Vince T. Nguyen

Class II transactivator and class II MHC gene expression in microglia: modulation by the cytokines TGF‐β, IL‐4, IL‐13 and IL‐10

Microglia are the resident macrophages of the brain, and when activated, have functions including cytokine production, phagocytosis and antigen presentation. The class II MHC genes encode proteins that present antigenic peptides to helper T cells, leading to T cell activation and the development of an antigen‐specific immune response. Class II MHC gene expression is strictly regulated by the class II transactivator (CIITA) transcription factor. In this study, we investigated the effects of various immunomodulatory cytokines on IFN‐γ induction of class II MHC and CIITA gene expression in microglia, both primary microglia and a murine microglial cell line, EOC 20. By flow cytometry analysis we show that IFN‐γ‐induced surface expression of class II MHC molecules on EOC 20 cells can be inhibited by the cytokines TGF‐β1, IL‐4 and IL‐10, but not IL‐13. Using a ribonuclease protection assay, we have found that TGF‐β1, IL‐4 and IL‐10 act by inhibiting the expression of IFN‐γ‐induced CIITA mRNA and, in turn, class II MHC mRNA. TGF‐β1, IL‐4, and IL‐10 inhibition of IFN‐γ‐induced CIITA mRNA accumulation was not due to destabilization of CIITA mRNA, suggesting an effect at the level of transcription. In primary murine microglia, IL‐10 and TGF‐β1 inhibited IFN‐γ‐induced CIITA and class II MHC expression. However, a discordant effect of IL‐4 was noted in that IL‐4 enhanced IFN‐γ‐induced CIITA and class II MHC expression in primary microglia. Although some differences are observed between EOC 20 cells and primary microglia in terms of responsiveness to TGF‐β, IL‐4 and IL‐10, CIITA and class II MHC gene expression are coordinately modulated.

Involvement of STAT-1 and Ets Family Members in Interferon-γ Induction of CD40 Transcription in Microglia/Macrophages

Cluster of differentiation (CD)-40 is a cell surface receptor belonging to the tumor necrosis factor receptor family that plays a critical role in the regulation of immune responses. We have previously shown that the cytokine interferon (IFN)-γ induces CD40 expression in microglia. Herein, we have elucidated the molecular mechanisms underlying IFN-γ induction of CD40 gene expression in microglia/macrophages. IFN-γ up-regulates CD40 expression at the transcriptional level, and this regulation involves the STAT-1α transcription factor. Microglia from STAT-1α-deficient mice were refractive to IFN-γ induction of CD40 expression, illustrating the importance of STAT-1α in this response. Functional analysis of the CD40 promoter indicates that two gamma activated sequence elements as well as two Ets elements are involved in IFN-γ induction of CD40 promoter activity. STAT-1α binds to the gamma activated sequence elements, whereas PU.1 and/or Spi-B bind to the Ets elements. The expression of PU.1 and Spi-B, in conjuction with STAT-1α activation, correlates with IFN-γ inducibility of CD40 expression. Collectively, our data demonstrate the involvement of STAT-1α, PU.1, and Spi-B in IFN-γ induction of CD40 gene expression in cells of the macrophage lineage.

Post‐transcriptional inhibition of CD40 gene expression in microglia by transforming growth factor‐β

Microglia are one of the major glial cell types within the central nervous system, and can function as immune effector cells upon activation. CD40 is a cell surface receptor belonging to the TNF receptor family that plays a critical role in the regulation of immune responses. In this study, we investigated the expression of CD40 on microglia, and the role of transforming growth factor‐β (TGF‐β), an immunosuppressive cytokine, in regulating CD40 expression. Microglia constitutively express very low levels of CD40, and IFN‐γ enhances CD40 mRNA and protein expression in these cells. IFN‐γ‐induced CD40 mRNA expression is partially sensitive to the protein synthesis inhibitor puromycin, suggesting that ongoing protein synthesis is necessary for optimal induction of CD40 mRNA by IFN‐γ. TGF‐β inhibits IFN‐γ‐induced CD40 protein and mRNA expression. Inhibition of IFN‐γ‐induced CD40 mRNA levels by TGF‐β in microglia is not due to inhibition of CD40 transcription; rather, inhibition is due to enhanced degradation of CD40 mRNA. These results indicate that TGF‐β can inhibit expression of an immunologically important receptor, CD40, in microglia, and does so at the post‐transcriptional level by destabilizing CD40 mRNA. TGF‐β inhibition of CD40 expression may be one of the mechanisms by which TGF‐β exerts its suppressive effects on immune responses.

