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Am J Physiol Gastrointest Liver Physiol 291: G532-G538, 2006; doi:10.1152/ajpgi.00524.2005
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HORMONES AND SIGNALING

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

Yahong Tu and Mary H. Perdue

Intestinal Disease Research Program, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada

Submitted 9 November 2005 ; accepted in final form 30 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously reported that CD23/Fc{epsilon}RII (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 µM 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.

low-affinity immunoglobulin E receptor; intestinal permeability; antigen

ALLERGIC DISEASES are the most common type of immune disorders and are increasing in incidence in the developed world (24). Food allergies affect 2–5% of adults and up to 8% of children (28). The symptoms of food allergies include nausea, vomiting, and diarrhea, as well as nonintestinal symptoms in the skin and airways. Currently, there are few effective treatments for these conditions apart from avoidance of offending foods. Elimination diets may result in lack of adequate nutrition, particularly in growing children. Therefore, it is important to understand the mechanisms involved in generating food allergic reactions to design novel therapeutic approaches.

An ingested antigen must cross the epithelium lining the intestinal tract to gain access to mucosal immune effector cells that are activated in intestinal anaphylactic reactions. A key immunoglobulin in allergies is IgE (31). There is evidence that in sensitized individuals, IL-4 is produced in excess and that IL-4 stimulates the production of IgE as well as expression of its receptors (10, 11, 34). The high-affinity receptor Fc{epsilon}RI is expressed on mast cells (21), and cross-linking of mast cell-bound IgE by antigen results in activation of these cells with release of potent mediators (36). In the gut mucosa, the action of these mediators on structural cells, such as epithelial cells and smooth muscle cells, causes the symptoms of food allergies (40). Interfering with antigen ingress into the mucosa might be a strategy to limit the activation of effector cells and thus reduce allergic reactions.

Our previous studies (4) in rodent models of food allergy demonstrated that transepithelial uptake of antigen from the intestinal lumen was enhanced in sensitized animals compared with controls. This enhanced uptake was specific for the antigen to which the animal was sensitized and dependent on IgE (37). Subsequent studies (39) in mice provided evidence that CD23 (the low-affinity receptor for IgE) expressed on the apical membrane of intestinal epithelial cells (enterocytes) was mediating this effect. Human epithelial cells, both in tissues as well as cultured cell lines, also express CD23 (34). We recently reported (34) that the expression of human intestinal epithelial CD23 is regulated by IL-4. In that study, we also demonstrated CD23-mediated IgE uptake and bidirectional transport across human epithelium; CD23 antibody concentration-dependently reduced IgE transport across HT29 cultured epithelial layers grown on filter supports. Transfection of these epithelial cells with antisense oligonucleotides inhibited CD23 expression; this strategy also reduced the transepithelial transport of IgE.

In human monocytes, expression of CD23 protein was reduced by inhibitors to p38 MAPK (22). Specifically, inhibition of phosphorylation of p38 MAPK protein and cascading phosphorylations of downstream proteins, such as MAPKAPK-2 (MK-2) by SKF86002 or SB203580, resulted in downregulated expression of CD23. These inhibitors were shown to reduce the surface expression of intact CD23, involving either transcriptional or translational regulation. The role of p38 MAPK in regulating epithelial CD23 expression has not been reported previously.

The current study was designed to continue our studies of the function of CD23 in human epithelial cells and to attempt to interfere with its function in an indirect manner. Although we had previously demonstrated CD23-mediated transport of IgE (34), we had not examined whether immune complexes can be carried across the intestinal barrier by this receptor. In this study, we used HT29 human epithelial cells as in our previous study (26), because they express CD23 in response to IL-4 (but do not develop decreased resistance) and form epithelial layers suitable for transport experiments. This study demonstrates that inhibition of p38 MAPK results in the downregulation of epithelial CD23 expression accompanied by reduced transepithelial transport of IgE and, more importantly, of IgE-antigen immune complexes across the epithelial barrier.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and antibodies. The CD23 antibody used in the study was mouse anti-human CD23, clone Tu1 (NovoCastra Laboratories, Newcastle upon Tyne, UK). Chimeric human IgE raised against 4-hydroxy-3-nitrophenylacetyl (NP) was a product of Serotec (Oxford, UK). NP(16)-ovalbumin (OVA) was purchased from Biosearch Technologies (Novato, CA). Rabbit polyclonal anti-human IgE antibody was purchased from DAKO Diagnostics (Mississauga, Ontario, Canada). Monoclonal anti-OVA antibody was purchased from Sigma-Aldrich (St. Louis, MO). All antibodies to phosphorylated and nonphosphorylated p38 MAPK, MK-2, and a kit for measuring p38 MAPK activity were from Cell Signaling Technology (Beverly, MA). Monoclonal anti-beta-actin was a product of Sigma-Aldrich. Recombinant human IL-4 was from R&D Systems (Minneapolis, MN). The p38 MAPK inhibitors SKF86002 and SB203580 were products of Calbiochem-Novabiochem (San Diego, CA).

