Intestinal epithelial CD23 mediates enhanced antigen transport in allergy: evidence for novel splice forms

Linda C. H. Yu,1 Guillaume Montagnac,2,3 Ping-Chang Yang,1 Daniel H. Conrad,4 Alexandre Benmerah,2,3 and Mary H. Perdue1

1Intestinal Disease Research Programme, McMaster University, Hamilton, Ontario, Canada L8N 3Z5; 2INSERM E9925, Faculté Necker, 75730 Paris; 3Department of Infectious Diseases, Cochin Institute, INSERM U567/CNRS UMP 8104, 75014 Paris, France; 4Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia 23298

Submitted 18 October 2002 ; accepted in final form 9 March 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously demonstrated enhanced transepithelial antigen transport in the intestine of allergic rodents associated with elevated expression of the low-affinity IgE receptor CD23 on enterocytes. Here, we examined the role of CD23 in the transport phenomenon using CD23/ mice and characterized the isoform of intestinal epithelial CD23. Jejunal segments of sensitized mice were challenged with antigen. Enhanced transepithelial antigen transport and transmucosal antigen flux were found in the intestine of sensitized CD23+/+ but not CD23/ mice. RT-PCR showed that enterocytes expressed only the isoform b of CD23. Sequencing revealed classic and alternative CD23b transcripts lacking exon 5 (b{Delta}5) or 6, all of which were translated into functional IgE receptors. The protein encoded by b{Delta}5 but not the classic b transcript was able to mediate the uptake of anti-CD23 or IgE, whereas both CD23 proteins were internalized after binding to IgE/antigen complexes. Our results suggest that the classic and alternative forms of CD23b display distinct endocytic properties, suggesting that they are likely to play different roles in transepithelial transport of IgE and allergens.

mouse; transgenic/knockout; mucosa; Fc receptors; antigen binding


ALLERGIC CONDITIONS, including food allergy, asthma, allergic rhinoconjunctivitis, and atopic dermatitis, are the largest group of immune disorders affecting 20–30% of the population in North America and Europe, and there is evidence that such conditions are increasing in prevalence (42, 45). Food allergy is a disorder that leads to gastrointestinal symptoms and, in some cases, extraintestinal symptoms in the skin and airways in sensitized individuals after food antigen ingestion. In severe cases, such as peanut allergy, systemic anaphylaxis can occur with fatal consequences (9). To date, the only effective treatment of food allergy is an elimination diet involving avoidance of suspected foods. However, particularly in young children, the diet may become so restricted that nutrition is compromised. Therefore, it is important to have a clear understanding of the pathophysiology of food allergic reactions to develop novel therapeutic approaches.

Intestinal anaphylactic symptoms develop extremely rapidly (1, 17). Such reactions are known to be mediated by mast cell activation induced by antigen cross-linking of IgE bound to the cell surface via high-affinity receptors (16). However, mast cells in the intestinal mucosa are located in the lamina propria beneath the epithelial lining of the gut, which, in theory, should prevent access of antigenic molecules to these effector cells (51). Epithelial cells (enterocytes) are held together at their apical poles by tight junctions that restrict molecules larger than 500 Da from passing through the paracellular pathways (24). In addition, although a small quantity of macromolecules is taken up into enterocytes by endocytosis, most proteins are degraded during transcytosis, thus reducing their antigenic properties (46). However, enhanced epithelial permeability and antigen uptake have been reported in food allergic patients and sensitized animals (7, 8, 23, 27, 48, 52). Therefore, several years ago, we began studies to examine the pathway and mechanism by which macromolecular antigens enter the body.

Our previous experiments in allergic rodents suggested that a unique mechanism was responsible for enhanced transport of the intact antigen across the epithelial barrier. Rats were sensitized to horseradish peroxidase (HRP) and subsequently, jejunal segments were challenged with antigen on the luminal surface. Enhanced antigen uptake (severalfold control values) into enterocyte endosomes and rapid transport across the cell were documented (8, 48). As early as 2 min, HRP antigen was already present in the lamina propria (8, 48), an extremely rapid rate of transcytosis compared with normal values of ~20–30 min (30). We termed this phase I of enhanced transepithelial antigen transport. Further studies revealed that phase I antigen transport was specific for the sensitizing antigen and was IgE dependent but mast cell independent, because similar findings were obtained in mast cell-deficient rats (7, 8, 48). Immunohistochemical staining demonstrated expression of the low-affinity IgE receptor [Fc{epsilon}RII/CD23, originally described in B cells (13)] on jejunal enterocytes (48, 52). Subsequently, we found that gut epithelial CD23 expression was associated with rapid antigen uptake into enterocytes in sensitized wild-type IL-4+/+ mice, but neither CD23 expression nor enhanced antigen uptake was demonstrated in sensitized IL-4/ mice (52). This finding implies that IL-4, a Th2 cytokine elevated in allergic conditions, regulates the expression of CD23 in intestinal epithelial cells. Finally, in confirmation of the role of CD23 in enhanced antigen uptake into enterocytes in phase I, results were negative in sensitized CD23/ mice (52).

A second phase of antigen penetration through the epithelium was evident >30 min after challenge in sensitized rats (8). This phase (termed phase II) involved antigen transport via the paracellular pathway as well as the transcellular pathway. HRP was visualized in the paracellular spaces and tight junctions, and a significant increase in the overall flux of antigen across the mucosa was documented. Mast cells were shown to be activated at this time as indicated by electron microscopy. phase II antigen transport did not occur in sensitized mast cell-deficient rats, implying that the augmented epithelial permeability in this phase was mast cell dependent (7).

