Volume 129, Issue 1, Pages 50-65 (July 2005) Characteristics of Intestinal Dendritic Cells in Inflammatory Bowel Diseases  Ailsa L. Hart, Hafid Omar Al-Hassi, Rachael J. Rigby, Sally J. Bell, Anton V. Emmanuel, Stella C. Knight, Michael A. Kamm, Andrew J. Stagg  Gastroenterology  Volume 129, Issue 1, Pages 50-65 (July 2005) DOI: 10.1053/j.gastro.2005.05.013 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Identification of blood DCs and expression of TLR2 and TLR4. (A) DCs were identified in blood by multicolor flow cytometry as HLA-DR+ lineage− (CD3−, CD14−, CD16−, CD19−, CD34−, CD56−) cells. Within this DC gate, CD11c+ and CD11c− DC subsets were identified. (B) Labeling of gated DC subsets with anti-TLR antibodies was quantified using SED normalized subtraction. In this example, 1-parameter histograms for staining with anti-TLR2 or its isotype-matched control are shown. The proportion of TLR2-positive DCs was determined by subtracting normalized cumulative histograms of labeling with the control antibody from similar histograms of labeling with anti-TLR2. In the histograms with subtraction percentages, the outline of the histogram indicates staining with anti-TLR2 and the shaded area represents the proportion of TLR2-positive cells after subtraction of isotype control binding. (C) The proportion of blood CD11c+ DCs expressing TLR2 and TLR4 in healthy controls (n = 8) was assessed using the SED normalized subtraction facility in WinList 5.0. Gastroenterology 2005 129, 50-65DOI: (10.1053/j.gastro.2005.05.013) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Identification of colonic DCs and expression of TLR2 and TLR4. (A) DCs were identified in colonic tissue by multicolor flow cytometry as HLA-DR+ lineage− (CD3−, CD14−, CD16−, CD19−, CD34−, CD56−). Within this gate, CD11c+ DCs were identified. (B) The proportion of CD11c+ DCs staining positive for TLR2 and TLR4 in tissue from healthy controls (n = 13), inflamed tissue from patients with ulcerative colitis (n = 15), and inflamed tissue from patients with Crohn’s disease (n = 7) is shown. Gastroenterology 2005 129, 50-65DOI: (10.1053/j.gastro.2005.05.013) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 TLR2 and TLR4 expression on intestinal DCs from healthy controls. (A) In 6 healthy controls, paired samples were taken from ileal and colonic tissue and the proportion of intestinal DCs expressing TLR2 and TLR4 was assessed. (B) Labeling of permeabilized and nonpermeabilized colonic DCs from healthy controls with antibodies to TLR2, TLR4, and β-actin. Intensity of staining was determined by subtraction using WinList, and data are presented as the ratio of labeling of permeabilized cells (surface plus intracellular staining) to labeling of nonpermeabilized cells (cell surface staining only). Values >1 indicate the presence of intracellular antigen. Gastroenterology 2005 129, 50-65DOI: (10.1053/j.gastro.2005.05.013) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 TLR2 and TLR4 expression on inflamed and noninflamed tissue from patients with ulcerative colitis. In 8 patients with ulcerative colitis in whom there was a demarcation between inflamed and noninflamed tissue, biopsy specimens were taken from both noninflamed and inflamed sections. The proportion of colonic DCs expressing TLR2 and TLR4 was assessed. (A) A representative example of the staining of gated DC from 1 patient. Open histograms show staining with an anti-TLR antibody or with an isotype-matched control as indicated. To the right of each of these pairs of histograms is a third histogram showing the result of the subtraction analysis. Here, the open histogram is the staining with the anti-TLR reagent and the shaded area represents the fraction of positive cells after subtraction of isotype control binding using SED normalized subtraction. Numerical values are the percentage of positive cells. (B) Pooled data for all 8 patients. Gastroenterology 2005 129, 50-65DOI: (10.1053/j.gastro.2005.05.013) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 TLR2 and TLR4 expression demonstrated by immunofluorescence on sorted colonic DCs from inflamed tissue. (A) The 2-parameter histograms of HLA-DR and lineage (CD3, CD14, CD16, CD19, CD34, CD56) indicate the cells before and after the sorting process. The single-parameter histogram of CD11c indicates the expression of CD11c on these cells before and after the sort. (B) Indirect immunofluorescence was performed on cytospins of sorted CD11c+ HLA-DR+ lineage− cells derived from inflamed tissue of a patient with Crohn’s disease. The right panels show immunofluorescent staining, and the left panels show the equivalent bright field images. Gastroenterology 2005 129, 50-65DOI: (10.1053/j.gastro.2005.05.013) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Flow cytometric data showing CD40 expression on DCs from patients with Crohn’s disease and controls. DCs were identified as HLA-DR+ lineage− cells in LPMCs (upper panel). In the lower panel, solid histograms represent staining of CD11c+ DCs with anti-CD40; open histograms show staining with the isotype control. Numerical values represent the MFI of anti-CD40 staining with the isotype control subtracted. Representative examples of DCs from patients with Crohn’s disease and controls are shown. Pooled data are presented in Figure 7. Gastroenterology 2005 129, 50-65DOI: (10.1053/j.gastro.2005.05.013) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 CD40 expression on DCs from patients with Crohn’s disease and controls. (A) CD40 expression on CD11c+ colonic DCs. Net MFI represents the MFI of anti-CD40 staining with isotype control binding subtracted. DCs from healthy controls and patients with Crohn’s disease, both inflamed (I) and noninflamed (NI), were assessed. (B) Expression of CD40 by colonic DCs assessed by net MFI before and after treatment of patients with Crohn’s disease with anti-TNF-α. Solid symbols represent DCs from inflamed tissue, and open symbols represent DCs from noninflamed tissue. Gastroenterology 2005 129, 50-65DOI: (10.1053/j.gastro.2005.05.013) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Cytokine production by DCs in patients with IBD and controls. Cytokine production by DCs was assessed by intracellular cytokine staining and SED normalized subtraction (see Materials and Methods). (A) Representative intracellular staining for IL-12, IL-6, and IL-10 in DCs from inflamed and noninflamed tissue. In each case, pairs of histograms are presented in which one histogram shows staining of cells cultured in the presence of monensin and the second histogram shows staining of DCs cultured without monensin. To the right of each pair is a third histogram showing the result of the subtraction analysis. Here the open histogram shows staining of DCs in the presence of monensin and the shaded area represents the fraction of cytokine-positive DCs after subtraction of staining of cells from the “no monensin” cultures. Numerical values show the percent cytokine-positive DCs. (B) Pooled data showing production of IL-12, IL-6, and IL-10 by DCs are shown for 5–10 patients with Crohn’s disease, patients with ulcerative colitis, and healthy controls. Gastroenterology 2005 129, 50-65DOI: (10.1053/j.gastro.2005.05.013) Copyright © 2005 American Gastroenterological Association Terms and Conditions