Epithelial cell culture. HT29-Cl 19A human intestinal epithelial cells were used for these studies, because we had previously determined (34) that of the three human epithelial cell lines tested, these HT29 cells produced the most consistent results, and the nonspecific permeability of HT29 epithelia was not affected by IL-4. The cells were cultured in McCoy's 5A modified medium without glucose (Invitrogen Canada, Burlington, Ontario, Canada), supplemented with 5% FBS, 0.0375% sodium bicarbonate, 0.2 mM L-glutamine, and penicillin/streptomycin, in an atmosphere of 5% CO2 at 37°C. The cells were split every 5 days, and media was changed every 2–3 days. Cells (5 x 105) were seeded into wells of 24-well plates, onto coverslips, or onto Transwell filters (Corning, Corning, NY) for transport studies. In the latter case, the cells were grown either on the normal or reverse side of the filter, which was then cultured in a conical tube with 25 ml media. The filter was returned to the Transwell compartment when the transepithelial resistance was at least 400 {Omega}/cm2 (~7 days), measured by an Ohm meter with chopstick electrodes (Millicell-RES; Millipore, Bedford, MA). Under these conditions, the cells formed differentiated monolayers. CD23 expression was stimulated by IL-4 (10 ng/ml) added 24 h before the experiment.

CD23 protein measurement by Western blot. Cells grown in plates were washed twice with cold PBS (pH 7.2) containing a cocktail of protease inhibitors (Sigma). The cells were harvested by scraping them into 500 µl of cold lysis buffer (100 mM NaCl, 10 mM Tris·HCl, 2 mM EDTA, 1.8% Triton X-100, and protease inhibitors) (pH 7.8). The total protein in the supernatant of the cell lysate was measured by the Bio-Rad protein assay (Bio-Rad Laboratories, Mississauga, ON, Canada). Then, 80 µg of protein for each sample were migrated on a 10% acrylamide gel and analyzed by SDS-PAGE. The proteins were transferred electrophoretically onto a nitrocellulose membrane and incubated with anti-human CD23 antibody (1:500) for 48 h at 4°C. After being washed with PBS, horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was added. An enhanced chemiluminescence detection method was used to detect the target CD23 protein using a LumiGlo substrate kit (KP Laboratories, Gaithersburg, MD) and X-ray film. The integrated intensity for the bands was quantified by scanning densitometry.

IgE binding/uptake into epithelial cells. IgE uptake into epithelial cells was determined using HT29 cells cultured on coverslips for 3 days. Cells were treated with IL-4 for 24 h and/or p38 MAPK inhibitors added 2 h before IL-4. Cells were then incubated for 90 min at 37°C with 10 µg/ml IgE [dissolved in IgE binding buffer, (in mM) 10 Tris·HCl (pH 7.35), 140 NaCl, 2 CaCl2, and 1 MgCl2, with 1.8 g/l glucose] (30). The cells were washed three times with cold PBS, including protease inhibitors, and then fixed with 50% acetone buffered in PBS for 15 min. After being washed, endogenous peroxidase activity was eliminated by 3% H2O2 for 15 min. The cells were blocked with 0.1 M Tris·HCl, 0.15 M NaCl, and 0.5% blocking reagent; pH 7.5 (TNB buffer) for 30 min. IgE was recognized by a rabbit polyclonal antibody against the {epsilon}-chain of human IgE (1:500) added to the cells for 30 min. The signal was amplified using streptavdin-HRP and tyramide fluorescein (TSA kit; NEN Life Science Products, Boston, MA). Images were observed under the confocal microscope and analyzed using LSM510 software.

Transepithelial transport of IgE or immune complexes. IgE transport across epithelial monolayers (after pretreatment with IL-4 and/or p38 MAPK inhibitors) was performed in Transwell compartments by adding 0.2 ml of the IgE transport solution (0.4 µM IgE in binding buffer) to either the apical or basal compartment (depending on whether the cells had been grown on the normal or reverse side of the filter). After a 2-h incubation at 37°C, the media from the opposite compartment was collected at 4°C. IgE was measured by Western blot using HRP-conjugated rabbit anti-human IgE antibody (1:20,000), details as for CD23. (The 2-h time was selected to accumulate enough IgE to measure accurately.)

To form immune complexes, NP-OVA (0.5 µM) was incubated with IgE anti-NP (0.2 µM) for 60 min. The complex was added to the apical or basal compartment. Media in the opposite compartment of the monolayer was collected for Western blot analysis using a secondary antibody to OVA (1:10,000) to detect the NP-OVA. Two bands were detected. The band with the larger molecular weight (>200 kDa), indicative of the complex, was quantified by densitometry.

Measurement of phosphorylated p38 MAPK. Phosphorylated p38 MAPK was analyzed by Western blot analysis of plate-grown cells after specific treatments. The cells were lysed in PBS containing (in mM) 20 HEPES (pH 7.5), 0.2 EDTA, 5 DTT, 1 PMSF, and 1.2 Na3VO4 added to the lysis buffer. Then cells were scraped off the culture dishes and centrifuged at 10,000 g for 30 min at 4°C. The supernatant was mixed with an equal volume of the same buffer including 40% glycerol (12). The samples were loaded onto 10% SDS-PAGE and transferred to a nitrocellulose membrane. The specific rabbit anti-phospho-p38 MAPK antibody against Thr180/Tyr182 sites, which does not crossreact with phosphorylated threonine/tyrosine of ERK 1/2 or JNK (Cell Signaling Technology, bulletin no. 9211), was incubated with the protein on the membrane at a dilution of 1:1,000 for 48 h at 4°C. The secondary HRP-conjugated anti-rabbit antibody was incubated with the membrane for 2 h at a 1:40,000 dilution. After the specific signal was captured, the membrane was stripped by 0.1 M acetic acid in PBS for 1 h. The membrane was incubated again with nonphosphorylated p38 MAPK antibody. This signal was used as an internal reference to semiquantify the bands for each loading sample. The ratio of the band-integrated intensity for the phospho-p38 MAPK to the band-integrated intensity for the nonphospho-p38 MAPK was used to indicate the change in phosphorylation.

