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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
Departments of 1Critical Care Medicine, 2Cell Biology and Physiology, 3Pathology, and 4Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Submitted 27 January 2005 ; accepted in final form 6 November 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, and IFN-
) 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. exosome; toll-like receptor; flagellin
HMGB1 also has been implicated in the pathogenesis of human disease. In the original report describing HMGB1 as a mediator of LPS-induced lethality, Wang et al. (1) reported that circulating levels of this protein are increased in patients with severe sepsis. Shortly thereafter, Ombrellino et al. (23) described a patient with high circulating levels of HMGB1 after an episode of hemorrhagic shock. More recently, increased levels of HMGB1 mRNA have been detected in whole blood samples from patients with septic shock, particularly among nonsurvivors (24). Similarly, persistently high serum levels of HMGB1 protein have been detected in patients with septic shock (34).
HMGB1 is actively secreted by immunostimulated macrophages (6, 11, 28, 38), natural killer cells (33), and pituicytes (39). This protein is also released by necrotic, but not apoptotic, cells (31). Because HMGB1 has been shown to modulate intestinal epithelial barrier function (30), we hypothesized that active secretion by enterocytes might be yet another source for this cytokine-like protein. Herein, we report that stimulating either Caco-2 cells or primary cultures of murine enterocytes with cytomix, a mixture of interferon (IFN)-, interleukin (IL)-1, and tumor necrosis factor (TNF), induced secretion of HMGB1. In addition, we show that the addition of a neutralizing polyclonal anti-HMGB1 antibody partially blocked the increase in permeability caused by incubating Caco-2 monolayers with cytomix. The data also indicate that HMGB1 is secreted in soluble form as well as a particulate form that is sequestered within exosomes. These data support the view that enterocyte-derived HMGB1 may be an autocrine amplifier of derangements in epithelial barrier function initiated by other proinflammatory stimuli.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Animals.
C57BL/6 mice (4–8 wk old; 
25 g) were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained at the University of Pittsburgh Animal Research Center with a 12:12-h light-dark cycle and free access to standard laboratory chow and water. The animals were not fasted before the experiments.
Materials. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Dulbecco's modified Eagle medium (DMEM) and phosphate-buffered saline (PBS) were from BioWhittaker (Walkersville, MD). Fetal bovine serum was from Hyclone (Logan, UT). Flagellin (LPS content <125 EU/mg) and a synthetic Toll-like receptor (Tlr)-2 agonist, FSL-1 (Pam2CGDPKHPKSF), were from Invivogen (San Diego, CA). Highly purified (TLRgrade) Salmonella minnesota R595 (Re) LPS was from Alexis Biochemicals (San Diego, CA).
Caco-2 cells.
Caco-2 cells were obtained from ATCC (Manassas, VA) and routinely maintained on collagen I-coated Biocoat tissue culture dishes (Becton-Dickinson, Bedford, MA) at 37°C in a 5% CO2 humidified atmosphere in DMEM supplemented with 10% fetal bovine serum, penicillin G (100 U/ml), streptomycin (100 g/ml), pyruvate (2 mmol/l), L-glutamine (4 mmol/l), and nonessential amino acids. For some studies, the cells were seeded onto 24-well plastic plates (100,000 cells/well). After being cultured for 7 days, some wells were stimulated with cytomix (10 ng/ml TNF, 10 ng/ml IL-1, and 1,000 U/ml IFN-), graded concentrations of flagellin, graded concentrations of FSL-1, or graded concentrations of LPS. Supernatants were harvested at the time points indicated in RESULTS.
For other experiments, Caco-2 cells (50,000 cells/well) were seeded onto permeable filters in 12-well transwell bicameral chambers (COSTAR, Corning, NY) and fed biweekly. After 21 days, some wells were stimulated with cytomix, which was added to the apical or the basolateral chamber. Supernatants were harvested from both the apical and basolateral chambers after 24 h.
To measure changes in the permeability of Caco-2 monolayers, the cells were plated onto permeable filters in 12-well Transwell chambers (105 cells/well) and fed biweekly. Permeability studies were performed using confluent monolayers between 21 and 28 days after being seeded. The permeability probe was fluorescein isothiocyanate-labeled dextran (average molecular mass = 4 kDa; FD4). A sterile stock solution of FD4 (25 mg/ml) was prepared by dissolving the compound in HEPES-buffered DMEM (pH 6.8) and passing it through a filter (0.45-µm pore size). For permeability studies, the medium was aspirated from the apical and basolateral sides of the Transwell chambers. FD4 solution (200 µl) was added to the apical compartments. The medium on the basolateral side of the Transwell chambers was replaced with 500 µl of control medium, medium containing cytomix, medium containing graded concentrations of anti-HMGB1 polyclonal antibody, or medium containing both cytomix and graded concentrations of anti-HMGB1 antibody. After 48 h of incubation, 30 µl of medium were aspirated from the basolateral compartments for spectrofluorometric determination of FD4 concentration as previously described (21). The permeability of monolayers was expressed as a clearance with the units (nl·cm–2·h–1), which was calculated as previously described (30).
