Biophysical and biological characterisation of a soluble human CD23 pigment

C3-antigen complexes induce B cell proliferation and the synthesis of specific antibodies in the immune response. The mechanism is understood to involve the co-ligation of antigen receptor (lgM) and CD21 on the membrane of antigen-specific B cells. CD23 is the low-affinity IgE receptor and also binds to CD21. It has been shown by NMR spectroscopy that the binding sites for IgE and CD21 on CD23 are distinct and nonoverlapping. CD23 is expressed as a homotrimer on the membrane of B cells and is cleaved by an endogenous metalloprotease to release soluble fragments that contain the f binding sites for both IgE and CD21. It is suggested that these fragments, like the C3antigen complexes, can co-ligate membrane IgE (mlgE) and CD21 on the surface of B cells to up-regulate IgE synthesis. This would enhance allergic responses in vivo. To test this hypothesis, a construct was generated to express a trimeric fragment of soluble human CD23 (LZ-CD23) and provide evidence for the protein's oligomeric state in solution using analytical ultracentrifugation. Surface plasmon resonance studies also showed that LZ-CD23 binds IgE with a high affinity. Confocal microscopy was used to demonstrate that LZ-CD23 caused the time-dependent re-distribution of fluorescentlylabelled mlgE and CD21 on human tonsillar B cells, stimulated with anti-CD40 and IL-4 to induce heavy-chain switching to 19B. As early as 15 minutes following incubation with LZ-CD23, rnIgE and CD21 were seen to co-cap in large aggregated clusters on the B cell surface. Additionally, the specificity of LZ-CD23 towards these markers was demonstrated as LZ-CD23 had no effect on the surface distribution of CD38. LZ-CD23 was shown to be biologically active by enhancing the secretion of IgE from B cells. Moreover, evidence is presented that CD23 exerts its IgE-potentiating action by specifically targeting cells that are pre-committed to 19B. The mechanism thus appears to resemble that of the C3-antigen complex in the immune response, except that it is isotype rather than antigen-specific. These results shed light on the role of CD23 in IgE homeostasis and suggest a novel means of targeting CD23 for intervention in the allergic response.EThOS - Electronic Theses Online ServiceGBUnited Kingdo