Precise regulation of MHC class II gene expression is crucial for development and function of the immune system. Class II trans-activator (CIITA) has been shown to be required for constitutive and IFN-γ-induced MHC class II transcription. TNF-α is commonly coexpressed with IFN-γ during immune-mediated inflammatory responses and modulates IFN-γ-stimulated MHC class II expression. The effect of TNF-α on MHC class II expression depends on cell type and cellular differentiation state. We show here that TNF-α suppresses IFN-γ-induced CIITA mRNA accumulation, resulting in decreased MHC class II expression in human fibrosarcoma HT1080 cells. TNF-α also inhibits CIITA mRNA accumulation and protein expression in a tetracycline-regulated system without affecting promoter activity. CIITA mRNA, regulated by either IFN-γ or tetracycline, was destabilized in the presence of TNF-α, suggesting that TNF-α utilizes a distinct mechanism to suppress MHC class II expression in HT1080 cells. Consistent with this interpretation, TNF-α blocked IFN-γ-induced CIITA and MHC class II expression in mutant cells that are unresponsive to TGF-β or IFN-β. This is the first instance in which MHC class II expression is inhibited by destabilizing CIITA mRNA.
The MHC class II genes encode heterodimeric cell surface glycoproteins that present processed exogenous Ags to T helper cells and participate in the thymic selection of T lymphocytes (1). Appropriate expression of MHC class II gene is crucial for normal immune function, whereas aberrant expression of MHC class II molecules occurs in a variety of immunopathologic disorders (2). Expression of MHC class II genes is tightly regulated, primarily at the level of transcription (1, 3). Conserved cis-acting motifs in the proximal regions of MHC class II gene promoter have been identified, and cognate DNA-binding proteins have been characterized (1, 4, 5, 6, 7, 8, 9, 10).
Constitutive expression of MHC class II genes is restricted primarily to APC and is regulated by developmental stimuli that govern the differentiation program of the cell. In MHC class II-negative cell types, expression can be induced by cytokines, most notably IFN-γ (1, 3).
IFN-γ is produced principally by cells of the lymphocytic lineage, including T cells and NK cells. IFN-γ can modulate or induce MHC class II expression in MHC class II-positive and -negative cells (1, 5). Induction of MHC class II genes by IFN-γ involves an indirect mechanism, requiring synthesis of a factor termed class II trans-activator (CIITA)3 that couples IFN-γ stimulation and MHC class II expression (11, 12, 13). MHC class II expression is quantitatively controlled by the level of CIITA, which itself is tightly regulated by IFN-γ (11, 14).
CIITA was cloned by genetic complementation and mutations in CIITA are responsible for bare lymphocyte syndrome, a primary immunodeficiency characterized by lack of MHC class II expression (1, 15). CIITA knockout mice lack both constitutive and inducible MHC class II expression (16). Overexpression of CIITA activates MHC class II transcription in the absence of IFN-γ (11, 12, 13, 15, 17, 18). These results indicate that CIITA plays a necessary and sufficient role in both constitutive and inducible MHC class II gene regulation.
Cytokines such as TNF-α, TGF-β, and IFN-αβ modulate IFN-γ-induced MHC class II expression in a cell type-specific manner (1, 13, 19, 20, 21). TNF-α, commonly coexpressed with IFN-γ during immune-mediated inflammatory responses, exerts additive effects to IFN-γ in numerous immunological events. Exposure to TNF-α typically does not induce MHC class II expression. However, TNF-α treatment augments or blocks MHC class II induction depending on cell type and cellular differentiation state (20, 21, 22, 23, 24, 25, 26, 27, 28, 29). Mechanisms of the antagonistic effects of TNF-α on induction of MHC class II expression remain obscure.
Here, we show that TNF-α suppresses IFN-γ-induced CIITA mRNA accumulation and MHC class II expression in human fibrosarcoma HT1080 cells. TNF-α also inhibits CIITA mRNA expression directed by a tetracycline (tet)-regulated system. CIITA mRNA, induced by either IFN-γ or withdrawal of tet, was significantly destabilized in the presence of TNF-α. TNF-α also blocks CIITA and MHC class II expression in mutant cells that are unresponsive to TGF-β or IFN-β. We conclude that TNF-α utilizes a distinct mechanism to modulate IFN-γ-induced MHC class II expression in HT1080 fibrosarcoma cells.
