Activation of MHC class I, II, and CD40 gene expression by histone deacetylase inhibitors.

Epigenetic mechanisms are involved in regulating chromatin structure and gene expression through repression. In this study, we show that histone deacetylase inhibitors (DAIs) that alter the acetylation of histones in chromatin enhance the expression of several genes on tumor cells including: MHC class I, II, and the costimulatory molecule CD40. Enhanced transcription results in a significant increase in protein expression on the tumor cell surface, and expression can be elicited on some tumors that are unresponsive to IFN-gamma. The magnitude of induction of these genes cannot be explained by the effect of DAIs on the cell cycle or enhanced apoptosis. Induction of class II genes by DAIs was accompanied by activation of a repressed class II transactivator gene in a plasma cell tumor but, in several other tumor cell lines, class II was induced in the apparent absence of class II transactivator transcripts. These findings also suggest that the abnormalities observed in some tumors in the expression of genes critical to tumor immunity may result from epigenetic alterations in chromatin and gene regulation in addition to well-established mutational mechanisms.

Chromatin-immune connections: regulation of MHC and other genes.

In cells, genes are contained within chromatin - a highly structured array of DNA wrapped around core histone proteins. Packaged genes are subject to a variety of regulatory modifications including, CpG methylation, histone acetylation and phosphorylation. These epigenetic mechanisms of gene regulation involve higher ordered protein complexes possessing enzymatic activities such as ATP hydrolysis and acetylation that are targeted to specific genes by transcription factors, coactivatorsand coreptessors. In this article, we endeavor to providean overview of current research on mechanisms of transcriptional regulation by chromatin remodeling of MHC and other genes that are of interest in reproductive immunology.

Effect of transforming growth factor-beta1 on microglial MHC-class II expression.

In the present report, the effects of IFN-gamma and transforming growth factor beta1 (TGF-beta1) on major histocompatibility complex class II (MHC-II) gene expression in isolated mouse brain microglial cells, in the MH-S macrophage cell line and in the primary mouse macrophage cultures were examined. IFN-gamma is a potent inducer of MHC-II gene and this induction was further elevated in microglia by TGF-beta1, while TGF-beta1 inhibited IFN-gamma, induction in macrophages. The enhancing effect of TGF-beta1 was also detected in microglia at the protein level. Transient transfection of microglia with 5' deletional mutants of the MHC-II IAalpha promoter linked to the chloramphenicol acetyltransferase reporter gene demonstrated that TGF-beta1 acts at the transcriptional level to enhance the MHC-II expression induced by IFN-gamma.

Differential regulation of major histocompatibility complex class II expression and nitric oxide release by beta-amyloid in rat astrocyte and microglia.

Astrocytes and microglial cells were examined for expression of two immunologically important molecules, major histocompatibility complex class II (MHC-II) and nitric oxide (NO) following treatment with IFN-gamma and beta-amyloid (betaA) peptides, betaA(1-42) and betaA(25-35). IFN-gamma is a potent inducer of both MHC-II gene expression and NO production. The induction of MHC-II was inhibited by both betaA peptides in astrocytes but they had little or no effect in microglia. betaA peptides had no effect on NO release in astrocytes but on microglia betaA(1-42) synergistically induced NO release with IFN-gamma. Transient transfection of astrocytes with 5' deletional mutants of MHC-II IAalpha promoter linked to the chloramphenicol acetyl transferase reporter gene (IAalpha-CAT), demonstrated that betaA acts at the transcriptional level to downregulate IFN-gamma induced MHC-II gene expression in astrocytes. In previous studies, the induction of MHC-II on glial cells were suggested to be involved in the pathogenesis of neurodegenerative diseases and MHC-II(+) microglial cells were observed at much higher frequency than astrocytes. This study provides information on the regulation of the MHC-II gene expression in astrocytes and in microglial cells by betaA and this pathway may be critically involved in the immune/inflammatory regulation within the central nervous system.

Absence of MHC class II antigen expression in trophoblast cells results from a lack of class II transactivator (CIITA) gene expression.