Phospho-p38 MAPK activity. The activity of phospho-p38 MAPK was performed using a p38 MAPK assay (Cell Signaling Technology). Briefly, the monoclonal phosphospecific antibody to p38 MAPK (Thr180/Tyr182) was employed to selectively immunoprecipitate active phospho-p38 MAPK from cell lysates. The resulting immunoprecipitate was then incubated with activating transcription factor-2 (ATF-2) fusion protein in the presence of ATP and kinase buffer. This allowed the immunoprecipitated active p38 MAPK to phosphorylate ATF-2 in vitro. The phosphorylation of ATF-2 at Thr71 was measured by Western blot using an anti-phospho-ATF-2 (Thr71) antibody. Thr71 of ATF-2 is a major ATF-2 phosphorylation site required for transcriptional activity (20).

Determination of the phosphorylation for MK-2. The phospho-MK-2 was also measured by Western blot. After treatments, the cells were immediately scraped off the culture dishes in a 1x SDS sample buffer and sonicated for 10–15 s. The samples were heated for 5 min at 100°C and centrifuged at 10,000 g for 5 min. Protein for each sample was loaded onto 10% SDS-PAGE and transferred to the nitrocellulose membrane. The protein on the membrane was incubated with a specific rabbit anti-phospho-MK-2 (Thr334) antibody overnight at 4°C, in a dilution of 1:2,000. This antibody detected MK-2 only when phosphorylated at Thr334. The signal elicited from the protein was visualized by HRP-conjugated secondary antibody and enhanced chemiluminescence. Then the antibodies were stripped (by adding 0.1 M acetic acid in PBS for 30 min followed by three washes in PBS), and the protein on the membrane was further incubated with rabbit anti-nonphospho-MK-2 antibody, which served as an internal control. The phosphorylation of MK-2 was indicated as the ratio of the phospho-MK-2 over the nonphospho-MK-2.

Statistical analysis. Statistical analysis was performed using a two-tail Student's t-tests or ANOVA. A P value <0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of p38 MAPK inhibitors on CD23 expression. We previously showed (5) that CD23 protein is constitutively expressed in epithelial HT29 cells and upregulated by IL-4. Here, we examined the effect of two p38 MAPK inhibitors, SKF86002 and SB203580. Both inhibitors at 10 µM significantly inhibited the constitutive expression of CD23 protein in human intestinal epithelial cells (Fig. 1). In addition, both inhibitors (added 2 h prior to IL-4) resulted in a significant diminution of the IL-4-enhanced CD23 protein expression. SB203580 was more effective, resulting in ~50% decrease in constitutive expression; IL-4-stimulated expression was reduced to ~33% by SKF86002 compared with ~50% by SB203580.


Figure 1
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  		Fig. 1. Inhibition of constitutive and IL-4-enhanced CD23 protein expression by p38 MAPK inhibitors. HT29 human intestinal epithelial cells (3 x 106) were cultured in 24-well plates for 3 days. Then cells were cultured for an additional 24 h with IL-4 (10 ng/ml) or vehicle. Each p38 MAPK inhibitor, SKF86002 (SKF) or SB203580 (SB), was added 2 h before IL-4 or vehicle. CD23 protein expression is indicated by Western blots. Integrated intensity was determined by scanning densitometry using beta-actin as an internal control. Bars indicate the percentage change compared with control (C) in the absence of IL-4. Values are means ± SE; n = 5 separate experiments. *P < 0.02, ***P < 0.0001 vs. control. ++P < 0.001, +++P < 0.0001 vs. IL-4 alone.

 
Effect of p38 MAPK inhibitors on IgE uptake. Immunohistochemistry of IgE uptake into epithelial cells was conducted using IL-4-treated cells. Confocal imaging showed that IgE was internalized into epithelial cytoplasmic compartments (Fig. 2). Both of the p38 MAPK inhibitors reduced fluorescence in a concentration-dependent manner, with partial inhibition at 10 µM and virtually complete elimination at 20 µM.


Figure 2
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  		Fig. 2. Reduction of IgE binding and uptake by p38 MAPK inhibitors. HT29 cells were grown on coverslips for 3 days. The cells were cultured for an additional 24 h with IL-4 (10 ng/ml). Each p38 MAPK inhibitor, SKF or SB at 10 or 20 µM, was added 2 h before IL-4. The culture media with the reagents were discarded. Then IgE (in IgE binding buffer), was added for 90 min at an atmosphere of 37°C, 5% CO2, allowing for IgE binding and uptake. After 3 washes, the cells were fixed with PBS-buffered 50% acetone. Endogenous peroxidase activity was eliminated by H2O2. A polycolonal rabbit anti-IgE {epsilon}-chain antibody was then incubated with the cells, and the signal was amplified using streptavdin-HRP and tyramide fluorescein. The upper panels are laser confocal images showing that the p38 MAPK inhibitors reduce IgE binding/uptake in a concentration-dependent manner. The lower panels are phase-contrast images.

