A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival.

The role of DNA methylation and of the maintenance DNA methyltransferase Dnmt1 in the epigenetic regulation of developmental stage- and cell lineage-specific gene expression in vivo is uncertain. This is addressed here through the generation of mice in which Dnmt1 was inactivated by Cre/loxP-mediated deletion at sequential stages of T cell development. Deletion of Dnmt1 in early double-negative thymocytes led to impaired survival of TCRalphabeta(+) cells and the generation of atypical CD8(+)TCRgammadelta(+) cells. Deletion of Dnmt1 in double-positive thymocytes impaired activation-induced proliferation but differentially enhanced cytokine mRNA expression by naive peripheral T cells. We conclude that Dnmt1 and DNA methylation are required for the proper expression of certain genes that define fate and determine function in T cells.

The roles of nuclear factor of activated T cells and ying-yang 1 in activation-induced expression of the interferon-gamma promoter in T cells.

Nuclear factor of activated T cells (NFAT) plays an important role in expression of many cytokine genes including interleukin-2 and interleukin-4. However, its role in interferon-gamma (IFN-gamma) expression is not well understood. In the current studies, two strong NFAT-binding sites in the IFN-gamma promoter were identified by DNase I footprint analysis at positions -280 to -270 and -163 to -155. NFATp bound independently to both sites and was required for the formation of a composite element with AP-1 spanning position -163 to -147. In Jurkat T cells and primary lymphocytes, activation-induced expression of IFN-gamma reporter constructs containing point mutations in either NFAT site or the AP-1 component of the composite site was decreased by approximately 40-65%. Despite elimination of both strong NFAT-binding sites, the IFN-gamma promoter remained completely sensitive to inhibition by cyclosporin. This suggests that other elements in the IFN-gamma promoter, such as the IFN-gamma proximal element, are sufficient for cyclosporin sensitivity of this gene. Ying-Yang 1 (YY1), a potential inhibitor of IFN-gamma expression, binds to sites located between the two NFAT sites. Mutation of the YY1 sites alone had little effect on IFN-gamma promoter activity. However, mutation of both the NFAT and YY1-binding sites abolished activation-induced expression in primary murine splenocytes but not in Jurkat T cells. This suggests that under some conditions, YY1 may play a positive role in activation-induced transcription of IFN-gamma.

The proximal regulatory element of the interferon-gamma promoter mediates selective expression in T cells.

Interferon-gamma (IFN-gamma) is produced by natural killer cells and certain subsets of T cells, but the basis for its selective expression is unknown. Within the region between -108 and -40 base pairs of the IFN-gamma promoter are two conserved and essential regulatory elements, which confer activation-specific expression in T cells. This report describes studies indicating that the most proximal of these two regulatory elements is an important determinant of its restricted expression. The proximal element is a composite site that binds members of the CREB/ATF, AP-1, and octamer families of transcription factors. Jun is essential for activation-induced transcription and binds preferably as a heterodimer with ATF-2. In contrast, CREB appears to dampen transcription from this element. The CpG dinucleotide in this element is selectively methylated in Th2 T cells and other cells that do not express IFN-gamma, and methylation markedly reduces transcription factor binding. As a target for DNA methylation and for binding of transcription factors that mediate or impede transcription, this element appears to play a central role in controlling IFN-gamma expression.

Two essential regulatory elements in the human interferon gamma promoter confer activation specific expression in T cells.

Like interleukin 2 (IL-2), interferon gamma (IFN-gamma) is an early response gene in T cells and both are prototypical T helper cell type 1 (Th-1) lymphokines. Yet IL-2 and IFN-gamma production are independently regulated, as demonstrated by their differential expression in certain T cell subsets, suggesting that the regulatory elements in these two genes must differ. To explore this possibility, the 5' flank of the human IFN-gamma gene was analyzed. Expression of IFN-gamma promoter-driven beta-galactosidase reporter constructs containing 538 bp of 5' flank was similar to that by constructs driven by the IL-2 promoter in activated Jurkat T cells; expression nearly as great was observed with the construct containing only 108 bp of IFN-gamma 5' flank. These IFN-gamma promoter constructs faithfully mirrored expression of the endogenous gene, in that expression required activation both with ionomycin and PMA, was inhibited by cyclosporin A, and was not observed in U937 or THP-1 cells. The region between -108 and -40 bp in the IFN-gamma promoter was required for promoter function and contained two elements that are conserved across species. Deletion of 10 bp within either element reduced promoter function by 70%, whereas deletions in nonconserved portions of this region had little effect on promoter function. The distal conserved element (-96 to -80 bp) contained a consensus GATA motif and a potential regulatory motif found in the promoter regions of the GM-CSF and macrophage inflammatory protein (MIP) genes. Factors binding to this element, including GATA-3, were found in Jurkat nuclear extracts by electromobility shift assays and two of the three complexes observed were altered in response to activation. One or both of these motifs are present in the 5' flank of multiple, other lymphokine genes, including IL-3, IL-4, IL-5, and GM-CSF, but neither is present in the promoter of the IL-2 gene. The proximal conserved element (-73 to -48 bp) shares homology with the NFIL-2A element in the IL-2 promoter; these elements compete for binding of factors in Jurkat nuclear extracts, although the NFIL-2A element but not the IFN-gamma element binds Oct-1. Factors binding to this element in the IFN-gamma gene were present in extracts from resting and activated Jurkat T cells. However, by in vivo footprinting of intact cells, this element was protected from methylation only with activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Ontogeny of T lymphocyte function in the neonate.

