Dontcho Z. Staynov

Cytokine Coexpression During Human Th1/Th2 Cell Differentiation: Direct Evidence for Coordinated Expression of Th2 Cytokines

We have developed an in vitro differentiation assay in which human naive CD4+ cells are driven toward either the Th1 or Th2 phenotype. We have examined the interrelationships among the expression of IL-2, IL-4, IL-5, IL-10, IL-13, GM-CSF, and IFN-γ in individual cells using intracellular cytokine staining at various times during the differentiation process. We provide direct evidence that the Th2 cytokines IL-4, IL-5, and IL-13, unlike the other cytokines, are regulated by a coordinated mechanism. We also show that IL-10 is expressed by a different subset of cells that is prevalent at early stages of Th2 differentiation, but then diminishes. Additionally we demonstrate that while naive cells can express IL-2 upon activation, they cannot express GM-CSF. Commitment to GM-CSF expression occurs during differentiation in a Th1/Th2 subset-independent manner. Furthermore, we have examined the levels of GATA3, c-Maf, T-bet, and Ets-related molecule during human Th1/Th2 differentiation and suggest that differences in the levels of these critical transcription factors are responsible for commitment toward the Th1 or Th2 lineage.

DNA Methylation Changes at Human Th2 Cytokine Genes Coincide with DNase I Hypersensitive Site Formation During CD4+ T Cell Differentiation

The differentiation of naive CD4+ T lymphocytes into Th1 and Th2 lineages generates either cellular or humoral immune responses. Th2 cells express the cytokines IL-4, -5, and -13, which are implicated in asthma and atopy. Much has been published about the regulation of murine Th2 cytokine expression, but studies in human primary T cells are less common. We have developed a method for differentiating human CD45RA+ (naive) T cells into Th1 and Th2 populations that display distinct cytokine expression profiles. We examined both CpG methylation, using bisulfite DNA modification and sequencing, and chromatin structure around the IL-4 and IL-13 genes before and after human T cell differentiation and in normal human skin fibroblasts. In naive cells, the DNA was predominantly methylated. After Th2 differentiation, DNase I hypersensitive sites (DHS) appeared at IL-4 and IL-13 and CpG demethylation occurred only around the Th2-specific DHS. Both DHS and CpG demethylation coincided with consensus binding sites for the Th2-specific transcription factor GATA-3. Although fibroblasts, like naive and Th1 cells, did not express IL-4 or IL-13, DHS and unmethylated CpG sites that were distinct from the Th2-specific sites were observed, suggesting that chromatin structure in this cluster not only varies in T cells according to IL-4/IL-13 expression but is also tissue specific.

Suppression of Granulocyte-Macrophage Colony-Stimulating Factor Expression by Glucocorticoids Involves Inhibition of Enhancer Function by the Glucocorticoid Receptor Binding to Composite NF-AT/Activator Protein-1 Elements

Increased expression of a number of cytokines including GM-CSF is associated with chronic inflammatory conditions such as bronchial asthma. Glucocorticoid therapy results in suppression of cytokine levels by a mechanism(s) not yet fully understood. We have examined regulation of GM-CSF expression by the synthetic glucocorticoid dexamethasone in human T cells. Transient transfection assays with reporter constructs revealed that dexamethasone inhibited the function of the GM-CSF enhancer, but had no effect on regulation of GM-CSF expression occurring through the proximal promoter. Activation of the GM-CSF enhancer involves cooperative interaction between the transcription factors NF-AT and AP-1. We demonstrate here that glucocorticoid-mediated inhibition of enhancer function involves glucocorticoid receptor (GR) binding to the NF-AT/AP-1 sites. These elements, which do not constitute recognizable glucocorticoid response elements, support binding of the GR, primarily as a dimer. This binding correlates with the ability of dexamethasone to inhibit enhancer activity of the NF-AT/AP-1 elements, suggesting a competition between NF-AT/AP-1 proteins and GR.

DNase I footprinting of the human interleukin-5 gene promoter.

A characteristic feature of allergic asthma is the overexpression of the T helper type 2 (Th2) cytokines interleukin‐4 (IL‐4), IL‐5 and IL‐13 by T lymphocytes. Of these cytokines, IL‐5 is critical for the growth, survival and recruitment of eosinophils which are thought to be responsible for the tissue damage observed in asthmatic airways. The expression of human IL‐5 is primarily regulated at the transcriptional level; however, little is known about the mechanisms that control its transcription. Using nuclear extracts from allergen‐specific human T‐cell clones we have performed DNase I footprinting of the human IL‐5 promoter in order to establish sites occupied by transcription factors. We show footprints covering the conserved lymphokine element 0 [(CLE0) – 60 to – 44 base pairs (bp)] and GATA (– 73 to – 62 bp) elements, which have previously been identified to be important in the regulation of the murine IL‐5 promoter. We also describe a footprint covering a considerably extended Octamer binding site (– 249 to – 217 bp), which encompasses two hitherto unidentified CCAAT/enhancer binding protein consensus binding sites. We have also identified a previously unknown Ets binding site (– 274 to – 264 bp). These novel data on the regions of the human IL‐5 promoter that are bound by transcription factors should allow dissection of the regulatory mechanisms involved in the transcription of IL‐5 in the T‐helper lymphocytes of asthmatics.

