Regulation of IgE production requires oligomerization of CD23.

Here we describe the production of a rabbit polyclonal Ab (RAS1) raised against the stalk of murine CD23. RAS1 inhibits release of CD23 from the surface of both M12 and B cells resulting in an increase of CD23 on the cell surface. Despite this increase, these cells are unable to bind IgE as determined by FACS. CD23 has previously been shown to bind IgE with both a high (4-10 x 10(7) M(-1)) and low (4-10 x 10(6) M(-1)) affinity. Closer examination by direct binding of (125)I-IgE revealed that RAS1 blocks high affinity binding while having no effect on low affinity binding. These data support the model proposing that oligomers of CD23 mediate high affinity IgE binding. These experiments suggest that RAS1 binding to cell surface CD23 results in a shift from oligomers to monomers, which, according to the model, only bind IgE with low affinity. These experiments also suggest that high affinity binding of IgE is required for IgE regulation by CD23 and is demonstrated by the fact that treatment of Ag/Alum-immunized mice treated with RAS1 results in a significant increase in IgE production similar to the levels seen in CD23-deficient mice. These mice also had significantly decreased levels of serum soluble CD23 and Ag-specific IgG1. RAS1 had no effect on IgE or Ag-specific IgG1 production in CD23-deficient mice.

The structure of the IgE Cepsilon2 domain and its role in stabilizing the complex with its high-affinity receptor FcepsilonRIalpha.

The stability of the complex between IgE and its high-affinity receptor, FcepsilonRI, on mast cells is a critical factor in the allergic response. The long half-life of the complex of IgE bound to this receptor in situ ( approximately 2 weeks, compared with only hours for the comparable IgG complex) contributes to the permanent sensitization of these cells and, hence, to the immediate response to allergens. Here we show that the second constant domain of IgE, Cepsilon2, which takes the place of the flexible hinge in IgG, contributes to this long half-life. When the Cepsilon2 domain is deleted from the IgE Fc fragment, leaving only the Cepsilon3 and Cepsilon4 domains (Cepsilon3-4 fragment), the rate of dissociation from the receptor is increased by greater than 1 order of magnitude. We report the structure of the Cepsilon2 domain by heteronuclear NMR spectroscopy and show by chemical shift perturbation that it interacts with FcepsilonRIalpha. By sedimentation equilibrium we show that the Cepsilon2 domain binds to the Cepsilon3-4 fragment of IgE. These interactions of Cepsilon2 with both FcepsilonRIalpha and Cepsilon3-4 provide a structural explanation for the exceptionally slow dissociation of the IgE-FcepsilonRIalpha complex.

IgE isotype determination: epsilon-germline gene transcription, DNA recombination and B-cell differentiation.

Immunoglobulin class switching is the process which determines whether a B-cell secretes antibodies of the IgM, IgG, IgA or IgE class (or isotype). IgE is the antibody that mediates the allergic response by sensitising mast cells to allergens at the mucosal barrier. Class switching proceeds by three successive steps, culminating in the synthesis and secretion of antibody: these are germline gene transcription, DNA recombination and B-cell differentiation. We review here the present state of knowledge concerning the mechanisms involved in each of these steps, with particular reference to IgE. Intervention in the mechanisms that specify the selection of IgE may offer a means to combat allergy.

Inhibition of IgE-receptor interactions.

Immunoglobulin E plays a central role in allergic disease and, as our understanding of the network of interactions between IgE and its receptors improves, new opportunities for therapeutic intervention emerge. IgE binding to its 'high-affinity' receptor, Fc epsilon RI, first identified on mast cells and now known to be expressed on a variety of other cell types, is the best characterised interaction, and has attracted most attention. The 'low affinity' receptor, Fc epsilon RII/CD23, first found on B-cells, appears to be part of a more complex network that has yet to be fully elucidated. Two recent advances concerning the IgE-Fc epsilon RI interaction are noteworthy. The first is the development of a monoclonal anti-IgE antibody, now in advanced clinical trials, which inhibits this interaction and certainly proves the viability of this approach. The second is the publication of the crystal structure of the complex between IgE and Fc epsilon RI, which opens the way for the first structure-based design of small molecule inhibitors.

Cleavage of the low-affinity receptor for human IgE (CD23) by a mite cysteine protease: nature of the cleaved fragment in relation to the structure and function of CD23.

