In vivo murine CD23 destabilization enhances CD23 shedding and IgE synthesis

To investigate the effects of in vivo CD23 destabilization on CD23 shedding and IgE production, an anti-CD23 stalk monoclonal (19G5), previously shown to enhance proteolysis of CD23 in vitro, was utilized. Compared to isotype control-treated mice, BALB/cJ mice injected with 19G5 displayed significantly enhanced serum soluble CD23 and IgE. Soluble CD23 and IgE levels were also increased in 19G5-treated C57BL/6J mice (intermediate IgE responders); however, the kinetics of the responses differed between the high (BALB/cJ) and intermediate responder mice, suggesting a potential role for CD23 in regulating IgE responder status. The 19G5-induced IgE response was dependent on IL-4 and independent of CD21 as demonstrated through use of IL-4Ralpha and CD21/35-deficient mice, respectively. Overall, the data provide a direct demonstration for CD23's role in regulating IgE production in vivo and suggest that therapies aimed at stabilizing cell surface CD23 would be beneficial in controlling allergic disease.     

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.     

129/SvJ mice have mutated CD23 and hyper IgE

CD23, the low affinity IgE receptor, is hypothesized to function as a negative regulator of IgE production. Upon discovering reduced CD23 surface levels in 129/SvJ inbred mice, we sought to further investigate 129/SvJ CD23 and to examine its influence on IgE levels. Five amino acid substitutions were found in 129/SvJ CD23. Identical mutations were also observed in CD23 from New Zealand Black and 129P1/ReJ mice. 129/SvJ B cells proliferated more rapidly than those from BALB/c after stimulation with IL-4 and CD40 ligand trimer. However, in vitro IgE levels in supernatants from stimulated 129/SvJ B cells were significantly reduced. Contrary to the in vitro findings, the 129/SvJ CD23 mutations correlated with a hyper IgE phenotype in vivo and 129/SvJ were able to clear Nippostrongylus brasiliensis infection more rapidly than either BALB/c or C57BL/6. Overall, this study further suggests that CD23 is an important regulatory factor for IgE production.     

Serum levels of total IgE and soluble CD23 in bronchial asthma

The aim of the present study was to compare, during the pollen season, serum levels of total IgE and soluble CD23 (sCD23) from patients with allergic bronchial asthma, with those from healthy subjects. Significantly higher levels of total IgE and sCD23 were found in patients with asthma compared to the control group. Both in normal controls and in asthmatic patients, a significant correlation was shown between the levels of these two molecules. In asthmatic patients, significant correlations were found for both total IgE and sCD23, with lung function measured as bronchial responsiveness to inhaled methacholine. These results suggest that in asthmatic patients, in addition to the study of total serum IgE levels, the assessment of sCD23 serum levels may be helpful in the evaluation of disease activity.     

Suppression of IgE responses in CD23-transgenic animals is due to expression of CD23 on nonlymphoid cells

Serum IgE is suppressed in CD23-transgenic (Tg) mice where B cells and some T cells express high levels of CD23, suggesting that CD23 on B and T cells may cause this suppression. However, when Tg B lymphocytes were compared with controls in B cell proliferation and IgE synthesis assays, the two were indistinguishable. Similarly, studies of lymphokine production suggested that T cell function in the Tg animals was normal. However, adoptive transfer studies indicated that suppression was seen when normal lymphocytes were used to reconstitute Tg mice, whereas reconstitution of controls with Tg lymphocytes resulted in normal IgE responses, suggesting that critical CD23-bearing cells are irradiation-resistant, nonlymphoid cells. Follicular dendritic cells (FDC) are irradiation resistant, express surface CD23, and deliver iccosomal Ag to B cells, prompting us to reason that Tg FDC may be a critical cell. High levels of transgene expression were observed in germinal centers rich in FDC and B cells, and IgE production was inhibited when Tg FDCs were cultured with normal B cells. In short, suppressed IgE production in CD23-Tg mice appears to be associated with a population of radioresistant nonlymphoid cells. FDCs that interface with B cells in the germinal center are a candidate for explaining this CD23-mediated IgE suppression.     