 
Effect of p38 MAPK inhibitors on transepithelial transport of IgE. Transepithelial transport of IgE across IL-4-treated cells occurred in both directions, from the apical-to-basal side of the epithelial monolayer and also from the basal-to-apical side of the monolayer (Fig. 3). However, the luminally directed transport (from basal-to-apical) was relatively greater, being approximately five times that in the opposite direction. The p38 MAPK inhibitors dose-dependently inhibited transepithelial IgE transport in both directions. At 10 µM, particularly for SB203580, transport of IgE was negligible.


Figure 3
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  		Fig. 3. Effects of p38 MAPK inhibitors on transepithelial IgE transport. HT29 cells were grown on transwell filters for 7 days until transepithelial resistance was >400 {Omega}/cm2. Monolayers with identical resistance were cultured for an additional 24 h with IL-4 (10 ng/ml) or vehicle. Each p38 MAPK inhibitor, SKF or SB, was added 2 h before IL-4 or vehicle. IgE (0.4 µM) was added to either the apical or basal compartment. After 2 h, the media in the opposite compartment was collected for Western blot analysis. The band intensity of the transported IgE was quantified by densitometry. Bars indicate the fold change compared with C in the absence of IL-4. Values are means ± SE; n = 4 separate experiments. *P < 0.05, **P < 0.01, ***P < 0.002 vs. control. +P < 0.05, ++P < 0.01, +++P < 0.001 vs. IL-4 alone.

 
Effect of the p38 MAPK inhibitors on the transepithelial transport of immune complex. Transepithelial transport of the IgE immune complex across IL-4-treated cells also occurred in both directions (Fig. 4). However, in this case, the serosally directed transport (from apical-to-basal) was relatively greater than the luminally directed transport. Again, the p38 MAPK inhibitors dose-dependently inhibited transepithelial immune complex transport in both directions.


Figure 4
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  		Fig. 4. Inhibition of specific antigen transport across HT29 monolayer by p38 MAPK inhibitors. HT29 cells were grown on transwell filters for 7 days until transepithelial resistance was >400 {Omega}/cm2. Monolayers with identical resistance were cultured for an additional 24 h with IL-4 (10 ng/ml) or vehicle. Each p38 MAPK inhibitor, SKF or SB, was added 2 h before IL-4 or vehicle. Immune complexes, formed by incubating 4-hydroxy-3-nitrophenylacetyl (NP)- ovalbumin (OVA; 0.5 µM) with IgE anti-NP (0.2 µM) for 60 min, were added to either the apical or basal compartment. After 2 h, the media in the opposite compartment was collected for Western blot analysis. The band intensity of the large molecular mass OVA complex was quantified by densitometry. Bars indicate the fold change compared with C in the absence of IL-4. Values are means ± SE; n = 4 separate experiments. ***P < 0.001 vs. control. +P < 0.05, ++P < 0.01, +++P < 0.001 vs. IL-4 alone.

 
Alteration of the p38 MAPK signal transduction pathway by IL-4. Figure 5A, shows that IL-4 treatment (10 ng/ml for 10 min) increased the phosphorylation of p38 MAPK in epithelial cells. Phosphorylation was inhibited by the p38 MAPK inhibitors at 10 µM (Fig. 5B). The inhibitors had no effect on phosphorylation in the absence of IL-4 (data not shown).


Figure 5
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  		Fig. 5. A: effect of IL-4 on phosphorylation of p38 MAPK. HT29 cells were cultured in plates. After treatment with IL-4 (10 ng/ml) or vehicle for 10 min, the cells were lysed in an ice-cold lysis buffer and scraped off the plates. Western blot analysis was performed. After the membrane was with specific antibody to phosphorylated p38 MAPK, the image was visualized. The membrane was stripped with 0.1 M acetic acid in PBS and further incubated with an antibody against the nonphosphorylated p38 MAPK. The result shown is representative of 3 separate experiments. B: effect of p38 MAPK inhibitors on MAPK activity. HT29 cells were cultured in plates. Each p38 MAPK inhibitor, SKF or SB, was added for 2 h before IL-4 or vehicle. After incubation with IL-4 (10 ng/ml) or vehicle for 10 min, the cells were lysed and the protein was extracted. The phospho-p38 MAPK activity was measured using a p38 MAPK assay as described in the MATERIALS AND METHODS. The result shown is representative of 3 separate experiments.

