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Am J Physiol Gastrointest Liver Physiol 293: G798-G808, 2007. First published August 2, 2007; doi:10.1152/ajpgi.00135.2007
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MUCOSAL BIOLOGY

Luminal antigens access late endosomes of intestinal epithelial cells enriched in MHC I and MHC II molecules: in vivo study in Crohn's ileitis

Gheorghe Hundorfean,1,2 Klaus-Peter Zimmer,3 Stephan Strobel,4 Andreas Gebert,2 Diether Ludwig,1 and Jürgen Büning1,2

1Department of Internal Medicine I, University Hospital of Schleswig-Holstein, Lübeck, 2Institute of Anatomy, University of Lübeck, Lübeck, Germany; 3Department of Pediatrics and Neonatology, University Hospital of Giessen and Marburg, Giessen, Germany; 4Peninsula Medical School, Universities of Plymouth and Exeter, Plymouth, United Kingdom

Submitted 24 March 2007 ; accepted in final form 23 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In contrast to healthy conditions, intestinal epithelial cells (IECs) stimulate proinflammatory CD4+ and CD8+ T cells during Crohn's disease (CD). The underlying regulatory mechanisms remain unknown. Here we investigated the epithelial expression of major histocompatibility complex (MHC) I and MHC II and its interference with endocytic pathways, in vivo. During ileoscopy, ovalbumin (OVA) was sprayed onto ileal mucosa of CD patients (ileitis and remission) and controls. The epithelial traffic of OVA and MHC I/II pathways were studied in biopsies using fluorescence and electron microscopy. We found MHC I and MHC II to accumulate within multivesicular late endosomes (MVLE) of IECs. Faint labeling for these molecules was seen in early endosomes and lysosomes. MVLE were entered by OVA 10 min after exposure. Exosomes carrying MHC I, MHC II, and OVA were detected in intercellular spaces of the epithelium. OVA trafficking and labeling patterns for MHC I and MHC II in IECs showed no differences between CD patients and controls. Independent of inflammatory stimuli, MHC I and MHC II pathways intersect MVLE in IECs, which were efficiently targeted by luminal antigens. Similar to MHC II-enriched compartments in professional antigen presenting cells, these MVLE might be critically involved in MHC I- and MHC II-related antigen processing in IECs and the source of epithelial-released exosomes. The access of luminal antigens to MHC I in MVLE might indicate that the presentation of exogenous antigens by IECs must not be restricted to MHC II but might also occur as "cross-presentation" via MHC I to CD8+ T cells.

Crohn's disease; intestinal epithelial cells; mucosa; antigen traffic; major histocompatibility complex molecules

THE FAILURE TO GENERATE a mucosal tolerance towards nonpathogenic luminal antigens (e.g., dietary or bacterial) seems to be a crucial event in the pathogenesis of Crohn's disease (CD). Inappropriately activated CD4+ Th1 and Th17 and CD8+ T cells proliferate and sustain mucosal inflammation by secretion of various proinflammatory cytokines (5, 6, 19, 23). The gut epithelium has traditionally been regarded as an absorptive tissue and a physical barrier positioned between the environment and the host. Previous work provides increasing evidence for an active role of intestinal epithelial cells (IECs) in sampling luminal antigens and modulating related immune responses as nonprofessional antigen presenting cells (APCs). Critically dependent on inflammatory influences, antigen presentation by IECs to CD4+ and CD8+ T cells might result in either regulatory or proinflammatory processes (1, 4, 18, 38, 41).

Antigen-specific stimulation of CD4+ and CD8+ T cells requires T cell receptor interaction with major histocompatibility complex (MHC)-antigen complexes (9, 40). Presentation of exogenous antigens has classically been attributed to MHC II molecules and CD4+ T cells. MHC I has been suggested to function in the presentation of endogenous, intracellularly derived antigens to CD8+ T cells. However, recent data indicated the presentation of exogenous antigens to CD8+ T cells via MHC I, processes termed "cross-presentation" (9). Studies on professional APCs, e.g., dendritic or B cells, yielded detailed insight into MHC I/II pathways in antigen presentation. In these cells, processing of internalized antigens and subsequent loading onto MHC II is predominantly ascribed to late endosomes, referred to as MHC II-enriched compartments (MIICs) (14, 21, 22). Moreover, MIICs were shown to play an important role in the MHC I-mediated presentation of exogenous antigens. MIICs can be identified by their MHC II content, the kinetics of antigen uptake (a late endocytic compartment), a characteristic multivesicular morphology and the lysosome-associated membrane protein (LAMP), a marker protein present in the majority of MIICs. The main part of hydrolytic degradation of exogenously acquired antigens occurs in lysosomes, the latest components of the endocytic routes. Lysosomes were also labeled for LAMP and were among others featured by an electron-dense morphology, partially enclosing membrane sheets (34).