The aim of the current study was to continue our examination of the role of epithelial CD23 in augmented intestinal antigen transport using CD23-deficient mice and to characterize the isoform of CD23 expressed on mouse intestinal epithelial cells. In humans, a and b isoforms of CD23 have been described on a wide range of cells (3, 11, 12, 18, 28, 49). However, in mice, expression of the a isoform of CD23 has been reported on B cells (13, 14, 40), but the existence of the b isoform remains controversial. Here, we provided further evidence that CD23 is required for enhanced intestinal antigen transport and demonstrated that intestinal epithelial cells express specific CD23 splice forms endowed with different endocytic properties.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. CD23/ mice (C57BL/6 background) were bred in the Central Animal Facility at McMaster University (original breeders obtained from M. Lamers, Max-Planck-Institut fur Immunbiologie, Freiburg, Germany) (53). CD23+/+ (C57BL/6) mice and BALB/c mice were purchased from Harlan breeding Laboratories (Indianapolis, IN). Mice were used at 7–14 wk of age. Mice were sensitized by intraperitoneal injection with 100 µg of HRP (Type II; Sigma, St. Louis, MO) in 0.2 ml of aluminum hydroxide [10% AlK(SO4)2·12 H2O; Sigma] and with 50 ng of Bordetella pertussis toxin as adjuvants to stimulate IgE synthesis as described previously (52). On days 7 and 14, mice were boosted intraperitoneally with 100 µg of HRP in PBS. Experiments were performed on day 21. Naive mice served as controls. Mice were anesthetized, and a blood sample was obtained for the measurement of IgE. Following with cervical dislocation, a laperotomy was performed, and a segment of jejunum was excised for experiments (see Ussing chambers). All animal experiments were conducted with the approval from the McMaster University Animal Care Committee.

Ussing chambers. A 15-cm segment of jejunum (beginning 5 cm distal to the ligament of Treitz) was excised and immediately placed in warmed Krebs buffer (in mM: 115.0 NaCl, 8.0 KCl, 1.25 CaCl2, 1.2 MgCl2, 2.0 KH2PO4, and 25.0 NaHCO3, pH 7.33–7.37). From each mouse, six to eight pieces of jejunal tissue (cut longitudinally into flat sheets exposing the luminal and serosal sides of intestine) were mounted in Ussing Chambers (WPI instruments, Narco Scientific, Mississauga, ON, Canada). Care was taken to avoid tissues with Peyer's patches. An area of 0.6 cm2 of intestine was exposed to 8 ml of circulating oxygenated Krebs buffer at 37°C. The serosal buffer contained 10 mM of glucose as an energy source osmotically balanced with 10 mM of mannitol in the luminal buffer. The tissue was clamped at 0 V using a WPI Instrument automatic voltage clamp (Narco Scientific). After an equilibration period of 20 min, antigen challenge was conducted by adding HRP (5 x 105 M) into the luminal buffer.

Antigen uptake and transport across enterocytes. Tissues were removed from the Ussing chambers at 60 min (in phase II) after HRP challenge and processed for electron microscopy to determine the route and rate of HRP transport across the epithelium (8, 48, 52). Jejunal segments were immediately fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at room temperature, washed, and left overnight at 4°C in the same buffer and washed three times for 5 min each in 0.05 M Tris buffer (pH 7.6). Tissues were incubated for 30 min in 5 mg of 3,3'-diaminobensidine tetrahydrochlorine (Sigma) in 10 ml of 0.05 M Tris buffer and 0.01% H2O2 (pH 7.6, 22°C) and subsequently processed for transmission electron microscopy and embedded in Epon. Ultrathin sections of midvillus epithelium (cut in the longitudinal plane) were placed on copper grids and stained with uranyl acetate and lead citrate, and photomicrographs of epithelial cells were taken at a magnification of x3,000 or x8,000. To quantify epithelial HRP uptake, the total area of HRP-containing endosomes in fixed-size windows (120 µm2) in the apical region (immediately below the microvilli) of enterocytes (Fig. 1) was measured in photomicrographs using a computerized image processing system as previously described (8, 48, 52). To assess the rate of transcytosis of HRP-containing endosomes, the distribution of endosomes within the cells was recorded as the percentage of cells containing endosomes in the apical (above the nucleus), mid (beside the nucleus), or basal (below the nucleus) regions of cells, and also in the lamina propria (Fig. 1) (8, 48, 52). This analysis was performed on 100 well-oriented enterocytes in tissues from four mice per group by one investigator (P. C. Yang) who was unaware of the origin of the tissues. As a negative control, intestine from mice sensitized to HRP, but not challenged with HRP, was fixed for electron microscopy, and the epithelium was examined for endogenous peroxidase activity. No HRP was evident in this group, indicating that endogenous peroxidase did not lead to artifactual results and was not a factor in these experiments.



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Fig. 1. Schema of endosomal horseradish peroxidase (HRP) quantification. Electron photomicrographs were prepared from midvillus epithelium. To quantify HRP uptake by enterocytes, the accumulated (total) area of HRP-containing endosomes was measured in fixed-size (120 µm2) windows beneath the microvilli. To examine the transcytotic rate of HRP-containing endosomes, the incidence (%) of enterocytes containing endosomes in the apical (above the nucleus), mid (beside the nucleus), or basal (below the nucleus) regions of cells and in the lamina propria was determined. This analysis was performed for 100 enterocytes from tissues of 4 mice per treatment group.