Materials and Methods
Cell culture and treatment
The human fibrosarcoma cell line HT1080 and derivatives, 2fTGH, U2A, and 903 cells (30, 31, 32), were grown in DMEM supplemented with 10% (v/v) heat-inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Hygromycin-resistant cells were maintained in medium containing 250 μg/ml hygromycin, G418-resistant cells were maintained in 250 or 500 μg/ml G418. HT1080.CIITA.tet cells, described previously (13), were maintained in same medium with 1 μg/ml tet. Recombinant human IFN-γ and TNF-α were obtained from Genentech (South San Francisco, CA) and Becton Dickinson Labware (Bedford, MA), respectively. Cells with 80% confluence were treated with IFN-γ (250 U/ml) and/or TNF-α (50 ng/ml) for 16 h, unless stated otherwise.
RNase protection assays
Total cellular RNA was prepared from monolayer cells using the TRIzol method according to the manufacturer’s specifications (Life Technologies, Gaithersburg, MD). RNase protections were performed using probes synthesized from SP6/T7 transcription vectors. Probes were labeled with [32P]UTP to a sp. act. of 2–5 × 108 cpm/μg of input DNA. Aliquots equivalent to ∼1 × 104 cpm (γ-actin) or 2.5 × 105 cpm (MHC class II DR and CIITA) of each probe and 15 μg of RNA were used in each assay. The CIITA probe protects 271 bp of fragment of CIITA mRNA, and MHC class II DR probe detects 568 bp of fragment of MHC class II mRNA. The actin probe was transcribed from a cDNA fragment of human γ-actin and yields a 130-bp fragment on protection.
After cytokine treatment or incubation of the indicated concentration of tet, cells were washed once with ice-cold PBS and harvested in ice-cold DMEM supplemented with 5% FBS. The cells (∼5 × 104 cells/reaction) were sedimented by brief centrifugation and washed twice with staining buffer (DMEM supplemented with 1% FBS and 0.02% NaN3) and resuspended in 100 μl staining buffer. Cells were incubated with a PE-conjugated MHC class II DR Ab (DAKO, Carpinteria, CA) on ice for 45 min. After three washes, cells were resuspended in 0.5 ml staining buffer and analyzed on a Becton FACScan instrument with the LYSYS II software package.
Luciferase activity assay
HT1080.CIITA.tet cells in the presence of tet (1 mg/ml) at ∼60% confluency in 100-mm plates were transfected with 2.5μg of pTO.luc plasmid DNA (33) for 6 h at 37°C using the calcium phosphate technique according to standard protocols. After recovery overnight, cells were equally redistributed in 60-mm plates, grown to 60% confluency, and then washed with serum-free medium and incubated in medium alone or with TNF-α (50 ng/ml) for 16 h. Luciferase activity was assayed using a luciferase assay kit (Promega, Madison, WI), in a Luminometer (Dynatech Laboratories, Chantilly, VA).
Cells on cover slides were rinsed twice in PBS and fixed in methanol-acetone (1:1) for 2 min. After two washes with TBST (10 mM Tris-HCI (pH 8.0), 150 mM NaCl, 0.02% Tween 20), nonspecific protein adsorption was blocked by incubation of cells for 40 min in TBST containing BSA (3%). Polyclonal anti-CIITA Abs (a gift from Jeremy M. Boss, Emory University, Atlanta, GA) was diluted 1:200 in blocking buffer and incubated with the fixed cells for 2 h at room temperature. Cells were washed in TBST, and fluorescein-conjugated goat anti-rabbit Ab (1:500 diluted in blocking buffer, Life Technologies) was added to the cells for 70 min at room temperature. After a final wash in TBST, the cells were mounted in Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA) and examined by fluorescence microscopy.
TNF-α inhibits IFN-γ-induced MHC class II expression in HT1080 cells
Modulation of IFN-γ-induced MHC class II expression by TNF-α has been observed in a variety of cells. We first monitored the effect of TNF-α on IFN-γ-induced MHC class II expression by flow cytometry assay. Fig. 1 shows that TNF-α suppressed IFN-γ-induced HLA-DR protein expression on the surface of HT1080 cells.