Regulation of IL-4, IL-5 and GM-CSF in T Lymphocytes

Publisher Summary The transcriptional regulation of IL-4, IL-5 and GM-CSF is a very complex process involving interactions between different cisacting elements and transcription factors. It is important to note that NF-AT appears to be required for the efficient activation of all of these genes in T cells. This is interesting because NF-AT is also essential for the activation of IL-2, a Thl cytokine. The NFAT complex is a family of related proteins (NFAT-1, -2, -3, -4) and it will be interesting to identify the role of each NF-AT family member in the co-ordinated expression of these cytokines, especially in the light of the results from the NFATp knock-out mouse showing that IL-2 is expressed normally. Similarly, elucidation of the functions of the palindromic elements has been recently described, and identification of the factors that bind to them, are central to determining the mechanisms that control the expression of these cytokines.

Regulation of the Cytokine Gene Cluster on Chromosome 5q

The role of cytokines in the inflammatory processes observed in asthma and atopy has become an area of intense research in recent years. This research has demonstrated that a large number of cytokine genes are overexpressed by inflammatory cells, including T lymphocytes, in patients with atopic disease. CD4+ T-helper lymphocytes have been divided into subsets based upon the cytokine genes that they express on activation [1]. T-helper (Th)1 cells express interleukin (IL)-2 and interferon-γ (IFN-γ) but not IL-4 or IL-5, whereas Th2 cells express IL-4, IL-5, IL-10 and IL-13, but not IL-2 or IFN-γ. Both cell types express IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). A third subset of cells termed Th0 has also been identified which express all of the aforementioned cytokines. These T cell subsets were originally identified in the mouse and a similar, though not identical, pattern of cytokine expression has since been observed in human T cells [2]. The cytokine genes encoding IL-3, IL-4, IL-5, IL-13 and GM-CSF are located in close proximity on human chromosome 5q with a similar gene cluster found on mouse chromosome 11q. This chapter looks at the evidence for the over-expression of a Th2-like cytokine profile in asthma, focusing on the gene cluster on chromosome 5q, and the mechanisms by which these genes are regulated in T cells.

Ricin-Sensitive Early Activated CD8+ T Cells Suppress IgE Responses and Regulate the Production of IFN-γ and IL-4 by Splenic CD4+, CD8+ T Cells

Immunization of rats with a bystander antigen and the toxic lectin, ricin, results in a dramatically potentiated IgE response which was only seen when ricin was administered together with antigen, or

Transcriptional regulation of interleukin‐5 gene expression in human T‐cells

Copyright D Munksgaard 1995 ALLERGY Supplement 25 ISBN 87-16-15155-0 ISSN 0108-1675 Transcriptional regulation of interleukin-5 gene expression in human T-cells D. Z. Staynov, D. J. Cousins, D. Richards, D. M. Kemeny, T. H. Lee Department of Allergy & Respiratory Medicine, UMDS, Guy’s Hospital, London, SEl 9RT, UK Cytokines are important molecules for cellular communication in asthma. Analysis of bronchial biopsies and lavage samples from asthmatic individuals has shown a characteristic eosinophilia and increased numbers of cells expressing Th2 cytokines including interleukin-5 (IL-5) mRNA. IL-5 promotes terminal differentiation of the committed eosinophil precursor and prolongs the survival of eosinophils in v i m . It is therefore pertinent to study the mechanisms of transcriptional control of IL-5 gene expression. Using DNase I footprinting and band retardation assays on nuclear extracts derived from human T-cell clones, we have found several cis acting elements in the promoter of the IL-5 gene that may have implications in T-cell differentiation and activation. These include: 1) A 15 nucleotide sequence ATTATTCATTTCCTC known as conserved lymphokine element 0 (CLEO). This element is fully conserved between mouse and human IL-5 and differs by only one nucleotide in granulocyte macrophage colonystimulating factor (GM-CSF). Although this sequence is not found in other genes, it binds transcription factors (CLEOa and b) found in many different cells. of many genes. These factors often work in concert with other nearby elements. In the IL-5 promoter, this sequence is adjacent to a CCAAT/enhancer binding protein (C/EBP) element. 3 ) TGGGGCAAT, this sequence binds members of the C/EBP family such as nuclear factor-IL-6 (NLIL6), which has been shown to be involved in the activation of several cytokine genes. 4) GAGGAA, which binds transcription factors of the ets proto-oncogene family. It is different to the consensus sequence for the ets-1 and ets-2 factors, found in activated T-cells that are involved in IL-2 regulation. 5 ) We have also discovered a new cytolune-specific element that binds several different tissue-specific nuclear proteins. It has related sequences in the promoters of GM-CSF, IL-4, and IL-13 genes, but not in the IL-9 and interferon y genes. The results show that the regulation of IL-5 gene expression involves complex interactions between several transcription factors. One pathway involved in this process has similarities to that of GM-CSF activation and another has similarities to IL-2 activation. 2) ATTTGCAT: this sequence binds proteins of the Octamer (Oct) family and is found in the promoters