 
Because MK-2 is a key downstream factor in the p38 MAPK pathway, we determined the phosphorylation of this protein after IL-4 stimulation. Figure 6A shows a time-dependent change in phosphorylated MK-2. Phosphorylation of the protein was evident at 15 min after treatment with IL-4 and lasted for ~6 h. Figure 6B, shows the inhibitory effect of the p38 MAPK inhibitor SB203580 (10 µM) on the IL-4-enhanced phosphorylation of the MK-2. In this study, both phosphorylated and nonphosphorylated MK-2 were determined. The percentage of the ratio of phosphorylated MK-2 over nonphosphorylated kinase was 100 ± 9.8% for the control (n = 5) and 163 ± 19.9% (n = 5) for IL-4-treated cells (P < 0.03 vs. control). The p38 MAPK inhibitor SB203580 significantly inhibited the effect of the IL-4 on phophorylation, reducing the percentage to 90 ± 12% (n = 5; P < 0.02 vs. IL-4 alone). Although SKF86002 inhibited the percentage from 163 ± 19.9% of the IL-4 treatment to 119 ± 30.7% of the IL-4 plus SKF86002 (n = 5), there was no significant difference between the two groups.


Figure 6
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  		Fig. 6. A: effect of IL-4 on phosphorylation of MAPKAPK-2. HT29 cells were grown in plates and treated with IL-4 (10 ng/ml) for different times. Western blot analysis was performed. After incubating the membrane with specific antibody to phosphorylated MAPKAPK-2, the image was visualized. The membrane was stripped with 0.1 M acetic acid in PBS and further incubated with an antibody against the nonphosphorylated MAPKAPK-2. The result shown is representative of 3 separate experiments. B: effect of p38 MAPK inhibitors on phosphorylation of MAPKAPK-2. HT29 cells were cultured in plates. Each p38 MAPK inhibitor, SKF or SB, was added for 2 h before IL-4 or vehicle. After incubation with IL-4 (10 ng/ml) or vehicle for 10 min, the cells were lysed and the protein was extracted. Western blot analysis was performed as described in MATERIALS AND METHODS. Bars indicate the ratio of phosphorylated MAPKAPK-2 compared with nonphosphorylated MAPKAPK-2, expressed as percent change compared with C in the absence of IL-4. Values are means ± SE; n = 5 separate experiments. **P < 0.01 vs. control, ++P < 0.01 vs. IL-4 alone.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We previously reported (34) that CD23, the low-affinity receptor for IgE, is expressed by human enterocytes. In addition, we showed that CD23 functions to bind, take up, and transport IgE across intestinal epithelial monolayers. Here, we showed that IgE immune complexes are also transported by epithelial CD23 in a bidirectional manner and that IgE is preferentially transported toward the lumen, whereas immune complexes are preferentially transported in the opposite (into the mucosa) direction across HT29 epithelial cell layers. We provided evidence that p38 MAPK is involved in the pathway-regulating expression of CD23 and that inhibition of this enzyme can virtually eliminate the transepithelial transport of both IgE and its immune complexes.

For many years we had observed that food antigen-induced changes in intestinal function (e.g., ion secretion) in sensitized rodents occurred extremely rapidly (within minutes) (4). The effector cells were shown to be mast cells located in the subepithelial mucosa (5, 9). Normally, the epithelial barrier (single cell layer of conjoined cells lining the intestinal tract) restricts penetration into the mucosa of antigen-sized molecules (13). To determine the route and rate of antigen uptake across the epithelial barrier, we sensitized rats to HRP and two wk later challenged intestinal tissues with HRP added to the luminal side of tissues mounted in Ussing chambers. HRP antigen was identified in the lamina propria at 2 min (20x faster than normal), and by 5 min there was evidence of mast cell activation associated with ion secretion (indicated by elevated short-circuit current) (3, 8). Because the accelerated responses were specific for the antigen to which the animal had been sensitized (4, 37), we postulated the involvement of an immunoglobulin recognition mechanism. Passive sensitization studies, in which IgE was depleted from immune serum transferred into naïve animals, demonstrated that IgE was required for enhanced transepithelial antigen uptake (39). We next showed that the enhanced antigen uptake was significantly inhibited by anti-CD23 antibody and was not present in CD23–/– mice (39). Expression of CD23 mRNA and proteins (the classic b isoform as well as 2 unique splice variants) in mouse enterocytes was identified to be regulated by IL-4 (41). Taken together, these studies implicated CD23 in the transport of IgE-antigen immune complexes across the epithelial barrier into the body.

In humans, CD23 expression has been demonstrated in epithelial cells as well as a host of immune/inflammatory cells (1, 15, 27, 33–35, 38). Intestinal CD23 expression was identified before birth, at ~16 wk of gestation (19). In enterocytes, CD23 protein was visualized on both the brush border luminal membrane and on the basolateral membrane (23, 34), suggesting that IgE or IgE immune complexes may be transported in both directions across the epithelium. In fact, we demonstrated bidirectional transepithelial IgE transport in our previous study (34), but we did not closely examine the relative amount of IgE transported in either direction. Here, we noted significantly greater transepithelial transport of IgE toward the lumen than in the opposite direction, whereas the opposite was true for immune complexes. Different isoforms/splice variants of CD23 are likely responsible for the preferential binding and transepithelial transport of IgE vs. its complexes (23, 41). This is an area of ongoing investigation.

