Deirdre R. Coombe

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. J. Leukoc. Biol. 63: 342–350; 1998.

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.

The Role of Stromal Cell Heparan Sulphate in Regulating Haemopoiesis

Intimate contact between haemopoietic progenitor cells and elements of the bone marrow stroma is required for progenitor cell proliferation and differentiation. It is believed that the stroma provides particular niches for the development of haemopoietic cells of different lineages. Cytokines, stromal cell surface molecules and molecules of the stromal extracellular matrix all contribute to defining these microenvironmental niches. Data obtained using an in vitro model of haemopoiesis support the view that progenitor cell adhesion to stroma is mediated by multiple receptor-ligand interactions. The possibility of a tethering step, mediated by the engagement of stromal cell heparan sulphate with its ligands on the progenitor cells, preceding stable cell adhesion is discussed. The role of stromal cell heparan sulphate is likely to include cytokine presentation to progenitors as well as the tethering of progenitors to stroma. It is proposed that intracellular signals induced by progenitor cell adhesion to stroma act in association with cytokine induced signals to regulate progenitor cell proliferation and differentiation.

Interaction Between Skeletal Muscle Cells and Extracellular Matrix Proteins Using a Serum Free Culture System

The ability to grow C2C12 myoblasts in a completely defined, serum free medium enables researchers to investigate the role of specific factors in myoblast proliferation, migration, fusion, and differentiation without the confounding effects of serum. The use of defined, animal free in vitro culture systems will improve reproducibility between research groups and may also enhance translation of tissue engineering techniques into clinical applications. Here, we describe the use and characterization of a serum free culture system for C2C12 myoblasts using standard tissue culture medium and readily available, defined growth factors and supplements.

Interleukin 5 binds to heparin/heparan sulphate: A model for an interaction with the 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-5dependent 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. J. Leukoc. Biol. 63: 342–350; 1998.