CD23 is a multifunctional receptor/ligand, found in a variety of cell types, such as human peripheral blood mononuclear cells (PBMCs), B-lymphoblastoid cell lines, mast cells and basophils. It is also found on a variety of haematopoietic cell lines. As the low-affinity receptor for immunoglobulin E (IgE), CD23 plays a role in antigen-presentation and macrophage activation. As a surface molecule cleaved from the cell membrane, soluble CD23 (sCD23) can act as an adhesion molecule and a cytokine. Perturbances of such molecular interactions may lead to various diseases such as allergies and other inflammatory diseases. It has been speculated that elevated levels of sCD23 may be used to bind secreted IgE, thus preventing it from binding to membrane CD23 on haematopoietic cells, preventing B cells from being activated into IgE producing cells. Signal transduction by sCD23 is dependent on cell subsets, ligands and co-factors required for its function. sCD23 plays a direct role in inducing tumour necrosis factor alpha (TNFα), interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1β) and soluble IL-1 receptor from activated human monocytes and PBMCs in vitro. Recombinant forms of 25 and 37 kDa human sCD23 were produced by polymerase chain reaction (PCR)-cloning into pET23a, a bacterial expression vector. The proteins were expressed and refolded, followed by purification by gel filtration chromatography. The purified proteins were biochemically characterized to ensure purity and biological activity, by observing the binding to human IgE both in enzyme-linked immunosorbant assay (ELISA) and surface plasmon resonance (SPR) spectroscopy. ELISA showed KD values of 7.23 x 10-9M and 8.12 x 10-9M for the 25 and 37 kDa proteins, respectively. These values were significantly lower than that of Hibbert et al., (2005). SPR data obtained for the 25 kDa CD23 was not of reliable quality but SPR for the 33kDa sCD23 showed a KD of 1.18 x 10-7M, close to that of Hibbert et al., (2005), J. Exp. Med, 202: 751-760. To test the therapeutic potential of the recombinant molecule, a B-lymphoblastoid cell line (Raji), a pre-monocytic cell line (U937), and PBMCs from normal and hyper-allergic individuals were used. All cells showed no change in production of cytokines. It is essential to investigate further cytokine functions and production implicated by recombinant forms of sCD23, as well as binding of sCD23 to CD21 and CD11b/c, and in vivo IgE regulation before a conclusion can be drawn as to whether recombinant sCD23 is a potential therapeutic target against allergic disease.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nmmu/vital:10305 |
| Date | January 2006 |
| Creators | Askew, Sandra Lyn |
| Publisher | Nelson Mandela Metropolitan University, Faculty of Science |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Masters, MSc |
| Format | xvi, 131 leaves, pdf |
| Rights | Nelson Mandela Metropolitan University |
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