Volume 132, Issue 1, Pages 139-153 (January 2007) Enhanced Recruitment of CX3CR1+ T Cells by Mucosal Endothelial Cell–Derived Fractalkine in Inflammatory Bowel Disease  Miquel Sans, Silvio Danese, Carol de la Motte, Heitor S.P. de Souza, Brenda M. Rivera–Reyes, Gail A. West, Manijeh Phillips, Jeffry A. Katz, Claudio Fiocchi  Gastroenterology  Volume 132, Issue 1, Pages 139-153 (January 2007) DOI: 10.1053/j.gastro.2006.10.010 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Enhanced up-regulation of FKN expression on HIMEC surfaces by proinflammatory cytokines. Control, UC, and CD HIMEC monolayers were left untreated or stimulated with (A) IL1-β, (B) IFN-γ, (C) TNF-α, or (D) TNF-α plus IFN-γ for different time periods, after which the percentage of FKN-expressing HIMECs was assessed by flow-cytometric analysis. Each bar represents 4–5 separate HIMEC lines. *P < .05 and #P < .01 for UC and CD compared with control HIMECs. □, Control; , UC; ■, CD. Gastroenterology 2007 132, 139-153DOI: (10.1053/j.gastro.2006.10.010) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 (A) Flow-cytometric analysis of FKN surface expression by HIMECs after cytokine stimulation. HIMEC monolayers were left untreated or stimulated with TNF-α, IFN-γ, or TNF-α plus IFN-γ for 48 hours, after which the percentage of FKN-expressing HIMECs was assessed by flow-cytometric analysis. Numbers in parentheses indicate the corresponding mean fluorescence intensity. The black curve represents the background signal from the isotype control. Figure is representative of 14 experiments (5 control, 5 UC, and 4 CD HIMECs). (B) Enhanced FKN production by IBD HIMECs. Control, UC, and CD HIMEC monolayers were left untreated or stimulated with IFN-γ plus TNF-α for 48 hours, after which cells were lysed, and total proteins were extracted for FKN assessment by immunoblotting. Figure is representative of 6 separate experiments. The molecular weight of recombinant human FKN (rhfkn) is slightly smaller than that of the native molecule because of differences in glycosylation. gapdh, glyceraldehyde-3-phosphate dehydrogenase. Gastroenterology 2007 132, 139-153DOI: (10.1053/j.gastro.2006.10.010) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Enhanced up-regulation of FKN expression on HIMEC surfaces by leukocyte contact. Control, UC, and CD HIMEC monolayers were left untreated or cultured with CD40L-negative Jurkat T cells, CD40L-positive D1.1 T cells, THP1 monocytic cells, or sCD40L for 24 hours, after which leukocytes were removed and the percentage of FKN-expressing (A) HIMECs and the (B) mean fluorescence intensity were assessed by flow-cytometric analysis. Where indicated, HIMECs were pretreated with IFN-γ for 48 hours before coculture. Each bar represents 4–5 separate HIMEC lines. *P < .05–.01 for stimulated compared with unstimulated (none) HIMECs; #P < .05 for CD and UC compared with control HIMECs. □, Control; , UC; ■, CD. Gastroenterology 2007 132, 139-153DOI: (10.1053/j.gastro.2006.10.010) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Leukocyte contact induced up-regulation of FKN surface expression by HIMECs. HIMEC monolayers were cultured with Jurkat T cells for 24 hours, after which the percentage of FKN-expressing HIMECs was assessed by flow-cytometric analysis. In some experiments (no contact), a .2-μm porous filter was placed between leukocytes and endothelial cells to prevent physical contact. Numbers in parentheses indicate the corresponding mean fluorescence intensity. The black curve represents the background signal from the isotype control. Figure is representative of 12 separate experiments (4 normal, 4 UC, and 4 CD HIMECs). Gastroenterology 2007 132, 139-153DOI: (10.1053/j.gastro.2006.10.010) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Cytokine-induced secretion of soluble FKN by HIMECs. Control, UC, and CD HIMEC monolayers were left untreated or stimulated with TNF-α, IFN-γ, or TNF-α plus IFN-γ. Supernatants were harvested at 48 hours and FKN content was analyzed by enzyme-linked immunosorbent assay. Each bar represents 4 separate HIMEC lines. *P < .05 for UC and CD compared with control HIMECs. □, Control; , UC; ■, CD Gastroenterology 2007 132, 139-153DOI: (10.1053/j.gastro.2006.10.010) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 (A) Cytokine-induced phosphorylation of p38 and ERK in HIMECs. HIMEC monolayers were left untreated or stimulated with IFN-γ, TNF-α, or IFN-γ plus TNF-α for 15 minutes, after which cells were washed, lysed, and total protein was extracted for hybridization with a phospho-p38– or phospho-ERK–specific antibody. Figure is representative of 3 separate experiments. (B) Decrease of FKN-expressing HIMECs by p38 and ERK inhibitors. Control, UC, and CD HIMEC monolayers were left untreated or pretreated for 1 hour with the p38 inhibitor SB203580 or the ERK-1 and ERK-2 inhibitor PD98059 before stimulation with IFN-γ plus TNF-α. After 24 hours, the percentage of FKN-expressing HIMECs was assessed by flow-cytometric analysis. Each bar represents 3–4 separate cell lines. *P < .05 compared with inhibitor-untreated cells. □, Control; , UC; ■, CD Gastroenterology 2007 132, 139-153DOI: (10.1053/j.gastro.2006.10.010) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 (A) Differential CX3CR1 expression by T cells from healthy and IBD subjects. PBMCs were isolated from healthy subjects (normal, n = 10) and patients with active CD (n = 8), inactive CD (n = 8), active UC (n = 9), and inactive UC (n = 7). Cells were costained for CX3CR1 and CD3, CD4, and CD8, and the percentage of positive cells for each subpopulation was calculated. *P < .05 for active UC and CD compared with normal and inactive UC and CD. ■, Active; ▩, inactive. (B) Flow-cytometry analysis showing differential surface expression of the FKN receptor CX3CR1 on peripheral blood T cells from healthy subjects (normal) and active CD patients. Values represent the percentage of CX3CR1-positive cells in each scatter plot. Figure is representative of 18 experiments (10 healthy subjects and 8 patients with active CD). Gastroenterology 2007 132, 139-153DOI: (10.1053/j.gastro.2006.10.010) Copyright © 2007 AGA Institute Terms and Conditions

