IL-1 beta inhibits IFN-gamma-induced class II MHC expression by suppressing transcription of the class II transactivator gene.

Class II MHC Ags are critical for the initiation of immune responses by presenting Ag to T lymphocytes, leading to their activation and differentiation. The transcriptional activation of class II MHC genes requires the induction of the class II transactivator (CIITA) protein, a master regulator that is essential for both constitutive and IFN-gamma-inducible class II MHC expression. The cytokine IL-1beta has been shown to inhibit IFN-gamma-induced class II MHC expression in various cell types. We investigated the underlying mechanism of this inhibitory effect of IL-1beta using human astroglioma cell lines. Our findings demonstrate that IL-1beta prevents the expression of class II MHC mRNA and protein upon treatment with IFN-gamma. Furthermore, we demonstrate that IFN-gamma induction of CIITA mRNA expression is inhibited by treatment of cells with IL-1beta. IL-1beta suppressed IFN-gamma activation of the type IV CIITA promoter in astroglioma cells, indicating that the inhibitory influence of IL-1beta is mediated by inhibition of CIITA transcription. IL-1beta did not interfere with IFN-gamma receptor signal transduction, since tyrosine phosphorylation, nuclear translocation, and DNA binding of STAT-1alpha to an IFN-gamma activation sequence of the type IV CIITA promoter were not affected by IL-1beta. As well, IL-1beta treatment did not affect the ability of IFN-gamma-induced interferon-regulatory factor-1 (IRF-1) to bind the IRF-1 element within the type IV CIITA promoter. This study suggests that IL-1beta may play a role in regulating immunoreactivity by inhibiting transcription of the CIITA gene, thereby reducing subsequent class II MHC expression.

Rohn W ，Tang LP ，Dong Y ，Benveniste EN ... -  《JOURNAL OF IMMUNOLOGY》

IFN-gamma regulation of the type IV class II transactivator promoter in astrocytes.

The transcriptional activation of class II MHC genes requires the class II transactivator (CIITA) protein, a regulator that is essential for both constitutive and IFN-gamma-inducible class II MHC expression. The CIITA gene is controlled by multiple independent promoters; two promoters direct constitutive expression, while another, the type IV CIITA promoter, mediates IFN-gamma-induced expression. We investigated the molecular regulation of IFN-gamma-induced type IV CIITA promoter activity in astrocytes. IFN-gamma inducibility of the type IV CIITA promoter is dependent on three cis-acting elements contained within a 154-bp fragment of the promoter; the proximal IFN-gamma activation sequence (GAS) element, the E box, and the proximal IFN regulatory factor (IRF) element. Two IFN-gamma-activated transcription factors, STAT-1alpha and IRF-1, bind the proximal GAS and IRF elements, respectively. The E box binds upstream stimulating factor-1 (USF-1), a constitutively expressed transcription factor. Furthermore, STAT-1alpha binding to the proximal GAS element is dependent on the binding of USF-1 to the adjacent E box. Functionally, the proximal IRF element is essential for IFN-gamma induction of type IV CIITA promoter activity, while the proximal GAS and E box elements contribute to the IFN-gamma inducibility of this promoter. In astrocytes, TNF-alpha enhances IFN-gamma-induced class II MHC transcription. Our results demonstrate that TNF-alpha does not enhance IFN-gamma-induced transcriptional activation of the type IV CIITA promoter, indicating that the enhancing effect of TNF-alpha is mediated downstream of CIITA transcription. These results define the molecular basis of IFN-gamma activation of the type IV CIITA promoter in astrocytes.

Dong Y ，Rohn WM ，Benveniste EN 《JOURNAL OF IMMUNOLOGY》

Stat1 alpha expression is involved in IFN-gamma induction of the class II transactivator and class II MHC genes.