Although the mechanism(s) underlying the failure of the maternal immune system to reject the semiallogeneic fetus have not been clearly defined, the absence of MHC class II antigen expression by fetal trophoblast cells very likely plays a critical role in the maintenance of normal pregnancy. However, the regulation of class II antigen expression in trophoblast cells is poorly understood. Class II transactivator (CIITA) is a transacting factor that is required for both constitutive and IFN-gamma-inducible class II gene transcription. In this report we demonstrate that the inability of trophoblast cells to express class II antigens is due to a lack of CIITA gene expression. Trophoblast cell lines derived from human, mouse, and rat do not express CIITA, and expression is not inducible by IFN-gamma. The absence of CIITA gene expression in trophoblasts treated with IFN-gamma does not result from a defect in the IFN-gamma receptor or the JAK/STAT pathway, because the classical IFN-gamma inducible gene encoding the guanylate-binding protein is expressed. Transfection of CIITA expression vectors into trophoblast cells results in activation of class II promoters, endogenous class II mRNA expression, and subsequent expression of class II antigens on the cell surface. In contrast, class I mRNA is not expressed in human trophoblast cells transfected with CIITA expression vectors. Thus, trophoblast cells contain all of the DNA binding factors necessary for class II transcription, and ectopic expression of CIITA is sufficient to activate class II, but not class I expression. The failure of trophoblast cells to express CIITA, and therefore class II antigens, provides a potential mechanism by which the fetus is protected from the maternal immune system during pregnancy.

Role of transforming growth factor-beta1 in the suppressed allostimulatory function of AIDS patients.

BACKGROUND: The T-cell stimulatory function of accessory cells isolated from peripheral blood lymphocytes of AIDS patients has been reported to be suppressed. These patients also have elevated levels of the immunosuppressive factor transforming growth factor (TGF)-beta1 in their serum and plasma. OBJECTIVE: To explore the role of TGF-beta1 in the loss of accessory cell function of peripheral blood lymphocytes from AIDS patients. METHODS: Fluorescent labeled anti-TGF-beta1 and confocal microscopy were used to detect the presence of TGF-beta1 on the cell membrane of dendritic cells. To assess the role of TGF-beta1 in the inhibition of accessory cell function in AIDS, antibodies against TGF-beta1 or the TGF-beta1 type III receptor, beta-glycan, were added to a mixed lymphocyte reaction. RESULTS: TGF-beta1 was detected on the cell membrane of dendritic cells isolated from AIDS patients. The addition of blocking antibodies against either TGF-beta1 or beta-glycan restored the T-cell stimulatory function to accessory cells from these patients. CONCLUSIONS: T-cell stimulatory function was not irreversibly lost in AIDS patients. Our data suggested that beta-glycan-TGF-beta1 immunosuppressive complexes may contribute to the suppression of accessory cell function in these patients.

Repression of MHC class II gene transcription in trophoblast cells by novel single-stranded DNA binding proteins.

The maintenance of the fetus during pregnancy has been attributed to the absence of major histocompatibility complex (MHC) class II antigens on fetal trophoblastic cells that make contact with the maternal immune system. However, the mechanism(s) by which class II genes are regulated in trophoblast cells is unclear. We have identified a negative regulatory element (IA alpha NRE) in the promoter of the mouse class II gene IA alpha that represses IA alpha transcription in trophoblast cells. IA alpha NRE, located from-839 to -828, binds transacting factors from rat, mouse and human trophoblast cells, but not from 18 other cell lines tested. These results indicate that IA alpha NRE binding proteins (IA alpha NRE BPs) are conserved in species with hemochordial placentas, and suggest that IA alpha NRE binding activity is restricted primarily to trophoblast cells. Interestingly, the IA alpha NRE BPs bind to the IA alpha NRE antisense strand in a sequence-specific manner. IA alpha NRE represses transcription from the IA alpha promoter in a position-dependent manner, and has a minor down-regulatory effect on the activity of the SV40 promoter/enhancer. Our results demonstrate that MHC class II gene transcription is repressed in fetal trophoblast cells by sequence-specific, single-stranded DNA binding proteins, and suggest a possible mechanism by which the conceptus is protected from immune rejection during pregnancy.

Differential regulation of TGF-beta 2 by hormones in rat uterus and mammary gland.

Previous work from this laboratory has shown that transforming growth factor beta 2 (TGF-beta 2) mRNA is abundant in the pregnant uterus. In the present study, we examined the synthesis and secretion of TGF-beta 1,2 and 3 in the rat uterus and mammary gland and show differential secretion and expression of TGF-beta 2 in a tissue specific manner. Elevated levels of TGF-beta 2 were detected in late pregnant maternal plasmas (> 100 pM), and in the milk (> 500 pM) during early lactation. High concentrations of TGF-beta 2 (> 200 pM) were also detected in uterine fluids collected from ovariectomized adult rats after high dose estrogen treatment. TGF-beta 2 mRNA levels were elevated in lobuloalveolar epithelial cells isolated from pregnant mammary gland. Three major transcripts of 3.5, 4.0, and 4.7 kb were seen, of which the 4.7 kb, dominates. Mammary glands of estrogen treated ovariectomized rats showed a similar pattern of TGF-beta 2 transcripts. In contrast, four major TGF-beta 2 mRNA transcripts of 5.7, 4.7, 4.0, and 3.5 kb, with the dominant species of 4.0 and 5.7 kb, were observed in uteri from the estrogen treated animals up to 48 h after the last estrogen injection. This suggests that TGF-beta 2 is regulated in a tissue specific manner. We conclude that the secretion of TGF-beta 2 is tightly regulated by hormones and that estrogen and prolactin are critical factors in the tissue-specific regulation of the local production of TGF-beta 2 in the mammary gland and female reproductive tract.