Mature IECs are known to constitutively express MHC II molecules. MHC II is expressed along with molecules necessary for functional antigen loading onto MHC II, e.g., invariant chain and HLA-DM (24). The constitutive expression of MHC II in IECs is upregulated during mucosal inflammation in the course of CD (26). In line with professional APCs, MHC II-restricted presentation of antigens by intestinal epithelial cell lines was suggested to involve acidic compartments and the activity of acidic hydrolases, features of late endosomes (16, 17). Recently, we established a new technique that rendered the opportunity to characterize epithelial traffic routes of luminal antigens in vivo in humans (8). Our results showed evidence of MIICs in colonic epithelial cells of CD patients, which were accessed by endoscopically exposed ovalbumin (OVA). Thus the data available indicate an important function of MIICs in MHC II-related antigen presentation by IECs, comparable to professional APCs. However, the mechanisms of antigen presentation via MHC II by ileal IECs, in particular the associated endocytic routes of exogenous antigens, remain unresolved. As bowel inflammation in CD characteristically affects the terminal ileum, the processes within ileal IECs are of particular interest. Furthermore, MHC I-restricted cross-presentation of exogenous antigens by IECs, presumably required for the stimulation of proinflammatory CD8+ T cells, has not been described so far. In this regard the subcellular expression of MHC I molecules in IECs and its intersections with endocytic routes of internalized antigens remain to be determined.

In this study, we aimed to identify the subcellular compartments of IECs involved in MHC I- and MHC II-related processing and presentation of exogenous antigens. We used our recently established technique and studied the epithelial trafficking of OVA, taken as soluble protein antigen, after exposure during ileoscopy. In view of the differential capacity of IECs to function as APCs, which might rely on differences in intracellular antigen sorting and MHC molecule expression, we investigated CD patients and healthy controls. Our results yield first in vivo insight into the transport process of luminal antigens in human ileal IECs and its linkage with MHC I and MHC II pathways.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Patients and ethics. The study included CD patients (n = 24) and controls (n = 13) undergoing ileocolonoscopy. The median age of CD patients (12 men, 12 women) was 39 yr (range 25–58) and those of controls (6 men, 7 women) 59 yr (range 24–85). CD patients were recruited on the basis of ileal disease, either active ileitis (n = 13) or ileitis in remission (n = 11). Patients undergoing ileocolonoscopy for carcinoma screening, gastrointestinal bleeding, constipation, or abdominal pain served as controls. The severity of inflammatory bowel disease was assessed by clinical, endoscopic, histopathological, and serological criteria. CD patients in remission and controls had no signs of bowel or systemic inflammation. Clinical history did not indicate food intolerance. Infectious ileitis was excluded by stool culture and serology. CD patient's specific medication consisted of corticosteroids (n = 8), azathioprine (n = 10), mesalazine (n = 10), sulfasalazine (n = 2), cyclophosphamide (n = 1), and methotrexate (n = 1). None of the control patients received immunosuppressive medication.

All subjects gave their written, informed consent. The study was approved by the Ethics Committee of the Medical Faculty, University of Lübeck (No. 02-073 and 03-043) and the German National Drug Administration (BfArM: 4021154) and was conducted according to the Declaration of Helsinki.

Tissue sampling and antigen exposure to ileal mucosa. For immunofluorescence investigation and resin embedding, specimens of ileal mucosa (size 5 mm) were obtained endoscopically. Biopsies were taken within the terminal ileum from macroscopic inflamed mucosa in CD ileitis and normal mucosa in CD patients in remission and controls. Histopathological examination confirmed CD-specific inflammation and healed/healthy mucosa, respectively.

For immunoelectron microscopy, in vivo exposure of OVA to the ileal mucosa was performed in those patients who consented to this supplemental procedure (CD ileitis n = 5, CD ileitis in remission n = 3, controls n = 4). During endoscopy, 10 ml of OVA solution (100 mg/ml in saline; fraction V, Sigma, Taufkirchen, Germany) was sprayed directly onto saline-cleaned inflamed or healthy ileal mucosa. Biopsies were obtained before (0 min) and after 5, 10, and 20 min of OVA incubation.

Antibodies. The primary antibodies used were affinity-purified polyclonal rabbit antibodies against OVA (7, 8, 43) and against MHC I and MHC II (both a gift from J. J. Neefjes, National Cancer Institute, Amsterdam, The Netherlands) (29) and monoclonal mouse antibodies against MHC I (clone HC10, gift from J. J. Neefjes) (22), against MHC II (clone CR3/43, gift from J. Cordell, John Radcliffe Hospital, Headington, Oxford, UK) (12), and lysosome-associated membrane protein-2 (LAMP-2, clone H4B4, BD Bioscience PharMingen, Hamburg, Germany).