 

To determine the overall luminal-to-serosal flux of HRP across the tissues, HRP (5 x 105 M) was added into the luminal buffer and 500 µl of serosal buffer samples were collected at 30-min intervals for 120 min and replaced with Krebs buffer. The concentration of HRP was measured by a kinetic enzymatic assay (8, 48). Briefly, 120 µl of sample were added to 800 µl of phosphate buffer containing 0.003% H2O2 and 80 µg/ml o-dianisidine (Sigma), and the enzymatic activity was determined from the rate of increase in optical density at 460 nm during a 1.5-min period. The luminal-to-serosal flux was calculated using a standard formula and expressed as picomoles per square centimeter per hour (8, 48). Eight tissues per group (2 from each of 4 mice) were used for the measurement of HRP flux.

Passive cutaneous anaphylaxis. The level of HRP-specific IgE in mouse serum was determined by passive cutaneous anaphylaxis. Sprague-Dawley rats were injected intradermally with 0.1 ml of mouse serum in duplicate dilutions from 1:1 to 1:1024. After 72 h, rats were challenged by intravenous injection with 0.5 ml of HRP (5 mg/ml) in 1% Evan blue. Positive reactions were evaluated as blue spots present after 30 min. The titer was the highest serum dilution showing a positive result. Heat treatment (56°C for 3 h) abolished the reaction indicating that the immunoglobulin was heat-labile IgE.

RT-PCR. RT-PCR was performed on RNA extracted from mouse jejunal segments, enterocytes isolated from mouse jejunum, or cultured mouse intestinal epithelial cells of the IEC-4 cell line. Segments of jejunum from control or sensitized BALB/c mice were washed in PBS and cut into 3-mm cubes for RNA extraction using a RNeasy Mini kit (Qiagen, Mississauga, ON, Canada). Enterocytes were isolated from the mouse small intestine by previously reported methods (33, 35) with modifications. The jejunal segment was slit open, washed, and incubated in RPMI 1640 media (Invitrogen, Carlsbad, CA) containing 1 mM DTT (Sigma) for 15 min at room temperature to remove mucus. Peyer's patches were removed. The tissues were then incubated with prewarmed isolation solution (0.05% trypsin, 0.53 mM EDTA in PBS; Invitrogen) for 20 min at 37°C and gently shaken every 5 min. The isolated cells were collected and washed in RPMI (as above without DTT), following with filtration through nylon mesh (Nytex, Tetko, Elmsford, NY). Epithelial cells were purified by density gradient centrifugation on a Percoll gradient (Amersham Pharmacia Biotech). Intestinal epithelial cells were collected, washed, and resuspended in RPMI. The viability of enterocytes (trypan blue negative) was >95%. The estimated purity of epithelial cells was determined to be ~90% by flow cytometry using cytokeratin as the epithelial cell marker (33, 35). RNA was extracted from the isolated enterocytes using the RNeasy Mini kit (Qiagen; see below).

IEC-4 cells were cultured in DMEM media (Invitrogen) supplemented with 5% FCS, 0.01 M HEPES, 20 mM L-glutamine, 0.1 U/ml penicillin G sodium, and 5 µg/ml streptomycin sulfate (34), and 106 cells per milliliter were seeded in a 60-mm diameter cell culture plate (Corning, Corning, NY) for 3 days until confluent. The RNA was extracted from cells using RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. RNA (2 µg) was reverse transcribed with oligo(dT)16 using Perkin-Elmer RNA PCR core kit (Perkin-Elmer, Mississauga, ON, Canada). The resulting cDNA (in 20 µl) was then subjected to PCR by the addition of 80 µl of a master mix containing 2 mM MgCl2 solution, 1 x PCR buffer, 2.5 U AmpliTaq DNA polymerase, 0.5 µM upstream primer, and 0.5 µM downstream primer.

To determine the isoform of CD23 expressed by intestinal epithelial cells, two sets of primers were used (32). As upstream primers, the a-isoform specific oligo-A (5'-CCTCATCACTGAAAGGATCCAAACAAG-3') and the b-isoform specific oligo-B (5'-GAAAGCCAATTTGAACGGGAACTTGG-3') were used. As a common downstream primer, oligo-E (5'GGAGCCCTTGCCAAAATAGTAGCAC-3') was used. The DNA thermal cycler (Teche PHC-3; Mandel Scientific Guelph, ON, Canada) was programmed to perform a protocol as follows: 94°C for 3 min for 1 cycle; 94°C for 1.5 min (denaturation), 60°C for 2 min (annealing), and 72°C for 3 min (extension) for 35 cycles; and 72°C for 7 min for final extension. To amplify full-length coding sequence of CD23b, we designed a new primer set including oligo-B' (5'-ATGAATTCTCAAAACCAGGGA-3') and oligo-F' (5'-TCAGGGTTCACTTTTTGGG-3'). The DNA thermal cycler was programmed as follows: 94°C for 5 min for 1 cycle; 94°C for 30 s, 58°C for 30 s and 72°C for 1 min for 35 or 40 cycles; and 72°C for 5 min. Negative controls were performed with samples lacking cDNA or samples with mRNA that were not reverse transcribed. RT-PCR products were then electrophoresed in a 0.8% agarose gel in the presence of 0.5 µg/ml ethidium bromide, visualized with an ultraviolet transilluminator, and photographs were taken. Molecular weight markers, Ready load {varphi}-X174 RF DNA/HaeIII fragments (Invitrogen) were used. The intensity of the DNA bands was analyzed using a densitometer with software from Kodak Digital Science 1D (GIBCO, Rockville, MD).

