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C J Sanderson

Publications and source records attributed to C J Sanderson.

At least 19 recordsLinked to original sources
PubMed1999

Identification of residues involved in binding of IL5 to betacom using betaIL3 and betacom chimeras.

In mice there are two forms of the beta chain used in the IL3 receptor system, betacom and betaIL3. betacom is used by the IL3, IL5 and GM-CSF receptors whereas betaIL3 is only used in the IL3 receptor. In this work an assay was developed to identify residues of beta1L3 that restrict IL5 activity. It was found that such residues reside within the 2nd CRM of the molecule. Furthermore, when residues in the betaIL3 B'-C' loop were replaced with betacom sequence a form of betaIL3 was produced that was able to respond to IL5. This region is also responsible for IL3 binding to betaIL3 in the absence of alpha chain. It is therefore an important structural motif of betacom and betaIL3 responsible for both ligand interaction and specificity.

Amino Acid Sequence↗
PubMed1999

The activity of the human interleukin-5 conserved lymphokine element 0 is regulated by octamer factors in human cells.

Interleukin-5 (IL-5) controls the development of eosinophilia and contributes to a number of disease states including asthma. Expression of IL-5 is inducible under tight transcriptional regulation. This requires the contribution of several promoter elements; however, the conserved lymphokine element 0 (CLE0) in particular, is essential for expression of IL-5. In this study, we report the nuclear factors which regulate human IL-5 CLE0 activity in the human cell line PER-117. Using specific antibodies, we identified the transcriptional factors Oct-1 and Oct-2 binding to the 5' region of the CLE0 element. The involvement of Oct factors with CLE0 has not been reported previously in any of the lymphokines. In addition, the CLE0 element also appeared to complex with the transcriptional activator AP-1, consisting of the family members Jun D and Fra-2. We observed the binding of Oct-1 to be constitutive in comparison to Oct-2 and AP-1, both of which were induced in response to cell activation by PMA/A23187. Although the interaction of all three factors with CLE0 was closely linked and overlapping, residues critical to their binding were identified. We demonstrate, using site-directed mutagenesis and cotransfection experiments, that the CLE0 element is indispensable for IL-5 promoter activity and that Octamer factors contribute to the positive regulation of the hIL-5 gene.

Base Sequence↗
PubMed1999

A human T-cell line with inducible production of interleukins 5 and 4. A model for studies of gene expression.

The production of interleukin-5 (IL5) and interleukin-4 (IL4) by activated T-cells is important in the pathogenesis of helminth infections and allergy. Human Jurkat cells express IL4 but one of the main factors restricting studies of human IL5 expression has been the lack of human T-cell lines which express significant levels of IL5 in an inducible fashion. We report that the human T-cell leukemia cell line (PER-117), previously shown to produce IL2, also produces IL5 and IL4, and is a useful model for the study of the regulation of IL5 and IL4 gene expression. We show that expression of IL5 and IL4 mRNAs in PER-117 cells is stimulation dependent. IL5 and IL4 reporter constructs are also transiently expressed in these cells in an inducible fashion. IL5 production in the PER-117 cell line can be activated by phorbol 12-myristate 13-acetate alone and further enhanced by calcium ionophore A23187, cyclic adenosine 3', 5'-monophosphate or anti-CD28 antibodies. The conditions used to stimulate the PER-117 cells determined whether IL5 production was inhibited by cyclosporin A or dexamethasone. These data indicate that the PER-117 cell line is a model to study signal transduction and transcriptional activation of the human IL5 gene in human T-cells.

Base Sequence↗
PubMed1999

Analysis of the 5' and 3'UTRs in the post-transcriptional regulation of the interleukin-5 gene.

