Volume 131, Issue 5, Pages 1475-1485 (November 2006) Eosinophilic Bowel Disease Controlled by the BB Rat-Derived Lymphopenia/Gimap5 Gene  Lesley Cousins, Margaret Graham, Reuben Tooze, Christine Carter, J. Ross Miller, Fiona M. Powrie, Gordon G. Macpherson, Geoffrey W. Butcher  Gastroenterology  Volume 131, Issue 5, Pages 1475-1485 (November 2006) DOI: 10.1053/j.gastro.2006.09.023 Copyright © 2006 AGA Institute Terms and Conditions

Figure 1 Pathology of PVG-RT1u, lyp/lyp rats. (A) Gross appearance of gastrointestinal tract lyp/lyp (Left) and WT (Right). (B and C) Cross sections of large intestine (original magnification, 100×). (B) WT, (C) lyp/lyp, and (D and E) cross sections of cecum (D) large intestine (E) of lyp/lyp rat (original magnification, 200×). (F and G) Sections of WT (F) and lyp/lyp small intestine (G). (H) Higher magnification of lyp/lyp small intestinal infiltrate (original magnification, 630×). (I and J) Alcian Blue staining of large intestine for goblet cells and mucus (original magnification, 200×) (I) lyp/lyp (J) WT. (K and L) Masson’s Trichrome staining for collagen (original magnification, 200×). (K) lyp/lyp large intestine and (L) WT large intestine. (B–H) H&E staining. Gastroenterology 2006 131, 1475-1485DOI: (10.1053/j.gastro.2006.09.023) Copyright © 2006 AGA Institute Terms and Conditions

Figure 2 Eosinophils and mast cells in PVG-RT1u, lyp/lyp rats. (A) Large intestine stained for eosinophils by Chromotrope 2R (original magnification, 630×) (Left panel) WT, (Right panel) lyp/lyp. (B) Large intestine stained for the mast cell-specific protease RCMPII (brown) (L panel) WT, (R panel) lyp/lyp. Counterstain was Methyl Green (original magnification, 100×). (C) Eosinophil numbers in peripheral blood. Eosinophils were identified in flow cytometry by expression of CD172 and high side scatter signal. Data are presented as means ± SEM. Gastroenterology 2006 131, 1475-1485DOI: (10.1053/j.gastro.2006.09.023) Copyright © 2006 AGA Institute Terms and Conditions

Figure 3 Cellular composition of secondary lymphoid organs of PVG-RT1u, lyp/lyp rats. (A) Total cell numbers; (B) total CD4+ T cells; (C) total CD8+ T cells; and (D) total B cell numbers in spleen, MLN, and superficial CLN of lyp/lyp rats and WT controls. (E) FACS plots showing lack of CD161+ TcRαβ+ NKT cells in the spleen of lyp/lyp rats. Data presented as mean ± SEM *P < .05, **P < .01. Gastroenterology 2006 131, 1475-1485DOI: (10.1053/j.gastro.2006.09.023) Copyright © 2006 AGA Institute Terms and Conditions

Figure 4 Characterization of T cells in PVG-RT1u, lyp/lyp rats. (A) Analysis of surface marker expression by CD4+ T cells from MLN. MLN cells were harvested from 6-month PVG-RT1u, lyp/lyp, and control WT animals and labelled for FACS analysis. Cells gated for CD4 and αβTCR expression were analyzed for expression of CD45RC, OX-40, and CD25. (B) T-cell–derived cytokines. Splenocytes and lymph node cells were pooled from 4- to 6-month-old rats and sorted for CD4+ T cells and CD45RClo CD4+ T cells. Cells were then activated in vitro for 4 hours and secreted IL-4 and IFN-γ assayed by ELISA in supernatants. IL-5 and IL-13 mRNA expression was assayed in cell lysates by QPCR and normalized to β-actin expression. mRNA levels were calculated relative to those in PVG CD4+ T cells. Values are displayed as the mean of triplicates ± SD. Data are representative of 3 experiments. Gastroenterology 2006 131, 1475-1485DOI: (10.1053/j.gastro.2006.09.023) Copyright © 2006 AGA Institute Terms and Conditions

Figure 5 B-cell responses in PVG-RT1u, lyp/lyp rats. (A) Serum IgG1, IgG2b, and IgE levels were measured in 6-month-old lyp/lyp rats and WT controls. (B) Time course of serum IgE levels in lyp/lyp rats. (C and D) Identification of IgE-positive B cells in MLN by costaining for CD45RA (green) and IgE (red) in lyp/lyp (C) and WT (D) rats. Original magnification 250×. (E and F) Labelling of IgE-positive (red) cells in the lamina propria of 6-month-old lyp/lyp rats (E) and WT (F) nuclear labelling with DAPI (original magnification, 400×). Gastroenterology 2006 131, 1475-1485DOI: (10.1053/j.gastro.2006.09.023) Copyright © 2006 AGA Institute Terms and Conditions

Figure 6 Intestine-specific autoantibodies in PVG-RT1u, lyp/lyp rats. (A) Serum from lyp/lyp rats was incubated on intestinal cryostat sections from athymic nude (rnu/rnu) rats. Autoantibody was detected using anti-rat IgG secondary antibody. Nuclear staining is with DAPI (original magnification, 200×). (B) Higher magnification of lyp/lyp autoantibody staining (original magnification, 400×). (C) Intestinal autoantibody staining was not seen using WT serum (original magnification, 200×). (D) Double staining of lyp/lyp autoantibodies (green) and CD45 (red) showing no colocalization of autoantibody-positive cells and CD45+ cells. (E) lyp/lyp Serum was incubated with lung tissue. (F) Autoantibody staining using intestinal tissue derived from a germ-free Wistar rat. Gastroenterology 2006 131, 1475-1485DOI: (10.1053/j.gastro.2006.09.023) Copyright © 2006 AGA Institute Terms and Conditions