Thomas M. Aune

Cutting Edge: Molecular Portrait of Human Autoimmune Disease

Autoimmune diseases affect 3–5% of the population, are mediated by the immune response to self-Ags, and are characterized by the site of tissue destruction. We compared expression levels of >4,000 genes in PBMC of control individuals before and after immunization to those of individuals with four distinct autoimmune diseases. The gene expression profile of the normal immune response exhibits coordinate changes in expression of genes with related functions over time. In contrast, each individual from all autoimmune diseases displays a similar gene expression profile unrelated to the pattern of the immunized group. To our surprise, genes with a distinct expression pattern in autoimmunity are not necessarily “immune response” genes, but are genes that encode proteins involved in apoptosis, cell cycle progression, cell differentiation, and cell migration.

Cutting Edge: Influence of Tmevpg1, a Long Intergenic Noncoding RNA, on the Expression of Ifng by Th1 Cells

The majority of the genome is noncoding and was thought to be nonfunctional. However, it is now appreciated that transcriptional control of protein coding genes resides within these noncoding regions. Thousands of genes encoding long intergenic noncoding RNAs (lincRNAs) have been recently identified throughout the genome, which positively or negatively regulate transcription of neighboring target genes. Both TMEVPG1 and its mouse ortholog encode lincRNAs and are positioned near the IFN-γ gene (IFNG). In this study, we show that transcription of both mouse and human TMEVPG1 genes is Th1 selective and dependent on Stat4 and T-bet, transcription factors that drive the Th1 differentiation program. Ifng expression is partially restored in Stat4−/−Tbx21−/− cells through coexpression of T-bet and Tmevpg1, and Tmevpg1 expression contributes to, but alone is not sufficient to, drive Th1-dependent Ifng expression. Our results suggest that TMEVPG1 belongs to the general class of lincRNAs that positively regulate gene transcription.

The Invasive Chytrid Fungus of Amphibians Paralyzes Lymphocyte Responses

Breaking Frog Defenses The first line of immune defense against most fungal infections consists of innate immune effector cells, including macrophages and neutrophils. However, Fites et al. (p. 366) have found that the fungus currently decimating the worlds amphibia, Batrachochytrium dendrobatidis, is readily engulfed by these cells, but that this does not effectively control the infection. The fungus releases cell-wall components that induce lymphocyte apoptosis and inhibit the proliferation of other nonlymphoid cell types, disarming lymphocyte-mediated responses to infection. The global spread of a fungal disease of frogs may be explained by its ability to inhibit immune clearance. The chytrid fungus, Batrachochytrium dendrobatidis, causes chytridiomycosis and is a major contributor to global amphibian declines. Although amphibians have robust immune defenses, clearance of this pathogen is impaired. Because inhibition of host immunity is a common survival strategy of pathogenic fungi, we hypothesized that B. dendrobatidis evades clearance by inhibiting immune functions. We found that B. dendrobatidis cells and supernatants impaired lymphocyte proliferation and induced apoptosis; however, fungal recognition and phagocytosis by macrophages and neutrophils was not impaired. Fungal inhibitory factors were resistant to heat, acid, and protease. Their production was absent in zoospores and reduced by nikkomycin Z, suggesting that they may be components of the cell wall. Evasion of host immunity may explain why this pathogen has devastated amphibian populations worldwide.

INHIBITION OF TUMOR CELL GROWTH BY INTERFERON-GAMMA IS MEDIATED BY TWO DISTINCT MECHANISMS DEPENDENT UPON OXYGEN TENSION: INDUCTION OF TRYPTOPHAN DEGRADATION AND DEPLETION OF INTRACELLULAR NICOTINAMIDE ADENINE DINUCLEOTIDE