Excessive IgE has been measured in the luminal contents/feces of food allergic individuals (6, 29), and it was suggested that IgE in the lumen is produced locally in the gastrointestinal mucosa (17). Whether produced in the mucosa or derived from the circulation, CD23-mediated IgE transport into the lumen would be a mechanism for clearance/secretion of the immunoglobulin. In studies of nematode infection in rats (25), excess IgE was also demonstrated in the gastrointestinal tract, where it was postulated to be involved in the host defense mechanism, that is, rapid expulsion of the parasite from an immunized host. Therefore, it is not surprising that our study indicated greater transport of IgE toward the lumen than in the opposite direction, whereas there was enhanced transport of immune complexes from the lumen toward the basal side of the monolayer (the physiologically relevant direction, because mucosal mast cells normally reside under the epithelium). The fate of CD23-bound IgE after transport is not clear. It might remain associated with its receptor or, because the receptor has relatively low affinity, be released into the lumen where it would be free to form immune complexes with ingested antigens. In this form, the conformation of the IgE molecule might be somewhat altered. It is possible that different splice variants of CD23b are expressed in human enterocytes and are responsible for the binding and transport of IgE vs. immune complexes. Different forms of CD23 may be directed differentially toward the basal or apical membrane. Studies need to address this issue.

In this study, we determined that the p38 MAPK inhibitors SKF86002 and SB203580 reduced the expression of CD23 in intestinal epithelial cells and inhibited its function. Both constitutive and IL-4-stimulated expression was inhibited, similar to the results of Marshall et al. (22) in monocytes. We used IL-4-treated epithelial cell monolayers for all the uptake/transport experiments, because IL-4 is elevated in allergic conditions, making that the more relevant situation. Both inhibitors caused a concentration-dependent reduction in uptake of IgE into the cells. In addition, transepithelial transport of either IgE or immune complexes was significantly diminished by the p38 MAPK inhibitors. With respect to transport, p38 MAPK inhibition was concentration dependent and resulted in virtual elimination of transport at 10 µM. The inhibition was similar, regardless of whether IgE was free or complexed to its antigen, and did not different for either direction of transport (although there were minor differences in the effective concentrations, possibly due to altered CD23 protein conformation). This suggests that the inhibition of function was indirect and due to reduced expression of CD23.

IL-4 has been shown to upregulate CD23 expression in murine lymphocytes (7) and rodent and human intestinal epithelial cells (22, 34, 41). IL-4 also induces activation of p38 MAPK in murine B- and T-lymphoid cell lines (14). In addition, Marshall et al. (22) showed in monocytes that IL-4 upregulated CD23 expression, and those inhibitors for p38 MAPK reduced the IL-4-increased CD23 protein expression. Here, we identified that IL-4 increased the phosphorylation of the p38 MAPK in epithelial cells but had no effect on the nonphosphorylated p38 MAPK enzyme. Neither inhibitor alone had any effect on the phosphorylation of p38 MAPK. This is in accordance with the theory that both inhibitors are acting at a step downstream from the kinase (18). We found that the activity of p38 MAPK with respect to phosphorylation of ATF-2 was upregulated by IL-4 and was inhibited by SKF86002 or SB203580 at 10 µM. Sundstrom et al. (32) also reported that p38 MAPK inhibitors attenuated stem cell factor-induced p38 MAPK activation cascade in a murine mast cell line. MK-2 is a substrate in the downstream signaling pathway of p38 MAPK. In this study, we observed that IL-4 stimulated the phosphorylation of MK-2, but did not alter the level of nonphosphorylated MK-2. Therefore, this result suggests that the IL-4-enhanced expression of CD23 involves signal transduction downstream of p38 MAPK.

In summary, our study indicates that inhibition of p38 MAPK inhibits the expression and function of CD23 in HT29 human intestinal epithelial cells. The p38 MAPK inhibitors exerted concentration-dependent inhibitory effects on the transport of IgE and IgE-antigen immune complexes across the intestinal barrier. We confirmed the involvement of the p38 MAPK signal transduction pathway. Recent publications (2, 16, 42) have provided evidence that the p38 MAPK pathway is involved in intestinal barrier damage after ischemia-reperfusion injury and after burn injury; inhibitors for p38 MAPK improved the integrity of the intestinal barrier and reduced the production of proinflammatory cytokines after damage. Because our studies were limited to only one line of cultured epithelial cells, we cannot extrapolate to all in vivo situations. However, our findings taken together with those of others suggest that p38 MAPK inhibitors deserve further study, because they may be potential candidates for the treatment of the food allergic conditions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by the Canadian Institutes for Health Research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. H. Perdue, Intestinal Disease Research Program, HSC-3N5C, McMaster University, 1200 Main St. West, Hamilton, ON, Canada, L8N 3Z5 (e-mail: perdue{at}mcmaster.ca)