Cytokine production by human soft tissue sarcomas: implications for immunosuppression within the tumour bed.

The local immune response to the presence of tumour is affected by the pattern of cytokine production within the tumour bed. The purpose of this study was to define the pattern of cytokine production in human soft tissue sarcomas, attempting to identify potential immune suppression. Total RNA was extracted from six human soft tissue sarcomas from which cDNA was generated. PCR was carried out in the presence of primer pairs for G3PDH, TGF-beta1, TGF-beta2, TNF-alpha, INF-gamma, IL-2 and IL-10. TGF-beta1 and IL-10 mRNA was detected in all tumours, however, mRNA for IL-2 and INF-gamma was detected in only two out of six sarcomas. Paraffin sections were incubated with alpha-hu-TGF-beta1 or alpha-hu-IL-10 antibodies to localize protein production. TGF-beta1 and IL-10 protein expression was only associated with the tumour cells. These findings suggest potential local immune suppression within the tumour bed.

Modeling of a possible conformational change associated with the catalytic mechanism in the hammerhead ribozyme.

Here we describe a possible model of the cleavage mechanism in the hammerhead ribozyme. In this model, the 2' hydroxyl of C17 is moved into an appropriate orientation for an in-line attack on the G1.1 phosphate through a change in its sugar pucker from C3' endo to C2' endo. This conformational change in the active site is caused by a change in the uridine turn placing the N2 and N3 atoms of G5 of the conserved core in hydrogen bonding geometry with the N3 and N2 atoms on the conserved G16.2 residue. The observed conformational change in the uridine turn suggests an explanation for the conservation of G5. In the crystal structure of H.M. Pley et al., Nature 372, 68-74 (1994), G5 is situated 5.3A away from G16.2. However, the uridine turn is sufficiently flexible to allow this conformational change with relatively modest changes in the backbone torsion angles (average change of 14.2 degrees). Two magnesium ions were modeled into the active site with positions analogous to those described in the functionally similar Klenow fragment 3'-5' exonuclease (L.S. Beese and T.A. Steitz, EMBO J. 10, 25-33 (1991)), the Group I intron (T.A. Steitz and J.A. Steitz, P.N.A.S. U.S.A. 90, 6498-6502 (1993); R.F. Setlik et al., J. Biomol. Str. Dyn. 10, 945-972 (1993)) and other phosphotransferases. Comparison of this model with one in which the uridine turn conformation was not changed showed that although the changes in the C17 sugar pucker could be modeled, insufficient space existed for the magnesium ions in the active site.

Activation of multiple transcription factors and fos and jun gene family expression in cells exposed to a single electric pulse.

We report that exposure of cells to a single electric pulse (250-1250 V/cm) results in the rapid and persistent activation of the DNA binding activities of a number of transcription factors, including AP-1, SP1, AP-2, and NF-kappa B, and the transient expression of select members of the fos and jun gene families. Induction of gene expression occurs primarily at the level of transcription, although c-jun expression also appears to be regulated posttranscriptionally. Interestingly, maximal induction of gene expression is detected at electrical field strengths that do not result in pore formation in the plasma membrane and that do not significantly affect cell viability. Exposure of cells to electric pulses does not result in the activation of HSF1 DNA binding activity, or the induction of hsp70 or p53 protein synthesis, indicating that the induction of fos and jun gene expression is not coincident with protein or DNA damage. The results of these studies suggest that electrical pulses may represent a novel mechanism for inducing the activities of multiple transcription factors and the expression of select members of the fos and jun gene families.

Effects of transforming growth factor-beta on bone marrow macrophage Ia expression induced by cytokines.

