Differential role for CD23 splice forms in apical to basolateral transcytosis of IgE/allergen complexes

The low affinity receptor for IgE (CD23) was recently implicated in the trans-epithelial transport of IgE-allergen complexes from the luminal side of enterocytes in animal models for intestinal allergy. Here, the respective functions of CD23 splice forms, b and bDelta5, in this apical to basolateral transport event have been investigated. First, the new bDelta5 splice form was further characterized, providing evidence that it binds IgE with high affinity, that its expression is induced by sensitization, and that bDelta5, unlike the classical b, undergoes constitutive internalization through clathrin-coated pits. These results suggested that the two CD23 splice forms were likely involved in different transcytotic events. MDCK cell lines expressing either b or bDelta5 were generated to directly test this hypothesis. In both cell lines, CD23 splice forms were localized at the apical membrane as in enterocytes from sensitized mice. Using mouse monoclonal IgE, we obtained evidence showing that bDelta5 mediates the apical to basolateral transport of free IgE, whereas classical b is much more efficient in mediating the transcytosis of IgE/allergen complexes. The present results shed new light on the role of CD23 species in IgE/allergen transepithelial transport and provide a new powerful physiological tool to study apical to basolateral transcytosis, a process which remains poorly characterized.     

Intracellular trafficking of CD23: differential regulation in humans and mice by both extracellular and intracellular exons

In mouse models of food allergy, we recently characterized a new CD23b-derived splice form lacking extracellular exon 5, bDelta5, which undergoes constitutive internalization and mediates the transepithelial transport of free IgE, whereas classical CD23b is more efficient in transporting IgE/allergen complexes. These data suggested that regulation of endocytosis plays a central role in CD23 functions and drove us to systematically compare the intracellular trafficking properties of human and murine CD23 splice forms. We found that CD23 species show similar endocytic behaviors in both species; CD23a undergoes constitutive clathrin-dependent internalization, whereas CD23b is stable at the plasma membrane. However, the mechanisms controlling these similar behaviors appeared to be different. In mice, a positive internalization signal was localized in the cytoplasmic region shared by all CD23 splice forms. This positive signal was negatively regulated by the intracellular CD23b-specific exon. In addition, the fact that alternative splice forms lacking exons of the extracellular region (5, 6, 7, and/or 8) were all constitutively internalized suggested that endocytosis of murine CD23 is regulated by a process similar to the outside-in signaling of integrins. In humans, the internalization signal was mapped in the CD23a-specific intracellular exon. Interestingly, this signal also behaved as a basolateral targeting signal in polarized Madin-Darby canine kidney cells. The latter result and the fact that human intestinal cell lines were found to coexpress both CD23a and CD23b provide a molecular explanation for the initial observations that CD23 was found at the basolateral membrane of intestinal epithelial cells from allergic patients.     

IgE-Mediated Enhancement of CD4+ T Cell Responses in Mice Requires Antigen Presentation by CD11c+ Cells and Not by B Cells

IgE antibodies, administered to mice together with their specific antigen, enhance antibody and CD4⁺ T cell responses to this antigen. The effect is dependent on the low affinity receptor for IgE, CD23, and the receptor must be expressed on B cells. In vitro, IgE-antigen complexes are endocytosed via CD23 on B cells, which subsequently present the antigen to CD4⁺ T cells. This mechanism has been suggested to explain also IgE-mediated enhancement of immune responses in vivo. We recently found that CD23⁺ B cells capture IgE-antigen complexes in peripheral blood and rapidly transport them to B cell follicles in the spleen. This provides an alternative explanation for the requirement for CD23⁺ B cells. The aim of the present study was to determine whether B-cell mediated antigen presentation of IgE-antigen complexes explains the enhancing effect of IgE on immune responses in vivo. The ability of spleen cells, taken from mice 1–4 h after immunization with IgE-antigen, to present antigen to specific CD4⁺ T cells was analyzed. Antigen presentation was intact when spleens were depleted of CD19⁺ cells (i.e., primarily B cells) but was severely impaired after depletion of CD11c⁺ cells (i.e., primarily dendritic cells). In agreement with this, the ability of IgE to enhance proliferation of CD4⁺ T cells was abolished in CD11c-DTR mice conditionally depleted of CD11c⁺ cells. Finally, the lack of IgE-mediated enhancemen of CD4⁺ T cell responses in CD23^-/- mice could be rescued by transfer of MHC-II-compatible as well as by MHC-II-incompatible CD23⁺ B cells. These findings argue against the idea that IgE-mediated enhancement of specific CD4⁺ T cell responses in vivo is caused by increased antigen presentation by B cells. A model where CD23⁺ B cells act as antigen transporting cells, delivering antigen to CD11c⁺ cells for presentation to T cells is consistent with available experimental data.     