Binding sites of the primary antibodies were visualized for electron microscopy by gold-conjugated goat antisera against rabbit and mouse IgG (6 or 12 nm in diameter; Dianova, Hamburg, Germany). For immunofluorescence microscopy, goat antisera against rabbit and mouse IgG conjugated to Alexa Fluor 488 and 555 (Molecular Probes, Eugene, OR) were used.

OVA antibodies did not cause nonspecific labeling on sections from ileal mucosa solely exposed to saline. The specificity of our polyclonal OVA antibodies has been demonstrated by preincubation of OVA antibodies with OVA in a previous project (7).

Immunofluorescence microscopy. Ileal specimens were frozen in liquid nitrogen. Sections (8 µm) were cut at –20°C, mounted on glass slides and air dried overnight. After fixation in acetone-methanol (1:1), they were successively incubated with primary antibodies, appropriate Alexa Fluor conjugates (Molecular Probes), and bis-benzimide (Sigma), for 60 min each. Double labeling was performed simultaneously, with primary antibodies derived from different species and appropriate fluorochrome conjugates. Cross-reactivity of antibodies applied in double-labeling experiments was excluded. Immunofluorescence analysis was done using a Zeiss LSM 510 Meta (Jena, Germany).

Immunoelectron microscopy of ultrathin cryosections. Cryosectioning and labeling of ultrathin sections was carried out according to the postembedding technique of Tokuyasu and Griffiths (13). Biopsies from ileal mucosa were fixed in 5% formaldehyde, cryoprotected in 0.03 M polyvinylpyrrolidone-1.6 M sucrose, and frozen in liquid nitrogen. Ultrathin cryosections (60 nm) were cut at –110°C, mounted on Formvar-coated copper grids, and consecutively incubated with primary and species-specific gold-conjugated secondary antibodies for 45 min each. Labeled grids were contrasted with uranyl acetate, embedded in 2% methylcellulose, and analyzed using a Philips EM 400 T transmission electron microscope (Kassel, Germany).

Double-labeling experiments were done applying primary antibodies from different species and corresponding 6 and 12 nm immunogold conjugates, respectively. Double-labeling was carried out for MHC I/LAMP-2, MHC II/LAMP-2, MHC I/MHC II, OVA/LAMP-2 and OVA/MHC I. Possible cross-reactivity of antibodies was excluded for each double-labeling step. Double-labeling for OVA/MHC II could not be performed owing to inefficacy of the monoclonal anti-MHC II antibodies available in immunolabeling of ultrathin cryosections.

Electron microscopy of resin-embedded sections. Specimens of ileal mucosa were fixed in 2% glutaraldehyde-0.6% formaldehyde in 0.06 M sodium cacodylate-HCl buffer (pH 7.3). After postfixation in OsO4, tissue was dehydrated in graded ethanol solutions and embedded in Araldite M (Fluka, Neu-Ulm, Germany). Ultrathin sections (60 nm) were prepared, stained with uranyl acetate and lead citrate, and examined with a Philips EM 400T transmission electron microscope.

Quantitation of MHC I and MHC II labeling in IECs. The relative distribution of the subcellular labeling for MHC I and MHC II in IECs was assessed separately in nine randomly chosen patients (3 patients of each group: CD ileitis, CD ileitis in remission, and control). Quantitation was done by an observer unaware of any clinical information and carried out according to the method introduced by Lucocq et al. (25). Ultrathin sections were used double labeled for MHC I/LAMP-2 and MHC II/LAMP-2, respectively. On one randomly taken grid per patient, 400 gold particles representing binding sites for MHC I or MHC II were counted and assigned to the different subcellular compartments within IECs. According to previous work on professional APCs, compartments were identified by their ultrastructural morphology and specific marker proteins (e.g., LAMP-2) (21, 34). In correspondence with Lucocq et al., the quantitation of 400 gold particles per antigen on one grid is evaluated with an error coefficient of up to 5% and considered sufficient for statistical validation. The percentage of gold counts for each compartment was established per antigen and patient. An arithmetic mean of the percentage was calculated for each patient group and antigen and used to elaborate a ranking of the labeling distribution for MHC I and MHC II.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
MHC I and MHC II in ileal IECs: expression in CD and healthy mucosa. Immunofluorescence experiments using monoclonal antibodies against MHC I (clone HC10) and MHC II (clone CR3/43) were designed to investigate regional differences in the epithelial expression related to the degree of mucosal inflammation (Fig. 1). Staining for MHC I and MHC II was seen in IECs and cells of the lamina propria. In IECs both MHC I and MHC II were predominantly detected at the basolateral cell surface and within cytoplasmic granules underlying the apical membranes. In all subjects, MHC I was found in crypt and villus IECs (Fig. 1, A–C), independent of mucosal inflammation. In contrast, inflammation influenced the staining pattern for MHC II along the crypt-villus axis. Although staining in crypt IECs was absent or faint in healthy mucosa (all controls and CD ileitis patients in remission) (Fig. 1, D and E), a strong expression was seen in crypts of inflamed mucosa (in 12 of 13 patients with CD ileitis) (Fig. 1F). Villus IECs showed MHC II expression in all patient groups.