The initiation of the immune response is regulated, in part, by the effect of cytokines on the level of expression of major histocompatibility complex (MHC) class II antigens on antigen-presenting cells (APC). The expression of class II antigens on B cell and macrophage APC is induced by IFN-gamma and IL-4, and GM-CSF induces class II expression on macrophages (M phi). Our results show that transforming growth factor-beta (TGF-beta) inhibits IL-4- and GM-CSF-induced Ia gene expression on bone marrow macrophages but enhances IFN-gamma-induced gene expression. Nuclear run-on experiments demonstrated that the inhibitory effects of TGF-beta on GM-CSF- and IL-4-induced Ia antigen expression were primarily posttranscriptional and augmentation of IFN-gamma by TGF-beta was largely transcriptionally regulated.

Secondary structure in solution of two anti-HIV-1 hammerhead ribozymes as investigated by two-dimensional 1H 500 MHz NMR spectroscopy in water.

Two hammerhead chimeric RNA/DNA ribozymes (HRz) were synthesized in pure form. Both were 30 nucleotides long, and the sequences were such that they could be targeted to cleave the HIV-1 gag RNA. Named HRz-W and HRz-M, the former had its invariable core region conserved, the latter had a uridine in the invariable region replaced by a guanine. Their secodary structures were determined by 2D NOESY 1H 500 MHz NMR spectroscopy in 90% water and 10% D2(0), following the imino protons. The data show that both HRz-M and HRz-W form identical secondary structures with stem regions consisting of continuous stacks of AT and GT pairs. An energy minimized computer model of this stem region is provided. The results suggest that the loss of catalytic activity that is known to result when an invariant core residue is replaced is not related to the secondary structure of the ribozymes in the absence of substrate.

TGF-beta 2 gene and protein expression in maternal and fetal tissues at various stages of murine development.

The transforming growth factor beta family of peptides have diverse actions on the reproductive tracts of primates and rodents. In this study we report the expression of high levels of mRNA of one member of this superfamily, TGF-beta 2, in the pregnant mouse uterus. Using Northern blot analysis and in situ hybridization techniques, we have examined the pattern of expression of TGF-beta 1, TGF-beta 2 and colony-stimulating factor (CSF-1) in mouse maternal and fetal tissue at specific days of gestation. We report here that TGF-beta 2 is synthesized primarily in maternal decidual and uterine epithelial tissues. We observed a shift in the major site of synthesis from decidua to uterus between days 8.5 and 10.5 of gestation. These data demonstrate that the expression of TGF-beta 2 is differentially regulated in the decidua and uterine epithelial cells at various times during gestation. Small amounts of TGF-beta 2 mRNAs were detected in the fetus, and none was detected in placenta, yolk sac, or amniotic membrane. The uterus is likely the major site of synthesis of the TGF-beta 2 found in mouse amniotic fluid. TGF-beta 1 mRNAs are expressed in the uterus at markedly lower levels when compared to TGF-beta 2 mRNAs in both the decidua and uterus. Our results suggest that there is a unique regulation of TGF-beta 2 during pregnancy which may depend on pregnancy hormone(s) and differentiates it from the other mammalian isoforms of the TGF-beta s. TGF-beta 2 may play an important, albeit unknown, role at the maternal/fetal interface.

Distinct IL-4 response mechanisms of the MHC gene A alpha in different mouse B cell lines.

Interleukin-4 (IL-4) is a multipotent cytokine which stimulates proliferation of B and T lymphocytes, induces B lymphocyte expression of major histocompatibility complex (MHC) class II molecules and Fc epsilon R II (CD23) molecules, and promotes immunoglobulin class switching to IgE and IgG1. The mechanisms by which IL-4 induces these changes are unclear. To study the basis for heterogeneity in induction of class II MHC proteins observed in splenic B cells, three mouse B cell lines were treated with IL-4, and the response of MHC class II A alpha mRNA was analyzed. Each of the three cell lines responded with a distinctive profile. In one line, 70Z/3, A alpha mRNA was induced greater than 10 fold by 65 hr of IL-4 stimulation. Additional studies showed that A alpha mRNA was stabilized by IL-4 treatment of 70Z/3 cells, and that changes in gene transcription accounted for little of the increase in mRNA levels. A second line, WEHI.231, was shown to increase A alpha mRNA levels 4 fold after 48 hr of IL-4 treatment. In contrast to 70Z/3, when A alpha mRNA stability in the IL-4 treated WEHI.231 cells was compared to untreated cells, no difference was observed, IL-4 treatment induced A alpha transcription. The third cell line, M12.4.1, expressed high basal levels of A alpha, and these levels increased only slightly following IL-4 stimulation. The small increase correlated with a comparable transcriptional response. These data shown that the nature of the A alpha gene response to IL-4 differs among B cell lines. This heterogeneity of response is consistent with responses in total splenic B cells, and with the existence of functionally distinct subpopulations of B cells.

