as determined by immunofluorescence analysis (Table 1). The reduced expression of the MHC class I antigens on C4-I cells is not due to a generalized inhibition of protein synthesis in DXM-treated cells, since expression of other membrane antigens, e.g., the receptor for the epidermal growth factor (EGF), is even enhanced [40] while still other antigens (e.g., the HEA125 marker) are not significantly affected by the hormone. Furthermore, DXM has strong growth-stimulating effects on C4-I cells 1471. Fluorescence microscopy of C4-I cells stained with the W6132 mAb and a fluorescence-labeled anti-mouse lg antibody revealed that most of the MHC class I antigens remain in the intracellular compartment. This is in line with reports that cell surface expression of MHC antigens is significantly disturbed in malignant cell lines [48]. Therefore, surface immunofluorescence studies on living cells might significantly underestimate the degree of reduction. Immunoprecipitation assays for the MHC class I antigens with the W6132 mAb confirmed the specific reduction of the 43-kDa MHC class I antigen in DXM-treated cells (Fig. 1). The antigen recognized by the HEA125 mAb [49] is not significantly influenced by DXM treatment.
Eur. J. Immunol. 1990. 20: 35-40
Glucocorticoids reduce MHC class I expression
Table 1. Reduction of MHC class I antigen expression on C4-I cellsa)
MI(CCII
37
Pmc
C4-I
Hours of DXM treatment
Mean fluorescence (channel numbers) W6/32 antibody
dex. U-1
(% )
dox.
c
.-0
4-
5
C4-1
0 18
48 62 120
1126 1008 859
100 89
738
65 59
660
76
dex. C4-1
n
dox. C4-1
dox.
a) C4-I cells were cultured for the time periods indicated in the presence of 1 p~ DXM.The cells were harvested and stained for 60 min with the W6132 mAb on ice. After three washes with PBS 0.1% BSA, bound mouse Ig was detected by staining for 60 min with a FITC-conjugated goat anti-mouse antiserum (Dianova, Hamburg, FRG). After three washes with PBS0.1% BSA mean fluorescence of cells was determined using a Becton Dickinson (Mountain View, CA) FACS III.
Immunoprecipitation assays, however, do not allow precise quantitation of specific proteins. Therefore, a quantitative immunoslot blot assay was used to determine the rate of reduction of the total cellular amount of MHC class I antigen in C4-I cells (Fig. 2). DXM treatment led to an about tenfold reduction of the total MHC class I antigen content in C4-I cells. A comparable reduction of the MHC class I antigen expression was observed in other epithelial cell lines (e.g., HT-3, HaCaT, SW756, SiHa, ME180; data not shown). Using the same assay, the HEA125 marker was not influenced by hormone treatment,while other antigens, e.g. the receptor for the EGF, were expressed at significantly increased rates [40].
dax.
Figure 2. Amount of MHC class I antigen on membranes of DXM-treated or untreated cells. Membrane proteins (10 pg) of C4-I cervical cancer cells either treated (1 p ~ B) , or not (0)with DXM for 1 week in five independent experiments was loaded on a nitrocellulose filter and incubated with the W6/32 mAb directed against the MHC class I antigens and subsequently with Iz5Ilabeled protein A. Densitometric evaluation of an autoradiogram of this filter revealed that upon DXM treatment about tenfold less radioactivity was bound to the membrane preparations in this assay. The average value and the standard deviation of five independent experiments are given. As positive control about 10 pg of membrane protein of A431 cells either treated or not with 1 p~ DXM was loaded on the same filter. No decrease of the MHC C1-I content upon DXM treatment was observed for this cell line. A second filter (Prot.) with identical amounts of the respective protein preparations was stained with Coomassie blue. It indicates that comparable amounts of protein were loaded in each slot.
3.2 DXM reduces the steady-state level of MHC class I mRNA
To investigate the influence of DXM on the MHC class I mRNA steady-state level, cytoplasmic RNA was extracted from treated and untreated cells and subjected to quantitative RNA slot blot analysis [50] using a 32P-labeledcDNA of the HL,A-B8 antigens as probe. DXM treatment for 1week led to decreased steady-state levels of MHC class I transcripts in C4-I, HT-3, HaCaT, SW 756, SiHa and ME180 cells. However, no reduction of MHC class I transcripts was observed in Caski and A431 cells (Fig. 3). Since other DXM-regulated proteins (e.g., the E GF receptor) are also not influenced in these cells, this is most likely due to the lack of glucocorticoid receptors in these cells.
