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Antigen Transport and Cytoskeletal Characteristics of a Distinct Enterocyte Population in Inflammatory Bowel Diseases  Sabine Kersting, Matthias Bruewer,

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Presentation on theme: "Antigen Transport and Cytoskeletal Characteristics of a Distinct Enterocyte Population in Inflammatory Bowel Diseases  Sabine Kersting, Matthias Bruewer,"— Presentation transcript:

1 Antigen Transport and Cytoskeletal Characteristics of a Distinct Enterocyte Population in Inflammatory Bowel Diseases  Sabine Kersting, Matthias Bruewer, Guido Schuermann, Axel Klotz, Markus Utech, Matthias Hansmerten, Christian F. Krieglstein, Norbert Senninger, Joerg-Dieter Schulzke, Hassan Y. Naim, Klaus-Peter Zimmer  The American Journal of Pathology  Volume 165, Issue 2, Pages (August 2004) DOI: /S (10) Copyright © 2004 American Society for Investigative Pathology Terms and Conditions

2 Figure 1 CD (ileum OVA incubation 10 minutes). A: Electron microscopy: three RACE cells surrounded by NE cells. Epon section. B: Quantitation of microvilli in RACE and NE cells. C: Immunofluorescence: rhodamine-labeled OVA in the cytosol of RACE cells. D: Immunoelectron microscopic localization of OVA (12-nm large gold particles) within the epithelial layer. A RACE cell is situated between NE cells. E: Higher magnification of the box in D: RACE cell with moderate OVA labeling (arrows) in the cytosol and in the nucleus. NE cell without any OVA labeling in the nucleus and the cytosol. F:Fluorescein isothiocyanate-labeled SI on microvilli of NE cells and RACE cells. Labeling for SI is present in the cytosol of all RACE cells and some NE cells. AB, Apoptotic body; AP, apical membrane; Lu, lumen; Mi, microvilli; V, vesicle; BL, basolateral membrane; SI, sucrase-isomaltase; N, nucleus; M, mitochondriae. Scale bars: 1 μm (A, D); 0.1 μm (F). The American Journal of Pathology  , DOI: ( /S (10) ) Copyright © 2004 American Society for Investigative Pathology Terms and Conditions

3 Figure 2 Quantitation of OVA at the paracellular space in IBD and controls. Graphs show mean values, error bars represent SEM. The American Journal of Pathology  , DOI: ( /S (10) ) Copyright © 2004 American Society for Investigative Pathology Terms and Conditions

4 Figure 3 Immunoelectron microscopic co-localization of OVA (OVA incubation 10 minutes, 6-nm gold particles) and SI or aminopeptidase (12-nm gold particles). A: Healthy control (ileum). SI and OVA (arrows) labeling on microvilli. B: CD (ileum). RACE cell with OVA labeling in the cytosol (arrows) and SI labeling on microvilli and in the cytosol. C: CD (ileum). RACE cell with OVA in the cytosol (arrows) and aminopeptidase labeling restricted to microvilli. D: Microvillous labeling density of SI. E: Cytosolic labeling densities of SI. Graphs show mean values, error bars represent SEM. N(HC), Nucleus of healthy controls; N(CD/UC), nucleus of CD and UC; HC-il, healthy ileal controls; HC-co, healthy colon controls; BG, background; Mi, microvilli; SI, sucrase-isomaltase; AP, aminopeptidase. Scale bars, 0.1 μm. The American Journal of Pathology  , DOI: ( /S (10) ) Copyright © 2004 American Society for Investigative Pathology Terms and Conditions