Monoclonal antibody to the interferon-inducible protein Leu-13 triggers aggregation and inhibits proliferation of leukemic B cells.

Interferon (IFN)-alpha inhibits DNA synthesis stimulated by low molecular weight B-cell growth factor (BCGF) in hairy cells in vitro, suggesting that the therapeutic efficacy of IFN-alpha in hairy cell leukemia (HCL) involves growth inhibition of malignant B cells. Evidence that the 16-Kd cell surface protein Leu-13 mediates an antiproliferative signal in T lymphocytes and is IFN-inducible in endothelial cells prompted us to examine the expression and functional role of this molecule in leukemic B cells. Leu-13 density, determined by flow cytometry, was upregulated in vitro and in vivo by IFN-alpha on malignant B cells from patients with HCL, chronic lymphocytic leukemia, and prolymphocytic leukemia. Monoclonal anti-Leu-13 triggered homotypic aggregation of leukemic B cells via an adhesion pathway that was not inhibited by antibodies to leukocyte function associated antigen-1 (LFA-1) or intercellular adhesion molecule-1 (ICAM-1). Moreover, anti-Leu-13 potentiated the inhibitory effects of IFN-alpha on BCGF-stimulated DNA synthesis, assessed by [3H]-thymidine and [3H]-deoxyadenosine incorporation into DNA. These results indicate that Leu-13 is part of a novel IFN-inducible signaling pathway which may modify the growth and adhesive properties of leukemic B cells under physiologic or therapeutic conditions.

A transforming growth factor beta 2 (TGF-beta 2)-like immunosuppressive factor in amniotic fluid and localization of TGF-beta 2 mRNA in the pregnant uterus.

This report describes a murine amniotic fluid (MAF) immunosuppressive factor that has properties similar to transforming growth factor beta (TGF-beta). The MAF factor exhibits TGF-beta-like activity in stimulating soft agar colony formation by AKR-2B cells and inhibiting thymidine uptake by Mv1Lu cells. We demonstrate that both the immunosuppressive and TGF-beta-like activities of the MAF factor are completely neutralized by anti-TGF-beta 2-specific antibodies and not by anti-TGF-beta 1-specific antisera. The immunosuppressive factor in MAF is novel in that it appears to be identical or very closely related to TGF-beta 2 and is active in its native state. This active and anti-TGF-beta 2-neutralizable factor chromatographs at approximately 70 kD on Sephadex at neutral pH and appears to be able to complex with alpha-fetoprotein in native amniotic fluid. Chromatography of native MAF under acidic conditions demonstrates a lower molecular mass protein that chromatographs on BioGel in the same position as the mature 25-kD TGF-beta. This protein has the biological properties of TGF-beta and is immunosuppressive. Both of these activities are neutralizable with anti-TGF-beta 2 but not with anti-TGF-beta 1 or other antisera. By Northern analysis, we find high levels of TGF-beta 2 mRNA (with little or no TGF-beta 1) in the pregnant uterus that peak around day 15 of gestation and then fall rapidly by day 19 as birth approaches. The TGF-beta 2-like factor could possibly play a role in maternal immunity, in the retention of the fetal allograft, as well as in regulating fetal and neonatal immunological competence.

Regulation of MHC class II gene expression in macrophages by hematopoietic colony-stimulating factors (CSF). Induction by granulocyte/macrophage CSF and inhibition by CSF-1.

CSF-1 and granulocyte/monocyte CSF (GM-CSF) were shown to modulate the levels of Ia gene and protein expression in bone marrow-derived macrophages (BMM). Recombinant GM-CSF induced high levels of Ia expression, similar to the levels induced by INF-gamma, while IL-3 had no effect. In contrast, recombinant CSF-1 not only suppressed the basal levels of Ia gene and protein expression in BMM, but also inhibited the induction of Ia by IFN-gamma and GM-CSF. Basal levels of Ia were not inhibited by recombinant CSF-1 until after 16-24 h of culture, suggesting an indirect mechanism of suppression. IFN-alpha/beta and PGE2 were shown not to be involved in the CSF-1 inhibition of basal levels of Ia expression. However, the CSF-1-mediated suppression of both the basal levels of Ia expression and the induction of Ia in BMM by IFN-gamma and GM-CSF did correlate with the induction of cellular proliferation. These data imply that in addition to regulating hematopoiesis, CSFs may regulate the initiation of the immune response through their effects on Ia expression in macrophages.