Figure 1. Immunoprecipitation of MHC class I antigens in hormone-treated and untreated cells. C4-I cells were cultured for 1 week in the absence or presence of 1 p~ DXM. After labeling of cellular proteins with [35S]methioninefor 90 min, the MHC class I antigens were immunoprecipitated as described in the method section with the W6132 mAb or the HEA125 mAb and analyzed on a polyacrylamide gel. In DXM-treated cells (dex.) significantly less labeled 43-kDa protein was precipitated with the W6132 mAb compared t o untreated controls. The P2m is linked to the MHC class I antigens and, therefore, coprecipitated with the MHC class I molecules.The same quantitative relation is, thus, observed for this 12-kDa protein. With the HEA125 rnAb about the same amount of antigen was precipitated in treated or untreated cells.
Down-regulation of the MHC class1 mFWA is dose dependent. Already at DXM concentrations of M there is a reduction of the mRNA detectable. At concentrations of M DXM in the culture medium reduction of MHC class I expression reaches its maximum extent. The new reduced equilibrium state state of MHC class I mRNA levels is reached approximately 36-48 h after onset of treatment. Twenty-four hours after hormone application the steady-state level was reduced by 50% (data not shown). Inhibition of transcription of some genes of DXM was shown to be mediated directly by cis-acting elements [51-551. However, inhibition of Ia antigen expression on murine B lymphocytes by DXM was reported to be mediated by a short-lived transacting factor [20].
Eur. J. Immunol. 1990.20: 35-40
M. Von Knebel Doeberitz, S. Koch, H. Drzonek and H. Zur Hausen
38 1
0
C4-1
HT-3
HaCaT
SW 756
SiHa
ME 180
Cask1 A431
Figure 3. Steady-state level of MHC class I mRNA in cells either ) or not ( 0 )with DXM. Cytoplasmic RNA of epithelial cell lines cultured with or without DXM (1 pM) for 1week was loaded on nitrocellulose filters and hybridized with a 3*P-labeled probe for the MHC class I HLA-B8 cDNA. A parallel filter of the same RNA was hybridized with a probe for ribosomal RNA [43]. The relative amount of MHC class I mRNA in DXM-treated cells was calculated by dividing the signal intensity of the hormone-treated cells by the untreated controls after standardization against ribosomal RNA.The relative reduction of the MHC class I transcripts in the hormone-treated cells is shown.
To investigate whether a short-lived transacting factor might also be involved in glucocorticoid-mediated reduction of MHC class I antigen expression, cells were treated with DXM and cycloheximide, a known protein-synthesis inhibitor acting at the level of translation. Cells were either cultured with DXM and cycloheximide for 6 h or treated for lweek with DXM and then for 6 h with DXM and cycloheximide. RNA was extracted from the respective cells and the amount of MHC class I transcripts was estimated from Northern blots (Fig. 4). Long-term treatment for 1 week with DXM reduces the amount of MHC class I transcripts. Short-term treatment for only 6 h has no significant influence on the MHC class I mRNA steady-state level. Cycloheximide treatment for 6 h leads to an increased MHC class I mRNA steady-state level in C4-I cells. In cells pretreated with DXM for 1 week application of cycloheximide for 6 h also led to an increase
xI xI 0
c (0
d 0
+
J
J
0
P
0 P
c
c
c
(D
P)
0
(0
0
c (0
+ J
9)
0 Y P)
t
of the cytoplasmic mRNA level compared to cells treated for 1 week with DXM. However, it did not reach the level of untreated cells. Cycloheximide is highly toxic if cells are treated for longer then 6 h. Therefore, cycloheximide experiments could not be extended and it is not possible to differentiate between direct or indirect effects of DXM on MHC class I mRNA levels mediated by proteins with a longer half-life time. The increased steady-state levels of MHC class I mRNA upon cycloheximide treatment of either hormone-treated or untreated cells suggests that expression of MHC class I genes might possibly be regulated by labile transacting factors in these cells. Evidence for negative regulating factors for MHC class I gene expression has been given by other experimental systems [56]. The steady-state level of other transcripts in C4-I cells is not affected by cycloheximide ([57] and Von Knebel Doeberitz, unpublished results).Thus, there is no evidence that cycloheximideleads to increased levels of class I transcripts by an unspecific stabilization of mRNA as has been described in other experimental systems [58]. 3.3 Stability of MHC class I transcripts in C 4 I cells in the presence or absence of DXM