Interactions between CD36 and global intestinal alkaline phosphatase in mouse small intestine and effects of high-fat diet

The mechanisms of the saturable component of long-chain fatty acid (LCFA) transport across the small intestinal epithelium and its regulation by a high-fat diet (HFD) are uncertain. It is hypothesized here that the putative fatty acid translocase/CD36 and intestinal alkaline phosphatases (IAPs) function together to optimize LCFA transport. Phosphorylated CD36 (pCD36) was expressed in mouse enterocytes and dephosphorylated by calf IAP (CIAP). Uptake of fluorescently tagged LCFA into isolated enteroctyes was increased when cells were treated with CIAP; this was blocked with a specific CD36 inhibitor. pCD36 colocalized in enterocytes with the global IAP (gIAP) isozyme and, specifically, coimmunoprecipitated with gIAP, but not the duodenal-specific isozyme (dIAP). Purified recombinant gIAP dephosphorylated immunoprecipitated pCD36, and antiserum to gIAP decreased initial LCFA uptake in enterocytes. Body weight, adiposity, and plasma leptin and triglycerides were significantly increased in HFD mice compared with controls fed a normal-fat diet. HFD significantly increased immunoreactive CD36 and gIAP, but not dIAP, in jejunum, but not duodenum. Uptake of LCFA was increased in a CD36-dependent manner in enterocytes from HFD mice. It is concluded that CD36 exists in its phosphorylated and dephosphorylated states in mouse enterocytes, that pCD36 is a substrate of gIAP, and that dephosphorylation by IAPs results in increased LCFA transport capability. HFD upregulates CD36 and gIAP in parallel and enhances CD36-dependent fatty acid uptake. The interactions between these proteins may be important for efficient fat transport in mouse intestine, but whether the changes in gIAP and CD36 in enterocytes contribute to HFD-induced obesity remains to be determined.     

CD23-mediated transport of IgE/immune complexes across human intestinal epithelium: role of p38 MAPK

We previously reported that CD23/FcepsilonRII (low-affinity IgE receptor) is expressed on human intestinal epithelial cells and is responsible for transepithelial transport of IgE. In this study, we compared the transport of IgE with that of immune complexes in both the apical-to-serosal and the serosal-to-apical directions across HT29 epithelial cell layers and examined the effects of two p38 MAPK inhibitors, SKF86002 and SB203580, on the expression and function of CD23. Our study showed that both p38 MAPK inhibitors at 10 microM significantly inhibited constitutive and IL-4-upregulated CD23 protein expression in epithelial cells. Both inhibitors, in a concentration-dependent manner, also significantly reduced IgE binding and uptake into cells. Transepithelial transport of IgE and immune complexes across the epithelial barrier were similarly inhibited. IL-4 upregulated the phosphorylation and activity of p38 MAPK and the phosphorylation of the downstream substrate MAPKAPK-2 (MK-2). The inhibitors exerted effects in the pathway post the p38 MAPK; SB203580 significantly inhibited the phosphorylation of MK-2. Our results indicate that CD23 expression in these human intestinal epithelial cells is mediated through the p38 MAPK pathway and that inhibition of p38 MAPK consequently interferes with the transport of IgE and immune complexes across the intestinal epithelial barrier.     