Isolation and culture of primary murine intestinal epithelial cells.
C57BL/6 black mice were euthanized, and the small intestine was removed for isolation of intestinal epithelial cells (IECs). The IECs were isolated using a modification of the method of Whitehead et al. (40), which has been reported as an effective method to avoid contamination with fibroblasts (32). Briefly, the tissue was flushed with ice-cold Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS) (Invitrogen, Carlsbad, CA) to remove intestinal contents. The tissue was cut into small pieces (1 mm x 1 mm) and decontaminated by soaking in 0.04% sodium hypochlorite for 20 min on ice. After being washed, the tissue pieces were soaked on ice for 90 min in 20 ml of Ca2+- and Mg2+-free HBSS containing 3 mM EDTA and 1 mM DTT. The tube was shaken vigorously to detach crypts, which were pelleted by centrifugation at 150 g for 3 min at 4°C. The pellet was washed twice with ice-cold Ca2+- and Mg2+-free HBSS and resuspended in DMEM/F12 culture medium (BioWhittaker), which was supplemented with 5% FBS, 100 U/ml penicillin G, 100 µg/ml streptomycin, 1x insulin-transferrin-selenium (Invitrogen), 20 mM HEPES, pH 7.4, 0.25 µg/ml amphotericin B, 1 µg/ml fibronectin, and 1 µg/ml hydrocortisone. Six-well plates were coated with collagen I and poly-L-lysine 1 h before the cells were seeded. The plates were seeded with 500 crypts/well and incubated at 37°C under a 5% CO2 atmosphere. Cellular viability was >95% by Trypan blue exclusion assay. Three days after being seeded, the cells were stimulated with cytomix, and supernatants were harvested after 24 and 48 h.
Lactate dehydrogenase release assay. Cells were incubated in the absence or presence of cytomix for 24 or 48 h. Samples of medium were collected, and the amount of lactate dehydrogenase (LDH) in the medium was measured with an assay kit (TOX-7) from Sigma, by following the protocol suggested by the manufacturer. To control for slight differences among wells in the total number of cells, we then lysed the cells, using two freeze-thaw cycles, and measured the total amount of LDH in each well. Results are expressed as the quantity of LDH in culture supernatants (before the freeze-thaw cycles) divided by the total amount of LDH in each well after cell lysis was induced by the freeze-thaw cycles.
Western blot analysis. Equal volumes of cell-culture supernatant were mixed with 6x loading buffer [375 mM Tris, pH 6.8, 50% glycerol, 0.03% bromophenol blue, and 10% SDS]. After being boiled, the samples were subjected to 10% SDS-PAGE electrophoresis. The resolved proteins were transferred to PVDF membrane (Amersham Pharamacia Biotech, Leicestershire, UK) and blocked with Blotto buffer (1x PBS, 5% nonfat milk, 0.05% Tween 20, and 0.2% NaN3) for 1 h. The membrane was then incubated with rabbit polyclonal anti-HMGB1 antibodies (BD Pharmingen, San Jose, CA) 1:2,000 diluted in blocking buffer overnight at 4°C. After being washed three times in 1x PBS with 0.3% Triton X-100, pH 7.4 (PBST), immunoblots were exposed for 1 h at room temperature to a 1:20,000 dilution of the HRP-conjugated goat anti-rabbit secondary antibody. After being washed three times with 1x PBST, the membrane was illuminated with the ECL reagents (Amersham Pharamacia Biotech) and X-ray film exposed, according to the manufacturer's instructions. HMGB1 expression was quantified densitometrically with the use of GelExpert version 3.5 software (Nucleotech, San Mateo, CA) and comparison to a standard curve generating by carrying out Western blot analysis with known quantities of recombinant human HMGB1.
