Journal
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
Volume 290, Issue 4, Pages C990-C999Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpcell.00308.2005
Keywords
toll-like receptor; flagellin
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Funding
- NCI NIH HHS [CA-76541] Funding Source: Medline
- NIGMS NIH HHS [GM-37631, GM-070738] Funding Source: Medline
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mobility group box 1 ( HMGB1), a cytokine-like proinflammatory protein, is secreted by activated macrophages and released by necrotic cells. We hypothesized that immunostimulated enterocytes might be another source for this mediator. Accordingly, Caco-2 cells or primary mouse intestinal epithelial cells ( IECs) were incubated with cytomix ( a mixture of TNF, IL-1 beta, and IFN-gamma)for various periods. HMGB1 in cell culture supernatants was detected by Western blot analysis and visualized in Caco-2 cells with the use of fluorescence confocal and immunotransmission electron microscopy. Caco-2 cells growing on filters in diffusion chambers were stimulated with cytomix for 48 h in the absence or presence of anti-HMGB1 antibody, and permeability to fluorescein isothiocyanate-dextran ( average molecular mass, 4 kDa; FD4) was assessed. Cytomix-stimulated Caco-2 cells secreted HMGB1 into the apical but not the basolateral compartments of diffusion chambers. Although undetectable at 6 and 12 h after the start of incubation with cytomix, HMGB1 was present in supernatants after 24 h of incubation. HMGB1 secretion by Caco-2 monolayers also was induced when the cells were exposed to FSL-1, a Toll-like receptor ( Tlr)-2 agonist, or flagellin, a Tlr5 agonist, but not lipopolysaccharide, a Tlr4 agonist. Cytomix also induced HMGB1 secretion by primary IECs. Cytoplasmic HMGB1 is localized within vesicles in Caco-2 cells and is secreted, at least in part, associated with exosomes. Incubating Caco-2 cells with cytomix increased FD4 permeation, but this effect was significantly decreased in the presence of anti-HMGB1 antibody. Collectively, these data support the view that HMGB1 is secreted by immunostimulated enterocytes. This process may exacerbate inflammation-induced epithelial hyperpermeability via an autocrine feedback loop.
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