5 Figure 4 Immunoelectron microscopic co-localization of OVA (OVA incubation 10 minutes; 6-nm gold particles) and villin (12-nm gold particles). A: Healthy control (ileum). Strong villin labeling on microvilli and in the terminal web. B: CD (ileum). RACE cell with OVA labeling (arrows) in the cytosol and weak villin labeling on microvilli and the terminal web. C: Microvillous labeling densities of villin. Graph shows mean values, error bars represent SEM. N(HC), Nucleus of healthy controls; N(CD/UC), nucleus of CD and UC; HC-il, healthy ileum controls; HC-co, healthy colon controls; BG, background; Lu, lumen; Mi, microvilli; V, vesicle; TW, terminal web. Scale bars, 0.1 μm. The American Journal of Pathology  , DOI: ( /S (10) ) Copyright © 2004 American Society for Investigative Pathology Terms and Conditions

6 Figure 5 Immunoelectron microscopic co-localization of OVA (OVA incubation 10 minutes; 6-nm gold particles, arrows) and actin (12-nm gold particles). A: Healthy control (ileum). High labeling density for actin on microvilli and terminal web. B: CD (ileum). RACE cell with OVA labeling in the cytosol (arrows) and low labeling density of actin on microvilli and terminal web. C: CD (ileum). Left: NE cell with high labeling density of actin near the basolateral membrane. Right: RACE cell with OVA labeling in the cytosol (arrows) and low labeling density of actin near the basolateral membrane. D: Labeling densities of actin in the terminal web. E: Labeling densities of actin near the basolateral membrane. Graphs show mean values, error bars represent SEM. N(HC), Nucleus of healthy controls; N(CD/UC), nucleus of CD and UC; HC-il, healthy ileum controls; HC-co, healthy colon controls; BG, background; Mi, microvilli; BL, basolateral membrane; TW, terminal web. Scale bars, 0.1 μm. The American Journal of Pathology  , DOI: ( /S (10) ) Copyright © 2004 American Society for Investigative Pathology Terms and Conditions

7 Figure 6 Immunoelectron microscopical co-localization of OVA (OVA incubation 10 minutes; 6-nm gold particles) and LAMP (12-nm gold particles). CD (ileum). A: RACE cell with OVA-loaded (arrows) LAMP-positive vesicle. B: Antigen transport in LAMP-positive vesicles. C: Labeling densities of OVA in antigen-loaded LAMP vesicles. Graphs show mean values, error bars represent SEM. N(HC), Nucleus of healthy controls; N(CD/UC), nucleus of CD and UC; HC-il, healthy ileum controls; HC-co, healthy colon controls; BG, background; Lu, lumen; Mi, microvilli; V, vesicle; M, mitochondrium. Scale bar, 0.1 μm. The American Journal of Pathology  , DOI: ( /S (10) ) Copyright © 2004 American Society for Investigative Pathology Terms and Conditions

8 Figure 7 Immunoelectron microscopic co-localization of OVA (OVA incubation 10 minutes; 6-nm gold particles) and UEA (12-nm gold particles). CD (ileum). A: NE cell with OVA (arrows)-loaded UEA-positive vesicle. B: Antigen transport in UEA-positive vesicles. C: Labeling densities of OVA in antigen-loaded UEA vesicles. Graphs show mean values, error bars represent SEM. N(HC), Nucleus of healthy controls; N(CD/UC), nucleus of CD and UC; HC-il, healthy ileum controls; HC-co, healthy colon controls; BG, background; Mi, microvilli; V, vesicle; M, mitochondrium. Scale bars, 0.1 μm. The American Journal of Pathology  , DOI: ( /S (10) ) Copyright © 2004 American Society for Investigative Pathology Terms and Conditions

9 Figure 8 Detection of caspase-3 by immunoelectron microscopy and Western blotting. A: Caspase-3 expression in HT29 monolayers. B: Subcellular detection of caspase-3 (12-nm gold particles) within apoptotic HT-29/B6 enterocytes. C: Absent caspase-3 labeling in the cytosol of RACE and NE. AB, Apoptotic body; BL, basolateral membrane; Lu, lumen; Mi, microvilli. Scale bars: 0.1 μm (B); 1 μm (C). The American Journal of Pathology  , DOI: ( /S (10) ) Copyright © 2004 American Society for Investigative Pathology Terms and Conditions

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