Gastroenterology

Gastroenterology

Volume 132, Issue 1, January 2007, Pages 139-153
Gastroenterology

Basic–alimentary tract
Enhanced Recruitment of CX3CR1+ T Cells by Mucosal Endothelial Cell–Derived Fractalkine in Inflammatory Bowel Disease

https://doi.org/10.1053/j.gastro.2006.10.010Get rights and content

Background & Aims: Fractalkine (FKN/CX3CL1) is a unique chemokine combining adhesive and chemotactic properties. We investigated FKN production by the mucosal microvasculature in inflammatory bowel disease (IBD), its capacity for leukocyte recruitment into the gut, and the number of CX3CR1+ cells in the circulation and mucosa of IBD patients. Methods: The expression of FKN by human intestinal microvascular endothelial cells (HIMECs) and CX3CR1 by circulating cells was evaluated by flow cytometry, and mucosal CX3CR1+ cells were enumerated by immunohistochemistry. The capacity of FKN to mediate leukocyte binding to HIMECs was assessed by immunoblockade, and to induce HIMEC transmigration by a Transwell system. Results: The spontaneously low HIMEC FKN expression was enhanced markedly by tumor necrosis factor-α plus interferon-γ stimulation, or direct leukocyte contact. This effect was significantly stronger in IBD than control HIMECs. Up-regulation of HIMEC FKN expression was dependent on p38 and extracellular signal-regulated kinase phosphorylation, as was abrogated by selective mitogen-activated protein kinase inhibitors. Circulating T cells contained significantly higher numbers of CX3CR1+ cells in active IBD than inactive IBD or healthy subjects, and IBD mucosa contained significantly more CX3CR1+ cells than control mucosa. Antibody-blocking experiments showed that FKN was a major contributor to T- and monocytic-cell adhesion to HIMECs. Finally, FKN enhanced the expression of active β1 integrin on leukocytes and mediated leukocyte HIMEC transmigration. Conclusions: In view of the capacity of FKN to mediate leukocyte adhesion, chemoattraction, and transmigration, its increased production by mucosal microvascular cells and increased numbers of circulating and mucosal CX3CR1+ cells in IBD point to a significant role of FKN in disease pathogenesis.

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Reagents, Antibodies, and Cell Lines

The following antibodies (Abs) were purchased from R&D Systems (Minneapolis, MN): blocking anti-FKN, biotinylated anti-FKN, anti-intercellular adhesion molecule 1 (ICAM-1) (CD54a), and anti–vascular cell adhesion molecule 1 (VCAM-1) (CD106). Antibodies against CX3CR1 were purchased from Torrey Pines Biolabs (Houston, TX) and from Abcam (Cambridge, MA). Phycoerythrin (PE)-conjugated anti-goat Ab was purchased from Caltag (Burlingame, CA) and fluorescein isothiocyanate–conjugated anti-rabbit Ab

Enhanced Cytokine-Induced FKN Surface Expression by IBD HIMECs

The production of chemokines by endothelial cells can be spontaneous or regulated by inflammatory mediators, and it usually is increased in inflammatory conditions such as IBD.44, 45 We initially investigated the capacity of control and IBD HIMECs to express FKN under resting and cytokine-stimulated conditions. Resting HIMECs from control, UC, and CD patients displayed low FKN surface expression, but this increased progressively in a time-dependent fashion on exposure to IL-1β, IFN-γ, or TNF-α (

Effect of FKN on HIMEC Transmigration

After showing the active contribution of FKN to leukocyte adhesion, we finally investigated the capacity of this chemokine to attract and transmigrate leukocytes through resting HIMEC monolayers. In preliminary experiments in which MCP-1 was used as a positive control, we showed that FKN was able to induce chemotaxis of TPH1 cells in a dose-dependent fashion (data not shown). Subsequently, using an optimal dose of FKN in a Transwell system, we tested its ability to induce transmigration of THP1

Discussion

This study shows that microvascular cells from IBD mucosa produce greater amounts of FKN than those from normal mucosa, and greater numbers of T cells express CX3CR1 in the circulation of IBD patients than healthy subjects. Thus, the study indicates that FKN is a major mediator of leukocyte-endothelial interaction in gut inflammation.

Both the surface and secreted form of FKN are up-regulated markedly by HIMECs on stimulation with Th1 cytokines, particularly the combination of TNF-α with IFN-γ.

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    Supported by grants from the Fulbright/Generalitat de Catalunya, Ministerio de Educación y Ciencia (Programa Ramón y Cajal, SAF2005-00280 and C03/02) and Fundación Ramón Areces (M.S.), and the National Institutes of Health (DK30399 and DK 50984) (to C.F.).

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