DNA sequence analysis. PCR products were extracted from the electrophoresed gel, cloned into pCR 3.1 plasmids, and amplified by transforming TOP 10F' competent cells using a eukaryotic bidirectional TA cloning Kit (Invitrogen). Transformed competent cells were plated on an Luria-Bertani (LB) agar plate containing 50 µg/ml ampicillin and incubated overnight. Individual colonies were grown in ampicillin-containing LB broth overnight, and plasmidic DNA was purified using the Qiaprep Miniprep kit (Qiagen). Clones containing CD23 cDNAs were sent for nucleotide sequencing (Eurogentec, Seraing, Belgium). Clones with cDNAs in the correct orientation were selected using appropriate restriction sites and used for transient transfection (see Transfection, immunofluorescence, and endocytosis).

Transfection, immunofluorescence, and endocytosis. HeLa cells were cultured in DMEM media supplemented with 10% FCS, 20 mM L-glutamine, and 5 µg/ml streptomycin sulfate to subconfluency on coverslips. HeLa cells were transfected with CD23 encoding plasmids using a calcium phosphate transfection kit (Invitrogen) and were processed for immunofluorescence studies the following day as previously described (5, 6).

Briefly, transfected HeLa cells were washed with PBS and fixed with 4% paraformaldehyde and 0.03 M sucrose at 4°C for 30 min and quenched with 50 mM NH4Cl in PBS at room temperature for 10 min. Cells were incubated with primary antibody B3B4 [20 µg/ml; rat IgG2a anti-mouse CD23 (39)] in a permeabilizing buffer [PBS containing 0.1% BSA and 0.05% saponin (Sigma)] at room temperature for 30 min, washed twice with the permeabilizing buffer, and then incubated with goat anti-rat IgG secondary antibody conjugated with Alexa Fluor 488 or 594 (1:100; Molecular probes, Eugene, OR) in permeabilizing buffer, and washed twice. Cells were mounted on microscope slides in 100 mg/ml Mowiol (Calbiochem, La Jolla, CA), 25% glycerol, 100 mM Tris·HCl, pH 8.5. Negative controls included staining in which the primary antibody was omitted as well as mock transfected HeLa cells.

For IgE binding studies, HeLa cells transiently transfected with CD23 isoforms were incubated with monoclonal mouse anti-dinitrophenyl (DNP) IgE (5 µg/ml; Sigma) in an IgE-binding solution [DMEM media containing 0.4 mM Ca(NO3)2·4H2O and 0.1% BSA (31, 41)] at 4°C for 1 h, washed twice in cold IgE binding solution, and then fixed with 4% paraformaldehyde as described above. To reveal membrane-bound IgE, cells were incubated with 10 µg/ml monoclonal rat IgG1 anti-mouse IgE antibodies (Southern Biotechnology, Birmingham, AL) in PBS containing 0.1% BSA (50 µl) at room temperature for 30 min, washed, and stained with secondary goat anti-rat IgG antibodies (1:100) conjugated with Alexa Fluor 488 or 594 (Molecular Probes).

Endocytosis of membrane-bound anti-CD23 or IgE, using transferrin as a marker of early endosomes, was examined 1 day after transfection in subconfluent HeLa cells grown on coverslips. The cells were first incubated for 20 min at 37°C in DMEM to eliminate receptor-bound transferrin, washed in cold PBS, and then incubated for 1 h at 4°C in the presence of the anti-CD23 antibody (50 µg/ml) in DMEM and 1 mg/ml BSA (DMEM-BSA) or monoclonal mouse anti-DNP IgE (5 µg/ml) in IgE-binding solution. Cells were washed two times in DMEM-BSA or IgE-binding solution and then incubated with 100 nM Alexa Fluor 594-conjugated transferrin (Molecular Probes). After incubation at 37°C for 30 min, the cells were rapidly cooled to 4°C using cold DMEM-BSA, washed twice in cold PBS, and then fixed for 1 h at 4°C. The internalized anti-CD23 and IgE were revealed using Alexa Fluor 488-labeled goat anti-rat IgG secondary antibody in permeabilizing buffer as described above.

To examine endocytosis of IgE/antigen immune complexes by the different proteins encoded by the CD23b splice variants, transfected HeLa cells were incubated with monoclonal mouse anti-DNP IgE (5 µg/ml) in IgE-binding solution for 1 h at 4°C, washed, and incubated with 0.01 µg/ml of DNP-BSA (Molecular Probes) and Alexa Fluor 594-conjugated transferrin at 37°C for 30 min. Cells were then fixed and stained with 10 µg/ml monoclonal rat IgG1 anti-mouse IgE antibodies secondary antibody in permeabilizing buffer as described above.

The samples were examined under an epifluorescence microscope (Axioplan II, Zeiss) attached to a cooled charge-coupled device camera (Spot-2, Diagnostic Instruments) or under a confocal microscope (LSM 510, Zeiss). Alexa Fluor 488 and 594 corresponding staining were observed using the classic FITC and rhodamine/Texas Red filters, respectively.

The number of cells showing IgE or anti-CD23 antibody colocalizing with transferrin in intracellular vesicles for 100 transfected cells (expressing CD23 protein) was determined, and the results are expressed as the percentage of cells showing endocytosis for each condition.

Statistics. Data are presented as means ± SE. Statistical significance was tested by ANOVA, with post hoc analysis using Newman-Keuls test or Student's t-test when appropriate. A P value <0.05 was considered to be significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sensitized CD23+/+ mice but not CD23/ mice demonstrate enhanced transepithelial antigen transport. We previously demonstrated enhanced transepithelial uptake of antigen in the intestine of sensitized CD23+/+ wild-type mice at 2 min after challenge (phase I), whereas the phenomenon was absent in sensitized CD23/ mice (52). Here, we further investigated the role of CD23 at 60 min after challenge (phase II) of transepithelial antigen transport.