Post-transcriptional regulation is emerging as an important control point in cytokine gene expression. However, the role that it plays in IL-5 gene expression is unclear with some conflicting reports. Here we investigate the importance of post-transcriptional regulation and the role of the 5' and 3' untranslated regions (UTRs) in mIL-5 gene expression. To do this, IL-5 expression from a panel of cDNA constructs was compared. We found it essential to remove the 5' synthetic oligonucleotide tails, introduced during the cloning of the mIL-5 cDNA, when studying IL-5 expression. The presence of these oligo(G) tails acted as potent inhibitors of translation of both SV40 and SP6 transcripts. Furthermore, the length of the tails was found to be critical to the translational efficiency. Taking this into account, we found no evidence to suggest that IL-5 is regulated at the level of mRNA stability or translation efficiency by either the 5' or 3'UTR. These results suggest that post-transcriptional control is not a major factor regulating IL-5 expression.

3' Untranslated Regions↗
PubMed1999

Binding of YY1 and Oct1 to a novel element that downregulates expression of IL-5 in human T cells.

BACKGROUND: IL-5 controls development of eosinophilia and has been shown to be involved in the pathogenesis of allergic diseases. In both atopic and nonatopic asthma, elevated IL-5 has been detected in peripheral blood and the airways. IL-5 is produced mainly by activated T cells, and its expression is regulated at the transcriptional level. OBJECTIVE: This study focuses on the functional analysis of the human IL-5 (hIL-5) promoter and characterization of cis -regulatory elements and transcription factors involved in the suppression of IL-5 transcription in T cells. METHODS: Methods used in this study include DNase I footprint assays, electrophoretic mobility shift assays, and functional analysis by mammalian cell transfection involving deletion analysis and site-directed mutagenesis. RESULTS: We identified 5 protein binding regions (BRs) located within the proximal hIL-5 promoter. Functional analysis indicates that the BRs are involved in control of hIL-5 promoter activity. Two of these regions, BR3 and BR4 located at positions -102 to -73, have not previously been described as regulators of IL-5 expression in T cells. We show that the BR3 sequence contains a novel negative regulatory element located at positions -90 to -79 of the hIL-5 promoter, which binds Oct1, octamer-like, and YY1 nuclear factors. Substitution mutations, which abolished binding of these proteins to the BR3 sequence, significantly increased hIL-5 promoter activity in activated T cells. CONCLUSION: We suggest that Oct1, YY1, and octamer-like factors binding to the -90/-79 sequence within the proximal IL-5 promoter are involved in suppression of IL-5 transcription in T cells.

Base Sequence↗
PubMed1999

Nuclear factor of activated T cells and YY1 combine to repress IL-5 expression in a human T-cell line.

BACKGROUND: IL-5 is an inducible T-cell cytokine with the unique ability to induce eosinophilia without increases in other cell compartments. Regulation of IL-5 expression is controlled primarily at the level of transcription. The role of eosinophilia in allergic disorders indicates IL-5 as a target for therapy. OBJECTIVE: This report aims to increase our understanding of IL-5 gene regulation by identifying distal control elements in the human (h) IL-5 promoter, determining the transcription factors that bind these elements and elucidating their role in control of hIL-5 gene expression. METHODS: Methods used in this study include deoxyribonuclease I footprint analysis, electrophoretic mobility shift assay, and functional analysis by transfection of PER-117 cells with site-directed mutants of the hIL-5 promoter. RESULTS: We have identified a protected region in the distal hIL-5 promoter that has sequence homology to the previously identified negative regulatory element within BR3. This protected region has not been previously reported and is shown to contain overlapping binding sites for YY1 and nuclear factor of activated cells. The binding sites exist between positions -447 and -459, and this sequence was named hPRE2-IL5. Substitution mutations that abolish binding of these proteins to hPRE2-IL5 result in a 2- to 3-fold increase in hIL-5 promoter activity in activated human T cells. CONCLUSION: We report the novel combination of YY1 and nuclear factor of activated T cells transcription factors binding to a distal hIL-5 promoter element where both factors are involved in down-regulation of hIL-5 gene expression in human T cell.

Base Sequence↗
PubMed1999

Role of nuclear factor of activated T cells (NFAT) in the expression of interleukin-5 and other cytokines involved in the regulation of hemopoetic cells.