To exclude an increased degradation of the MHC class I mRNA in the cytoplasm under DXM treatment, the half-life time of the mature MHC class I transcripts in the cytoplasm of C4-I cells was analyzed by inhibiting the “de novo” synthesis of mRNA with actinomycin D.The amount of MHC class I mRNA was then analyzed at various time intervals after application of actinomycin D. Cells were seeded in 10-cm culture dishes and cultured either in the presence or absence of the hormone for 1week. Actinomycin D was then added and RNA was extracted 90, 180, 320 and 420 min after actinomycin D treamtent. After > 420 min following application of the RNA synthesis inhibitor toxic effects on the cells became detectable, thus no further extension of actinomycin D treatment was possible. Northern blots were prepared and hybridized with the MHC class I probe. To control for the actinomycin D effect, the same blots were washed and rehybridized with a 32P-labeledprobe for the third exon of the c-myc gene [59] since the c-myc mRNA has a relatively short half-life time [60]. No significant reduction of the
r
CHX 50 pp/ml
dex. 1 pY a ea
180 320 420
a
90 180
320 420
0
80 180 320 420 mln. acllnomycln D
MHC CI I
pmyc exm 111
MHC CI I
Figure 5. Stability of MHC class I transcripts in hormone-treated and untreated cells. RNA was extracted from C4-I cells either Figure 4. Effect of cycloheximide (CHX.) on DXM (dex.)-treated treated with DXM (dex.) (1 p ~ or ) cycloheximide (CHX., and untreated cells. Cytoplasmic RNA of C4-I cells treated with 50 pg/ml) or not at 0, 90, 180, 320, 420 min after application of cycloheximide (50 pg) for 6 h, DXM (1 p ~for ) 6 h, DXM (1 p ~ ) actinomycin D (10 pglml). Northern blots were prepared as deand cycloheximide (50 pg/ml) for 6 h, DXM for 1 week as well with scribed in Sect. 2.4 and hybridized first with the MHC class1 DXM (1 PM) for 1week and cycloheximide (50 pg) for 6 h was probe. After autoradiography, the probe was completely washed extracted and anlyzed on a Northern blot after electrophoresis in a off at the melting temperature of DNA (Tm -0°C) as proposed by 1% nondenaturing MOPS agarose gel. The filter was hybridized the manufacturer, and the filters were rehybridized with a probe for with a probe for the MHC class I transcripts. the third exon of the c-myc gene.
Eur. J. Immunol. 1990. 20: 35-40
Glucocorticoids reduce MHC class I expression
MHC class I transcripts could be observed within 420 min (Fig. 5). DXM or cycloheximide treatment did not alter the half-life time of the MHC class I mRNA to the detectable extent. However, in DXM-treated cells the MHC class I probe gave a weaker signal compared to cycloheximide or untreated controls. Hybridization of the same filters with the c-myc probe revealed as expected that no c-myc transcripts could be detected after 90 min of actinomycin D treatment in DXM or untreated cells. In cycloheximidetreated cells, however, the half-life time of the c-myc gene was prolonged to about 120 min leading to increased levels of the steady-state level of c-myc mRNA.This is consistant with similar observations made in other cell systems
WI. These findings indicate that the MHC class I transcripts in the cytoplasm are very stable.The DXM- or cycloheximideinduced alterations of the MHC class I mRNA steady-state level is, thus, not due to significant alterations of the half-life time as far as it can be judged within the time range of 420 min. Alterations of the half-life time beyond 420 min cannot be excluded with this assay. 3.4 DXM treatment leads to decreased transcription of
the MHC class I genes To investigate the influence of DXM on the transcription rate of the MHC class I genes nuclear run-on experiments were performed. C4-I cells were cultured for 1week either in the presence or absence of DXM. Nuclei were prepared and nuclear run-on experiments were performed. 32P-labeledand in vitro synthesized RNA of nuclei of C4-I cells which were either treated or not with the hormone for 1week was hybridized to nitrocellulose filters with 10 pg of the respective plasmid DNA. Autoradiograms of these filters were scanned. In three independent experiments DXM treatment reduced transcription of the MHC class I gene by about 50% compared to untreated controls (Fig. 5). Despite the high steady-state level of MHC class I transcripts in the cytoplasm, the transcription rate as determined with nuclear run-on experiments in our hands appeared to be relatively low. 200,
dex.
