Elsevier

Journal of Neuroimmunology

Volume 99, Issue 2, 29 October 1999, Pages 195-204
Journal of Neuroimmunology

Differential selectivity of CIITA promoter activation by IFN-γ and IRF-1 in astrocytes and macrophages: CIITA promoter activation is not affected by TNF-α

https://doi.org/10.1016/S0165-5728(99)00117-4Get rights and content

Abstract

During demyelinating disease of the central nervous system (CNS), locally elevated cytokine levels may induce upregulation of MHC class II molecules on otherwise low expressing or negative cell types such as microglia and astrocytes, since IFN-γ has been shown to induce MHC class II expression on these cell types in vitro. While many transcription factors are involved with MHC class II expression, only the class II transactivator (CIITA) is tightly coordinated with IFN-γ-inducibility. Control of CIITA gene expression is complex, involving four distinct promoters, two of which (promoters III and IV) are IFN-γ-inducible in certain cell types. Here we demonstrate that IFN-γ treatment of rat astrocytes induces only CIITA promoter IV activity in contrast to the murine macrophage cell line RAW 264.7 that uses both IFN-γ-inducible promoters. In contrast to previously published reports, promoter IV activation is completely dependent upon an intact interferon regulatory factor-1 (IRF-1) but not STAT1 binding site using promoter constructs specifically mutated at these positions. Importantly, while TNF-α is able to synergize with IFN-γ to increase astrocyte MHC class II expression in vitro, we show that treatment of rat astrocytes with TNF-α has no effect on CIITA promoter activity. These data demonstrate that TNF-α augments MHC class II expression through a mechanism downstream or independent of CIITA induction.

Introduction

Diseases of the central nervous system (CNS) are often characterized by the presence of activated glial cells around areas of demyelination. Several published reports have demonstrated a direct correlation between the level of MHC class II expression on cells of the CNS and the susceptibility to demyelinating disease in a number of different laboratory models (Massa et al., 1987aMassa et al., 1987b; Male and Pryce, 1989; Birnbaum and Kotilinek, 1990; Borrow and Nash, 1992). Massa et al. (1987b)demonstrated that rat strains exhibiting hyperinducible MHC class II expression on astrocytes were more susceptible to the induction of experimental autoimmune encephalomyelitis. This correlation has been demonstrated for cerebral vascular endothelial cells in the Theiler's murine encephalomyelitis virus-induced demyelinating model (Male and Pryce, 1989; Welsh et al., 1993), as well. Data from our laboratory has shown that the role of MHC class II expression in disease severity can also be extended to a non-T-cell-mediated model for demyelination (Matsushima et al., 1994). When Twitcher mice (a model for globoid cell leukodystrophy exhibiting spontaneous demyelination) were bred onto a MHC class II knockout background they exhibited significantly reduced twitching and demyelination providing evidence that MHC class II may perform functions other than simply presenting antigen to T-cells (Matsushima et al., 1994). Together, these data demonstrate a diverse role for MHC class II molecules during CNS disease pathogenesis.

The ability to therapeutically downregulate MHC class II during CNS demyelinating disease is an issue of great importance. While several groups have demonstrated that the systemic administration of anti-MHC class II antibodies resulted in improved prognosis for animals in which demyelination had been induced (Sriram and Steinman, 1983; Sriram et al., 1987; Friedmann et al., 1987; Jonker et al., 1988; Steinman, 1990; Smith et al., 1994), this treatment regimen did not specifically target MHC class II expressed in the CNS. The ability to specifically downregulate MHC class II expression on cells of the CNS would be a powerful tool for improving disease prognosis in individuals affected by demyelinating disease without affecting peripheral immune function. Thus, defining the molecular control of MHC class II expression on brain glial cells such as astrocytes is central to our understanding of how to therapeutically target MHC class II expression in the CNS.

Astrocytes are parenchymal cells constituting the largest population of cells of the CNS. A number of different in vitro stimuli are capable of inducing an activated phenotype in astrocytes including the proinflammatory cytokines IFN-γ and TNF-α (Wong et al., 1984; Fierz et al., 1985; Fontana et al., 1986; Benveniste et al., 1989). Damage to the CNS in vivo caused by ischemia, viral infection, or injury also serves to activate astrocytes leading to increased production of immune effector molecules such as TNF-α, TGF-β, IL-1, IL-3, PGE2, LTB4, IL-6, and IFN α/β (Fontana et al., 1983; Frei et al., 1985, Frei et al., 1989; Tedeschi et al., 1986; Robbins et al., 1987; Hartung et al., 1988; Sawada et al., 1989; Hailer et al., 1998). Similar to many other cell types, surface expression of MHC class II is upregulated on astrocytes following in vitro exposure to IFN-γ (Wong et al., 1984; Moses et al., 1991) and this expression is further augmented in the presence of TNF-α (Benveniste et al., 1989). Most studies of MHC class II gene regulation have been conducted using B-cell or macrophage cell lines, however, the molecular control of expression is not completely understood. It is possible that cell type-specific regulation of MHC class II genes may exist as reflected by the varied activation of MHC class II by different cytokines.