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   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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  1. Abdelilah SG, Bouchaib L, Morita M, Delphine A, Marika S, Andre C, and Monique C. Molecular characterization of the low-affinity IgE receptor Fc {epsilon}RII/CD23 expressed by human eosinophils. Int Immunol 10: 395–404, 1998.[Abstract/Free Full Text]
  2. Badger AM, Olivera DL, Talmadge JE, and Hanna N. Protective effect of SK&F86002, a novel dual inhibitor of arachidonic acid metabolism, in murine models of endotoxic shock: inhibition of tumor necrosis factor as a possible mechanism of action. Circ Shock 27: 51–61, 1989.[Web of Science][Medline]
  3. Barrett KE. Immune-related intestinal chloride secretion. III. Acute and chronic effects of mast cell mediators on chloride secretion by a human colonic epithelial cell line. J Immunol 147: 959–964, 1991.[Abstract]
  4. Berin MC, Kiliaan AJ, Yang PC, Groot JA, Taminiau JA, and Perdue MH. Rapid transepithelial antigen transport in rat jejunum: impact of sensitization and the hypersensitivity reaction. Gastroenterology 113: 856–864, 1997.[CrossRef][Web of Science][Medline]
  5. Berin MC, Kiliaan AJ, Yang PC, Groot JA, Kitamura Y, and Perdue MH. The influence of mast cells on pathways of transepithelial antigen transport in rat intestine. J Immunol 161: 2561–2566, 1998.[Abstract/Free Full Text]
  6. Brown WR and Lee EH. Studies on IgE in human intestinal fluids. Int Arch Allergy Appl Immunol 50: 87–94, 1976.[Web of Science][Medline]
  7. Conrad DH, Keegan AD, Kalli KR, Dusen RV, Rao M, and Levine AD. Superinduction of low affinity IgE receptors on murine B lymphocytes by lipopolysaccharide and IL-4. J Immunol 141: 1091–1097, 1988.[Abstract]
  8. Crowe SE, Sestini P, and Perdue MH. Allergic reactions of rat jejunal mucosa. Ion transport responses to luminal antigen and inflammatory mediators. Gastroenterology 99: 74–82, 1990.[Web of Science][Medline]
  9. Crowe SE and Perdue MH. Functional abnormalities in the intestine associated with mucosal mast cell activation. Reg Immunol 4: 113–117, 1992.[Medline]
  10. Gascan H, Gauchat JF, Roncarolo MG, Yssel H, Spits H, and De Vries JE. Human B cell clones can be induced to proliferate and to switch to IgE and IgG4 synthesis by interleukin 4 and a signal provided by activated CD4+ T cell clones. J Exp Med 173: 747–750, 1991.[Abstract/Free Full Text]
  11. Gosset P, Lamblin-Degros C, Tillie-Leblond I, Charbonnier AS, Joseph M, Wallaert B, Kochan JP, and Tonnel AB. Modulation of high-affinity IgE receptor expression in blood monocytes: opposite effect of IL-4 and glucocorticoids. J Allergy Clin Immunol 107: 114–122, 2001.[CrossRef][Web of Science][Medline]
  12. Haddad JJ and Land SC. Redox/ROS regulation of lipopolysacccharide-induced mitogen-activated protein kinase (MAPK) activation and MAPK-mediated TNF{alpha} biosynthesis. British J Pharmacol 135: 520–536, 2002.[CrossRef][Web of Science][Medline]
  13. Hollander D. The intestinal permeability barrier. A hypothesis as to its regulation and involvement in Crohn's disease. Scand J Gastroenterol 27: 721–726, 1992.[Web of Science][Medline]
  14. Hunt AE, Williams LM, Lali FV, and Foxwell BMJ. IL-4 regulation of p38 MAPK signaling is dependent on cell type. Cytokine 18: 295–303, 2002.[CrossRef][Web of Science][Medline]
  15. Kaiserlian D, Lachaux A, Grosjean I, Graber P, and Bonnefoy JY. Intestinal epithelial cells express the CD23/Fc{epsilon}RII molecule: enhanced expression in enteropathies. Immunology 80: 90–95, 1993.[Web of Science][Medline]
  16. Kita T, Yamaguchi H, Sato H, Kasai K, Tanaka T, and Tanaka N. Role of p38 mitogen-activated protein kinase pathway on renal failure in the infant rat after burn injury. Shock 21: 535–542, 2004.[CrossRef][Web of Science][Medline]
  17. Kolmannskog S and Haneberg B. Immunoglobulin E in feces from children with allergy. Evidence of local production of IgE in the gut. Int Arch Allergy Appl Immunol 76: 133–137, 1985.[Web of Science][Medline]
  18. Lee JC and Young PR. Role of CSBP/p38/RK stress response kinase in LPS and cytokine signaling mechanisms. J Leukoc Biol 59: 152–157, 1996.[Abstract]
  19. Lima JO, Zhang L, Atkinson TP, Philips J, Dasanayake AP, and Schroeder HW. Early expression of I{epsilon}, CD23 (Fc{epsilon}RII), IL-4R{alpha}, and IgE in the human fetus. J Allergy Clin Immunol 106: 911–917, 2000.[CrossRef][Web of Science][Medline]
  20. Livingstone C, Patel G, and Jones N. ATF-2 contains a phosphorylation-dependent transcriptional activation domain. EMBO J 14: 1785–1797, 1995.[Web of Science][Medline]
  21. Macglashan D. IgE and FceRI Regulation. Clin Rev Allergy Immunol 29: 49–60, 2005.[CrossRef][Web of Science][Medline]
