1. Volume 140, Issue 5, Pages 1597-1607 (May 2011)
Eosinophils Express Muscarinic Receptors and Corticotropin-Releasing Factor to Disrupt the Mucosal Barrier in Ulcerative Colitis 
Conny Wallon, Mats Persborn, Maria Jönsson, Arthur Wang, Van Phan, Maria Lampinen, Maria Vicario, Javier Santos, Philip M. Sherman, Marie Carlson, Ann–Charlott Ericson, Derek M. Mckay, Johan D. Söderholm 
Gastroenterology 
Volume 140, Issue 5, Pages (May 2011)
DOI: /j.gastro
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2. Figure 1
Increased HRP flux but unaltered paracellular permeability in colonic mucosa of UC. Transmucosal permeability in Ussing chambers in endoscopic biopsies of controls and inactive UC, respectively, as assessed by flux of the protein antigen, (A) HRP, and permeability to the paracellular probes (B) 51Cr-EDTA and (C) fluorescein isothiocyanate−dextran (D) TER and (E) baseline short circuit current (Isc at start; t = 0 minute) were also determined. Data are presented as mean ± standard error of mean of n = 15 individuals in each group (except for FD4000, n = 3 individuals in each group); *P < .05 vs controls; unpaired t test.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
CCh effects on HRP flux are mediated via muscarinic pathways and mast cells. Assessment of Isc (A), (C), (E), and HRP flux (B), (D), (F), in endoscopic biopsies of healthy volunteers after exposure to CCh alone or after pre-exposure to muscarinic receptor antagonists (atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide [4-DAMP]) or nicotinic antagonist (hexamethonium [Hex]) (C, D), mast cell stabilizer, Lod, or fast neural Na+ channel blocker, tetrodotoxin (TTX) (E, F). Data are presented as mean ± standard error of mean of 16 individuals for each modulation; *P < .05 vs vehicle control; #P < .01 vs CCh-challenged tissues; analysis of variance with Tukey's post-hoc tests.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

4. Figure 3
Increased transmucosal flux of HRP in UC is dependent on muscarinic receptors, CRF, and mast cells. (A) HRP flux in UC biopsies after 20 minutes pretreatment with vehicle, the muscarinic receptor blocker, atropine, the CRF receptor antagonist, CRF(9−41), and the mast cell stabilizer, Lod. Bars represent mean ± standard error of mean; n = 15 UC patients; *P < .05 vs vehicle; analysis of variance (ANOVA) with Tukey's post-hoc tests. (B) HRP flux in biopsies of healthy controls after pretreatment with vehicle (n = 8 individuals), atropine, CRF(9–41), and Lod, respectively (n = 4; 3 biopsies per antagonist), with no significant differences between treatments, ANOVA P = .84.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions

5. Figure 4
Muscarinic receptor expression in human colonic mucosa. Photomicrographs of colonic mucosa from 2 UC patients. Muscarinic receptors were stained in red (M1 in panels A, B; M2 in panels C, D; M3 in panels E, F). Double-staining was done in green for eosinophil peroxidase (EPO) (E) in panels A, C, E, and for mast cell (MC) tryptase in panelsB, D, F. Bar = 50 μm. (A) Note low co-staining of eosinophils and M1; (C) all eosinophils positive for M2; (E) a majority of eosinophils positive for M3 (64% of cells in UC vs 95% in controls). (B, D, F) MC showed no co-staining with muscarinic receptors.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Increased numbers of activated eosinophils in colonic mucosa of UC. (A–D) Eosinophils (staining for eosinophil peroxidase [EPO]; green) in (A, C) colonic mucosa of controls and (B, D) UC patients. Images are representative of 8 controls and 9 UC patients (see text for quantification). (D) Note activated and degranulated eosinophils surrounding the crypts in UC mucosa, but (C) lower numbers and intact eosinophils in controls. Bars = 100 μm. Transmission electron photomicrographs showing (E) a normal eosinophil from a control subject and (F) an activated, partially (piecemeal) degranulated eosinophil from a UC patient; arrows: emptied granules; arrowheads: partially emptied granules with a shift to lighter staining in the periphery. Bars = 1 μm.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Eosinophils of human colonic mucosa contain CRF. (A, B) Serial sections of immunostaining for (A, red) CRF and (B, green) eosinophil peroxidase (EPO). Circles and arrows indicate expression in the same cells of both anti-EPO and anti-CRF. Bar = 50 μm. (C, D) Higher magnification of serial sections with double-stained cells demonstrating distinct morphological features of eosinophils.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Eosinophil 15HL-60 cells express muscarinic receptors and release CRF on exposure to carbachol to activate mast cells that affect T84 epithelial barrier function. (A) Reverse-transcription polymerase chain reaction messenger RNA expression of muscarinic receptor subtypes M1, M2 and M3, and CRF under basal conditions (vehicle, VE), stimulation with butyric acid (BA) only, or co-stimulation with BA and granulocyte-macrophage colony-stimulating factor for 7 days. β-actin was used as positive control. Results are typical of 3 separate experiments. (B) CRF release to culture supernatant after exposing differentiated 15HL-60 cells to vehicle (negative control), CCh (0.1 mM) for 6 or 24 hours, or lipopolysaccharide (LPS; 1 μg/mL for 24 hours; positive control). Mean ± standard error of mean (SEM); n = 6 for each treatment in 1 representative experiment of 2 experiments; **P < .01 compared to negative control, analysis of variance (ANOVA) with Tukey's post-hoc tests. (C) Degranulation of HMC-1 cells (release of β-hexosaminidase) exposed to culture medium (C, negative control), A23187 (300 nM; positive control), CCh in culture medium (0.1 mM), supernatant of nontreated eosinophils (Eos), or supernatant of eosinophils pretreated with CCh for 6 hours or 24 hours, respectively. Mean ± SEM; n = 4 for each treatment in each experiments; results typical for 3 experiments; **P < .01 vs negative control, ANOVA with Tukey's post-hoc tests. (D, E) horseradish peroxidase (HRP) flux in human colonic T84 monolayers: (D) Monolayers exposed on basolateral side for 24 hours to medium (control), CCh 0.1 mM (CCh), or supernatant of untreated 15HL-60 (EO sup) or 15HL-60 pretreated for 24 hours to CCh (EO/CCh sup), respectively. (E) Monolayers exposed on basolateral side for 24 hours to medium (control), or cocultured with HMC-1 cells alone (HMC), HMC cells with medium from untreated 15-HL60 (HMC + EO sup), or HMC cells exposed to conditioned medium of 15HL-60 pretreated for 24 hours with CCh (HMC + EO/CCh sup). Mean ± SEM; n = 8 for each treatment in each experiment with results typical for 3 separate experiments. **P < .01 vs negative control, ANOVA with Tukey's post-hoc tests.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions