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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 22, 2005 as doi:10.1096/fj.05-4807fje. |
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,1
* Medizinische Klinik I, Universitätsklinikum Schleswig-Holstein, Lübeck, Germany;
Institut für Anatomie, Universität zu Lübeck, Lübeck, Germany;
Klinik und Poliklinik für Kinderheilkunde, Universitätsklinikum Münster, Münster, Germany; and
Peninsula Medical School, Universities of Plymouth and Exeter, Plymouth, UK
1 Correspondence: Medizinische Klinik I and Institut für Anatomie, Universitätsklinikum Schleswig-Holstein, Ratzeburger Allee 160, Lübeck D-23538, Germany. E-mail: buening{at}anat.uni-luebeck.de
SPECIFIC AIMS
The purpose of the present study was to investigate the subcellular expression of MHC class II in colonic epithelial cells (CECs) and its modulation during Crohn’s disease (CD). We further aimed to determine whether endocytic pathways of internalized antigens in CECs intersect a MHC class II-enriched compartment and, if so, to characterize its nature. A major objective of this work was to establish a new experimental technique in humans that provides insight into the epithelial uptake and trafficking of antigens in vivo.
PRINCIPAL FINDINGS
1. Targeting of ovalbumin to late endosomes in CECs of CD patients and healthy controls
Mucosal biopsies were taken from patients with acute Crohn’s colitis (n=12), colonic CD in remission (n=6), and healthy controls (n=10) undergoing colonoscopy. In vivo exposure of antigens to colonic mucosa was carried out in patients who gave the informed consent for this additional procedure (Crohn’s colitis n=6; CD in remission n=2; controls n=5). Ovalbumin (OVA) was directly sprayed onto inflamed or normal mucosa and biopsies were obtained before and 5, 10, and 20 min after administration. We performed immunolabeling on ultrathin cyrosections to localize OVA during trafficking across the epithelium. Electron microscopic analysis showed that the transepithelial passage of OVA occurred within 5 min. At this time, OVA was detected in the intercellular space of the epithelium and the lamina propria. Double-labeling for OVA and LAMP-2, a marker protein for late endocytic compartments, was used to characterize the intracellular localization of OVA in CECs. At 5 min after exposure, OVA labeling was restricted to early endosomes. However, after 10 and 20 min OVA was found in late endosomes, which revealed LAMP-2 labeling and contained some electron-dense vesicular material. In contrast, lysosomes, which appeared homogeneous electron-dense, lacked OVA at all times examined. The time-related subcellular distribution of OVA within CECs showed no difference between CD (acute colitis or remission) and control patients.
2. Ovalbumin accumulates in MHC class II-enriched late endosomes in CECs of Crohn’s colitis patients
Immunofluorescence staining on cryostat-sections showed that in CD patients in remission (not shown) and healthy controls MHC class II was absent from the epithelium but detected on lamina propria cells. MHC class II was strongly up-regulated in CECs of Crohn’s colitis patients and seen at basolateral membranes and within cytosolic granules.
To further investigate the subcellular localization of MHC class II induced in CECs during CD inflammation, we performed double-labeling for LAMP-2 and MHC class II on ultrathin sections. Quantitative analysis of immunogold labeling showed that the majority of intracellular MHC class II induced in CECs was observed in late endosomes, which are likely to represent the cytosolic granules seen in immunofluorescence staining. Compared with late endosomes, less MHC class II was found in lysosomes. MHC class II was further detected in vesicles situated close to basolateral membranes, within the Golgi complex and early endosomes.
Class II-associated antigen processing requires that internalized antigens gain access to MHC class II-loaded compartments. Thus, double-labeling for OVA and MHC class II was used to elucidate intersections of the endocytic pathways of OVA with MHC class II pathways in CECs. We found that 10 and 20 min after administration, OVA accumulated in MHC class II-enriched late endosomes of CECs affected by CD inflammation (Fig. 1
).
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3. MHC class II molecules reside in the intercellular space between CECs during CD inflammation
Ultrathin sections of specimen taken from control and CD patients consistently revealed flocullar electron-dense material in the widened intercellular spaces of the epithelium. Our immunofluorescence data demonstrated MHC class II expression in basolateral position within the colonic epithelium during CD inflammation, whereas epithelial expression of MHC class II was absent in remission and controls. Due to immunolabeling on ultrathin sections, cell surface labeling for MHC class II was abundant at the basolateral membranes and only faint at the apical cell surface. The basolateral labeling was not only found to be membrane associated, but was additionally seen on the flocullar electron-dense material in the intercellular spaces. Vesicles containing OVA, MHC class II, and LAMP-2 were frequently close to the basolateral membranes, and fusion events were identified. Quantitative analysis showed that labeling for LAMP-2 was only faint at basolateral membranes of CECs and within intercellular spaces while abundant in late endosomes and lysosomes.
CONCLUSIONS AND SIGNIFICANCE
The present study provides in vivo insight into the processes of antigen uptake and trafficking within the colonic epithelium of CD patients and healthy controls. We found that, independent of the inflammatory state of the mucosa, the passage of OVA across the epithelial barrier was a fast event occurring within 5 min. Transcytosis and paracellular flux might have contributed to the transepithelial trafficking of OVA, the paracellular route most likely confined to mucosal inflammation. Since intracellular OVA in CECs is almost entirely restricted to LAMP-2-negative compartments after 5 min, the transcellular route within this period seems to be mediated by early endosomes. This pathway may facilitate the epithelial passage of intact proteins or peptides, as previously observed in vitro by others using intestinal epithelial cells.