NFAT (nuclear factor of activated T cells) is a transcription factor that plays a role in the regulation of various cytokines, including those involved in the regulation of hemopoetic cells such as granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL4), interleukin-3 (IL3), interleukin-13 (IL13) and interleukin-5 (IL5). In this report we provide a summary of the various locations in the promoters of each of these cytokines where NFAT has been shown or suggested to bind, and at which sites NFAT has been shown to be involved in transcriptional regulation. We also provide experimental data to show that the binding of NFAT to the nucleotides GAA at positions -113 to -111 of the human IL5 promoter is associated with functional activity in human T cells.

Animals↗
PubMed1998

Expressed luciferase viability assay (ELVA) for the measurement of cell growth and viability.

An expressed luciferase viability assay (ELVA) has been developed for cell viability and cell number based on detecting the expression of luciferase transfected into the cells. Stable transfectants were produced that expressed luciferase constitutively. Like many endogenous enzymes, luciferase is rapidly degraded following cell death, so that the enzyme can be used as a measure of cell viability. A modified luciferase assay was used in which the reagents were added directly to the cells in a microplate. The main advantages compared to other cell viability assays are the wide dynamic range, high sensitivity, low background, and the absence of any requirement to wash or harvest the cells. Stable transfectants of three factor-dependent cell lines (B13, Ba/F3 and CTLL) were produced and used in cytokine assays. Three strategies of selection after electroporation were tested: (1) using a plasmid containing both the genes encoding firefly luciferase and a selectable marker (neo), (2) cotransfection of a plasmid containing luciferase and a plasmid containing a selectable marker (puromycin resistance), and (3) cotransfection of a plasmid containing luciferase and a plasmid containing the human IL-5Ralpha-chain, and selecting in IL-5. This latter strategy produces an IL-5 responsive cell line expressing luciferase in a single step without the need for antibiotic selection.

Animals↗
PubMed1998

Interleukin-5 binds to heparin/heparan sulfate. A model for an interaction with extracellular matrix.

Interleukin-5 (IL-5) is the major cytokine regulating eosinophil production. In allergic disease tissue damage is primarily caused by eosinophils. Heparan sulfate proteoglycans are components of the bone marrow stroma, which supports hemopoietic cell differentiation and proliferation. We show that at low IL-5 concentrations heparan sulfate enhances the proliferation of an IL-5-dependent cell line. To investigate a mechanism for this effect we used an artificial proteoglycan to establish an enzyme-linked immunosorbent assay for the binding of heparin to proteins. Using this assay we demonstrate that IL-5 binds to heparin. The IL-5/heparin interaction is inhibited by ethylenediaminetetraacetate and enhanced by low concentrations of zinc ions. IL-5 interacts with iduronic acid containing glycosaminoglycans, and heparan sulfate preparations that have numerous N-sulfated domains per chain are especially efficient at inhibiting heparin binding. Both IL-5/heparin binding and the synergistic effect of IL-5 and heparan sulfate on cell proliferation were inhibited by an anti-IL-5 monoclonal antibody. These data suggest that the binding of IL-5 to heparan sulfate modulates IL-5 activity.

Animals↗
PubMed1998

Identification of two novel palindromic regulatory elements in the murine interleukin-5 promoter.

Interleukin-5 has remarkable specificity for the eosinophil lineage. This fact, combined with the biological specificity of eosinophilia suggests tight and independent regulation of IL-5 expression. Here we report two novel palindromic regulatory elements (PRE) which contain positive regulatory motifs (PRM) that control transcription of the murine IL-5 gene. The first element, mPRE1-IL5 (-79 to -90) contains the mPRM1 at positions -87 to -89 which operates as a positive regulatory element with mutation of this motif resulting in a 64% decrease in gene activity. Gene expression was reduced by 67% when a similar mutation was introduced into the mPRM2 (-467 to -469) of mPRE2-IL5 (-459 to -470). Both elements specifically bind proteins from EL4-23 cell nuclear extracts forming constitutive DNA-protein complexes. EMSA experiments utilising mutated mPRE-IL5 oligonucleotides indicate that in both elements, the mPRMs are essential for protein binding.

Animals↗
PubMed1998

Biological and molecular characteristics of interleukin-5 and its receptor.

Interleukin-5 (IL5) is a T cell-derived cytokine involved in the pathogenesis of atopic diseases. It specifically controls the production, the activation and the localization of Eosinophils. The Eosinophils are the major cause of tissue damage resulting in the symptoms of asthma and related allergic disorders. T cells purified from bronchoalveolar lavage and peripheral blood of asthmatics secrete elevated amount of IL5. Therefore IL5 emerges to be an attractive target for the generation of new anti-allergic drugs. Agents which inhibit either the production or the activity of IL5 could be expected to ameliorate the pathological effects of the allergic response. A better understanding of the biology of IL5 and the regulation of its expression is, however, a prerequisite for the development of new therapeutic agents. This review covers the major biological, molecular and structural aspects of IL5 research since the identification of this cytokine ten years ago.

Eosinophils↗
PubMed1998

New directions in understanding interleukin-5 gene expression.

Specific regulation of the interleukin-5 (IL-5) gene is implied by the unique control of eosinophilia which is regulated by IL-5. In studies of IL-5 gene expression, the only control elements identified for the IL-5 gene have been transcriptional elements in the 5' untranslated region (UTR). Significant differences exist in the arrangement of the murine and human IL-5 promoters, which is surprising considering the tight regulation of the gene. Novel palindromic regulatory elements involved in transcriptional regulation have been found in the 5' UTR and new results show the presence of transcriptional elements in the 3' UTR. Post-transcriptional control mechanisms in both the 5' and 3' UTRs have also been described.

Animals↗
PubMed1997

Eosinophilic interleukin 5 (IL-5) transgenic mice: eosinophil activity and impaired clearance of Schistosoma mansoni.

Eosinophilia is a feature common to many invasive helminth infections and eosinophils are often considered to be effector cells in immunity to helminths. This study examined the possible influence of constitutive eosinophilia on the clearance of Schistosoma mansoni infections in mice. Eosinophils from interleukin-5 transgenic mice exhibit normal ultrastructure and function with regard to phagocytosis and killing of bacteria and responses to chemotactic stimuli. IL-5 transgenics and non-transgenic littermates were immunized once or four (hyperimmunization) times with irradiated cercariae of S. mansoni. Animals were challenged percutaneously with unirradiated cercariae one month after their last exposure to irradiated parasites. One month after challenge transgenic animals, whether unimmunized, vaccinated or hypervaccinated, carried significantly more liver-stage parasites than non-transgenic animals. These results suggest that although eosinophils from IL-5 transgenic mice are functional for a number of key parameters, large numbers of eosinophils and/or high levels of IL-5 may in some way impair clearance of S. mansoni. A re-evaluation of the roles of eosinophils and IL-5 in infections with this and other parasites may therefore be warranted.

Animals↗
PubMed1997

Anti-interleukin 5 strategies as a potential treatment for asthma.

A paradigm shift, championed largely by cellular immunologists, has redefined asthma as an immune mediated phenomenon characterised by an interleukin 5 (IL-5) driven eosinophilic bronchitis. This change in emphasis has provoked intense interest in the possibility that inhibitors of IL-5 production, or antagonists of its activity, will provide a new generation of anti-asthma drugs.

Asthma↗
PubMed1996

Two pathways can activate the interleukin-5 gene and induce binding to the conserved lymphokine element 0.

Eosinophilia is a uniquely specific phenomenon regulated by interleukin-5 (IL-5), suggesting specific control for IL-5 gene expression. Using a transient-transfection reporter assay and DNA mobility-shift experiments in EL4 mouse lymphoma cells, reporter expression and binding of transcription factors to the conserved lymphokine element 0 (CLE0) in the mouse (mIL-5) promoter was investigated. Activation of the IL-5 promoter required costimulation of T cells with phorbol ester (phorbol 12-myristate 13-acetate [PMA]) and cyclic adenosine 3',5'-monophosphate (cAMP), but was blocked by the immunosuppressive drug, cyclosporin A (CsA). Binding to CLE0 was induced under conditions optimal for IL-5 transcription but was not blocked by CsA. CD28-induced signals could partly substitute for cAMP. However, the effects of cAMP, but not of CD28, were sensitive to the cAMP-dependent protein kinase inhibitor, H89, suggesting that CD28 does not involve a cAMP mechanism. It therefore appears that IL-5 expression can be induced by at least two distinct stimulatory pathways. Although CLE0 contains sequences similar to AP-1 and NF-AT, only the AP-1 moiety of the CLE0 element could be demonstrated to have inducible binding. Experiments with antisera to the AP-1 family of transcription factors indicated that c-fos and JunB bind to the IL-5 CLE0 in activated lymphoma cells. The role of the NF-AT-like element was less clear. A constitutively expressed protein showed a weak band that was inhibited by mIL-2 NF-AT competitor sequences. However, this protein did not react with an anti-NF-ATp antiserum. On the other hand, transcription was partially inhibited by an oligonucleotide containing the intact NF-AT-like element from CLE0, suggesting that the element is important for optimal transcription, but the nature of the protein binding to it remains unknown. The fact that these factors are induced in a subclone of EL4 that does not express IL-5 and bind to a number of other cytokine gene promoters suggests that although binding to CLE0 appears to be necessary for IL-5 transcription, other factors must control the specific expression of the gene.

Animals↗
PubMed1996

How is expression of the interleukin-5 gene regulated?

Eosinophilia is a uniquely specific phenomenon regulated by interleukin-5 (IL-5), suggesting specific control for IL-5 expression. However in eosinophilia IL-5 is often co-expressed with other lymphokines such as IL-4, indicating that common, as well as independent, control mechanisms must exist. IL-5 gene expression is regulated at the transcriptional level. The molecular analysis of the IL-5 promoter region reveals the presence of positive regulatory sites that are common to many lymphokine genes. Results from immunosuppression studies suggest that the key control mechanism of IL-5 regulation may not depend on specific regulatory factors but on how gene expression is activated.

Animals↗
PubMed1996

Diagnostic criteria for selenium toxicosis in aquatic birds: dietary exposure, tissue concentrations, and macroscopic effects.

A feeding study with mallard ducks (Anas platyrhynchos) was conducted during March to July 1988 in Laurel, Maryland (USA), to identify diagnostic criteria for selenium toxicosis in birds. One-year-old male mallards in groups of 21 were fed diets containing 0, 10, 20, 40, or 80 parts per million (ppm) selenium, as seleno-DL-methionine, for 16 weeks. All ducks receiving 80 ppm died. Ducks receiving 40 or 80 ppm selenium consumed less feed than ducks in the other treatment groups. Body weights of ducks receiving 40 or 80 ppm selenium declined during the study. The post-breeding molt was delayed in ducks receiving 40 ppm; most ducks receiving 80 ppm selenium died prior to the onset of molt. At necropsy, numerous abnormalities were observed in ducks that died but only a small number of abnormalities were observed in ducks surviving to the end of the study in the 40 ppm group. Weights of the heart, spleen, and pancreas were mostly lower and weights of the kidney were higher for ducks dying during the study than for euthanized ducks. Liver weights were unaffected. Selenium accumulated in soft tissues approximately in proportion to dietary concentrations. Selenium concentrations in tissues of all ducks that died were different from those of surviving ducks in the 0, 10, and 20 ppm groups, but were not different from those of surviving ducks in the 40 ppm group. Proposed diagnostic criteria for fatal chronic selenosis were derived from body weight, macroscopic abnormalities, organ weights, and concentrations of selenium in the liver. Proposed diagnostic criteria for non-fatal chronic selenosis were derived from body weight, plumage condition, macroscopic abnormalities, concentrations of selenium in the liver, reproductive failure, and alterations of blood and tissue chemistries. Lead or dioxin poisoning have diagnostic criteria most similar to selenium toxicosis.

Administration, Oral↗