TNF-α inhibits IFN-γ induction of CIITA and MHC class II mRNA in HT1080 and mutant cells unresponsive either to TGF-β or IFN-β
It was uncertain how TNF-α inhibited IFN-γ-induced MHC class II expression in HT1080 cells. Because CIITA plays a necessary and sufficient role in MHC class II gene expression and inhibition of CIITA expression by cytokines results in decreased MHC class II expression, we examined the effects of TNF-α on IFN-γ-induced CIITA and MHC class II mRNA expression. In HT1080 cells, CIITA mRNA expression became detectable 1 h after IFN-γ stimulation, peaked by 16 h, gradually declined, but remained detectable at low levels for 48 h. IFN-γ-induced MHC class II DRA mRNA expression was detected 4 h after IFN-γ stimulation, reaching maximal levels at 24 h (Fig. 2,A). IFN-γ-induced CIITA mRNA accumulation preceded that of DRA mRNA expression by ∼3 h, as observed in other cell types (13). TNF-α inhibited both IFN-γ-induced CIITA and MHC class II DR mRNA by ∼68% and ∼85%, respectively (Fig. 2,B). Pretreatment of these cells with TGF-β1 for 12 h also suppressed IFN-γ-induced CIITA and MHC class II DR mRNA accumulation by ∼52% and ∼78%, respectively (Fig. 2,B), as shown in our previous studies. Pretreatment of cells with cycloheximide, a potent protein synthesis inhibitor, abolished the ability of TNF-α to inhibit IFN-γ-induced CIITA mRNA accumulation (Fig. 2 C). These results suggested that TNF-α inhibition of IFN-γ-induced DRA mRNA expression may be mediated through suppression of CIITA mRNA accumulation and requires a new or ongoing protein synthesis.
Previously, we showed that either TGF-β or IFN-β efficiently suppressed MHC class II transcription induced by IFN-γ in HT1080 or its derivatives (13, 18). TNF-α-mediated induction or activation of these cytokines has been reported. To differentiate the inhibitory mechanisms of these cytokines, CIITA and MHC class II mRNA expression were examined in mutant 903 and U2A cells unresponsive to TGF-β (32) and IFN-β (31), respectively. TNF-α suppressed IFN-γ-induced CIITA and MHC class II mRNA accumulation in both 903 and U2A cells (Fig. 2, D and E). These data indicated that both TGF-β and IFN-β signaling are dispensable for TNF-α-mediated inhibition of MHC class II and CIITA mRNA accumulation.
TNF-α inhibits tet-regulated CIITA and MHC class II expression in HT1080.CIITA.tet cells
Our initial results suggested that TNF-α acted at a posttranscriptional level to suppress CIITA mRNA accumulation. To further address the mechanism of this inhibition, we determined the effect of TNF-α treatment on tet-regulated CIITA expression in a cell line designated HT1080.CIITA.tet (13). HT1080.CIITA.tet cells were stably transfected with pTA.hygro, which directs expression of the tet-regulated trans-activator and pTO.CIITA.neo, which contains the complete CIITA coding region and 58 nucleotides of 3′-untranslated region (UTR) governed by the tet-regulated trans-activator (Fig. 3,A). In these cells, MHC class II expression could be controlled through either addition of IFN-γ or withdrawal of tet (13). CIITA mRNA accumulation could be regulated by tet in this cell line, and MHC class II mRNA in turn was directly regulated by CIITA (Fig. 3,B). In HT1080.CIITA.tet cells incubated in tet (to repress the transfected CIITA gene), IFN-γ treatment resulted in robust HLA-DR surface expression. TNF-α suppressed IFN-γ-induced CIITA and MHC class II expression in HT1080.CIITA.tet cells in the presence of tet (data not shown), as previously shown in wild-type HT1080 cells. After tet withdrawal, the cells became CIITA and MHC class II positive in the absence of IFN-γ (Fig. 3, B and C) and TNF-α inhibited tet-regulated CIITA mRNA (Fig. 3,D), MHC class II DRA mRNA (Fig. 3,D), and protein expression (Fig. 3,C). TNF-α also inhibited tet-regulated CIITA and MHC class II expression after withdrawal of tet for 3 days, when CIITA and MHC class II expression were constitutive (data not shown). As reported before (13), TGF-β did not affect tet-induced CIITA and MHC class II expression (Fig. 3,D). The inhibitory effect of TNF-α was maximal at 0.05–0.5 μg/ml tet (data not shown). We analyzed the distribution and abundance of CIITA protein by immunohistochemistry, to ascertain that there was a predictable relationship between accumulation of the CIITA mRNA and levels of cognate protein. CIITA protein was undetectable in tet-treated cells and appeared in the perinuclear cytoplasm and nucleus after tet withdrawal. TNF-α pretreatment abrogated induction of CIITA protein after tet withdrawal (Fig. 3,E), demonstrating that CIITA protein levels were determined by mRNA abundance. Finally we considered the unlikely possibility that TNF-α blocked transcription of tet-regulated promoters. As shown in Table I, TNF-α did not affect tet-regulated promoter activity in a transient transfection assay.