Soluble CD23 controls IgE synthesis and homeostasis in human B cells

CD23, the low-affinity receptor for IgE, exists in membrane and soluble forms. Soluble CD23 (sCD23) fragments are released from membrane (m)CD23 by the endogenous metalloprotease a disintegrin and metalloprotease 10. When purified tonsil B cells are incubated with IL-4 and anti-CD40 to induce class switching to IgE in vitro, mCD23 is upregulated, and sCD23 accumulates in the medium prior to IgE synthesis. We have uncoupled the effects of mCD23 cleavage and accumulation of sCD23 on IgE synthesis in this system. We show that small interfering RNA inhibition of CD23 synthesis or inhibition of mCD23 cleavage by an a disintegrin and metalloprotease 10 inhibitor, GI254023X, suppresses IL-4 and anti-CD40-stimulated IgE synthesis. Addition of a recombinant trimeric sCD23 enhances IgE synthesis in this system. This occurs even when endogenous mCD23 is protected from cleavage by GI254023X, indicating that IgE synthesis is positively controlled by sCD23. We show that recombinant trimeric sCD23 binds to cells coexpressing mIgE and mCD21 and caps these proteins on the B cell membrane. Upregulation of IgE by sCD23 occurs after class-switch recombination, and its effects are isotype-specific. These results suggest that mIgE and mCD21 cooperate in the sCD23-mediated positive regulation of IgE synthesis on cells committed to IgE synthesis. Feedback regulation may occur when the concentration of secreted IgE becomes great enough to allow binding to mCD23, thus preventing further release of sCD23. We interpret these results with the aid of a model for the upregulation of IgE by sCD23.     

Comparative binding of soluble fragments (derCD23, sCD23, and exCD23) of recombinant human CD23 to CD21 (SCR 1-2) and native IgE, and their effect on IgE regulation

IgE, responsible for type I hypersensitivities, is regulated by interactions between its receptor, CD23, and co-receptor CD21. To examine comparative binding of recombinant human CD21 SCR 1-2 and native human IgE to CD23 plus the effect of CD23 on IgE production, we engineered recombinant soluble human CD23 fragments; (1) derCD23, (2) sCD23 and (3) exCD23, formed in vivo by proteolysis. SPR analysis revealed a progressive increment in affinity of soluble fragments for IgE, upon increasing length of CD23 “stalk” domain, exCD23 > sCD23 > derCD23. Soluble CD23 fragments and their oligomeric state are shown to fine-tune the immune response. Oligomers appear more important in enhancing IgE synthesis and monomers lacking the tail residues fail to bind CD21 yet bind membrane IgE and down-regulate IgE synthesis. Co-ligation of membrane IgE and CD21 through soluble CD23 monomers is disturbed. This study supports anti-allergic therapies involving stabilizing membrane CD23, or preventing shedding of soluble CD23.     

Production and characterization of a monoclonal antibody specific for the human lymphocyte low affinity receptor for IgE: CD 23 is a low affinity receptor for IgE

In the present study a gamma 1 kappa monoclonal antibody, Mab 25, specific for the receptor for the Fc fragment of IgE on lymphocytes (Fc epsilon RL) was established. This antibody was generated after fusion of spleen cells from mice immunized with the EBV-transformed lymphoblastoid cell line RPMI 8866, which is known to express Fc epsilon RL at high density. Mab 25 inhibits strongly the binding of IgE to RPMI 8866 cells and to other Fc epsilon RL-positive EBV-transformed lymphoblastoid cell lines. A 50% inhibition of IgE binding was observed at a Mab 25 concentration of 10 ng/ml. The binding of IgE was also inhibited by Fab fragments of Mab 25, suggesting that the inhibition is not simply due to steric hindrance or to an eventual binding through its Fc portion. Mab 25 only binds to cell lines expressing Fc epsilon RL. Mab 25 immunoprecipitated a single polypeptide with an apparent m.w. of 42 Kd, pI 4.9. The membrane molecule bound to and eluted from a Mab 25 immunoabsorbent had the same apparent m.w. and pI as the Fc epsilon RL purified from an IgE immunoabsorbent. Additionally, when RPMI 8866 cell lysates were cleared with Mab 25, no Fc epsilon RL could be bound to or eluted from an IgE immunoabsorbent. Mab 25 was found to weakly bind to a minor proportion of blood (1 to 4%), tonsil (2 to 9%) and spleen (4 to 5%) mononuclear cells with a low intensity. By double fluorescence analysis, most of the Fc epsilon RL-positive cells were found to be CD 20-positive B lymphocytes. The staining pattern of Mab 25 and the biochemical characteristics of the antigen detected by Mab 25 were comparable to those of the CD 23 Mab. The four CD 23 Mab MHM 6, PL 13, HD 50, and Tü 1 were found to inhibit the binding of IgE. PL 13 was found to totally inhibit the binding of Mab 25 to RPMI 8866 cells, whereas Tü 1 and MHM 6 only partially inhibited Mab 25 binding. HD 50 was unable to block the binding of Mab 25. The finding that different CD 23/Fc epsilon RL-specific monoclonal antibodies recognizing distinct epitopes have in common the capacity of inhibiting the binding of IgE suggests that upon binding they induce a conformational alteration of the Fc epsilon RL resulting in a loss of the IgE binding capacity. In conclusion, our data demonstrate that the CD 23 antigen is a low affinity receptor for IgE on lymphocytes.     