Figure 1
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  		Fig. 1. Expression of major histocompatibility complex (MHC) I and MHC II in the ileal mucosa of Crohn's disease (CD) patients and healthy controls. MHC I (A–C) and MHC II antigens (D–F) were visualized by green fluorescence on cryosections. The blue fluorescence stained nuclei. Ileal mucosa was taken from a control subject (A and D), a CD patient in remission (B and E), and a CD ileitis patient (C and F). MHC I is found in villus and crypt (Cr) intestinal epithelial cells (IECs) independent of the inflammatory state of the mucosa. In contrast, epithelial staining for MHC II in the noninflamed mucosa of healthy controls (D) and CD patients in remission (E) is restricted to villi. During CD ileitis (F), both villus and crypt IECs show staining for MHC II. Cells of the lamina propria (LP) reveal MHC I and MHC II in all patient groups.

 
Double-labeling was performed to unravel the assumed late endocytic nature of the granular staining for MHC I and MHC II in IECs. In these experiments, LAMP-2 and either MHC I or MHC II were simultaneously visualized. Merged images of double-labeled sections consistently revealed colocalization for LAMP-2/MHC I (Fig. 2) and LAMP-2/MHC II (Fig. 3) in IECs. Colocalization was predominantly found in the supranuclear part of IECs and indicated the presence of MHC I and MHC II molecules within late endocytic structures of IECs. Beyond this, some intracellular staining in particular for MHC I and to a lesser extent for MHC II was seen in LAMP-2-negative regions. MHC I and LAMP-2 colocalized within crypt and villus IECs regardless of the degree of mucosal inflammation (Fig. 2). In line with our results obtained with the monoclonal antibodies against MHC II, colocalization for MHC II and LAMP-2 in crypt IECs was seen in inflamed mucosa, whereas those in villus IECs was found in all subjects (Fig. 3).


Figure 2
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  		Fig. 2. MHC I resides in late endocytic compartments of IECs. MHC I (green fluorescence) and lysosome-associated membrane protein (LAMP)-2 (red fluorescence) were double labeled on cryosections. Sections were made from ileal mucosa obtained from a control subject (A), a CD patient in remission (B and D), and a CD ileitis patient (C). The merged images show colocalization (yellow fluorescence) for MHC I and LAMP-2 in the supranuclear part of IECs. Cell surface staining for MHC I in IECs is predominantly seen at the basolateral membranes. No differences are found regarding villus (A–C) and crypt (D) IECs or dependent on mucosal inflammation. Colocalization of MHC I and LAMP-2 is additionally detected in cells of the LP. Lu, lumen.

 

Figure 3
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  		Fig. 3. Intracellular MHC II accumulates in late endocytic compartments of IECs. The merged images show a double staining for MHC II (green fluorescence) and LAMP-2 (red fluorescence) on cryosections. Biopsies from ileal mucosa were taken from a control subject (A), a CD patient in remission (B), and a CD ileitis patient (C and D). Colocalization of MHC II and LAMP-2 (yellow fluorescence) is seen in the supranuclear part of villus IECs (A–C) throughout. Colocalization of both molecules in crypt IECs is restricted to mucosa affected by CD ileitis (D). Cell surface staining of MHC II in IECs is mainly observed on basolateral membranes. Cells of the lamina propria reveal MHC II/LAMP-2 colocalization in all patients.

 
Multivesicular late endosomes: major components of MHC I and MHC II pathways in IECs. To identify compartments that accounted for colocalization of MHC I/II and LAMP-2 within IECs in our immunofluorescence experiments, we performed a double labeling on ultrathin sections. The subcellular distribution of immunogold labeling for MHC I (Fig. 4) and MHC II (Fig. 5) was quantitatively assessed to characterize the pathways of these molecules in IECs.


Figure 4
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  		Fig. 4. Subcellular distribution of MHC I in IECs of CD patients and controls. The subcellular distribution of MHC I in IECs was assessed on immunogold-labeled ultrathin sections as described in detail in MATERIALS AND METHODS. The percentages of total particle counts for MHC I within the subcellular compartments in IECs were determined and presented as means of 3 patients per group. Consistent for all patients examined, the majority of MHC I in IECs is identified on the basolateral membranes (BLM, 42%) and in late endosomes (LE, 32%). Some MHC I is additionally seen in small vesicles (Ves), early endosomes (EE), lysosomes (Lys), and the Golgi complex and on apical membranes (APM). Labeling detected on mitochondria (Mit) and nuclei (Nuc) and within the cytosol (Cyt) represents background staining and is negligible.