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.     

Antigen Transfer from Exosomes to Dendritic Cells as an Explanation for the Immune Enhancement Seen by IgE Immune Complexes

IgE antigen complexes induce increased specific T cell proliferation and increased specific IgG production. Immediately after immunization, CD23⁺ B cells capture IgE antigen complexes, transport them to the spleen where, via unknown mechanisms, dendritic cells capture the antigen and present it to T cells. CD23, the low affinity IgE receptor, binds IgE antigen complexes and internalizes them. In this study, we show that these complexes are processed onto B-cell derived exosomes (bexosomes) in a CD23 dependent manner. The bexosomes carry CD23, IgE and MHC II and stimulate antigen specific T-cell proliferation in vitro. When IgE antigen complex stimulated bexosomes are incubated with dendritic cells, dendritic cells induce specific T-cell proliferation in vivo, similar to IgE antigen complexes. This suggests that bexosomes can provide the essential transfer mechanism for IgE antigen complexes from B cells to dendritic cells.     

Generation of polymeric immunoglobulin receptor-deficient mouse with marked reduction of secretory IgA.

We generated mouse lacking exon 2 of polymeric Ig receptor (pIgR) gene by a gene-targeting strategy (pIgR-deficient mouse; pIgR-/- mouse) to define the physiological role of pIgR in the transcytosis of Igs. pIgR-/- mice were born at the expected ratio from a cross between pIgR+/- mice, indicating that disruption of the pIgR gene in mice is not lethal. pIgR and secretory component proteins were not detected in pIgR-/- mice by Western blot analysis. Moreover, immunohistochemical analysis showed that pIgR protein is not expressed in jejunal and colonic epithelial cells of pIgR-/- mice, whereas IgA+ cells are present in the intestinal mucosa of pIgR-/- mice as well as wild-type littermates. Disruption of the pIgR gene caused a remarkable increase in serum IgA concentration and a slight increment of serum IgG and IgE levels, leaving serum IgM level unaltered. In contrast, IgA was much reduced but not negligible in the bile, feces, and intestinal contents of pIgR-/- mice. Additionally, IgA with a molecular mass of 280 kDa preferentially accumulated in the serum of pIgR-/- mice, suggesting that transepithelial transport of dIgA is severely blocked in pIgR-/- mice. These results demonstrate that dIgA is mainly transported by pIgR on the epithelial cells of intestine and hepatocytes, but a small quantity of IgA may be secreted via other pathways.     

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.     

Structural changes in the lectin domain of CD23, the low-affinity IgE receptor, upon calcium binding

CD23, the low-affinity receptor for IgE (Fc epsilonRII), regulates IgE synthesis and also mediates IgE-dependent antigen transport and processing. CD23 is a unique Fc receptor belonging to the C-type lectin-like domain superfamily and binds IgE in an unusual, non-lectin-like manner, requiring calcium but not carbohydrate. We have solved the high-resolution crystal structures of the human CD23 lectin domain in the presence and absence of Ca2+. The crystal structures differ significantly from a previously determined NMR structure and show that calcium binding occurs at the principal binding site, but not at an auxiliary site that appears to be absent in human CD23. Conformational differences between the apo and Ca2+ bound structures suggest how IgE-Fc binding can be both calcium-dependent and carbohydrate-independent.     

