title: HMGB1 is secreted by immunostimulated enterocytes and contributes to cytomix-induced hyperpermeability of Caco-2 monolayers.
abstract: High-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-1beta, 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.
authors.affiliation: Department of Critical Care Medicine, Univ. of Pittsburgh School of Medicine, 616 Scaife Hall, 3550 Terrace St., PA 15261, USA.
authors.forename: Shiguang
authors.initials: S
authors.lastname: Liu
authors.forename: Donna B
authors.initials: DB
authors.lastname: Stolz
authors.forename: Penny L
authors.initials: PL
authors.lastname: Sappington
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Macias
authors.forename: Meaghan E
authors.initials: ME
authors.lastname: Killeen
authors.forename: Jyrki J
authors.initials: JJ
authors.lastname: Tenhunen
authors.forename: Russell L
authors.initials: RL
authors.lastname: Delude
authors.forename: Mitchell P
authors.initials: MP
authors.lastname: Fink
mesh_terms: D000818:Animals; D019898:Autocrine Communication; D018938:Caco-2 Cells; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology*; D020895:Enterocytes / Q000166:cytology / Q000276:immunology* / Q000648:ultrastructure; D024243:HMGB1 Protein / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007114:Immunization; D051379:Mice; D008810:Mice, Inbred C57BL
chemical_list: D016207:Cytokines; D024243:HMGB1 Protein