 

Figure 5
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  		Fig. 5. Subcellular distribution of MHC II in IECs of CD patients and controls. Quantitation of MHC II labeling in IECs was done on immunogold-labeled sections as stated in MATERIALS AND METHODS. Here percentages of total particle counts for MHC II in the different compartments were shown as means covering 3 patients per group. Independent of mucosal inflammation, the bulk of MHC II labeling in IECs is detected on the basolateral membranes (31–36%) and within late endosomes (36–45%). Some amounts of MHC II are further observed on apical membranes and in the Golgi complex, vesicles, early endosomes, and lysosomes. Negligible background staining is seen on mitochondria and nuclei and within the cytosol.

 
Cell surface labeling of MHC I and MHC II in IECs was abundantly seen at basolateral membranes. Faint labeling for both molecules was additionally detected on microvilli. All components of the endocytic tract revealed labeling for MHC I and MHC II. The majority of MHC I (Fig. 6A) and MHC II (Fig. 6B) molecules in the endocytic tract accumulated within late endosomes, particularly in multivesicular late endosomes (MVLE). The remainder were seen in early endosomes, lysosomes, and small vesicles. Endo-/lysosomal labeling for MHC I and MHC II was localized on the external and the enclosed membranes throughout. MHC I and MHC II were further detected on membranes of the endoplasmic reticulum and the Golgi complex, representing the biosynthetic pathway. The cytosol, mitochondria, and nuclei showed negligible labeling for both MHC I and MHC II.


Figure 6
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  		Fig. 6. Multivesicular late endosomes are major components of MHC I and MHC II pathways in IECs. A–C: ultrathin cryosections were immunolabeled for MHC I, MHC II, and LAMP-2 with 6- or 12-nm gold particles. Sections were prepared of ileal mucosa taken from a CD ileitis patient (A) and a CD patient in remission (B, C). Multivesicular late endosomes (MVLE), characterized by labeling for LAMP-2, accumulate MHC I (arrowheads) (A) and MHC II molecules (B). Both molecules are localized on the limiting membranes and internal vesicles of MVLE. Colocalization of MHC I and MHC II (arrowheads) was detected in MVLE (C) and identifies intersections of the two pathways in these compartments. Of note, A and C depict fusion events of MVLE. The double-labeling results featured in the present figure are representative for all 12 patients that underwent ovalbumin (OVA) exposure. D shows the ultrastructural morphology of a MVLE in IECs after resin embedding of ileal mucosa (obtained from a CD patient in remission). Bars = 100 nm.

 
To provide evidence for a supposed intersection of MHC I and MHC II pathways in IECs, double labeling for MHC I and MHC II was carried out on ultrathin sections. Colocalization was identified throughout the endocytic tract, again most prominent in MVLE (Fig. 6C). As expected, both molecules intensively colocalized at basolateral membranes, whereas faint colocalization was further seen on microvilli and within the biosynthetic pathway (not shown).

Immunogold labeling confirmed the distinct expression of MHC II in IECs along the crypt-villus axis, in accordance with our immunofluorescence experiments. Except for the absence of MHC II labeling in crypts of noninflamed mucosa, the labeling patterns of MHC I and MHC II within IECs did not differ dependent on the maturity of IECs or mucosal inflammation.

To confirm the distinct morphology of MVLE identified on ultrathin sections processed for immunogold labeling, we analyzed ultrathin sections of resin-embedded ileal mucosa. In line with our immunogold experiments we consistently found multivesicular endosomes in the supranuclear areas of IECs in all patient groups (Fig. 6D).

Antigen trafficking in IECs of CD patients and controls. The epithelial transport of the model protein antigen OVA was studied on mucosal biopsies taken from ileal mucosa after in vivo incubation with luminally applied OVA. The subcellular localization of OVA in the epithelial compartment was analyzed by double labeling for OVA and LAMP-2 on ultrathin sections. Five minutes after exposure, OVA labeling was detected on microvilli, in the widened intercellular spaces and the lamina propria (not shown). At this time, internalized OVA was almost entirely localized in LAMP-2-negative early endosomes situated close to the apical membranes (not shown). In rare occasions LAMP-2-positive late endosomes were labeled for OVA after 5 min. OVA accumulated in late endosomes and in particular within MVLE 10 min after exposure and was equally seen within these compartments after 20 min (Fig. 7, A–C). Lysosomes showed faint labeling for OVA at 10 and 20 min (not shown). The time-related subcellular distribution of OVA was similar in crypt and villus IECs and did not differ between CD (CD ileitis, CD ileitis in remission) and healthy controls. Table 1 summarizes traffic routes of OVA in IECs, representative for all 12 patients exposed to OVA, analyzing at least 100 IECs per patient. Specimens taken from saline-cleaned ileal mucosa before OVA administration (0 min period) did not show any labeling for OVA (not shown).