Temperature effect on IgE binding to CD23 versus Fc epsilon RI

A chimeric soluble CD23, consisting of the extracellular domain of mouse CD23 and a modified leucine zipper (lz-CD23), has been shown to inhibit IgE binding to the FcepsilonRI. A similar human CD23 construct was also shown to inhibit binding of human IgE to human FcepsilonRI. In both systems, the inhibition was found to be temperature dependent; a 10-fold molar excess of lz-CD23 gave 90-98% inhibition at 4 degrees C, dropping to 20-30% inhibition at 37 degrees C. Surface plasmon resonance analysis of lz-CD23 binding to an IgE-coated sensor chip suggested that the effective concentration of lz-CD23 was lower at the higher temperatures. Analysis of (125)I-IgE binding to CD23(+)-Chinese hamster ovary cells also indicated that increased temperature resulted in a lower percentage of IgE capable of interacting with CD23. In contrast, IgE interacts more effectively with FcepsilonRI(+)-rat basophilic leukemia cells at 37 degrees C compared with 4 degrees C. The results support the concept that the open and closed IgE structures found by crystallography interact differently with the two IgE receptors and suggest that temperature influences the relative percentage of IgE in the respective structural forms. Changes in CD23 oligomerization also plays a role in the decreased binding seen at physiological temperatures.     

CD23-bound IgE augments and dominates recall responses through human naive B cells

Human peripheral blood BCRμ(+) B cells express high levels of CD23 and circulate preloaded with IgE. The Ag specificity of CD23-bound IgE presumably differs from the BCR and likely reflects the Ag-specific mix of free serum IgE. CD23-bound IgE is thought to enhance B cell Ag presentation to T cells raising the question of how a B cell might respond when presented with a broad mix of Ags and CD23-bound IgE specificities. We recently reported that an increase in CD23(+) B cells is associated with the development of resistance to schistosomiasis, highlighting the potential importance of CD23-bound IgE in mediating immunity. We sought to determine the relationship between BCR and CD23-bound IgE-mediated B cell activation in the context of schistosomiasis. We found that crude schistosome Ags downregulate basal B cell activation levels in individuals hyperexposed to infectious worms. Schistosome-specific IgE from resistant, occupationally exposed Kenyans recovered responses of B cells to schistosome Ag. Furthermore, cross-linking of CD23 overrode intracellular signals mediated via the BCR, illustrating its critical and dominating role in B cell activation. These results suggest that CD23-bound IgE augments and dominates recall responses through naive B cells.     

Expression of Human Recombinant CD23 in Insect Cells

Abstract

Human CD23 (low affinity receptor for IgE) has been expressed in insect cells (Sf9) using the baculovirus expression system and the baculovirus transfer vector pAc373. Insect cells infected with a recombinant baculovirus coding for CD23 synthesized a polypeptide not found in wild-type infected insect cells that had antigenic properties similar to natural CD23 produced in RPMI 8866 cells. Surface expression of recombinant CD23 was demonstrated by its ability to bind IgE. Recombinant CD23 expressed in insect cells had a slightly lower molecular weight (4 3 kDa) than that of natural CD23 (4 5 kDa) from RPMI 8866 cells as detected by SDS-PAGE followed by Western-blotting. Affinity-purified recombinant CD23 from in-fected insect cells showed B-cell growth promoting activity. These observations demonstrate for the first time that biologically active recombinant CD23 can be produced by the baculovirus expression system, thus providing a useful source of recombinant material to elucidate the biological functions of CD23.     

IgE enhances antibody and T cell responses in vivo via CD23+ B cells

IgE Abs, passively administered together with their specific Ag, can enhance the production of Abs recognizing this Ag by >100-fold. IgE-mediated feedback enhancement requires the low affinity receptor for IgE, CD23. One possible mechanism is that B cells take up IgE-Ag via CD23 and efficiently present Ag to Th cells, resulting in better Ab responses. To test whether IgE Abs have an effect on Th cells in vivo, mice were adoptively transferred with CD4+ T cells expressing a transgenic OVA-specific TCR, before immunization with IgE anti-TNP (2,4,6-trinitrophenyl) plus OVA-TNP or with OVA-TNP alone. IgE induced a 6- to 21-fold increase in the number of OVA-specific T cells. These cells acquired an activated phenotype and were visible in splenic T cell zones. The T cell response peaked 3 days after immunization and preceded the OVA-specific Ab response by a few days. Transfer of CD23+ B cells to CD23-deficient mice rescued their ability to respond to IgE-Ag. Interestingly, in this situation also CD23-negative B cells produce enhanced levels of OVA-specific Abs. The data are compatible with the Ag presentation model and suggest that B cells can take up Ag via "unspecific" receptors and activate naive T cells in vivo.     