|Conditions .||Luciferase Activity (×10) ± SD .|
|CON||3.3 ± 1.9|
|TNF-α||3.4 ± 2.1*|
|Conditions .||Luciferase Activity (×10) ± SD .|
|CON||3.3 ± 1.9|
|TNF-α||3.4 ± 2.1*|
pTO.luc, in which luciferase expression is negatively regulated by the tet operator, was transiently transfected into HT1080.CIITA.tet cells, and promoter activity was monitored by luciferase assay after tet withdrawal, as described in Materials and Methods. Values reflect mean luciferase activity ± SD from three independent experiments. ∗, p > 0.5 compared with CON (paired t test).
Stability of CIITA mRNA in parental HT1080 and HT1080.CIITA.tet cells exposed to IFN-γ and TNF-α
To investigate the effect of TNF-α on CIITA mRNA stability, HT1080 cells were treated with TNF-α and IFN-γ for 10 h (time 0) and then incubated in actinomycin D (ACT-D) to arrest transcription, for an additional 8 h. At various time points, CIITA mRNA was quantitated by nuclease protection assay. The t1/2 of IFN-γ-induced CIITA mRNA was ∼6 h. In the presence of TNF-α, CIITA mRNA t1/2 was reduced to ∼2 h (Fig. 4,A). The observations were reproducible, with variability of ≤20% for the individual values plotted in the CIITA RNA decay curve in two experiments, with regulation by IFN-γ and TNF-α. Similar results were observed in HT1080.CIITA.tet cells (Fig. 4 B). These data indicated that TNF-α-mediated inhibition of CIITA mRNA accumulation was associated with destabilization of the message.
Precise regulation of MHC class II gene expression is a crucial for physiological control of the immune response. Positive and negative defects in the regulation of MHC class II expression are associated with a variety of immune disorders (1, 3, 34). Physiological and pathological MHC class II gene expression can be fine-tuned by a large number of different stimuli, including inflammatory cytokines, such as IFN-γ and TNF-α (1, 5). CIITA has been demonstrated as a master control factor for both constitutive and inducible MHC class II expression (1, 6, 12, 17). Furthermore, CIITA governs expression of invariant chain and HLA-DM genes, the products of which are involved in Ag processing. In this study, we address the mechanism by which TNF-α inhibits tet-regulated and IFN-γ-induced MHC class II expression in human fibrosarcoma cells, with an emphasis on the effect of TNF-α on CIITA mRNA accumulation. Our results demonstrate that TNF-α suppresses constitutive and IFN-γ-induced CIITA mRNA, MHC class II mRNA, and MHC class II protein in HT1080 cells. For additional experiments, we utilized a tet-regulated CIITA-expressing cell line, in which MHC class II expression was stringently dependent on CIITA. We demonstrated TNF-α–mediated inhibition of CIITA regardless of whether induction proceeded by addition of IFN-γ or withdrawal of tet. This result indicated that TNF-α suppression of CIITA mRNA accumulation could be observed without transcription being directed by the endogenous CIITA promoter or the IFN-γ-induced JAK/STAT signaling components. Further, ACT-D chase experiments were used to show that TNF-α inhibits CIITA mRNA accumulation by destabilizing CIITA mRNA. Furthermore, using TGF-β- and IFN-β-unresponsive cells (31, 32), it was demonstrated formally that TNF-α-mediated inhibition of IFN–γ-induced MHC class II expression did not require TGF-β or IFN-β signaling.