Figure 7
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  		Fig. 7. Targeting of luminally administered OVA into multivesicular late endosomes of IECs. Ultrathin cryosections were immunolabeled for OVA, LAMP-2, and MHC I with 6- or 12-nm gold particles. Sections were made of ileal mucosa taken from a control subject (A and D), a CD patient in remission (B) and a CD ileitis patient (C) 20 min after endoscopic exposure. A–C: at this period, OVA is consistently localized in MVLE, labeled for LAMP-2. OVA targeting into MVLE of IECs is not dependent on the inflammatory state of the mucosa. D shows colocalization of OVA and MHC I in a MVLE 20 min after OVA administration. These results are representative for all patients studied. V, vesicles. Bars = 100 nm.

 

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  		Table 1. Dynamics of subcellular OVA trafficking in IECs

 
Targeting of OVA into MHC I-enriched late endosomes of IECs. A speculated cross-presentation of exogenous antigens would require antigen access to MHC I molecules. Here we studied whether and where luminally administered antigens meet MHC I molecules in the endocytic tract of IECs. Double labeling for OVA and MHC I was carried out on ultrathin sections.

In all patients endoscopically exposed to OVA (CD ileitis, CD ileitis in remission, and controls), MHC I pathways intersect the endocytic traffic of OVA in IECs. At 5 min, some OVA was seen in faintly MHC I-positive endosomes, presumably early endosomes (not shown). In accordance with the subcellular labeling for OVA and MHC I described above, colocalization was consistently found in late endosomes (especially in MVLE) at the later periods (10 and 20 min) (Fig. 7D). Lysosomes revealed faint colocalization for OVA and MHC I at these times (not shown). Differences between patient groups were not observed.

Exosomes in the intercellular spaces of the ileal epithelium. Likewise in professional APCs (10), recent data suggested the release of MHC I/II-loaded vesicles from intestinal epithelial cells, referred to as exosomes (20, 39). However, exosomes derived from the epithelial compartment of the gut have not been described in vivo yet. Here we used an ultrastructural analysis of resin-embedded tissue to unravel exosomal structures in ileal epithelium. We found electron-dense content in the widened intercellular spaces, which mainly consisted of amorphous material (Fig. 8A). In addition, intercellular vesicles (40–100 nm in diameter), distinct from basolateral membranes of IECs, were regularly identified within the epithelium (Fig. 8A). These vesicles were seen independent of mucosal inflammation.


Figure 8
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  		Fig. 8. Exosomes bearing OVA, MHC I, and MHC II are identified in the intercellular spaces between IECs. Ultrathin sections were made of resin-embedded tissue (A) or were processed for immunolabeling to visualize OVA, MHC I, MHC II, and LAMP-2 with 6- and 12-nm gold particles (B and C). Sections were prepared of ileal mucosa from a CD remission (A and B) and a CD ileitis patient (C). A: exosomes (arrows) are localized in the intercellular spaces (ICS) of the ileal epithelium. Beyond this, the intercellular spaces contain some amorphous electron-dense material. B and C: exosomes (arrows), identified extracellularly between IECs, carry OVA (20 min after exposure), MHC I (B), and MHC II molecules (C), but not LAMP-2. BLM, basolateral membrane. The pictures shown here are representative for all 12 subjects whose biopsies were analyzed by electron microscopy. Bars = 100 nm.

 
To elucidate MHC protein and LAMP-2 expression on exosomes found in the ileal epithelium, we performed immunogold labeling. By using double labelings for MHC I/LAMP-2, MHC I/OVA and MHC II/LAMP-2, both MHC I (Fig. 8B) and MHC II molecules (Fig. 8C) were detected membrane bound on extracellular vesicles between IECs. Inflammation did not change the labeling pattern for MHC I and MHC II on the intercellularly situated vesicles. Labeling for LAMP-2 was only exceptionally observed on these structures. As mentioned earlier, OVA was abundantly present within the intercellular spaces of the epithelium already 5 min after luminal exposure. Of note, part of this intercellular labeling for OVA was localized on the limiting membranes of intercellular vesicles (Fig. 8B). Although these OVA-labeled vesicles were regularly found 10–20 min after OVA administration, their detection was a rare event at the 5-min period.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In accordance with our previous data on jejunal and colonic mucosa, the epithelial crossing of OVA in the terminal ileum occurs within 5 min after endoscopic exposure (7, 8, 43). Independent of the inflammatory status, OVA in IECs is almost entirely restricted to LAMP-2-negative early endosomes at this period. Thus the transcellular traffic in this period seems to be a uniform process that might be responsible for the transepithelial passage of unmodified peptides or proteins, as shown by others (3). A paracellular flux of OVA might also account for the epithelial translocation, although preferentially occurring in inflamed mucosa (30, 36).