Functional in vitro data indicated that processing of internalized antigens in CECs requires the involvement of acidic endosomes. Our in vivo data provide evidence that luminal exposed OVA is targeted to late endocytic compartments in CECs of healthy and inflamed mucosa. OVA accumulated in late endosomes already 10 min after administration. Late endosomes were identified due to their vacuolar morphology and the transport kinetics detected. Since late endosomal delivery of OVA in CECs was found in noninflamed (controls and CD patients in remission) and inflamed mucosa, inflammatory stimuli seem not to be essential for antigen sorting in these transport processes. This finding is in line with previous in vivo studies in mice and ex vivo studies in humans that demonstrated the routing of OVA to late endocytic compartments of duodenal and jejunal enterocytes. Antigen trafficking to late endosomes has also been described in other polarized epithelial cells such as MDCK cells and was reported to occur in a period of 15 min. The observed lack of OVA targeting to lysosomes, identified by LAMP-2 labeling and homogeneous electron-dense morphology, might be due to the incubation period of up to 20 min, which was likely insufficient for transfer of OVA to these latest components of the endocytic pathway.
It has been suggested that the OVA-specific stimulation of CD4+ T cells by CECs is mediated via MHC class II-associated antigen presentation. Our immunofluorescence experiments showed lack of MHC class II expression in CECs of normal mucosa (CD in remission and controls). MHC class II was strongly up-regulated in the colonic epithelium of Crohn’s colitis patients and mainly seen at the basolateral membranes and in cytosolic granules within CECs. In professional antigen presenting cells, MHC class II molecules were shown to be stored in late endosomes, termed MIICs, which are responsible for antigen processing and MHC class II/peptide loading. Using immunolabeling on ultrathin sections we now demonstrate that intracellular MHC class II molecules in CECs induced during CD inflammation also reside in late endosomes. These CEC’s MIICs showed similar morphological features when compared with MIICs identified in professional antigen-presenting cells.
Processing and loading of antigens onto MHC class II require that internalized antigens meet the MHC class II pathway. Previous in vitro studies using intestinal epithelial cell lines indicated that a common late endosome, accessible for apical and basolateral pathways, might be responsible for MHC class II-associated antigen processing in enterocytes. We now demonstrate that internalized OVA is efficiently targeted to MIICs in CECs affected by CD inflammation. Thus, our results prove that the endocytic pathway of OVA in CECs of Crohn’s colitis patients intersects MIICs. Although our experiments could not unravel whether the apical, the basolateral, or both pathways contribute to the transfer of OVA to MIICs, these compartments might represent the suggested common endosome crucially involved in MHC class II-mediated antigen processing in enterocytes.
Ultrastructural analysis showed that the cell surface expression of induced MHC class II in CECs was predominantly found at the basolateral and only faintly at the apical membrane. This polarized expression is in good accordance with the CEC’s ability to present peptides via MHC class II solely at the basolateral surface. Remarkably, basolateral labeling for MHC class II was not only detected as membrane-associated, but was also found on floccular electron-dense material in the widened intercellular spaces. The appearance of MHC class II molecules in the intercellular spaces was strictly related to the up-regulation of MHC class II in CECs during CD inflammation. Therefore, we hypothesize the intercellular MHC class II is derived from CECs rather than lamina propria cells. Cleavage of cell surface bound MHC class II and/or the exocytic release of intracellular MHC class II might account for the intercellular residing MHC class II molecules. The extracellular release of MHC class II/peptide complexes by intestinal epithelial cells was recently demonstrated in vitro and mediated by the exocytic release of small vesicles, termed exosomes. LAMP-2-positive vesicles containing MHC class II and OVA were consistently observed to fuse with the basolateral membranes. Thus, we suggest these structures might function as transport intermediates between CEC’s MIICs and the basolateral cell surface. They may be responsible for the delivery of MHC class II complexes generated in MIICs, then displayed on the cell surface, and facilitate its extracellular release. Our inability to identify vesicular structures and LAMP-2 labeling (exosomes were reported to reveal LAMP labeling) within the intercellular spaces argues against exosomes being a major source of the intercellularly detected MHC class II molecules and in favor of a soluble form. Further projects should elucidate the fate of MHC class II/antigen complexes generated within CECs.
We established a new experimental technique to investigate the trafficking of luminal antigens in the intestinal epithelium of humans in vivo. Our data yield insight into the endocytic pathways of OVA in human CECs and its associations with MHC class II expressed during CD inflammation (Fig. 2
). We identified MHC class II-enriched late endosomes in CECs of Crohn’s colitis patients that were absent in CD patients in remission and healthy controls and most likely equivalent to MIICs identified in professional antigen presenting cells. Our findings provide in vivo evidence that luminal antigens efficiently access MIICs in CECs and support the suggested function of CECs in MHC class II-associated antigen presentation during CD inflammation.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4807fje;
This article has been cited by other articles:
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  G. Hundorfean, K.-P. Zimmer, S. Strobel, A. Gebert, D. Ludwig, and J. Buning Luminal antigens access late endosomes of intestinal epithelial cells enriched in MHC I and MHC II molecules: in vivo study in Crohn's ileitis Am J Physiol Gastrointest Liver Physiol, October 1, 2007; 293(4): G798 - G808. [Abstract] [Full Text] [PDF]
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