Restoration of the antibody response to IgE/antigen complexes in CD23-deficient mice by CD23+ spleen or bone marrow cells

Mice immunized with IgE/Ag complexes produce significantly more Ag-specific Abs than mice immunized with Ag alone. The enhancement is mediated via the low-affinity receptor for IgE (FcepsilonRII or CD23), as shown by its complete absence in mice pretreated with mAbs specific for CD23 and in CD23-deficient mice. Because the constitutive expression of murine CD23 is limited to B cells and follicular dendritic cells (FDCs), one of these cell types is likely to be involved. One of the suggested modes of action of IgE/CD23 is to increase the ability of B cells to present Ag to T cells, as demonstrated to take place in vitro. Another possibility is that FDCs capture the IgE/Ag complexes and present these directly to B cells. The purpose of the present study was to determine whether CD23+ B cells or FDCs are responsible for the IgE/CD23-mediated enhancement of specific Ab responses in vivo. We show that the enhancement is completely restored in irradiated CD23-deficient mice reconstituted with CD23+ spleen or bone marrow cells. In these mice, the B cells are CD23+ and the FDCs are presumably CD23- because the FDCs are radiation resistant and are reported not to be replaced by donor cells after this type of cell transfer. In contrast, enhancement was not restored in irradiated wild-type mice reconstituted with CD23- cells. These results indicate that CD23+ B cells, and not FDCs, are the cells that capture IgE/Ag complexes and induce enhancement of Ab responses in vivo.     

CD23-dependent transcytosis of IgE and immune complex across the polarized human respiratory epithelial cells

IgE-mediated allergic inflammation occurs when allergens cross-link IgE on the surface of immune cells, thereby triggering the release of inflammatory mediators as well as enhancing Ag presentations. IgE is frequently present in airway secretions, and its level can be enhanced in human patients with allergic rhinitis and bronchial asthma. However, it remains completely unknown how IgE appears in the airway secretions. In this study, we show that CD23 (FcεRII) is constitutively expressed in established or primary human airway epithelial cells, and its expression is significantly upregulated when airway epithelial cells were subjected to IL-4 stimulation. In a transcytosis assay, human IgE or IgE-derived immune complex (IC) was transported across a polarized Calu-3 monolayer. Exposure of the Calu-3 monolayer to IL-4 stimulation also enhanced the transcytosis of either human IgE or the IC. A CD23-specific Ab or soluble CD23 significantly reduced the efficiency of IgE or IC transcytosis, suggesting a specific receptor-mediated transport by CD23. Transcytosis of both IgE and the IC was further verified in primary human airway epithelial cell monolayers. Furthermore, the transcytosed Ag-IgE complexes were competent in inducing degranulation of the cultured human mast cells. Because airway epithelial cells are the first cell layer to come into contact with inhaled allergens, our study implies CD23-mediated IgE transcytosis in human airway epithelial cells may play a critical role in initiating and contributing to the perpetuation of airway allergic inflammation.     

Hyper IgE in New Zealand black mice due to a dominant-negative CD23 mutation

Immunoglobulin E (IgE) plays a critical role in both resistance to parasitic infection and allergy to environmental antigens. The IgE response is in turn regulated by the B-cell co-receptor CD23, and CD23-deficient mice show exaggerated IgE responses and airway hyper-responsiveness. In this report, we show that New Zealand black (NZB) mice express a variant CD23 allele, with mutations in both the C-lectin-binding domain and stalk region, which fails to bind IgE at high affinity and has reduced expression on the cell surface. Expression of the variant CD23 chain interferes with trimerisation of the receptor and has a dominant-negative effect leading to reduced IgE binding in crosses between NZB and other strains. Genetic mapping shows that the variant CD23 leads to an exaggerated primary IgE response, which is independent of other strain-specific effects. These results suggest that NZB mice or mice carrying the variant allele will be useful models for studying both allergy and quantitative traits associated with atopy. The exaggerated IgE response provides an explanation for the natural resistance of NZB mice to parasitic infection by Leishmania.     