The titer of anti-HRP IgE in serum from sensitized wild-type CD23+/+ mice was 1:128 (median value) and from sensitized CD23/ mice serum was 1:256 (median values), whereas no IgE was detectable in nonsensitized CD23+/+ or CD23/ mice.

At 60 min after HRP challenge, antigen uptake by enterocytes was enhanced in sensitized CD23+/+ mice compared with controls; however, no significant difference was found between sensitized and nonsensitized CD23/ mice (Fig. 2). A threefold increase (P < 0.01) in the total area of HRP-containing endosomes in enterocytes of sensitized CD23+/+ mice was demonstrated compared with nonsensitized wild-type mice or sensitized or nonsensitized CD23/ mice (Fig. 3A). The values for the percentage of cells containing endosomes in cell regions were: apical 61%, mid 21%, basal 15%, and lamina propria 20% in sensitized CD23+/+ mouse intestine vs. apical 28%, mid 1%, basal 1%, and lamina propria 0% in nonsensitized controls (Fig. 3B). Rapid transcytosis of antigen across enterocytes was not evident in sensitized CD23/ mice. Values for the percentage of enterocytes containing HRP-containing endosomes in apical, basal, and midregions and lamina propria were 24, 1, 1, and 0%, respectively, in sensitized CD23/ mice vs. 14, 1, 0, and 1%, respectively, in nonsensitized CD23/ mice (Fig. 3B).



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Fig. 2. Electron photomicrographs showing HRP uptake into enterocyte endosomes and paracellular spaces in the intestinal epithelium. Jejunal tissues mounted in Ussing chambers were challenged with HRP added to the luminal buffer. Tissues were fixed at 60 min after challenge and processed for electron microscopy to visualize HRP-containing endosomes or paracellular spaces. Representative photomicrographs show HRP-containing endosomes (arrows) in tissues obtained from HRP-sensitized CD23+/+ mice (A and B), control CD23+/+ mice (C), HRP-sensitized CD23/ mice (D). B: enlargement of A, showing HRP-containing endosomes (circled with dotted lines). Bars indicate 2 µm, except in B (bar indicates 1 µm). E and F: the paracellular spaces (arrows) between enterocytes, which are filled with HRP in tissues of HRP-sensitized CD23+/+ mice (E) but not in HRP-sensitized CD23/ mice (F). Tight junctions are indicated by arrowheads. Bars indicate 1 µm. These photomicrographs are representative of those used for quantitative measurements of endosomal area (see Fig. 3).

 


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Fig. 3. HRP transport across the intestinal epithelium of nonsensitized and sensitized (SENS) CD23+/+ and CD23/ mice. Intestinal tissues from control (CON) and SENS mice were challenged with HRP from the luminal side and then fixed at 60 min after challenge and processed for electron microscopy. A: the accumulative area of HRP-containing endosomes in enterocytes measured in a fixed-sized window in electron photomicrographs (n = 12 photomicrographs from 4 mice per group). B: the percentage of enterocytes containing HRP endosomes in the apical, mid, and basal regions of cells and in the lamina propria (LP) (n = 100 well-oriented enterocytes from 4 mice per group). C: HRP flux across intestine from control and SENS CD23/ and CD23+/+ mice (n = 12 tissues from 4 mice per group). Values represent means ± SE; *P < 0.05 compared with control CD23+/+ mice.

 

HRP was also present in the tight junctions and paracellular spaces between enterocytes at 60 min after challenge in sensitized CD23+/+ mice (Fig. 2E) but was not observed in the paracellular spaces in sensitized CD23/ mice (Fig. 2F) or in nonsensitized mice (not shown). Furthermore, a significant increase in overall HRP flux across the tissues (2.3-fold; P < 0.05) postantigen challenge was demonstrated in sensitized CD23+/+ mice compared with controls (Fig. 3C). However, the transmucosal flux of HRP was similar for sensitized and control CD23/ mice at values to nonsensitized CD23+/+ mice.

The b isoform of CD23 was the only transcript expressed by mouse intestinal epithelial cells. CD23 is expressed as two major isoforms, a and b, that show differences in their expression pattern and cellular functions (11, 18, 49). To better understand the role of CD23 in enhanced antigen transport, we characterized the isoform(s) expressed by mouse intestinal epithelial cells. A well-characterized mouse epithelial cell line, IEC-4 (34), was used in this study. RT-PCR was performed on mRNA using two different pairs of primers that were designed to amplify specifically isoform a or b of murine CD23 (32). A band of the expected size was obtained only with the b isoform specific primers (Fig. 4A). In contrast, mRNA isolated from mouse spleen cells yielded a positive result only for isoform a, as would be expected from published studies (14, 40). These results suggested that the b isoform was the only subtype expressed constitutively by intestinal epithelial cells. Moreover, the b isoform was also demonstrated in RNA prepared from both full jejunum and isolated enterocytes (Fig. 4B), confirming its expression in vivo.



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Fig. 4. Characterization of the isoforms of CD23 transcripts in mouse intestinal epithelial cells. A: identification of CD23 isoform. RNA was extracted from untreated and IL-4-treated IEC-4 cells and reverse transcribed. The cDNA was subjected to respective primer sets for CD23a and CD23b transcripts (for detail see MATERIALS AND METHODS), and the resulting PCR products were loaded onto a gel (labeled as a and b in A). Spleen cell RNA was used as template for the positive control of subtype a. B: expression of subtype b of CD23 transcripts in IEC-4 cells, full mouse jejunum, and isolated mouse enterocytes. Primer sets used for RT-PCR were oligo B' and oligo F' for 40 cycles. M, molecular weight markers; –, the negative control where no template was added. This figure is representative of 3 individual experiments.

