Basic–alimentary tractEnhanced Recruitment of CX3CR1+ T Cells by Mucosal Endothelial Cell–Derived Fractalkine in Inflammatory Bowel Disease
Section snippets
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.).