Although the priming of naive T cells in the gut is attributed to dendritic cells, IECs are thought to participate in antigen presentation of luminal antigens towards primed T cells (27). In the healthy organism, MHC II-restricted antigen presentation by IECs is considered to result in a clonal anergy of CD4+ T cells or an activation of CD4+ regulatory T cells. Recent data demonstrated the induction of CD4+ regulatory T cells by IECs in vivo by use of a transgenic mouse model (41). Influenced by proinflammatory mediators such as in CD, IECs are potent activators of proinflammatory CD4+ T cells (11). The IEC-mediated activation of CD8+ regulatory T cells, occurring constitutively, was shown to involve gp180 and the nonclassical MHC protein CD1d (1, 38). In contrast, the stimulation of cytotoxic CD8+ T cells during mucosal inflammation in CD is suggested to involve classical MHC I proteins (4, 18, 31). However, the underlying regulatory mechanisms in IECs that critically determine the functional outcome of antigen presentation still remain ill defined.

In the present work, we show that MHC I is constitutively expressed in the ileal epithelium along the entire crypt-villus axis, whereas MHC II expression is predominantly restricted to villus IECs. CD inflammation upregulates epithelial MHC II expression in crypts, which is reversed during mucosal healing in remission. This expression pattern of MHC II proteins in ileal IECs differs from the epithelial expression within the colon. In the healthy colon, epithelial cells were found to lack MHC II staining or to show merely faint expression, dependent on the antibodies applied. However, affected by CD inflammation, colonic epithelial cells revealed a strong cell surface and endosomal staining for MHC II (8, 26). In professional APCs, MHC II molecules were shown to be expressed throughout the endocytic tract, and, conforming with this, class II antigen loading was demonstrated in early and late endocytic compartments. However, the majority of MHC II proteins accumulated in MIICs and MHC II-restricted antigen processing and peptide loading is basically considered to be the characteristic feature of MIICs (15, 21, 40). As described in professional APCs, we find MHC II localized throughout the endocytic tract, including early endosomes, late endosomes, and lysosomes. A quantitative analysis shows the majority of intracellular MHC II molecules in MVLE, morphologically similar to MIICs in professional APCs. Of note, we demonstrate that intracellular MHC I molecules are not restricted to the biosynthetic pathway but additionally are found in all components of the endocytic pathway. Similar to MHC II and in line with the data on professional APCs, most of endosomal MHC I labeling is seen in the MIICs of IECs (9). Our findings using double labeling for MHC I and MHC II confirm the assumed intersection of MHC I and MHC II pathways in the endocytic tract of IECs, which predominates in MIICs.

Access of internalized antigens to MIICs is a prerequisite for processing and binding to either MHC I or MHC II within these compartments (40). Late endosomal targeting of exogenous antigens has been reported in polarized epithelial cells such as MDCK cells after a 15-min pulse (33). We have previously described a uniform traffic pathway of antigens such as OVA or gliadin into epithelial MIICs in the noninflamed upper small intestine (7, 42, 43). Consistent with these former studies, the present data show that endoscopically administered OVA enters MIICs of ileal IECs within a period of 10–20 min, independent of additional inflammatory stimuli. Colonic epithelial cells featured late endosomal targeting of OVA under constitutive conditions in our previous endoscopy project (8). Of note, antigen transport into MIICs was confined to mucosal inflammation in Crohn's colitis because of lack of epithelial class II staining in the healthy colon. Thus the epithelial uptake of luminal antigen into MIICs strikingly differs with respect to the quantity of late endosomal MHC II expression between the small bowel and the colon. The functional relevance of this rather quantitative aspect remains unknown. In the present study MHC I molecules colocalize with OVA in all components of the endocytic pathway in IECs. The most intensive labeling of both proteins is seen in MIICs. Our findings strongly suggest that MHC I and MHC II pathways meet the endocytic routes of internalized antigens in MIICs of ileal IECs. Although these morphological data do not provide definitive evidence for complex binding of exogenous antigens to MHC I or MHC II, they might indicate that these MIICs are responsible for MHC I- and MHC II-restricted presentation of exogenous antigens by IECs. Further projects will need to clarify the function of the different MHC I- and MHC II-bearing compartments of IECs in antigen processing and presentation.