Exosome isolation from culture medium. Caco-2 cells were grown in 75 cm2 culture flasks until confluent. The cells were incubated under control conditions or stimulated with cytomix for 24 h. The medium was collected and centrifuged at 400 g at 4°C for 5 min to remove debris. The supernatants were subjected to ultracentrifugation at 100,000 g at 4°C for 1 h to pellet exosomes. The supernatant was collected and assayed for the presence of HMGB1 by Western blot analysis and the pellet was washed with 1x PBS and pelleted again by ultracentrifugation as described above. The final pellet was resuspended in 200 µl of PBS for immuno-transmission electron microscopy (TEM) and Western blot analysis.
Immunofluorescence confocal microscopy. Caco-2 cells were grown on filter inserts as described and fixed in 2% paraformaldehyde in PBS for 1 h. Cells were washed three times in PBS, then three times (5 min each) with PBS supplemented with 0.5% bovine serum albumin (PBB). Cells were permeabilized in 0.1% Triton X-100 in PBB for 30 min, then washed once with PBB. Cells were blocked with 2% bovine serum albumin in PBS for 40 min then washed once with PBB. Rabbit anti-HMGB1 antibodies (1:2,000) were added to cells in PBB incubated at room temperature for 1 h. Cells were washed four times in PBB, then secondary antibodies (goat anti-rabbit Alexa 488; 1:500 dilution; Molecular Probes, Eugene, OR), rhodamine-phalloidin (1:250 dilution; Molecular Probes), and the nuclear dye, Draq5 (1:1,000 dilution; Biostatus, Leicestershire, UK) were added for 1 h at room temperature. Cells were washed three times in PBB and then three times in PBS. Cells were mounted with the use of gelvatol (23 g polyvinyl alcohol 2,000, 50 ml glycerol, and 0.1% sodium azide to 100 ml PBS), then viewed on a confocal scanning fluorescence microscope (model Fluoview 1000, Olympus, Malvern, NY). Confocal stacks (0.5 µm slices) were obtained and rendered with the use of Metamorph analytical software (Molecular Devices, Sunnyvale, CA). X,Y, X,Z, and Y,Z stacks were obtained using Imaris software (BitPlane, St. Paul, MN).
Immunoelectron microscopy. Caco-2 cells were grown on filter inserts as described and fixed in cryofix (2% paraformaldehyde, 0.01% glutaraldehyde in 0.1 M PBS) for 1 h. Cells on filters were coated on both the top and bottom of the filter with 3% gelatin in PBS for 1 h at 37°C. The gelatin was solidified at 4°C for 1 h, and then fixed for an additional 15 min in cryofix. Washed exosome pellets were likewise encapsulated in 3% gelatin. Gelatin-cell filter and gelatin-exosome pellet blocks were cryoprotected in polyvinylpyrollidine cryoprotectant (25% polyvinylpyrollidine, 2.3 M sucrose, 0.055 M Na2CO3, pH 7.4) overnight at 4°C, as previously described (35). Blocks were situated on ultracryotome stubs that were cross-sectioned, frozen with liquid nitrogen, and stored in liquid nitrogen until use. Ultrathin sections (70 nm) were cut with the use of a Reichert Ultracut U ultramicrotome with a FC4S cryo-attachment, lifted on a small drop of 2.3 M sucrose in PBS, and mounted on Formvar-coated copper grids. Sections were washed three times with PBS, then three times with PBG, followed by a 30-min blocking incubation with 5% normal goat serum in PBG. Sections were labeled with rabbit anti-HMGB1 (1:100 dilution in PBG) for 1 h. Sections were washed four times in PBG and labeled with goat anti-rabbit antibody conjugated with 5 nm of colloidal gold (Amersham, Piscataway, NJ) at a dilution of 1:25 for 1 h. Sections were washed three times in PBG, three times in PBS, and then fixed in 2.5% glutaraldehyde in PBS for 5 min. The sections were washed two times in PBS, and then washed six times in double-distilled H2O (ddH2O). Sections were poststained in 2% neutral uranyl acetate for 7 min, washed three times in ddH2O, stained for 2 min in 4% uranyl acetate, and then embedded in 1.25% methyl cellulose. Labeling was observed with a TEM (model JEM 1210, Jeol, Peabody, MA) at 80 kV.