Growth of a variety of human tumor cell lines is inhibited by interferon-gamma (IFN-gamma) in vitro. This mechanism is not well understood. The present experiments identify two separate mechanisms which account for the growth inhibitory activity of IFN-gamma. Cell lines most sensitive to IFN-gamma (inhibited by 10-30 U/ml IFN-gamma in 3 d) were stimulated by IFN-gamma to oxidize tryptophan in media to kynurenine and completely eliminated tryptophan from the culture media after 48-72 h. Addition of L-tryptophan, but not other aromatic amino acids, other essential amino acids, or D-tryptophan, prevented inhibition of cell growth by IFN-gamma. The amount of IFN-gamma required to yield 50% inhibition of cell growth was directly related to the concentration of L-tryptophan in culture media and increased from approximately 3 to 600 U/ml as the concentration of tryptophan in the media was increased from 25 to 1,000 microM. By contrast, inhibition of growth of the cell lines, BT20 and HT29, was not prevented by addition of tryptophan. Inhibition by IFN-gamma (100-300 U/ml after 5-6 d) was, however, completely prevented by addition of two inhibitors of adenosine diphosphate-ribosyl transferase (ADP-RT), 3-aminobenzamide or nicotinamide. Activity of ADP-RT was increased in these cell lines after addition of IFN-gamma. ADP-RT catalyzes the incorporation of the ADP moiety of nicotinamide adenine dinucleotide (NAD) into proteins and causes depletion of intracellular NAD. All tumor cell lines tested had reduced levels of intracellular NAD after treatment with IFN-gamma and loss of NAD preceded inhibition of cell growth by 12-24 h. Inhibitors of IFN-gamma-mediated inhibition of cell growth prevented loss of levels of intracellular NAD. Generation of reactive oxygen species lead to DNA strand breaks which result in activation of ADP-RT. Increased DNA strand breaks were induced in BT20 and HT29 cells but not ME180 and A549 cells after culture with IFN-gamma. The two enzymes known to catalyze the decyclization of tryptophan to kynurenine require superoxide anion for activity. Increased amounts of superoxide anion were released from ME180 and A549 cells after culture with IFN-gamma. Reduced oxygen concentration decreased the ability of IFN-gamma to inhibit tumor cell growth in vitro. Intracellular glutathione has been shown to protect cells against oxidative damage by various agents. Elevation or reduction of intracellular glutathione concentrations lowered or raised sensitivity of cell lines to IFN-gamma, respectively. These data indicate that at least two distinct mechanisms can account for IFN-gamma-madiated inhibition of tumor cell growth. Both mechanisms appear to be sensitive to oxygen tension and to changes in intracellular glutathione concentrations, and both mechanisms lead to loss of intracellular NAD.

Differential transcription directed by discrete gamma interferon promoter elements in naive and memory (effector) CD4 T cells and CD8 T cells.

Acquisition of the ability to produce gamma interferon (IFN-gamma) is a fundamental property of memory T cells and enables one subset (T helper 1 [TH1]) to deliver its effector functions. To examine regulation of IFN-gamma gene expression in a model system which recapitulates TH1 differentiation, we prepared reporter transgenic mice which express the luciferase gene under the control of proximal and distal regulatory elements (prox.IFN gamma and dist.IFN gamma) from the IFN-gamma promoter. Memory T cells, but not naive T cells, secreted IFN-gamma and expressed both prox.IFN gamma and dist.IFN gamma transcriptional activities. Naive T cells required priming to become producers of IFN-gamma and to direct transcription by these elements. While both CD4+ and CD8+ T cells produced IFN-gamma, only CD4+ T cells expressed prox.IFN gamma transcriptional activity. Induction of transcriptional activity was inhibited by known antagonists of effector T-cell populations. Cyclosporin A inhibited transcriptional activity directed by both elements in effector T cells. Elevated cyclic AMP inhibited transcriptional activity directed by prox.IFN gamma in primed CD4+ T cells but enhanced transcriptional activity directed by dist.IFN gamma in primed CD8+ T cells. Taken together, these data show that prox.IFN gamma and dist.IFN gamma transcriptional activities mirror IFN-gamma gene expression in naive and memory CD4+ T cells but suggest that differences exist in regulation of IFN-gamma gene expression in CD4+ and CD8+ T-cell subsets.

Cell contact between T cells and synovial fibroblasts causes induction of adhesion molecules and cytokines.

Human activated T cells adhere to synovial fibroblast‐like cells in vitro. The present study was conducted to investigate the consequences of T cell‐synovial fibroblast interactions with regard to induction of adhesion molecules and proinflammatory cytokines. A sensitive Western blot technique, polymerase chain reaction (PCR) amplification, and fluorescence‐activated cell sorter (FACS) analysis were used to analyze the induction of VCAM‐1 and ICAM‐1 expression in T cell‐synovial fibroblast cocultures. VCAM‐1 and ICAM‐1 expression could be induced in synovial fibroblast‐like cells by 2 h. PCR amplification showed that both forms of VCAM‐1 mRNA are found after the interaction of synovial fibroblasts with T cells. Up‐ regulation of VCAM‐1 and ICAM‐1 was confined to synovial fibroblasts; T cells did not express VCAM‐1 or increased ICAM‐1. In contrast to the T cell‐synoviocyte interaction, the interaction between T cells and dermal fibroblasts resulted in the up‐regulation of ICAM‐1 but not VCAM‐1, suggesting tissue‐specific regulation of VCAM‐1. The T cell‐synovial fibroblast interaction also resulted in increased levels of tumor necrosis factor (TNF), interferon‐ 7, and interleukin‐6 in coculture supernatant. Of the neutralizing antibodies used against these cytokines, only anti‐ TNF could significantly inhibit VCAM‐1 and ICAM‐1 expression. When T cells were separated from synoviocytes by a chamber that allowed medium exchange but no cell contact, VCAM‐1 and ICAM‐1 failed to be up‐regulated and cytokine accumulation in cocultures was drastically reduced. Our results demonstrate mutual cell activation of T cells and synoviocytes upon cell contact as shown by the release of T cell‐ and synoviocyte‐specific cytokines and suggest a cell contact‐mediated and T cell‐initiated mechanism for the chronic accumulation and retention of mononuclear cells via VCAM‐l/ICAM‐1 by synovial fibroblasts in the rheumatoid synovium.