Recent in vitro data demonstrated the release of immunocompetent, MHC I- and MHC II-carrying exosomes (30–90 nm in diameter) by intestinal epithelial cell lines (20, 39). Here we provide human in vivo evidence for the presence of epithelial exosomes (40–100 nm in diameter) in the gut. Consistent with exosomes derived from intestinal epithelial cell lines, we identify MHC I and MHC II molecules on their limiting membranes. OVA labeling demonstrated on exosomes in the intercellular spaces of the epithelium (especially 5 min after exposure) does not prove the presence of processed and MHC-bound OVA-fragments. Nevertheless, the OVA/MHC-bearing vesicles at the later times (10 and 20 min) might represent immunocompetent exosomes derived from OVA-targeted MIICs and account for a function of IECs in adaptive immunity not only confined to the gut mucosa. Although the function of IEC-derived exosomes remains inconclusive, a direct interaction of exosomal MHC-antigen complexes with mucosal T cells as well as an uptake and processing by dendritic cells of the gut mucosa were discussed. Exosomes secreted from MIICs of professional APCs were shown to be capable of activating CD4+ and cytotoxic CD8+ T cells (35, 44). Under healthy conditions, the dissemination of exosomes from IECs might be involved in the homeostatic regulation of the response to luminal antigens as suggested in murine models of oral tolerance (32). The immunocompetence of IEC-released exosomes during mucosal inflammation has to be elucidated.

We are unable to detect inflammation-related changes in the subcellular expression of MHC I and MHC II or the intracellular trafficking of OVA that might explain the dichotomy in the antigen presenting capacity of IECs. Crypt IECs affected by CD inflammation show similar trafficking of OVA and expression of MHC II compared with villus IECs. Because a distinct function of immature IECs in antigen uptake has been reported, antigen presentation by crypt IECs might specifically contribute to the inflammatory processes in CD ileitis (37). Furthermore, we suggest that the IECs' function as APCs might crucially depend on antigen processing properties and the differential involvement of inflammation-related costimulatory molecules such as B-7 proteins (2, 16, 28). Inflammatory conditions were shown to regulate the activity of proteases involved in the MHC-associated processing of exogenous antigens. Thus, dependent on inflammatory stimuli, antigen processing in the IECs MIICs might create MHC-antigen complexes with different immunogenic properties. Further projects will need to address the influence of inflammation on the generation of MHC-antigen complexes within the MIICs of IECs and the related functional consequences in antigen presentation. Our results provide evidence that luminal antigens access MHC I molecules in MIICs of IECs, and functional studies should unravel the impact of an assumed cross-presentation by IECs via MHC I. It is tempting to speculate that the MHC I-restricted cross-presentation of exogenous antigens by IEC might be involved in the stimulation of cytotoxic CD8+ T cells and thus promote the inflammatory processes in CD.

In conclusion, the present study yields in vivo insight into trafficking routes of luminal antigens in human IECs (Fig. 9). We show that MHC I and MHC II pathways in IECs comprise MIICs, which were efficiently targeted by exogenous antigens and might act similar to MIICs identified in professional APCs. We provide the structural basis for the in vivo production of MHC-bearing exosomes by IECs. Our findings support the suggested function of IECs in antigen presentation to CD4+ T cells and might moreover point to a possible cross-presentation of exogenous antigens to cytotoxic CD8+ T cells considered in CD.


Figure 9
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  		Fig. 9. Scheme for MHC I- and MHC II-restricted presentation pathways of luminal antigens in IECs. Undegraded antigens will pass through the ileal epithelium transcellularly via early endosomes and/or via paracellular flux. The latter route will most likely be confined to inflamed mucosa. Antigens, internalized at apical membranes, will initially enter early endosomes. Inflammatory stimuli may additionally account for antigen uptake into the endocytic pathway via the basolateral membranes. Passing early endosomes, antigens will be delivered into multivesicular late endosomes. Analogous to MHC II-enriched compartments (MIICs) in professional antigen presenting cells, antigens will be processed and their peptides loaded onto MHC I and MHC II molecules. MHC-antigen complexes generated will subsequently be displayed on the basolateral membranes accessible to adjacent lymphocytes. In addition, MHC-antigen complexes will be released, carried on exosomes, and thus may exert systemic immune functions. Part of the internalized antigens will proceed to lysosomes and undergo complete proteolytic degradation.

 

   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This research was supported by a postgraduate scholarship from the Land Schleswig-Holstein GS Schl.-H. II, G.I.221-0-2-1 (to G. Hundorfean) and by grants from the Medical Faculty, University of Lübeck, JU04-2004 (to J. Büning).


   ACKNOWLEDGMENTS
 
The authors thank Doris Stöckmann, Doreen Unmack, Heidi Schlichting, Harry Manfeldt, and Christo Örün for excellent technical assistance and Dr. P. J. Peters, Dr. J. J. Neefjes, and Dr. J. Cordell for the antibodies against MHC-I and MHC-II.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Büning, Dept. of Internal Medicine I, Univ. Hospital of Schleswig-Holstein, Ratzeburger Allee 160, D-23538 Lübeck, Germany (e-mail: juergen.buening{at}uk-sh.de)

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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 MATERIALS AND METHODS
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 DISCUSSION
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