 

Intestinal epithelial cells expressed classic CD23b and novel alternative splice forms lacking exons 5 and 6. To further confirm that the amplification products corresponded to CD23b, they were purified, subcloned into a PCR3.1 vector, and individual clones were sequenced. The majority of the clones displayed the exact sequence of CD23b (clone pERB452, GenBank no.: X64223 [GenBank] ; see Ref. 32), denoted here as the classic CD23b transcript or classic b isoform. In addition, several clones demonstrated internal deletions, between bases 229 and 291 or bases 291 and 354 (base numbers according to the sequence of pERB452). The missing regions corresponded to the entire sequences of exon 5 or 6, respectively, which are part of the extracellular stalk region (Fig. 5A). To determine whether these deletions corresponded to functional splice events and to examine whether other alternative splice forms could be found, mRNA from IEC-4 cells were subjected to RT-PCR using a new set of primers designed to amplify the full-length coding region of CD23b. The PCR products were processed as described above, and a total of 34 clones was analyzed from four individual experiments. The sequencing results confirmed the presence of full-length alternative splice forms lacking only exon 5 or 6, designated b{Delta}5 (GenBank accession no. AY069980 [GenBank] ) or b{Delta}6 (GenBank Accession no. AY069981 [GenBank] ), respectively. From the 34 analyzed clones, 23 contained classic b transcripts (68%), whereas eight clones corresponded to b{Delta}5 (24%), and three corresponded to b{Delta}6 (9%) (Fig. 5B). In addition, we identified the presence of classic b and b{Delta}5 transcripts in full-thickness jejunum and isolated enterocytes.



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Fig. 5. Classic and alternative CD23b transcripts expressed in IEC-4 cells. A: schematic sequences of the classic b and alternative b{Delta}5 and b{Delta}6 CD23 transcripts. TM, transmembrane region. B: number of clones expressing classic b, b{Delta}5, and b{Delta}6 CD23 transcripts obtained from IEC-4 cells.

 

Classic and alternative transcripts of CD23b are translated into IgE receptors. To investigate whether the novel alternative transcripts of CD23b encode for functional IgE receptors, plasmids containing the cDNA of the alternative and classic forms were used to transiently transfect HeLa cells. Immunostaining (using permeabilizing buffer) revealed that the different transcripts were translated into proteins and correctly folded because they were recognized by a well-characterized anti-CD23 antibody (Fig. 6) (39). The protein encoded by the classic CD23b transcript was localized mainly on the cell surface at steady state (Fig. 6A), whereas the proteins encoded by both the b{Delta}5 (Fig. 6B) and b{Delta}6 (Fig. 6C) forms were found on intracellular vesicular structures (arrows) as well as on the cell surface. These findings were confirmed by confocal microscopy (data not shown). No staining was seen in neighboring nontransfected cells and mock-transfected cells (Fig. 6D). To confirm that the expressed CD23 proteins were functional, CD23- and mock-transfected cells were incubated with monoclonal mouse IgE at 4°C and stained with a secondary anti-IgE antibody. Bright membrane staining was observed in cells transfected with the classic b isoform (Fig. 6E) as well as in cells transfected with the alternative b{Delta}5 and b{Delta}6 forms (Fig. 6, F and G). IgE binding was specific because it was not observed in mock-transfected cells (Fig. 6H). Similar results were obtained in MDCK cells (not shown). Together, these results indicate that all of the CD23b transcripts expressed in intestinal epithelial cells are translated into functional IgE receptors.



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Fig. 6. Expression of and IgE binding to proteins encoded by different CD23b transcripts in transfected HeLa cells. HeLa cells were transfected with plasmids encoding classic CD23 b (A, E), b{Delta}5 (B, F), or b{Delta}6 (C, G) transcripts or with empty vector (D, H). The location of CD23 protein (A-D) expression was examined on these cells by staining with monoclonal rat anti-mouse CD23 antibodes in permeabilizing buffer and was revealed using Alexa Fluor 488-labeled secondary antibody. Proteins encoded by the classic CD23b transcripts were located mainly on the cell surface, whereas those of b{Delta}5 and b{Delta}6 were located in intracellular vesicles (arrows) as well as on the cell surface. Neighboring nontransfected cells (top 3 panels) and mock-transfected cells (negative controls) did not show staining. The binding of IgE by CD23 proteins (E-H) was examined by incubation of transfected cells with monoclonal mouse IgE at 4°C for 1 h. After cells were fixed, IgE binding was revealed by staining with anti-IgE antibodies and Alexa Fluor 488-conjugated secondary antibody. All 3 forms of CD23b proteins bind to IgE (E, F, G). No staining was seen in nontransfected or mock-transfected cells.

 

Classic and alternative transcripts of CD23b display different endocytic properties. Because our results suggested a model in which CD23 mediates IgE/allergen uptake at the apical surface, we next examined whether the b{Delta}5 protein demonstrated functional differences when compared with the classic CD23b protein. HeLa cells transfected with the classic b or the b{Delta}5 transcripts were tested for their ability to mediate internalization after ligation with anti-CD23 antibodies or IgE. Confocal microscopy of b{Delta}5 expressing cells after 30 min incubation at 37°C showed membrane bound anti-CD23 located in intracellular vesicles (Fig. 7D). These vesicles were identified as early endosomes by colocalization with internalized transferrin (Fig. 7E) as shown by the yellow staining observed in the combined image (Fig. 7F). This was not the case in classic b-expressing cells where the bound anti-CD23 antibodies remained on the plasma membrane (Fig. 7A), whereas transferrin was internalized (Fig. 7B), resulting in a lack of yellow staining in the combine image (Fig. 7C). Similar results were obtained for IgE uptake (data not shown and Fig. 8). The quantification of transfected cells showing anti-CD23 or IgE internalization confirmed the difference between the two CD23b isoforms (Fig. 8).



