CD23, the low affinity IgE receptor, is expressed by various cell types and has numerous functions depending on the form of the protein, its interaction with various ligands and the type of cell involved. CD23 is pivotal in the regulation of IgE, with the soluble form involved in up-regulation, while the membrane bound form is involved in the down-regulation. It is clear why it is believed to be a central molecule in allergic responses, and a therapeutic target for the treatment of allergic disease. In this study a recombinant form of the entire extracellular domain of the protein, exCD23, was produced by PCR cloning and expressed in E. coli. His•Tag™s were introduced onto the C-terminus and N-terminus, respectively, in order to simplify the purification procedure. After renaturation and purification, the recombinant exCD23 bound IgE, indicating its activity. From the IgE binding studies it was established that the position of the tag did not influence the binding. GST•Tagged™ exCD23 was also produced in an attempt to increase the solubility of the recombinant protein, but this proved unsuccessful. Butyrate differentiated EoL-1 cells were treated with the Nterminal His•Tagged™ exCD23, and the protein appeared to suppress the secretion of the constitutively expressed cytokines, especially IL-8 and IFN- , when compared to untreated cells. In addition, treatment of the EoL-1 cells with exCD23 had a significant proliferative effect, but could not induce differentiation of this cell line into mature eosinophilic-like cells.

The low affinity IgE receptor, CD23, is expressed by a wide variety of cells and cleaved from its original 45 kDa size to several smaller soluble CD23 proteins. Soluble CD23 function depends on the form of the protein and its interaction with various ligands. CD23 is believed to play an important role in regulating allergic responses and in inflammation, amongst others. β2 integrins are important in a variety of cell-adhesion reactions during immune-inflammatory mechanisms and the binding of their natural ligands generates outside-in cellular signalling, leading to cell activation. Although the binding of CD23 to β2 integrins contributes to this signalling in monocytes, the interaction site for CD23 is unknown. This study focused on the interaction of three soluble CD23 proteins with the β2 integrins CD11b/CD18 and CD11c/CD18. Differentiated HL60, THP1 and U937 monocytic cells were used to demonstrate the binding of three recombinant CD23 constructs (corresponding to 16, 25 and 33 kDa human soluble CD23) to upregulated CD11b/CD18 and CD11c/CD18. This binding was partially blocked by an antibody specific for the CD11b/CD18 αI domain, demonstrating that αI domains are involved in binding to CD23. Recombinant αI domain proteins of CD11b and CD11c were demonstrated to bind CD23 using ELISA and in surface plasmon resonance spectroscopy. The dissociation constants for CD23-CD11b/CD18 and CD23-CD11c/CD18 are comparable to other integrin ligands. This study has shown that CD23 interacts directly with the αI domains of β2 integrins and that the interaction surface likely spans the lectin domain as well as either the stalk and/or C-terminal tail of CD23. This study also looked at the effect that soluble CD23 proteins had on monocyte biology. It appears that iv sCD23 proteins have little effect on the phagocytic or chemotactic ability of monocytes, while an increase in oxidative burst was shown with the 16 kDa and 25 kDa CD23 proteins. Signalling pathways for the production of reactive oxygen species were investigated and it appears that the CD23 proteins signal mainly through the phosphoinositide-3 kinase pathway, although the mitogen activated protein kinase and Src kinase pathways may also play a role. These data suggest that sCD23 proteins induce outside-in signalling of β2 integrins and are able to change the activation state of CD11b/CD11c by stimulating oxidative burst. This needs to be further investigated by determining how the three sCD23 proteins are binding the CD11 proteins and investigating further leukocyte function and inflammatory responses by the cells.

Both CD23 and the β2 integrins (also known as CD11/CD18) have very important immunological functions, especially during the allergic response where the binding of CD23 to β2 integrins contributes to various types of signalling in monocytes which can result in drastic sensitivities experienced by some allergic individuals. CD23, also known as the low affinity receptor for immunoglobulin E or (FcεRII), is a type II transmembrane glycoprotein which is synthesized by haematopoietic cells and has biological activity in both membrane-bound and freely soluble forms. It acts via a number of receptors, including the β2 integrins. β2 integrins are specifically found on leukocytes and they play important roles in cell–cell or cell–matrix adhesion via their ability to bind multiple ligands. These molecules occur as heterodimers consisting of an alpha (α) and beta (β) subunit. The α-subunits of β2 integrins contain an approximately 200-amino-acid inserted domain or I-domain which is implicated in ligand binding function. There are four different types of β2 integrins, namely CD11a, CD11b, CD11c and CD11d, all dimers with the common beta subunit, CD18. CD23 and CD11/18 are natural ligands of each other; however the interaction site for CD23 is unknown. It is postulated that the integrin recognizes a tripeptide motif in a small disulfide-bonded loop at the N-terminus of the lectin head region of CD23, which is focussed around Arg172, Lys173 and Cys174 (RKC). This study thus focused on the interaction between the I-domain of CD11 (b and c) and a recombinant 25kDa construct of sCD23. In order to understand the characteristics of ligand binding between the relevant proteins of interest, alanine substitutions on the RKC motif of CD23 were made via site-directed mutagenesis. Consequently, a recombinant form of the I-domain of CD11 (b and c) as well as a wild type (containing the RKC motif) and mutant form (containing an AAC motif) of sCD23 were expressed and purified. The CD11 recombinant proteins were purified via affinity chromatography and the CD23 recombinant proteins via gel filtration chromatography. In addition, synthetic (CD23 derived) peptides, one containing the RKC sequence and the other the AAC sequence, were designed and custom synthesized. The synthetic peptides as well as the recombinant CD23 proteins were then analyzed for their interaction with the CD11 I-domain via ELISA. Subsequent ELISA analyses showed that the native sCD23 and the RKC peptide were able to bind to the integrin α I-domain whereas the mutant sCD23 and the corresponding synthetic AAC peptide failed to bind. This interaction was also analysed via flow cytometry using differentiated U937 cells, yielding similar results. ELISA analyses for the sCD23-CD11b I-domain interaction showed a Kd of 0.36 ± 0.14 μM whereas the RKC-CD11b I-domain interaction yielded a Kd of 1.75 ± 0.58 μM. Similarly, the sCD23-CD11c I-domain interaction yielded a Kd of 0.39 ± 0.09 μM and 1.53 ± 0.72 μM for the RKC-CD11c I-domain interaction. Peptide inhibitory analysis, analysed via ELISA and flow cytometry, reinforced the fact that the RKC motif on sCD23 is a prerequisite for ligand binding of the CD11b/c I-domain.

In order to further elucidate the interaction between CD23 and β2 integrins (CD11b/CD18) the following objectives were established: Expression and purification of CD11b I-domain as a GST-fusion protein using Escherichia coli; Cloning, synthesis and expression of CD18 I-Like domain.CD11b I-domain has previously been expressed as a GST-fusion protein (Daniels, 2010) and consequently led to comparable expression of CD18 I-like domain as a GST-fusion protein; Preparation of two site-directed mutants of CD18 I-Like domain in order to study the function of the serine residue involved in the S116P mutation. The serine was mutated to proline, as in LAD patients, as well as alanine, a non-polar alternative, in order to contrast and compare binding characteristics.  Expression, refolding and purification of sCD23, and a double mutatedsCD23 (RKΔAA) from E. coli; This was performed according to the method described by Daniels et al. (2005); Investigation of the CD23-CD11b I-like domain interaction through surface plasmon resonance spectroscopy.