Previous studies from our laboratory and others have indicated that the regulation of MHC class II is primarily controlled at the transcriptional level by a number of different DNA binding proteins including NF-Y and RFX (reviewed in Glimcher and Kara (1992)). These factors are ubiquitously expressed and are not regulated by IFN-γ. Another molecule, the class II transactivator (CIITA), is coordinately regulated with MHC class II in response to IFN-γ (Steimle et al., 1994). The CIITA is a master regulator of all MHC class II genes including Ii and H-2M gene products important for the exogenous pathway of antigen processing (Chang and Flavell, 1995). CIITA per se is not a DNA-binding protein, rather it is thought to act indirectly on the MHC class II promoter by interacting with an as yet undefined DNA binding protein, although recent evidence has suggested interactions with RFX5, CBP, and Bob1 (Riley et al., 1995; Fontes et al., 1996; Scholl et al., 1997; Kretsovali et al., 1998). The expression of CIITA is regulated at the transcriptional level through the action of four distinct promoters (Muhlethaler-Mottet et al., 1997) that play a significant role in determining tissue-specific expression of CIITA and therefore of MHC class II. CIITA promoter I-driven transcripts are found in dendritic cells while promoter III drives constitutive expression in B-cells (Lennon et al., 1997; Muhlethaler-Mottet et al., 1997). Promoter IV was initially thought to be solely responsible for the IFN-γ-inducible expression of CIITA (Muhlethaler-Mottet et al., 1997) and is active in many non-professional antigen presenting cells (APCs) leading to IFN-γ-inducible expression of MHC class II. More recent work from our laboratory has demonstrated an IFN-γ-inducible element in promoter III as well, located 2.5-kb upstream of the transcriptional start site, indicating a role for this promoter in the cytokine induction of MHC class II activation (Piskurich et al., 1998). Finally, we also found that TGF-β downregulates the IFN-γ activation of both promoters III and IV (Piskurich et al., 1998).

Less is known regarding the regulation of MHC class II gene expression in the CNS at the molecular level. Several lines of evidence have demonstrated differential MHC class II protein expression in response to various cytokines and effector molecules by astrocytes and microglia, suggesting that there are cell-specific differences in MHC class II gene regulation even within the same tissue (Hellendall and Ting, 1997; Sasaki et al., 1989, Sasaki et al., 1990). Here we investigate the regulation of CIITA expression in primary rat astrocytes in an effort to define these differences. Using various CIITA promoter/luciferase reporter constructs we have determined that promoter IV is the only active promoter in primary rat astrocytes following IFN-γ stimulation. This is in contrast to the murine macrophage cell line RAW 264.7 in which IFN-γ-inducible promoter III is also active. We extend these findings to demonstrate that CIITA promoter IV activity in rat astrocytes is dependent upon the interferon regulatory factor-1 (IRF-1) binding site but is not dependent upon the STAT 1-binding site, in contrast to previously published reports of other cells (Muhlethaler-Mottet et al., 1998). The inclusion of TNF-α or neutralizing anti-TGF-β antibodies in the astrocyte cultures had minimal effect on CIITA promoter activity suggesting that: (a) augmentation of MHC class II induction by TNF-α must occur at a point downstream of CIITA induction or is independent of CIITA induction, and (b) endogenous TGF-β does not account for the lack of CIITA promoter III activity in astrocytes.

Section snippets

Rats

Timed pregnant Sprague–Dawley rats were purchased from Charles River (Wilmington, MA). Rats were maintained in a specific pathogen-free environment in the University of North Carolina animal facility until delivery of the pups. One-day-old rat pups were used for isolation of astroglial cultures.

Media and cell lines

Astroglial cells were maintained in Dulbecco's modified Eagles medium (DMEM)/Ham's F12 (1:1) medium (Gibco BRL, Gaithersburg, MD) supplemented with 10% FBS (Sigma, St. Louis, MO), 100 U/ml penicillin

IFN-γ-inducible CIITA expression in RAW 264.7 macrophages uses both CIITA promoters III and IV

Previous work has demonstrated that CIITA promoter usage determines tissue-specific expression of CIITA and ultimately MHC class II expression. Muhlethaler-Mottet et al. (1997)have shown that IFN-γ-inducible MHC class II expression is dependent upon the activity of CIITA promoter IV. More recent work from our laboratory has demonstrated that while CIITA promoter III is constitutively active in B-cells, there is an IFN-γ-inducible element that is located over 2.5-kb upstream of the

Discussion

MHC class II gene regulation in different cell types can be separated into two basic categories: those that exhibit constitutive expression and those that exhibit cytokine-inducible expression. The control of MHC class II expression lies at the transcriptional level and is dependent in part upon the activities of the non-DNA binding, transcriptional transactivator, and master regulator, CIITA. The expression of CIITA is complex, depending upon the activity of four distinct promoter regions (

Acknowledgements

This work was supported by National Multiple Sclerosis Society (NMSS) Grant RG-1785, NIH Grant NS34190, and NMSS Fellowships FA-1307-A-2, FG-1173-A-1, and FA-1109-A-1. K.M.N., J.F.P., and R.P.H. are postdoctoral fellows of the National Multiple Sclerosis Society. J.P.-Y.T. is an American Cancer Society Faculty Awardee.

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