Der p I, a cysteine protease representing a major allergen of the house dust mite Dermatophagoides pteronyssinus, has recently been shown to cleave CD23 from the surface of cultured human B cells (RPMI 8866 B cell line). We have now undertaken a detailed investigation of CD23 cleavage by Der p I. We demonstrate that Der p I cleaves CD23 at two sites (Ser155-Ser156 and Glu298-Ser299) to produce a 17-kDa fragment containing the lectin domain and only part of the C-terminal tail. No such effect was demonstrable with mouse CD23, a finding which was anticipated based on its lack of the cleavage sites identified on human CD23. Based on the cleavage pattern and the model of CD23, we propose a sequence of events leading to the liberation of the 17-kDa soluble CD23 fragment. The biological significance of such cleavage is underlined by the demonstration that Der p I-treated B lymphocytes lose their ability to bind IgE, and that the 17-kDa fragment (amino acids 156-298) contains the minimum structural requirement (amino acids 156-288) for binding to both IgE and CD21.

Interaction of the low-affinity receptor CD23/Fc epsilonRII lectin domain with the Fc epsilon3-4 fragment of human immunoglobulin E.

CD23/Fc epsilonRII, the low-affinity receptor for IgE, is a multifunctional protein of importance in blood cell development and the immune system. We have studied the interaction of CD23 with IgE in solution using hydrodynamic methods applied to recombinant fragments of both ligands: sCD23, corresponding to the soluble lectin domain of CD23, and Fc epsilon3-4, a dimer of the C epsilon3-C epsilon4 sequence of IgE. The hydrodynamic, spectroscopic, and biological properties of these fragments suggest that they have a fully native structure. Sedimentation equilibrium studies on mixtures of sCD23 and Fc epsilon3-4 indicate that IgE has two binding sites for CD23, each characterized by affinities of approximately 10(5) M(-1). Analysis of the sedimentation as a function of temperature allows conclusions to be drawn about the thermodynamics of binding at the two sites. Binding at the first site is characterized by large changes in enthalpy (delta H(degree)To = -2.1 +/- 3.3 kcal mol(-1)) and heat capacity (delta Cp(degree) = -320 +/- 320 cal mol(-1) K(-1)), whereas binding at the second site is characterized by small changes in enthalpy (delta H(degree)To = 0.1 +/- 5.6 kcal mol(-1)) and heat capacity (delta Cp(degree) = -140 +/- 550 cal mol(-1) K(-1)). In native CD23, there are two or three lectin domains, associated through an alpha-helical coiled-coil stalk. The predicted structure of the CD23 oligomers and symmetry considerations rule out the possibility of two lectin domains from one oligomer binding to identical sites in IgE. The notion of two types of interaction in the 2:1 complex between CD23 and IgE is consistent with the thermodynamic data presented.

Automated hydrodynamic modelling of a complex between a human IgE fragment (Fc epsilon 3-4) and the IgE high affinity receptor Fc epsilon RI alpha-chain.

The binding of IgE to its high affinity receptor Fc epsilon RI plays an important role in the allergic response. The interaction between soluble Fc epsilon RI alpha-chain (sFc epsilon RI alpha) and Fc epsilon 3-4, a fragment of IgE consisting of the C epsilon 3 and C epsilon 4 heavy chain constant domains, has been studied using analytical ultracentrifugation (Keown et al. this volume). Here we describe the development of a simple automated hydrodynamic modelling technique and its application to this interaction. This procedure utilises sphere models of the two molecules and performs an automated systematic translational search of sFc epsilon RI alpha relative to Fc epsilon 3-4. The result of this is the generation of 40,359 individual models of how the receptor can be placed relative to Fc epsilon 3-4. These are then assessed for consistency by comparing the sedimentation coefficients generated for the models to the experimentally determined sedimentation coefficients, and are displayed graphically to show allowed and disallowed complexes. From this analysis, it is clear that the complex between sFc epsilon RI alpha and Fc epsilon 3-4 is compact, with the most elongated models being excluded. In addition, sFc epsilon RI alpha appears not to interact with the C-terminal end of Fc epsilon 3-4, and probably binds either to the sides or face, observations which are consistent with other experimental data on the Fc epsilon RI alpha/IgE interaction. Automated hydrodynamic modelling also has the potential to be used for other interactions, providing a simple way of looking at a large number of models, and making rigorous studies of interacting components more feasible.

CD23/Fc epsilon RII and its soluble fragments can form oligomers on the cell surface and in solution.

Human CD23 (also known as Fc epsilon RII) is a 45,000 MW glycoprotein with homology to C-type animal lectins. It is involved in B-cell differentiation and IgE regulation, and is naturally cleaved to give soluble products of 37,000, 33,000, 29,000, 25,000 and 16,000 MW. Previous work has suggested that the region between the transmembrane sequence and the extracellular lectin head is capable of forming an alpha-helical coiled coil, one of the main consequences of which would be formation of dimers or trimers. Here we present protein-protein cross-linking data showing that CD23 forms trimers on the cell surface and hexamers in solution, and we use several different fragments to determine the regions of the protein involved in this self-association. The region of the putative coiled coil is indeed responsible for trimerization, with additional interactions between the lectin heads resulting in the formation of hexamers observed in solution.