In Situ Expression of Interleukin-10 in Noninflamed Human Gut and in Inflammatory Bowel Disease  Frank Autschbach, Jutta Braunstein, Burkhard Helmke, Ivan Zuna, Guido Schürmann, Zofia I. Niemir, Reinhard Wallich, Herwart F. Otto, Stefan C. Meuer  The American Journal of Pathology  Volume 153, Issue 1, Pages 121-130 (July 1998) DOI: 10.1016/S0002-9440(10)65552-6 Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 1 IL-10 mRNA expression in noninflamed gut. A: In situhybridization of noninflamed colon demonstrates IL-10 mRNA expression by intestinal epithelial cells with accumulation of white autoradiographic grains. Only a few lamina propria MNCs display positive signals. B: Negative control with use of the sense cRNA probe. Dark-field micrographs; magnification, ×40. The American Journal of Pathology 1998 153, 121-130DOI: (10.1016/S0002-9440(10)65552-6) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 2 IL-10 protein expression in noninflamed gut. A:Immunohistochemical detection of IL-10 protein with strong staining (red) of intestinal epithelia in noninflamed normal colon. Only a few lamina propria MNCs are faintly stained. B: Corresponding negative control with omission of the primary antibody. PSP method; alkaline phosphatase; magnification, ×40. The American Journal of Pathology 1998 153, 121-130DOI: (10.1016/S0002-9440(10)65552-6) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 3 Expression of IL-10 mRNA (A) and IL-1β mRNA (B) by nonepithelial cells of noninflamed and inflamed gut. The number of positive cells expressing mRNA was independently assessed by two observers. Fractions of 500 nucleated cells were counted in two different representative areas in the lamina propria and the submucosa and within dense lymphoid aggregates, and the relative percentage of mRNA-expressing cells to the total number was determined. The box plot diagrams represent median values of the respective results (median, 25th and 75th percentile). Minimum and maximum are indicated by dotted lines. The American Journal of Pathology 1998 153, 121-130DOI: (10.1016/S0002-9440(10)65552-6) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 4 IL-10 mRNA expression in IBD. A: Only a few MNCs in the inflamed mucosa express IL-10 mRNA (white grains) in a case of UC (arrowheads), whereas epithelia diffusely express the signal. B andC: In comparison, many scattered inflammatory MNCs in the adjacent submucosa, including perivascular and perineural MNCs strongly express IL-10 mRNA in the same case. D to F:Immunohistology of the same area as in B and C reveals that many inflammatory cells express the macrophage marker CD68, suggesting that IL-10 mRNA is expressed by such cells (D). However, some CD3-positive T cells (E), but virtually no CD20-positive B-cells (F), are present at this site. Aand B: In situ hybridizations; dark-field micrographs; magnification, ×40. C: Corresponding bright-field of B (grains hardly visible at this magnification, ×40). D: CD68;E: CD3; F: CD20; alkaline phosphatase; magnification, ×40. The American Journal of Pathology 1998 153, 121-130DOI: (10.1016/S0002-9440(10)65552-6) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 5 Expression of IL-10 mRNA and protein in dense lymphoid aggregates in IBD. A and B: Corresponding bright-field (A) and dark-field (B) micrographs of a submucosal dense lymphoid aggregate in a case of CD (ileum) hybridized for IL-10 mRNA demonstrate strong expression of white autoradiographic signals (B) on inflammatory MNCs at the periphery of the focus, whereas most cells inside the lymphoid aggregate do not express the signal. In situ hybridization; magnification, ×40.C: Immunohistochemistry with demonstration of CD14-positive MNCs predominantly at the periphery of a lymphoid aggregate in the same case (red) suggests that macrophages produce the IL-10 mRNA at such sites. D andE: However, despite the lack in mRNA expression, most MNCs inside a dense lymphoid aggregate are stained for IL-10 protein in the same case (D). Such aggregates are shown to contain many CD20-positive B cells (E). C: CD14; D: IL-10 protein; E: CD20; alkaline phosphatase; magnification, ×40. The American Journal of Pathology 1998 153, 121-130DOI: (10.1016/S0002-9440(10)65552-6) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions

Figure 6 IL-10 protein expression in IBD. A: Many submucosal inflammatory cells in a case of CD strongly stain for IL-10 protein (red), whereas only few MNCs in the adjacent lamina propria are weakly positive. Note that the gut epithelium also stains for IL-10 protein in inflamed gut. PSP method, alkaline phosphatase; magnification, ×25. B andC: Immunohistochemical double staining of inflammatory cells in a case of UC at a perivascular site in the submucosa. Weak to moderate stainings for IL-10 can be observed on most inflammatory cells (blue color, no nuclear counterstain), which contain both CD3-positive T cells (B) as well as CD68-positive macrophages (C) (brown color). PSP method; alkaline phosphatase and peroxidase; magnification, ×100. The American Journal of Pathology 1998 153, 121-130DOI: (10.1016/S0002-9440(10)65552-6) Copyright © 1998 American Society for Investigative Pathology Terms and Conditions