Regulation of mRNA stability in higher eukaryotes is not well characterized at either the cis or the trans level (35, 36). TNF-α inhibited CIITA mRNA accumulation in HT1080.CIITA.tet cells, in which the CIITA expression construct contains the coding region and 58 nucleotides of 3′ UTR sequences. The destabilizing cis determinants in 3′ UTR of CIITA have not been reported. Therefore, it is pertinent to consider whether CIITA may bear a coding region stability determinant that mediates these effects of TNF-α treatment. Coding region determinants in c-myc and c-fos mRNA have been identified; both contain a leucine-rich motif (37, 38, 39). A coding region stability determinant of appropriate 320 nucleotides located near the center of the c-fos mRNA encodes the basic and leucine zipper regions of fos protein. The coding region stability determinant of c-myc mRNA specifies the C-terminal 60 amino acids of the protein, including part of the helix-loop-helix and all of the leucine zipper motif (39). This mRNA cis element interacts with a protein that shields the mRNA from endonucleolytic attack in vitro (40). Interestingly, the C-terminus of CIITA contains a leucine-rich region that is important for CIITA function (15, 41, 42, 43, 44, 45). Small in-frame deletions in this region result in nonfunctional CIITA, which was responsible for bare lymphocyte syndrome in two patients (15, 44).
Mach and colleagues (46) recently reported that CIITA transcription is controlled by at least four independent promoters, two of which direct constitutive expression in dendritic cells and B lymphocytes, respectively, while a third mediates IFN-γ-induced expression. In addition to the IFN-γ response element, this inducible CIITA promoter contains at least two TNF-α response elements, NFκB and IRF1/IRF2 binding motifs (46). NFκB and IRF1 are involved in positive regulation of many TNF-α responding genes. In HT1080 cells, TNF-α induces and activates both NFκB and IRF1 proteins and synergizes with IFN-γ to augment IFN-γ activation site (GAS)-binding activity, activation, and GAS directed gene transcription. These observations imply that TNF-α destabilization of IFN-γ-induced CIITA mRNA predominates over potential positive transcriptional effects.
Many other cytokines and growth factors, such as TGF-β, IFN-β, LPS, and IL-10, are also able to suppress IFN-γ-induced MHC class II expression (1, 19, 47, 48, 49). We previously demonstrated that TGF-β and IFN-β act upstream and downstream of CIITA mRNA accumulation, respectively (13, 18). Cells defective in TGF-β and IFN-β signaling remained sensitive to TNF-α-mediated inhibitory effects on IFN-γ-induced CIITA and MHC class II expression. These results, together with other previously reported findings, indicate the mechanism of TNF-α inhibition of IFN-γ-induced MHC class II expression is distinct. In particular, TNF-α acts on CIITA mRNA stability, TGF-β suppressed CIITA transcription and IFN-β acts downstream of CIITA mRNA accumulation.
In summary, we showed that TNF-α treatment of cells resulted in destabilization of CIITA mRNA in HT1080 cells. This observation adds to our understanding of the complex regulation of MHC class II expression by cytokines.
We thank Drs. George R. Stark and Philip H. Howe (Cleveland Clinic Foundation) and Ian M. Kerr (Imperial Cancer Research Fund Laboratories) for providing cell lines.
This research was supported by the National Multiple Sclerosis Society (RG 2362), the National Institutes of Health (RO1-NS 32151; PO1-CA 62220), and the Williams Family Fund for Multiple Sclerosis Research.
Abbreviations used in this paper: CIITA, class II trans-activator; tet, tetracycline; RPA, RNase protection assay; IRF, IFN-regulatory factor; UTR, untranslated region; ACT-D, actinomycin D.