Exosome staining. Copper grids (200 mesh) were coated with 0.125% Formvar (Ted Pella, Redding, CA) in chloroform. Washed exosomes (1–10 µl of suspension) were loaded onto grids by centrifugation in an Airfuge Ultracentrifuge (Beckman, Palo Alto, CA) using the EM-90 rotor. After centrifugation at 100,000 g for 5 min, the grid was removed and excess sample solution was wicked away with filter paper. For negative staining, exosomes were fixed with 2.5% glutaraldehyde in PBS for 10 min, washed in PBS, and then negatively stained with 1% aqueous uranyl acetate. For immunostaining, exosomes were fixed with 2% paraformaldehyde in PBS for 5 min. The exosomes were washed three times with PBS, and then three more times with PBG. Some samples were permeabilized with 0.1% Triton X-100 in PBG for 5 min, followed by 30-min incubation with 5% normal goat serum in PBG. Exosomes were labeled with rabbit anti-HMGB1 for 1 h at room temperature. Exosomes were washed four times with PBG and then labeled with 5 nm of colloidal gold by being incubated with gold-conjugated goat anti-rabbit antibody at 1:25 dilution at room temperature for 1 h. Sections were washed three times in PBG and three times in PBS. The sections were fixed in 2.5% glutaraldehyde in PBS for 5 min, washed two times in PBS, and then washed six times in ddH2O. The grid was placed on a drop of 0.45 µm filtered 2% phosphotungstic acid, pH 6.0 in milli-Q H2O for 30–60 s. Excess stain was wicked away and the samples were viewed with a JEM 1210 TEM at 80 kV.
Statistical methods. Clearance data from the Transwell experiments using FD4 are expressed as means ± SE. Data were analyzed by ANOVA, followed by Fisher's protected least-significant difference test. Significance was declared for P < 0.05.
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RESULTS |
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Cytomix-stimulated Caco-2 cells growing on filters in diffusion chambers or on plastic plates release similar quantities of HMGB1.
By generating a standard curve with known amounts of recombinant human HMGB1, we used Western blot analysis to quantify the release of HMGB1 from control and immunostimulated Caco-2 cells. Irrespective of whether the cells were grown on filters in diffusion chambers or in plastic wells, stimulation with cytomix resulted in the release of similar amounts of HMGB1 (Fig. 1D). For example, after 24 h of incubation with cytomix, the cells released 800 ng of HMGB1 per 106 cells. For comparison, murine peritoneal macrophages stimulated with IFN- (100 or 1,000 U/ml) for 16 h release 100 ng of HMGB1 per 106 cells (28).
Tlr2 and Tlr5 agonists induce HMGB1 secretion by Caco-2 cells.
Previous studies indicate that several human enterocyte-like cells lines (including Caco-2) express pathogen-associated molecular pattern (PAMP) recognition receptors, including Tlr2 (9) and Tlr5 (5). Under normal conditions, expression of Tlr4 is downregulated in Caco-2 cells (3, 22) but expression of this PAMP is increased when these cells are stimulated with IFN- and/or TNF (2). Prompted by these observations, we carried out studies to determine whether LPS (Tlr4 agonist), FSL-1 (synthetic Tlr2 agonist), or flagellin (Tlr5 agonist) can stimulate Caco-2 cells to secrete HMGB1. Accordingly, Caco-2 cells growing in 24-well plastic plates were incubated under control conditions or stimulated with graded concentrations of LPS, FSL-1, or flagellin. After 48 h, supernatants were harvested and assayed for HMGB1 by Western blot analysis. Both FSL-1 (Fig. 2A) and flagellin (Fig. 2B) clearly stimulated HMGB1 in a concentration-dependent fashion, whereas highly purified LPS (0.5 to 10 µg/ml) was without effect (data not shown).
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-actin. Although both unstimulated and immunostimulated Caco-2 cells released HMGB1, Western blots of supernatants (before ultracentrifugation) revealed that the cultures incubated with cytomix released a great deal more of this protein (see the gels labeled "whole medium" in Fig. 9). This finding recapitulates the data presented in Fig. 1. When the particulate fractions from ultracentrifuged Caco-2 supernatants were assayed by Western blot analysis, the samples from unstimulated and cytomix-stimulated cells contained similar amounts of the cytoskeletal protein, -actin (see the gels labeled "pellet" in Fig. 9). However, the exosome-containing 100,000 g particulate phase from the cytomix-stimulated cultures contained large amounts of HMGB1, whereas this protein was just barely detectable in the pellet samples from unstimulated Caco-2 cultures. When the soluble fractions from ultracentrifuged culture supernatants were assayed, the samples from cytomix-stimulated cells contained considerably more HMGB1 than did the samples from control cells, but HMGB1 was nonetheless clearly present even in the samples from unstimulated cultures (see the gels labeled "supernatant" in Fig. 9). Collectively, these data support the view that under basal conditions, Caco-2 cells release small quantities of HMGB1 predominantly in a soluble form, whereas immunostimulated cells release large quantities of HMGB1 both in a soluble form and associated with -actin-containing particles (presumably exosomes).