Evidence for multiple complementary pathways for efficient cholesterol absorption in mice

Apolipoprotein B (apoB)-dependent and apoB-independent pathways for cholesterol transport have been described in cultured cells. Here, we show that the apoB-independent pathway involves apoA-I-containing high density lipoproteins (HDLs). Cholesterol secretion by the HDLs, but not by the apoB pathway, was significantly reduced in primary enterocytes isolated from chow- and cholesterol-fed apoA-I(-/-) mice. These enterocytes were capable of cholesterol efflux when apoA-I was provided extracellularly. In apoA-I(-/-) mice, the absorption of a bolus of cholesterol was similar in control and apoA-I(-/-) mice fed chow or high-cholesterol diet. However, short-term studies revealed that cholesterol absorption was occurring over longer lengths of the intestine, and cholesterol but not triglyceride transport to the plasma and liver in chow- and cholesterol-fed apoA-I(-/-) mice was significantly reduced. These studies indicate that in apoA-I deficiency, there is a delay in cholesterol absorption, but cholesterol is eventually absorbed because of the compensatory apoB pathway. Nonetheless, long-term studies involving multiple feedings showed significant reduction in cholesterol absorption after 4 days. We propose that multiple compensatory mechanisms ensure efficient cholesterol absorption in mice.     

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.     

Complex formation of CD23/surface immunoglobulin and CD23/CD81/MHC class II on an EBV-transformed human B cell line and inferable role of tetraspanin

CD23, a low-affinity IgE receptor, is a type II transmembrane protein having a C-type lectin domain and it associates noncovalently with MHC class II on B cells. The results of our immunoprecipitation analysis suggest that CD23 co-exists with at least two additional molecules, surface immunoglobulin (sIg) and CD81 (and/or CD9), on the cell surface of L-KT9 cells (an Epstein-Barr virus (EBV)-transformed human B cell line). When both CD23 and sIg molecules were stimulated simultaneously by the corresponding antibodies, a large increase in CD81 in the immunoprecipitation was observed as compared with the case of stimulation by only one antibody. Simultaneous stimulation by anti-CD23 and anti-Ig may mimic the situation of B cells stimulated by an antigen/IgE complex. In addition, a large increase in MHC class II in the immunoprecipitation was also observed by cross-linking of CD23 with anti-CD23 and its second antibody as compared with the case of stimulation by anti-CD23 alone. The cross-linking of CD23 with anti-CD23 and its antibody may mimic the situation of B cells stimulated by an IgE/antigen/IgE complex. Therefore, the complex formation among CD23, sIg, MHC class II, and CD81 on the cell surface of L-KT9 cells by the antigen/IgE or IgE/antigen/IgE complex is most likely to be closely related to B cell regulatory events by signaling through sIg or MHC class II. Tetraspanins such as CD81 and CD9 are thought to be involved in the formation and the preservation of various different membrane complexes consisting of several functional proteins.     

B-lymphocytes as Key Players in Chemical-Induced Asthma

T-lymphocytes and B-lymphocytes are key players in allergic asthma, with B-lymphocytes producing antigen-specific immunoglobulins E (IgE). We used a mouse model of chemical-induced asthma and transferred B-lymphocytes from sensitized animals into naïve wild type mice, B-lymphocyte knock-out (B-KO) mice or severe combined immunodeficiency (SCID) mice. On days 1 and 8, BALB/c mice were dermally sensitized with 0.3% toluene diisocyanate (TDI) (20µl/ear). On day 15, mice were euthanized and the auricular lymph nodes isolated. B-lymphocytes (CD19⁺) were separated from the whole cell suspension and 175,000 cells were injected in the tail vein of naïve wild type, B-KO or SCID mice. Three days later, the mice received a single oropharyngeal challenge with 0.01% TDI (20µl) or vehicle (acetone/olive oil (AOO)) (controls). Airway reactivity to methacholine and total and differential cell counts in the bronchoalveolar lavage (BAL) fluid were measured 24 hours after challenge. B-lymphocytes of AOO or TDI-sensitized mice were characterized for the expression of surface markers and production of cytokines. We found that transfer of B-cells obtained from mice dermally sensitized to toluene diisocyanate (TDI) into naïve wild type mice, B-KO mice or SCID mice led, within three days, to an acute asthma-like phenotype after an airway challenge with TDI. This response was specific and independent of IgE. These B-lymphocytes showed antigen presenting capacities (CD80/CD86 and CD40) and consisted of B effector (Be)2- (IL-4) and Be1-lymphocytes (IFN-γ). The transferred B-lymphocytes were visualized near large airways, 24 hours after TDI challenge. Thus, B-lymphocytes can provoke an asthmatic response without the action of T-lymphocytes and without major involvement of IgE.     

