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
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ABSTRACT |
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mouse; transgenic/knockout; mucosa; Fc receptors; antigen binding
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 2030 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
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.
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MATERIALS AND METHODS |
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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 7.337.37). 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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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 -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.
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RESULTS |
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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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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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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
b5 (GenBank accession no. AY069980
[GenBank]
) or
b
6 (GenBank Accession no. AY069981
[GenBank]
), respectively.
From the 34 analyzed clones, 23 contained classic b transcripts
(68%), whereas eight clones corresponded to b
5 (24%),
and three corresponded to b
6 (9%)
(Fig. 5B). In
addition, we identified the presence of classic b and
b
5 transcripts in full-thickness jejunum and isolated
enterocytes.
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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
b5 (Fig.
6B) and b
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
5 and b
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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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 b5 protein demonstrated functional differences
when compared with the classic CD23b protein. HeLa cells transfected
with the classic b or the b
5 transcripts
were tested for their ability to mediate internalization after ligation with
anti-CD23 antibodies or IgE. Confocal microscopy of
b
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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We further examined whether classic CD23b or
b5 proteins were able to mediate uptake of
IgE-antigen complexes. Transfected cells expressing either classic
CD23b or b
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
5 transcripts have distinct endocytic
properties, suggesting different functions in epithelial cells.
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DISCUSSION |
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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 (9097%) 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
(b5) or exon 6 (b
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 b5 and
b
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
b5 transcripts displayed different endocytic
properties in transfected cells. The b
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
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 b5 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
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.
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ACKNOWLEDGMENTS |
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L. C. H. Yu was the recipient of an Rx&D (Canada) Scholarship.
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FOOTNOTES |
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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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REFERENCES |
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