WC class I cell. DNA
dex.
Figure 6. Nuclear transcription rate of the MHC class I genes in DXM (dex.)-treated or untreated cells. Nuclear run-on assays were performed as outlined in Sect. 2.5. 32P-labeledin vitro synthesized nuclear RNA was hybridized to nitrocellulose filters with 10 pg of plasmid DNA of the MHC class I B8 cDNA and 2 pg of total cellular DNA (HeLa).The signal intensity of the respective slots was scanned. To calculate the reduction or increase of the respective signals, the intensity of the signal obtained with total cellular DNA served as reference. The signal for class I transcription in untreated cells was set as 100 and the degree of downregulation in hormone-treated cells was calculated.The SD of three independent experiments is given.
39
4 Discussion The experiments described here reveal that DXM reduced MHC class I gene expression in various human epithelial cell lines. The phenomenon was observed in all lines tested which express functional glucocorticoid receptors. Downregulation of MHC class I antigens by DXM was accompanied by a reduced steady-state level of the mRNA in the cytoplasm. After application of the hormone it took a rather long-time interval of about 48 h until the final reduced steady-state level was reached. Cycloheximide treatment led to an about twofold increase of the MHC class I mRNA steady-state level suggesting that labile protein factors either repressing transcription or enhancing degradation might possibly be involved in the regulation of MHC class I antigen expression. Inhibition of the synthesis of such a factor by cycloheximide might lead to increased mRNA levels. In cells treated for 1week with DXM addition of cycloheximide for 6 h also led to an about twofold increase of the MHC class I mRNA. This might be due to inhibition of synthesis of a DXM-induced transacting repressor acting either on the transcription or degradation of MHC class1 mRNA or to a DXM-independent mechanisms as observed in hormone-untreated cells. The half-life time of the MHC class I mRNA in DXMtreated cells and untreated controls is rather long, since no significant reduction was observed even more than 6 h after application of actinomycin D. DXM or cycloheximide had no detectable influence on the stability of the MHC class I mRNA within this time range. These data suggested that alterations of the MHC class I mRNA steady-state level might rather be due to variations of the transcription initiation or elongation rates than to a significantly decreased stability of the mRNA in the cytoplasm upon DXM treatment. Repeated nuclear run-on experiments clearly indicated a reproducible reduction of about 50% of the transcription rate of the MHC class I genes in DXM-treated C4-I cells. The transcription rate determined by nuclear run-on assays appeared to be relatively low compared to other genes.The high steady-state level of the mRNA in the cytoplasm can, therefore, be attributed to a slow degradation rate of mature transcripts. This is in good agreement with the longevity of the MHC class I mRNA. Small alterations of the transcription rate may, thus, lead to significant changes of the steady-state level of mRNA after a prolonged period of time. Similarly, small variations of the degradation rate might also lead to significantly reduced steady-state levels. Such changes might not be detectable with the actinomycin D assay and can, therefore, not be excluded on the basis of these experiments. Isolation and detailed characterization of the glucocorticoid-responsive regulatory squences of MHC class I genes is necessary to analyze the exact mechanism of DXM-mediated down-regulation of MHC class I antigen expression. The data presented here may explain some aspect of the immunosuppressive actions of glucocorticoid hormones, since expression of a critical threshold level of MHC class I antigens on epithelial target cells is an obligatory prerequisite for MHC class I-mediated immune functions [14]. Even if there is no complete down-regulation of the MHC class I
40
M.Von Knebel Doeberitz, S. Koch, H. Drzonek and H. Zur Hausen
expression, these findings clearly demonstrate t h e potential role of glucocorticoid hormones t o modulate t h e expression level of MHC antigens on epithelial cells, thereby maybe interfering with MHC class I-independent reactions, e.g., t h e attack of specific CTL. For critical reading of the manuscript and helpful suggestions we wish to thank E. Schwarz, J. Wolf and H . Jakobsen.
Received September 8, 1989.
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