Expression of CD23 by human bone marrow stromal cells.

CD23 is a surface antigen expressed by a variety of human hematopoietic cells and shown to display multiple biological functions. In present work, we assayed CD23 expression by human bone marrow (BM) or by stromal cells derived from this tissue. While freshly isolated BM-cells showed low CD23 expression, a subset of long term BM-culture (LTBMC)-derived stromal cells expressed CD23 mRNA at high levels in their steady state and secreted soluble CD23 in their culture supernatants. To assay the role of CD23 in LTBMC, these cultures were initiated in the presence of neutralizing anti-CD23 mAb. A dramatic decrease in total numbers of hematopoietic cells and CFU-GM recovery was observed in these cultures as compared to controls. These data suggest a role of CD23 expression in stroma cell functions and further confirm the ability of this antigen to regulate human hematopoietic cell development.     

The low affinity IgE receptor (CD23) is cleaved by the metalloproteinase ADAM10

The low affinity IgE receptor, FcepsilonRII (CD23), is both a positive and negative regulator of IgE synthesis. The proteinase activity that converts the membrane-bound form of CD23 into a soluble species (sCD23) is an important regulator of the function of CD23 and may be an important therapeutic target for the control of allergy and inflammation. We have characterized the catalytic activity of ADAM (a disintegrin and metalloproteinase) 10 toward human CD23. We found that ADAM10 efficiently catalyzes the cleavage of peptides derived from two distinct cleavage sites in the CD23 backbone. Tissue inhibitors of metalloproteinases and a specific prodomain-based inhibitor of ADAM10 perturb the release of endogenously produced CD23 from human leukemia cell lines as well as primary cultures of human B-cells. Expression of a mutant metalloproteinase-deficient construct of ADAM10 partially inhibited the production of sCD23. Similarly, small inhibitory RNA knockdown of ADAM10 partially inhibited CD23 release and resulted in the accumulation of the membrane-bound form of CD23 on the cells. ADAM10 contributes to CD23 shedding and thus could be considered a potential therapeutic target for the treatment of allergic disease.     

Cytokine effects of CD23 are mediated by an epitope distinct from the IgE binding site.

Human CD23 and its soluble forms (sCD23) display various biological activities, in addition to their IgE binding function (IgE/BF). The IgE binding domain was recently mapped to residues between Cys163 and Cys282 but its involvement in IgE-independent, CD23 functions remains unknown. In order to clarify this point, a series of N-terminal, C-terminal and internal deletion mutants of CD23 or sCD23 were expressed in CHO cells and tested for their ability (i) to bind to IgE, (ii) to induce colony formation by human myeloid precursor cells, (iii) to promote mature T cell marker expression by early prothymocytes, and (iv) to regulate IgE synthesis. The present study indicates that cytokine activities require the presence of Cys288, while this amino acid is not necessary for IgE/BF. Blocking experiments using various conformation-sensitive monoclonal antibodies further suggest that active epitope(s) of CD23 in cytokine assays is(are) distinct from those involved in IgE/BF.     

Possible role of human lymphocyte receptor for IgE (CD23) or its soluble fragments in the in vitro synthesis of human IgE

The present study demonstrates that human rIL-4 is capable of inducing the secretion of IgE by PBMC. At a concentration of 200 U/ml, an IgE response was observed in 11/26 cultures of PBMC from normal donors and in 12/15 cultures from allergic individuals. The same rIL-4-stimulated cells released significant amounts of IgE-binding factors (IgE-BF) in their culture supernatant. These IgE-BF were shown for the first time to bind simultaneously to some mAb against Fc epsilon R II (mAbER) and to soluble IgE. The lack of correlation between the rIL-4-induced secretion of IgE-BF and IgE indicates that the production of IgE-BF or the expression of Fc epsilon R II is not the only factor involved in the induction of IgE synthesis by rIL-4. However, the observation that mAbER suppressed the rIL-4-induced IgE synthesis strongly suggests that either Fc epsilon R II or IgE-BF are necessary for an IgE response. Finally, the spontaneous in vitro synthesis of IgE by enriched B cell preparations isolated from atopic donors was suppressed in an isotype-specific manner by the same mAbER or by their F(ab')2 fragments. These observations suggest that the ongoing IgE synthesis by in vivo pre-activated B cells is also regulated by IgE-BF or Fc epsilon R II. It is concluded that Fc epsilon R II or IgE-BF play an essential role both in the induction of IgE synthesis by normal B cells and in the ongoing IgE synthesis by in vivo activated B cells from allergic donors.