T cell precursors are first detected in the thymus at eight weeks of gestation. By 15 to 20 weeks of gestation, T-cell precursors expressing alpha beta and gamma delta T-cell receptors are present in the thymus in numbers relatively similar to those found in postnatal life. However, recent data suggest that T-cell receptor diversity is more limited during fetal and neonatal life than in adults. Additionally, the functional capacity of T cells in the fetus and neonate is immature, in that neonatal T cells express a limited repertoire of lymphokines in response to activation. Specifically, the production of the lymphokines, interferon-gamma and interleukin-4, which participate in the maturation of cytotoxic cells, activation of macrophages, and the maturation and modulation of B cell function and isotype expression, is reduced more than tenfold compared to cells from adults. This appears to result primarily from the lack of memory T cells in the fetus and neonate, reflecting their antigenic naivete. The difference in lymphokine expression is due to diminished transcription of these genes in neonatal T cells in response to activation. Preliminary data indicate that differences in essential promoter elements regulating transcription of these lymphokine genes plays a role in their differential expression in T cells.

Differential regulation of lymphotoxin and tumor necrosis factor genes in human T lymphocytes.

Lymphotoxin (LT) and tumor necrosis factor (TNF) are related cytokines that share many biological effects. The genes for LT and TNF are adjacent to each other on chromosome 6 in man, but previous data indicate that the kinetics of their production differ markedly. To explain the mechanisms for this difference, we compared the regulation of these two genes in human T lymphocytes, isolated from peripheral blood, after stimulation with the mitogens concanavalin A and phorbol myristate acetate. Differences in the kinetics of protein secretion were paralleled by differences in cognate mRNA accumulation. TNF mRNA accumulated rapidly after stimulation, peaked by 6 h, and returned to unstimulated (base-line) levels by 24 h. In contrast, LT mRNA accumulated slowly after stimulation, usually peaked at approximately 18 h, and remained increased above base-line levels at 48-72 h. By nuclear transcription run-on assays, increased transcription of TNF mRNA and LT mRNA was demonstrated after stimulation. However, TNF transcription peaked earlier and appeared to be 4-10 times greater than that of the LT gene. In contrast, the half-life of LT mRNA was 8-10-fold longer than that of TNF mRNA as demonstrated by actinomycin D pulse-chase experiments. Cycloheximide did not block LT or TNF mRNA accumulation, indicating that new protein synthesis was not required for induction of either gene. These results suggest strongly that the LT and TNF genes are regulated differently in human T lymphocytes after mitogen stimulation. TNF mRNA accumulates rapidly primarily because of increased transcription and decreases rapidly related to its brief half-life. In contrast, LT mRNA accumulates more slowly but persists much longer; the accumulation of this mRNA appears to be controlled largely by post-transcriptional mechanisms.

Regulation of tumor necrosis factor/cachectin and IL-1 secretion in human mononuclear phagocytes.

To determine whether the production and secretion of TNF and IL-1 by human mononuclear phagocytes could be independently modulated, we examined secretion of TNF and IL-1 by fresh monocytes and monocytes pretreated with IFN-gamma or granulocyte macrophage CSF before LPS stimulation. TNF and IL-1 secretion were in part differentially modulated. Fresh monocytes secreted large amounts of TNF and IL-1 after LPS stimulation and less than 6% as much without LPS. The capacity to secrete TNF in response to LPS decreased slightly in cultured monocytes but was markedly augmented by IFN-gamma (approximately five-fold more than fresh monocytes). In contrast, cultured monocytes secreted less than 5% as much IL-1 as fresh monocytes and, although augmented by IFN-gamma, IL-1 secretion remained much less than by fresh monocytes. These differences in modulation were reflected by differences in the molecular mechanisms regulating TNF and IL-1 secretion. TNF secretion was regulated primarily by changes in the duration of increased transcription and by an apparent increase in translation or protein stability in response to LPS; greater than 95% TNF produced was secreted under all conditions. In contrast, the changes in IL-1 secretion reflected primarily post-transcriptional regulation of IL1-alpha mRNA, transcriptional and post-transcriptional regulation of IL-1 beta mRNA and a decrease in the fraction of IL-1 secreted by cultured compared with fresh monocytes (10 and 60%, respectively). Changes in translational efficiency or protein processing or stability appeared not to be important mechanisms regulating IL-1 secretion. Additional evidence that TNF and IL-1 can be differentially modulated was the selective decrease in TNF secretion and the failure of IFN-gamma to enhance TNF secretion by cultured monocytes from neonates, whereas results for IL-1 were similar with adult and neonatal monocytes. Results with tissue macrophages were similar to those with cultured monocytes. These results indicate that TNF and IL-1 production and secretion by mononuclear phagocytes can be differentially modulated, reflecting in part different mechanisms of regulation; this may allow them to play partially independent roles in the host immune response.