Identification of the lymphokine soluble immune response suppressor in urine of nephrotic children.

Patients with minimal change nephrotic syndrome (MCNS) frequently have suppressed in vivo and in vitro immune responsiveness of uncertain etiology. Because increased suppressor cell activity has been associated with this disease, urines from MCNS patients were screened for activity of the lymphokine soluble immune response suppressor (SIRS), a product of concanavalin A- or interferon-activated suppressor T cells. Urines from untreated MCNS patients suppressed polyclonal plaque-forming cell responses of cultured splenocytes. This suppressive activity was identified as human SIRS by the following functional and physical criteria: molecular weight estimated by gel filtration; kinetics of suppression; inhibition of suppression by catalase, levamisole, and 2-mercaptoethanol; abrogation of activity by acid or protease treatment; elution pattern on high performance liquid chromatography; and cross-reactivity with monoclonal antimurine SIRS antibodies. Suppressive activity disappeared from urine after initiation of treatment but before remission of symptoms. Urines were tested from 11 patients with MCNS, all of whom excreted SIRS. In addition, two nephrotic patients with acute glomerulonephritis and three nephrotic patients with membranoproliferative disease excreted SIRS, but other nephrotics and all nonnephrotic patients did not. These results indicate that excretion of SIRS occurs in certain cases of nephrotic syndrome and that the presence of SIRS in the urine is not accounted for solely by the presence of proteinuria or nephrosis. Serum from four nephrotic patients also contained SIRS, whereas neither serum nor urine from six normal subjects contained SIRS activity. The systemic presence of SIRS in these four patients, and the identification of SIRS in urines from a larger group of patients, suggest a possible role for SIRS in the suppressed immune responses often found in nephrotic syndrome.

Epigenetics and T helper 1 differentiation

Naïve T helper cells differentiate into two subsets, T helper 1 and 2, which either transcribe the Ifng gene and silence the Il4 gene or transcribe the Il4 gene and silence the Ifng gene, respectively. This process is an essential feature of the adaptive immune response to a pathogen and the development of long‐lasting immunity. The ‘histone code’ hypothesis proposes that formation of stable epigenetic histone marks at a gene locus that activate or repress transcription is essential for cell fate determinations, such as T helper 1/T helper 2 cell fate decisions. Activation and silencing of the Ifng gene are achieved through the creation of stable epigenetic histone marks spanning a region of genomic DNA over 20 times greater than the gene itself. Key transcription factors that drive the T helper 1 lineage decision, signal transducer and activator 4 (STAT4) and T‐box expressed in T cells (T‐bet), play direct roles in the formation of activating histone marks at the Ifng locus. Conversely, STAT6 and GATA binding protein 3, transcription factors essential for the T helper 2 cell lineage decision, establish repressive histone marks at the Ifng locus. Functional studies demonstrate that multiple genomic elements up to 50 kilobases from Ifng play critical roles in its proper transcriptional regulation. Studies of three‐dimensional chromatin conformation indicate that these distal regulatory elements may loop towards Ifng to regulate its transcription. We speculate that these complex mechanisms have evolved to tightly control levels of interferon‐γ production, given that too little or too much production would be very deleterious to the host.

Dynamic alterations in the conformation of the Ifng gene region during T helper cell differentiation

Gene expression and silencing in eukaryotic systems can be controlled by regulatory elements acting over a distance. Here, we analyze chromatin conformation of the 24-kb region of the Ifng gene during CD4+ T helper (Th) cell differentiation. We find that chromatin within this region is a highly flexible structure that undergoes dynamic changes during the course of transcriptional activation and silencing of the Ifng gene. Each Th subset displays a common core conformation in this gene region and unique features that distinguish neutral and effector Th1 and Th2 lineages. This chromatin configuration brings distal regions into close proximity to the gene. Th1 cells that produce high levels of IFN-γ display the most open conformation. In contrast, IFN-γ silent Th2 cells have a tightly closed conformation. Therefore, we postulate that there is a direct structure–function relationship between the spatial organization of the chromatin around the Ifng gene and its transcriptional potential.

Gene expression signatures for autoimmune disease in peripheral blood mononuclear cells

The relatively new technology of DNA microarrays offers the possibility to probe the human genome for clues to the pathogenesis and treatment of human disease. While early studies using this approach were largely in oncology, many new reports are emerging in other fields including infectious diseases and pharmacology, and applications in autoimmunity have been recently reported by our group and others. Some of these investigations have examined animal models of autoimmune disease, but a number of human studies have also been carried out. Of special interest are those that have used peripheral blood samples because, unlike tissue biopsies, these are readily available from all subjects. Using this approach, patterns of gene expression can be detected that distinguish patients with autoimmune conditions from normal subjects. Furthermore, the genes that are identified provide clues to possible pathogenetic mechanisms and are likely to be useful in developing tests to establish diagnostic categories and predict therapeutic responses.