IgE mediates killing of intracellular Toxoplasma gondii by human macrophages through CD23-dependent, interleukin-10 sensitive pathway

Background

In addition to helminthic infections, elevated serum IgE levels were observed in many protozoal infections, while their contribution during immune response to these pathogens remained unclear. As IgE/antigen immune complexes (IgE-IC) bind to human cells through FcεRI or FcεRII/CD23 surface molecules, the present study aimed to identify which functional receptor may be involved in IgE-IC interaction with human macrophages, the major effector cell during parasite infection.

Methodology/Principal Findings

Human monocyte-derived macrophages were infected with Toxoplasma gondii before being incubated with IgE-IC. IgE receptors were then identified using appropriate blocking antibodies. The activation of cells and parasiticidal activity were evaluated by mediator quantification and direct counting of infected macrophages. RNAs were extracted and cell supernatants were also collected for their content in tumor necrosis factor (TNF)-α, interleukin-10 (IL-10) and nitrites. Sera from symptomatic infected patients were also tested for their content of IgE, IL-10 and nitrites, and compared to values found in healthy donors. Results showed that IgE-IC induced intracellular elimination of parasites by human macrophages. IgE-mediated effect was FcεRI-independent, but required cross-linking of surface FcεRII/CD23, cell activation and the generation of nitric oxide (NO). Although TNF-α was shown to be produced during cell activation, this cytokine had minor contribution in this phenomenon while endogenous and exogenous IL-10 down-regulated parasite killing. Inverse relationship was found between IL-10 and NO expression by infected human macrophages at both mRNA and mediator levels. The relationship between these in vitro data and in vivo levels of various factors in T. gondii infected patients supports the involvement of CD23 antigen and IL-10 expression in disease control.

Conclusion

Thus, IgE may be considered as immune mediator during antiprotozoal activity of human macrophages through its ability to trigger CD23 signaling. Increased cell activation by IgE-IC may also account for chronic inflammatory diseases observed in some patients.     

Regional variation in ontogeny of class II antigens in enterocytes of mouse small intestine.

The ontogeny of major histocompatibility class II antigens in small intestine enterocytes of postnatal C3H/He mice was investigated. Cryosections of duodenal, jejunal, and ileal segments from 7-, 14-, 16-, 20-, 21-, 23-, 25-, 27-, 28-day-old and 7-week-old mice were stained for the class II antigens with MRC OX6 monoclonal antibodies by peroxidase-antiperoxidase labelling. In adults, the duodenum exhibited least expression of class II antigens that increased progressively towards the ileum. The expression in the villous epithelium was first seen in the duodenum and jejunum 21 days after birth but the ileal enterocytes did not exhibit any class II antigens. The earliest appearance (21 days postnatal) of class II antigens in the enterocytes coincides with the age of weaning which suggests that immunologic stimulation by ingested antigens after weaning may influence expression of these antigens. At day 28 after birth, the duodenum and jejunum expressed levels comparable to those in the adults. The first expression of the antigens seen in the ileum was at day 28 postpartum. Crypt epithelium of the three regions of the small intestine showed expression similar to that of corresponding regional villous enterocytes. We conclude that there is an age-dependent regional variation in the expression of class II antigens in enterocytes, and the expression increases with age. The variation in expression of the class II antigens in enterocytes of postnatal mice is attributed to the developmental status of the tissue. The nature of postnatal expression of the antigens is important since an early appearance of these antigens may have implications in autoimmunity.     