Figure 8 Detection of CX3CR1+ cells in normal and IBD mucosa. The panels show dark brown immunohistochemical staining for CX3CR1 in mononuclear cells of the colonic lamina propria of histologically normal control, active CD, and active UC tissue sections. The insert in UC shows CX3CR1+ cells in the lumen of or adherent to the wall of the mucosal microvasculature in actively inflamed areas of the same UC specimen. Figure is representative of 14 control, 9 CD, and 11 UC samples. Gastroenterology 2007 132, 139-153DOI: (10.1053/j.gastro.2006.10.010) Copyright © 2007 AGA Institute Terms and Conditions

Figure 9 Relative contribution of FKN to HIMEC T-cell adhesion. Confluent HIMEC monolayers were left unstimulated or stimulated with TNF-α and IFN-γ. After 24 hours, calcein-labeled Jurkat T cells were cocultured with the HIMEC monolayers for 1 hour, after which the monolayers were rinsed and retained leukocytes were quantified using a computerized imaging system on an inverted fluorescence microscope. To assess the relative contribution of FKN, CX3CR1, VCAM-1, and ICAM-1 to adhesion, antibodies specific for each molecule were added alone (gray bars) or in combination to HIMEC or Jurkat cells (shaded bars). Each bar represents 4 separate HIMEC lines. *P < .05 for antibody-treated compared with antibody-untreated HIMECs. Gastroenterology 2007 132, 139-153DOI: (10.1053/j.gastro.2006.10.010) Copyright © 2007 AGA Institute Terms and Conditions

Figure 10 FKN-induced enhancement of active β1 integrin expression on leukocytes. PBMCs isolated from healthy subjects, Jurkat cells, and THP1 cells were cultured with 50 nmol/L of human recombinant FKN. After 30 minutes the cells were washed, stained with 12G10, a monoclonal antibody that specifically recognizes the activated form of β1 integrin, and assessed by flow cytometry. Each bar represents 4 separate experiments. *P < .05 compared with FKN-untreated cells. □, Unstimulated; ■, FKN. Gastroenterology 2007 132, 139-153DOI: (10.1053/j.gastro.2006.10.010) Copyright © 2007 AGA Institute Terms and Conditions

Figure 11 FKN-induced transmigration of THP-1 cells through HIMEC monolayers. HIMEC monolayers were grown to confluence on filter inserts separating the upper and lower chambers of a Transwell system. Calcein-labeled THP1 cells were placed in the upper chamber overlaying the HIMEC monolayer, whereas medium alone, MCP-1, or FKN were added to the lower chamber. After a 4-hour incubation period, migrated cells were quantified using a computerized imaging system on an inverted fluorescence microscope. Each bar represents 3 separate HIMEC lines. *P < .05 for FKN compared with unstimulated, and MCP-1 compared with FKN. □, Unstimulated; ■, FKN; , MCP-1. Gastroenterology 2007 132, 139-153DOI: (10.1053/j.gastro.2006.10.010) Copyright © 2007 AGA Institute Terms and Conditions