IFN-γ Regulation of Class II Transactivator Promoter IV in Macrophages and Microglia: Involvement of the Suppressors of Cytokine Signaling-1 Protein

The discovery of the class II transactivator (CIITA) transcription factor, and its IFN-γ-activated promoter (promoter IV), have provided new opportunities to understand the molecular mechanisms of IFN-γ-induced class II MHC expression. Here, we investigated the molecular regulation of IFN-γ-induced murine CIITA promoter IV activity in microglia/macrophages. In the macrophage cell line RAW264.7, IFN-γ inducibility of CIITA promoter IV is dependent on an IFN-γ activation sequence (GAS) element and adjacent E-Box, and an IFN response factor (IRF) element, all within 196 bp of the transcription start site. In both RAW cells and the microglia cell line EOC20, two IFN-γ-activated transcription factors, STAT-1α and IRF-1, bind the GAS and IRF elements, respectively. The E-Box binds upstream stimulating factor-1 (USF-1), a constitutively expressed transcription factor. Functionally, the GAS, E-Box, and IRF elements are each essential for IFN-γ-induced CIITA promoter IV activity. The effects of the suppressors of cytokine signaling-1 (SOCS-1) protein on IFN-γ-induced CIITA and class II MHC expression were examined. Ectopic expression of SOCS-1 inhibits IFN-γ-induced activation of CIITA promoter IV and subsequent class II MHC protein expression. Interestingly, SOCS-1 inhibits the constitutive expression of STAT-1α and its IFN-γ-induced tyrosine phosphorylation and binding to the GAS element in CIITA promoter IV. As well, IFN-γ-induced expression of IRF-1 and its binding to the IRF element is inhibited. These results indicate that SOCS-1 may be responsible for attenuating IFN-γ-induced CIITA and class II MHC expression in macrophages.

Regulation and function of class II major histocompatibility complex, CD40, and B7 expression in macrophages and microglia: Implications in neurological diseases

The ability of microglia, the brain’s resident macrophage, to present antigen through the class II major histocompatibility complex (MHC) to T cells allows these normally quiescent cells to play a critical role in shaping the outcome of many neurological diseases. The expression of class II MHC antigens and the costimulatory molecules CD40 and B7 on microglia and infiltrating macrophages is regulated through a complex network of cytokines in the inflamed brain. In this review, we describe the molecular mechanisms underlying class II MHC, CD40 and B7 regulation in microglia and macrophages. Our focus is on the cis-elements in the promoters of their genes and the transcription factors activated by cytokines that bind them. The functional implications of aberrant class II MHC, CD40 and B7 expression by microglia and macrophages as related to the diseases of Multiple Sclerosis and Alzheimer’s Disease are discussed.

IL-4-Activated STAT-6 Inhibits IFN-γ-Induced CD40 Gene Expression in Macrophages/Microglia

The antagonism between the cytokines IFN-γ and IL-4 is well documented, but the mechanism by which IL-4 inhibits IFN-γ-induced gene expression is not clearly understood. CD40 is a type I transmembrane protein that is critical for proper functioning of the immune system. We have previously shown that IFN-γ is the most potent inducer of CD40 expression by macrophages and microglia. In this report, we describe the molecular mechanisms by which IL-4 inhibits IFN-γ-induced CD40 expression. IL-4 suppresses IFN-γ-induced CD40 gene expression in both macrophages and microglia, and such inhibition is dependent on the activation of STAT-6. Nuclear run-on and transfection studies indicate that IL-4-mediated repression is at the transcriptional level. Furthermore, IL-4 inhibition of IFN-γ-induced CD40 expression is specific, since IL-4 does not inhibit IFN-γ-induced IFN-responsive factor-1 gene expression. Site-directed mutagenesis studies demonstrate that two STAT binding sites, named proximal and distal IFN-γ-activated sequences, in the human CD40 promoter are important for IL-4 inhibition of IFN-γ-induced CD40 promoter activity. Moreover, EMSAs indicate that IL-4-activated STAT-6 binds to these two STAT binding sites. These results suggest that IL-4 inhibition of IFN-γ-induced CD40 gene expression is mediated by direct STAT-6 binding to the CD40 promoter.

Suppressor of cytokine signaling 1 inhibits cytokine induction of CD40 expression in macrophages.

CD40 is a type I membrane-bound molecule belonging to the TNFR superfamily that is expressed on various immune cells including macrophages and microglia. The aberrant expression of CD40 is involved in the initiation and maintenance of various human diseases including multiple sclerosis, arthritis, atherosclerosis, and Alzheimer’s disease. Inhibition of CD40 signaling has been shown to provide a significant beneficial effect in a number of animal models of human diseases including the aforementioned examples. We have previously shown that IFN-γ induces CD40 expression in macrophages and microglia. IFN-γ leads to STAT-1α activation directly and up-regulation of NF-κB activity due to the secretion and subsequent autocrine signaling of TNF-α. However, TNF-α alone is not capable of inducing CD40 expression in these cells. Suppressor of cytokine signaling 1 protein (SOCS-1) is a cytokine-inducible Src homology 2-containing protein that regulates cytokine receptor signaling by inhibiting STAT-1α activation via a specific interaction with activated Janus kinase 2. Given the important role of CD40 in inflammatory events in the CNS as well as other organ systems, it is imperative to understand the molecular mechanisms contributing to both CD40 induction and repression. We show that ectopic expression of SOCS-1 abrogates IFN-γ-induced CD40 protein expression, mRNA levels, and promoter activity. Additionally, IFN-γ-induced TNF-α secretion, as well as STAT-1α and NF-κB activation, are inhibited in the presence of SOCS-1. We conclude that SOCS-1 inhibits cytokine-induced CD40 expression by blocking IFN-γ-mediated STAT-1α activation, which also then results in suppression of IFN-γ-induced TNF-α secretion and subsequent NF-κB activation.