Class II MHC Ags are critical in the regulation of immune responses by presenting Ag to T lymphocytes, resulting in their activation and differentiation. Class II expression is rare in the normal central nervous system, but elevated expression on glial cells has been observed in several neurologic diseases. We have previously demonstrated that IFN-gamma-induced class II expression in glial cells involves activation of both tyrosine kinase and protein kinase C. IFN-gamma induces tyrosine phosphorylation of the tyrosine kinases Jak1 and Jak2 and of Stat1 alpha. In addition, IFN-gamma enhances expression of Stat1 alpha mRNA and protein. We utilized antisense oligonucleotides against Stat1 alpha to determine directly whether IFN-gamma-induced activation and/or enhancement of Stat1 alpha is involved in class II expression. Antisense oligonucleotides complementary to Stat1 alpha mRNA were introduced in CH235-MG astroglioma cells by transient transfection; such treatment inhibited both constitutive and IFN-gamma-enhanced expression of Stat1 alpha. IFN-gamma-induced class II MHC expression was also inhibited in cells exposed to Stat1 alpha antisense oligonucleotides. The fact that the class II promoter does not contain IFN-gamma-activated sequences for binding Stat1 alpha suggests that Stat1 alpha must activate another protein that is directly involved in class II expression. A likely candidate is the class II MHC transactivator (CIITA). IFN-gamma induction of CIITA mRNA was also inhibited in cells treated with antisense oligonucleotides against Stat1 alpha. These findings demonstrate that Stat1 alpha is involved in IFN-gamma induction of CIITA expression, resulting in class II MHC expression.

Lee YJ ，Benveniste EN 《JOURNAL OF IMMUNOLOGY》

TGF-beta suppresses IFN-gamma induction of class II MHC gene expression by inhibiting class II transactivator messenger RNA expression.

Recently, a non-DNA binding protein, class II transactivator (CIITA), has been shown to be required for constitutive and IFN-gamma-inducible class II MHC transcription. The cytokine TGF-beta inhibits IFN-gamma-induced class II MHC expression at the transcriptional level. In this study, we provide evidence that TGF-beta blocks IFN-gamma-induced CIITA mRNA accumulation. TGF-beta down-regulates class II MHC and CIITA mRNA accumulation in human astroglioma and fibrosarcoma cell lines, but TGF-beta does not destabilize the CIITA message, suggesting an effect at the transcriptional level. In cells that stably overexpressed CIITA, leading to a constitutive class II MHC-positive phenotype, the inhibitory effect of TGF-beta on class II MHC was abrogated, but the cells remained responsive for expression of TGF-beta-inducible genes. Cell lines that possessed defects in TGF-beta signaling also became refractory to inhibition of IFN-gamma-induced CIITA and class II MHC expression. Our data indicate that TGF-beta suppresses IFN-gamma-induced class II MHC expression by inhibiting accumulation of CIITA mRNA.

Lee YJ ，Han Y ，Lu HT ，Nguyen V ，Qin H ，Howe PH ，Hocevar BA ，Boss JM ，Ransohoff RM ，Benveniste EN ... -  《JOURNAL OF IMMUNOLOGY》

Identification of distinct regions of 5' flanking DNA that mediate constitutive, IFN-gamma, STAT1, and TGF-beta-regulated expression of the class II transactivator gene.

Class II transactivator (CIITA) is a master regulator required for constitutive and IFN-gamma-inducible expression of class II MHC genes. Although the role of CIITA is greatly appreciated, the mechanisms underlying constitutive and IFN-gamma-induced expression of CIITA are not understood. The study of CIITA induction is extremely important, but has been fraught with difficulty. This study describes for the first time a large (7-kb) fragment of 5' flanking sequences that mediates the B cell-specific, IFN-gamma-induced, and TGF-beta-suppressed expression of CIITA. This pattern of expression matches the authentic expression of the endogenous gene. Within the 7-kb fragment, sequences that lie between nucleotides -545 and -113 relative to the transcriptional start site are critical for constitutive promoter expression in B cells. In contrast, inducible activation of CIITA by IFN-gamma requires sequences contained in an additional 4 kb of upstream DNA. This region mediates an IFN-gamma response when linked to either the endogenous CIITA promoter or a heterologous promoter. A role for STAT1 in regulation of the CIITA promoter is shown by the rescue of IFN-gamma induction by expression of STAT1 in STAT1-defective U3A cells. TGF-beta significantly inhibits IFN-gamma-mediated induction of the CIITA promoter in 2fTGH fibroblasts, which indicates that the promoter is a target for TGF-beta. This inhibition is achieved by suppression of the basal promoter. This study provides a focal point for understanding the mechanism of B cell-specific, IFN-gamma-induced, and TGF-beta-suppressed expression of CIITA.

Piskurich JF ，Wang Y ，Linhoff MW ，White LC ，Ting JP ... -  《JOURNAL OF IMMUNOLOGY》