Abstract
The low-affinity IgE receptor FcϵRII (CD23) is part of the regulatory system controlling IgE synthesis in human B cells and exists in membrane and soluble forms. Binding of IgE to CD23 has been described to have stabilizing effects and to prevent cleavage of CD23. Previous experiments using anti-CD23 antibodies reduced IgE synthesis but were difficult to interpret as the antibody Fc part might also mediate feedback mechanisms. The purpose of this study was to investigate the regulatory role of CD23, by using designed ankyrin repeat proteins (DARPins) that specifically recognize CD23. Anti-CD23 DARPins were isolated by ribosome display and were produced as monovalent and bivalent constructs. Affinities to CD23 were measured by surface plasmon resonance. IgE synthesis and up-regulation of CD23 in human peripheral B cells were induced by IL-4 and anti-CD40 antibody. We assessed CD23 expression and its stabilization by FACS and used an ELISA for detecting soluble CD23. IgE synthesis was measured by ELISA and real-time PCR. Surface plasmon resonance revealed affinities of the DARPins to CD23 in the pico-molar range. Anti-CD23 DARPins strongly inhibited binding of IgE to CD23 and share thus a similar binding epitope as IgE. The DARPins stabilized membrane CD23 and reduced IgE synthesis in an isotype specific manner. Furthermore, the anti-CD23 DARPins decreased IgE transcript through inhibition of mature Cϵ RNA synthesis suggesting a posttranscriptional control mechanism. This study demonstrates that targeting CD23 alone is sufficient to inhibit IgE synthesis and suggests that a negative signaling occurs directly through the CD23 molecule.
Highlights
FceRII (CD23) is the low affinity receptor for IgE
It has been hypothesized that binding of IgE or anti-CD23 antibodies to the lectin domain renders CD23 less susceptible to cleavage by proteases leading to a reduced IgE synthesis
In order to investigate whether the two designed ankyrin repeat proteins (DARPins) molecules D86 and D89 recognize different epitopes on CD23, we performed an assay on surface plasmon resonance
Summary
FceRII (CD23) is the low affinity receptor for IgE It is a type II transmembrane glycoprotein consisting of a short cytoplasmic N-terminus followed by an extracellular part with a stalk region and a C-type lectin-domain at the C-terminus, where binding of IgE occurs [1]. CD23a is constitutively expressed in B cells while CD23b, expressed upon stimulation with IL-4, is present on monocytes, follicular dendritic cells as well as intestinal epithelial cells [1, 4] Both isoforms were described to have different functions. Specific inhibition of the protease ADAM 10 has demonstrated a reduction in the synthesis of IgE [8,9,10,11]. These data suggest that surface CD23 may be involved in a negative feedback inhibition of IgE synthesis
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