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Fig. 7. Endocytic properties of proteins encoded by classic b and b{Delta}5 CD23 transcripts. To examine the uptake of anti-CD23 antibody, HeLa cells transfected with plasmids encoding classic b (A-C) and b{Delta}5 (D-F) CD23 transcripts were incubated with anti-CD23 at 4°C for 1 h, washed, and then incubated in the presence of a marker for early endosomes, transferrin, at 37°C for 30 min to allow for internalization. Fluorescence images were obtained by confocal microscopy. CD23 is indicated by the green staining (A and D), transferrin by the red staining (B and E), colocalization of anti-CD23 with the endosomal marker, transferrin, is indicated by the yellow color in the superimposed images (C and F).

 


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Fig. 8. Percentage of transfected cells showing endocytosis after binding to ligands or IgE/antigen. HeLa cells transfected with plasmids encoding classic b (open bar) and b{Delta}5 (striped bar) CD23 transcripts were examined for protein internalization after binding to ligands, i.e., anti-CD23 or IgE, or with IgE/antigen. For the study of endocytosis after IgE/antigen binding, transfected cells were incubated with monoclonal anti-DNP IgE at 4°C for 1 h for surface binding and then with DNP-conjugated BSA at 37°C for 30 min to determine CD23 internalization in the presence of transferrin. Internalization was calculated as the percentage of cells showing CD23 or IgE internal staining colocalized with internalized transferrin in 100 counted cells expressing CD23. The data (means ± SE) presented here are the values obtained from at least 4 independent experiments.

 

We further examined whether classic CD23b or b{Delta}5 proteins were able to mediate uptake of IgE-antigen complexes. Transfected cells expressing either classic CD23b or b{Delta}5 proteins were incubated with monoclonal anti-DNP IgE following by DNP-BSA antigen. Our results showed that both isoforms were capable of mediating the uptake of immune complexes (Fig. 8). Together, these results provide evidence that the proteins encoded by classic CD23b and b{Delta}5 transcripts have distinct endocytic properties, suggesting different functions in epithelial cells.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously reported (7, 8, 48, 52) increased transepithelial antigen transport (~20-fold normal) in the intestine of sensitized rodents resulting in rapid production of allergic symptoms. In this study, we confirmed that CD23 is critical for phase II as well as phase I of enhanced transepithelial antigen transport, because even after 60 min, there was no enhanced antigen transport in enterocytes of CD23/ mice. In addition, we characterized the murine epithelial CD23 isoform as type b and identified classic as well as alternative transcripts of CD23b. Proteins encoded by these CD23b transcripts displayed distinct endocytic properties.

The high serum titer of HRP-specific IgE verified that both CD23+/+ and CD23/ mice were similarly sensitized. CD23/ mice have been reported to retain normal differentiation and phenotypes of lymphocyte populations and to mount normal antibody responses to immunization and parasite infection (21, 43). We previously demonstrated that uptake and transepithelial transport of HRP antigen were significantly enhanced in jejunal enterocytes of sensitized CD23+/+ mice but not CD23/ mice, compared with those of nonsensitized controls at 2 min after challenge (phase I). A further increase in antigen uptake was seen in sensitized CD23+/+ mice at 60 min after challenge, and HRP-containing endosomes were widely distributed within the enterocytes as well as in the lamina propria. In contrast, in nonsensitized CD23+/+ and nonsensitized and sensitized CD23/ mice, HRP was only found in endosomes in the apical region of the cells. Several studies have shown that soluble proteins internalized from the apical side of polarized epithelial cells are transported to the late endosomes/lysosomes found in the supranuclear region (10, 25, 46). Our results showing that HRP in basal enterocyte endosomes at 60 min in CD23+/+ mice suggest that binding of antigen to IgE/CD23 protects it from intracellular degradation in late endocytic compartments.

At 60 min after challenge, HRP was also found in the tight junctions and paracellular spaces between intestinal epithelial cells in sensitized CD23+/+ mice but not in sensitized CD23/ mice. In addition, there was a significant increase in the overall transmucosal HRP flux in sensitized CD23+/+ mice compared with the other groups. The similar results for HRP fluxes in sensitized and control CD23/ mice suggest that the lack of CD23 abolished the enhanced transcytosis of antigen induced by sensitization. There was a low but measurable HRP flux in the intestine of control CD23+/+ mice and sensitized and control CD23/ mice, despite the fact that by electron microscopy we did not see HRP in the lamina propria at 60 min after challenge (Fig. 3B). This discrepancy suggests that the majority of internalized HRP is degraded during transcellular transport and thus is not visualized in the electron microscope. In addition, the kinetic enzymatic assay is an extremely sensitive method able to detect low concentrations of HRP accumulated in the serosal buffer over several hours. This is in agreement with previously reported studies showing small quantities of intact HRP being transported across nonsensitized intestinal epithelium (15, 47), whereas the majority of the protein (~90–97%) is degraded (22, 44). The enhanced overall flux in sensitized CD23+/+ mice probably involves HRP transported via both the paracellular and transcellular pathway. Together, these results suggest that the lack of epithelial CD23 prevented the enhanced antigen uptake, initially by transcytosis across enterocytes, and subsequent paracellular antigen flux.