Comparative susceptibility of group B streptococci and Staphylococcus aureus to killing by oxygen metabolites.

Group B streptococci (GBS) lack catalase, and they produce and release H2O2;thus, they should be readily killed by phagocytes with a diminished respiratory burst. Surprisingly, although strains of Staphylococcus aureus were killed at H2O2 concentrations greater than 0.5 mM, GBS strains were killed only at concentrations greater than 5mM. In contrast, GBS were killed by hydroxyl radicals generated by the xanthine oxidase-acetaldehyde system at O2 fluxes greater than or equal to 3.5 nmol/ml per min, whereas O2 fluxes greater than or equal to 10 nmol/ml per min were required to kill the S. aureus strains. Results with virulent and laboratory strains of GBS were similar. The differences in susceptibility of GBS and S. aureus seemed to correlate with differences in content of endogenous oxygen-metabolite scavengers. GBS contained approximately 100-fold more glutathione and approximately 20-fold more glutathione reductase than did S. aureus, whereas S. aureus was rich in catalase that GBS lacked. GBS that were grown in buthionine sulfoximine, however, contained 87% less glutathione than did controls but were not more susceptible to killing by H2O2 or the xanthine oxidase-acetaldehyde system. Similarly, the relative susceptibility of GBS to tert-butyl hydroperoxide and H2O2 paralleled that of S. aureus. Thus, inherent differences in susceptibility of vital cellular functions to oxidative damage rather than content of oxygen-metabolite scavengers may account for the differences in susceptibility of GBS and S. aureus.

Phenotypic and genetic parameters for growth, puberty and composition traits in gilts.

A total of 12 Landrace and 12 Duroc boars were mated randomly to crossbred sows to produce 210 gilts over six farrowing seasons. Each sire was used in only one farrowing season. Biweekly measurements were taken on the live gilts for height, weight, loin depth, shoulder backfat, 10th rib backfat and stifle backfat from 30 to 105 kg. Gilts were removed from confinement units and placed in pasture lots. Estrous detection began when the gilts reached 68 kg and continued until 75% of gilts in a test group had exhibited estrous. Compositional traits were regressed to 105 kg prior to being analyzed. Estimates of genetic parameters were determined using paternal half-sib analysis. Heritability estimates for age at first detectable estrous (ACYCL), tenth rib backfat at 105 kg (BFB) and average daily gain (ADG) were .29, .28 and greater than 1, respectively. Phenotypic correlations between ACYCL and backfat traits were approximately zero, those between ACYCL and growth traits were moderate and desirable and those between growth and backfat traits were low. Genetic correlation between ACYCL and ADG was estimated to be -.67 indicating faster gaining gilts were cycling at a younger age. Estimated genetic correlation between ACYCL and BFB was -.77 indicating that leaner gilts were cycling at a later age. Also, genetic correlation estimated between BFB and ADG was .64 inferring that faster gaining gilts were depositing more backfat.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation, purification and characteristics of mononuclear phagocytes from human placentas.

Methods for the isolation of mononuclear phagocytes from human placentas by digestion with collagenase or with trypsin are described. Mononuclear phagocytes were approximately 35% of the cells obtained. Preparations were enriched for mononuclear phagocytes by sequential density gradient centrifugation over Ficoll-Hypaque and Percoll, adherence to plastic and removal of contaminating trophoblastic cells with brief trypsin exposure. Monolayers obtained by these methods were greater than 85% mononuclear phagocytes. These cells were predominantly of fetal origin; most were mature macrophages but up to 20% appeared to be recently derived from blood monocytes as determined by ultrastructural peroxidase cytochemistry.

Effects of muramyl dipeptide on superoxide anion release and on anti-microbial activity of human macrophages.

Exposure of human monocyte derived macrophages to muramyl dipeptide in vitro increased phorbol myristate acetate-stimulated O2- release by these cells; the activity of such macrophages against Toxoplasma gondii or against Staphylococcus aureus was not increased. The failure of muramyl dipeptide to enhance human macrophage anti-microbial activity correlated with the failure of muramyl dipeptide to enhance O2- release in response to phagocytic stimuli. These results and those previously published by others indicate that muramyl dipeptide may enhance release of certain inflammatory mediators (O2- and endogenous pyrogen) without enhancing the anti-microbial activity of human macrophages.