Intestinal dysplasia induced by simian virus 40 T antigen is independent of p53

Transgenic mice expressing simian virus 40 large T antigen in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. Hyperplasia is dependent on T antigen binding to the retinoblastoma (pRb) family of tumor suppressor proteins. Mice expressing a truncated T antigen that inactivates the pRb-family, but is defective for binding p53, exhibit hyperplasia but do not progress to dysplasia. We hypothesized that the inhibition of the pRb family leads to entry of enterocytes into the cell cycle, resulting in hyperplasia, while inactivation of p53 is required for progression to dysplasia. Therefore, we examined T antigen/p53 complexes from the intestines of transgenic mice. We found that T antigen did not induce p53 stabilization, and we could not detect T antigen/p53 complexes in villus enterocytes. In contrast, T antigen expression led to a large increase in the levels of the cyclin-dependent kinase inhibitor p21. Furthermore, mice in which pRb was inactivated by a truncated T antigen in a p53 null background exhibited intestinal hyperplasia but no progression to dysplasia. These data indicate that loss of p53 function does not play a role in T antigen-induced dysplasia in the intestine. Rather, some unknown function of T antigen is essential for progression beyond hyperplasia.     

Overexpression of CD97 in Intestinal Epithelial Cells of Transgenic Mice Attenuates Colitis by Strengthening Adherens Junctions

The adhesion G-protein-coupled receptor CD97 is present in normal colonic enterocytes but overexpressed in colorectal carcinoma. To investigate the function of CD97 in colorectal carcinogenesis, transgenic Tg(villin-CD97) mice overexpressing CD97 in enterocytes were generated and subjected to azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis-associated tumorigenesis. Unexpectedly, we found a CD97 cDNA copy number-dependent reduction of DSS-induced colitis in Tg compared to wild-type (WT) mice that was confirmed by applying a simple DSS protocol. Ultrastructural analysis revealed that overexpression of CD97 strengthened lateral cell-cell contacts between enterocytes, which, in contrast, were weakened in CD97 knockout (Ko) mice. Transepithelial resistance was not altered in Tg and Ko mice, indicating that tight junctions were not affected. In Tg murine and normal human colonic enterocytes as well as in colorectal cell lines CD97 was localized preferentially in E-cadherin-based adherens junctions. CD97 overexpression upregulated membrane-bound but not cytoplasmic or nuclear β-catenin and reduced phospho-β-catenin, labeled for degradation. This was associated with inactivation of glycogen synthase kinase-3β (GSK-3β) and activation of Akt. In summary, CD97 increases the structural integrity of enterocytic adherens junctions by increasing and stabilizing junctional β-catenin, thereby regulating intestinal epithelial strength and attenuating experimental colitis.     

Uncoupling of Natural IgE Production and CD23 Surface Expression Levels

CD23, the low affinity receptor for immunoglobulin E (IgE), has been proposed to play a critical role in the regulation of IgE production, based on altered IgE levels in CD23-deficient mice and transgenic mouse models, as well as in mouse strains with mutations in the CD23 gene, e.g. 129 substrains. Here, we have investigated a mouse line termed LxT1 that expresses reduced CD23 surface levels on B cells, and its influence on natural IgE production. Extensive phenotypic analysis showed that CD23 surface expression was reduced in LxT1 compared to the control, without affecting B cell development in general. This CD23^low surface level in LxT1 mice is not as a result of reduced CD23 mRNA expression levels or intracellular accumulation, but linked to a recessive locus, a 129-derived region spanning 28 Mb on chromosome 8, which includes the CD23 gene. Sequence analysis confirmed five mutations within the CD23 coding region in LxT1 mice, the same as those present in New Zealand Black (NZB) and 129 mice. However, this CD23^low phenotype was not observed in all 129 substrains despite carrying these same CD23 mutations in the coding region. Moreover, serum IgE levels in LxT1 mice are as low as those in the C57BL/6 (B6) strain, and much lower than those in 129 substrains. These data indicate that the CD23 surface level and serum IgE level are uncoupled and that neither is directly regulated by the mutations within the CD23 coding region. This study suggests that caution should be taken when interpreting the immunological data derived from mice with different genetic background, especially if the gene of interest is thought to influence CD23 surface expression or serum IgE level.     

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.