Expression of murine CD23 has been reported in B cells, T cells, and follicular dendritic cells (13), and we recently demonstrated (48, 52) CD23 protein expression by immunostaining in intestinal epithelial cells in sensitized rats and mice. In B cells, CD23 facilitates antigen uptake and focusing (29, 37). Results presented here and in our previous studies (48, 52) show that CD23 plays a similar role in facilitating antigen entry into and transport across enterocytes. We previously showed (48) in sensitized rats by immunogold labeling of enterocyte CD23 that ligand binding induced internalization of both CD23 and antigen within the same endosomal compartment. Moreover, transcytosis of IgA and IgG across intestinal epithelial cells is mediated by specific Fc receptors, i.e., pIgR and FcRn, and binding of immunoglobulin to its receptor circumvents its breakdown during intracellular endosomal transport (19, 26). Therefore, it is likely that CD23 is responsible for transepithelial transport of intact antigens by internalization of IgE/antigen complexes at the apical surface and intracellular transport of these complexes across the cell.

Two isoforms of CD23, a and b, have been reported in humans. The a isoform is constitutively expressed in B cells, whereas IL-4 induces the expression of isoform b in B cells and non-B cells, including monocytes, eosinophils, and keratinocytes (11, 18). The amino acid sequences of the CD23 proteins encoded by the different isoforms a and b differ only in their 6/7 NH2-terminal residues, a region that corresponds to the cytoplasmic domain (49), suggesting that this region regulates divergent intracellular trafficking and/or signaling pathways. In mice, B cells express CD23a (13), but the existence of an IL-4-inducible b-like isoform remains controversial (14, 40). Only one group has reported a murine b isoform (32). In our studies, we found that spleen cells isolated from sensitized mice expressed only the CD23a transcript, whereas intestinal epithelial cells expressed exclusively isoform b. Moreover, we also detected the presence of CD23b transcripts in both murine intestine and isolated enterocytes, indicating that isoform b exists not only in cultured mouse intestinal epithelial cells but also in vivo.

In addition to the classic transcript of CD23b, we identified two novel alternative transcripts lacking the entire sequence of exon 5 (b{Delta}5) or exon 6 (b{Delta}6). In human and mouse B cells, there have been reports of alternative splice forms of CD23 transcripts, mainly lacking exon 3 encoding the transmembrane region of the protein (36, 50) but, to our knowledge, no reports of the novel transcripts we identified. The repeated heptad amino acid sequences derived from exons 5 to 8 in the mouse CD23 transcript make up the hydrophobic core of the stalk region of the protein and were shown to be important (at least exons 6, 7, and 8) for regulating the affinity of IgE binding (2, 20). The existence of a number of alternative transcripts of CD23 may imply functional discrepancies for the different forms of the protein.

Immunofluorescence studies using transfected cells demonstrated that the classic and novel CD23b transcripts were translated into functional proteins. The localization of the classic CD23b protein was mainly on the cell surface, whereas the b{Delta}5 and b{Delta}6 proteins were found on the cell surface and also in intracellular vesicular structures. The intracellular location of the alternative CD23b proteins at steady state may represent either retention of the newly synthesized proteins in intracellular compartments or increased turnover of membrane proteins due to constitutive endocytosis (see below). We identified that both the classic and alternative CD23b proteins expressed on the cell surface were functional IgE receptors.

We demonstrated that the proteins encoded by the classic CD23b and b{Delta}5 transcripts displayed different endocytic properties in transfected cells. The b{Delta}5 but not the classic b isoform was able to mediate the internalization of noncomplexed ligands, the anti-CD23 antibody B3B4, previously shown to attach to the IgE binding site on the lectin domain of the CD23 receptor (39), and IgE. These data are in agreement with the results obtained with human CD23, showing that the classic b isoform does not mediate the internalization of membrane-bound anti-CD23 antibody (49). In addition, we demonstrated that both classic CD23b and b{Delta}5 proteins were endocytosed similarly on cross-linking of membrane-bound IgE by the antigen, indicating that the cross-linking of IgE may be involved in the initiation of antigen uptake in vivo at the apical side of the enterocytes.

Overall, these results suggest that b{Delta}5 proteins were internalized constitutively as well as on ligand binding, whereas the classic CD23b proteins were expressed mainly on the cell surface and were endocytosed only after binding to IgE/antigen. These distinct endocytic properties may suggest different functions for each protein. Increased levels of IgE in the intestinal lumen have been reported in food allergic individuals and patients infected with parasites, suggesting transepithelial transport of IgE alone (4, 38). Therefore, the alternative b{Delta}5 and the classic form of CD23b proteins may play different roles accounting for enhanced transepithelial transport of IgE and IgE-allergen complexes following sensitization.

In summary, our study demonstrated a functional role for CD23b in facilitating the IgE-mediated enhanced antigen transport across mouse intestinal epithelium. Our findings suggest that antigen binding to IgE/CD23 bypasses the lysosomal degradative pathway, resulting in large quantities of antigen penetrating the epithelial barrier. We further provided evidence for the presence of classic and alternative CD23b transcripts in mouse intestinal epithelial cells, with the encoded proteins displaying different endocytic properties.


    ACKNOWLEDGMENTS
 
This research was supported by a grant from Canadian Institutes of Health Research (to M. H. Perdue) and grants from INSERM and Nutricia Research Foundation (to A. Benmerah).

L. C. H. Yu was the recipient of an Rx&D (Canada) Scholarship.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. H. Perdue, IDRP, HSC 3N5C, McMaster Univ., 1200 Main St. W., Hamilton, ON, Canada L8N 3Z5 (E-mail: perdue{at}mcmaster.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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