  22. Marshall LA, Hansbury MJ, Bolognese BJ, Gum RJ, Peter RY, and Mayer RJ. Inhibitors of the p38 mitogen-activated kinase modulate IL-4 induction of low affinity IgE receptor (CD23) in human monocytes. J Immunol 161: 6005–6013, 1998.[Abstract/Free Full Text]
  23. Montagnac G, Molla-Herman A, Bouchet J, Yu LC, and Conrad DH. Intracellular trafficking of CD23: differential regulation in humans and mice by both extracellular and intracellular exons. J Immunol 174: 5562–5572, 2005.[Abstract/Free Full Text]
  24. Mowat AM, Parker LA, Beacock-Sharp H, Millington OR, and Chirdo F. Oral tolerance: overview and historical perspectives. Ann N Y Acad Sci 1029: 1–8, 2004.[CrossRef][Web of Science][Medline]
  25. Negrao-Correa D, Adams LS, and Bell RG. Intestinal transport and catabolism of IgE: a major blood-independent pathway of IgE dissemination during a Trichinella spiralis infection of rats. J Immunol 157: 4037–4046, 1996.[Abstract]
  26. Parkos CA, Delp C, Arnaout MA, and Madara JL. Neutrophil migration across a cultured intestinal epithelium. Dependence on a CD11b/CD18-mediated event and enhanced efficiency in physiological direction. J Clin Invest 88: 1605–1612, 1991.[Web of Science][Medline]
  27. Rosenthal KL, Ishizaka T, Befus AD, Dennert G, Hengartner H, and Bienenstock J. Expression of IgE receptors and histamine in cloned natural killer cell lines. J Immunol 133: 642–646, 1984.[Abstract]
  28. Sampson HA and Burks AW. Mechanisms of food allergy. Annu Rev Nutr 16: 161–177, 1996.[Web of Science][Medline]
  29. Sasai K, Furukawa S, Sugawara T, Kaneko K, Baba M, and Yabuta K. IgE levels in faecal extracts of patients with food allergy. Allergy 47: 594–598, 1992.[Web of Science][Medline]
  30. Sato T, Konishi A, Yasuno S, Arai J, Kamei M, Bitoh M, and Yamaguchi T. A new method for studying the binding of human IgE to CD23 and the inhibition of this binding. J Immunol 209: 59–66, 1997.
  31. Scurlock AM, Lee LA, and Burks AW. Food allergy in children. Immunol Allergy Clin North Am 25: 369–388, 2005.[CrossRef][Web of Science][Medline]
  32. Sundstrom M, Alfredsson J, Olsson N, and Nilsson G. Stem cell factor-induced migration of mast cells requires p38 Mitogen-activated protein kinase activity. Exp Cell Res 267: 144–151, 2001.[CrossRef][Web of Science][Medline]
  33. Thornton CA, Hollowaym JA, Popplewell EJ, Shute JK, Boughton J, and Warner JO. Fetal exposure to intact immunoglobulin E occurs via the gastrointestinal tract. Clin Exp Allergy 33: 306–311, 2003.[CrossRef][Web of Science][Medline]
  34. Tu Y, Salim S, Bourgeois J, Di Leo V, Irvine EJ, Marshall JK, and Perdue MH. CD23-mediated IgE transport across human intestinal epithelium: inhibition by blocking sites of translation or binding. Gastroenterology 129: 928–40, 2005.[CrossRef][Web of Science][Medline]
  35. Yamaoka KA, Arock M, Issaly F, Dugas N, Le Goff L, and Kolb JP. Granulocyte macrophage colony stimulating factor induces Fc epsilon RII/CD23 expression on normal human polymorphonuclear neutrophils. Int Immunol 8: 479–490, 1996.[Abstract/Free Full Text]
  36. Yamasaki S and Saito T. Regulation of mast cell activation through Fc{epsilon}RI. Chem Immunol Allergy 87: 22–31, 2005.[Medline]
  37. Yang PC, Berin MC, Yu LC, Conrad DH, and Perdue MH. Enhanced intestinal transepithelial antigen transport in allergic rats is mediated by IgE and CD23 (Fc{epsilon}RII). J Clin Invest 106: 879–886, 2000.[Web of Science][Medline]
  38. Yokota A, Kikutani H, Tanaka T, Sato R, Barsumian EL, Suemure M, and Kishimoto T. Two species of human Fc{epsilon} receptor II (Fc{epsilon}RII/CD23): tissue-specific and IL-4-specific regulation of gene expression. Cell 55: 611–618, 1988.[CrossRef][Web of Science][Medline]
  39. Yu LC, Yang PC, Berin MC, Di Leo V, Conrad DH, McKay DM, Satoskar AR, and Perdue MH. Enhanced transepithelial antigen transport in intestine of allergic mice is mediated by IgE/CD23 and regulated by interleukin-4. Gastroenterology 121: 370–381, 2001.[CrossRef][Web of Science][Medline]
  40. Yu LC and Perdue MH. Role of mast cells in intestinal mucosal function: studies in models of hypersensitivity and stress. Immunol Rev 179: 61–73, 2001.[CrossRef][Web of Science][Medline]
  41. Yu LC, Montagnac G, Yang PC, Conrad DH, Benmerah A, and Perdue MH. Intestinal epithelial CD23 mediates enhanced antigen transport in allergy: evidence for novel splice forms. Am J Physiol Gastrointest Liver Physiol 285: G223–G234, 2003.[Abstract/Free Full Text]
  42. Zheng SY, Fu XB, Xu JG, Zhao JY, Sun TZ, and Chen W. Inhibition of p38 mitogen-activated protein kinase may decrease intestinal epithelial cell apoptosis and improve intestinal epithelial barrier function after ischemia-reperfusion injury. World J Gastroenterol 11: 656–660, 2005.[Medline]




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