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, and IFN-. To test this hypothesis, we incubated Caco-2 monolayers under control conditions or with graded concentrations of polyclonal anti-HGMB1 antibody or with cytomix or with cytomix in the presence of graded concentrations of anti-HMGB1 antibody. As expected, incubating the cells with cytomix significantly increased the permeability of the epithelial sheet to the fluorescent probe, FD4 (Fig. 10). In the absence of cytomix, anti-HMGB1 antibody was without effect, but treatment with the antibody significantly attenuated the development of hyperpermeability induced by the mixture of TNF, IL-1, and IFN-.
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DISCUSSION |
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In the present work, we extended the investigation of HMGB1 secretion by immunostimulated cells to a different cell type, namely enterocytes. Although Kuniyasu et al. (18) recently reported that WiDr human colon cancer cells constitutively release HMGB1 into culture supernatants, we observed only very low levels of this protein in the media of unstimulated Caco-2 transformed human enterocyte-like cells. However, after stimulation of the cells with cytomix, the synthetic Tlr2 ligand, FSL-1, or the Tlr5 ligand, flagellin, we observed a large increase in the amount of HMGB1 released into the culture media. Because it is known that HMGB1 is released by necrotic (but not apoptotic) cells (31), it is important that we were able to document that incubating Caco-2 cells with cytomix for 48 h was neither associated with an increase in the number of cells taking up the vital dye, Trypan blue, nor increased release of the intracellular enzyme LDH. These observations confirm findings previously reported by our laboratory, wherein we showed that incubation of Caco-2 cells with cytomix fails to increase staining with the fluorescent dye, ethidium homodimer-1, which penetrates only into dead cells (29).
Because Kuniyasu et al. (18) showed that colon cancer cells release HMGB1 and Caco-2 cells are also cancer cells, it is noteworthy that we showed that cytomix-stimulated (but not resting) primary murine enterocyte cultures release HMGB1. We isolated and cultured murine enterocytes, using the approach described by Whitehead et al. (40). The cells grown using this method previously were identified as epithelial cells (not fibroblasts) (32, 40). Thus we believe that our findings support the view that immunostimulated enterocytes (and not just colon cancer cells) secrete HMGB1, and the release of this protein by these cells is the result of an active process rather than secondary to cell death.
Data obtained by Gardella et al. (11) support the notion that the secretion of HMGB1 by stimulated monocytes occurs when secretory lysosomes undergo exocytosis. While our data cannot exclude that a similar mechanism is responsible for the secretion of HMGB1 by Caco-2 cells, our findings support the view that one pathway for the release of HMGB1 from these cells depends on the release of exosomes into the extracellular environment on exocytic fusion of multivesicular endosomes with the cell surface. The data supporting this view are biochemical, immunohistochemical, and immuno-ultrastructural in nature. Specifically, we showed that when supernatants from immunostimulated Caco-2 cultures are subjected to ultracentrifugation, HMGB1 can be detected in the exosome-containing pellet, even after being washed extensively. By the same token, HMGB1 also can be detected in the exosome-depleted supernatant from this ultracentrifugation process, suggesting that the protein may be released from Caco-2 cells by more than one process.
Using confocal fluorescence microscopy, we visualized immunoreactive HMGB1 within cytoplasmic vesicles, which were predominantly localized toward the apical ends of cells near regions of cell-cell contact. We also observed punctuate surface staining for HMGB1, especially in cells exposed to cytomix. Using immuno-TEM, we showed that Caco-2 cells contain MVBs, a finding that confirms previously reported observations (17, 19). Electron-dense gold particles, indicative of binding of the anti-HMGB1 antibody, were localized within MVBs and smaller vesicles within the cells. Immuno-TEM images also revealed HMGB1 associated with exosomes from cytomix-stimulated Caco-2 cells. Because electron-dense gold particles were most apparent after extracting the exosome preparations with 0.1% Triton X-100, a process that renders the membrane-bound particles permeable to the anti-HMGB1 antibody, our findings suggest that HMGB is probably localized inside the vesicles. The immunohistochemical and immuno-ultrastructural studies, however, clearly revealed the presence of extracellular HMGB1 that was not associated with exosomes. This finding is consistent with Western blot analysis, which shows the presence of HMGB1 in the soluble fraction of ultracentrifuged culture supernatants from cytomix-stimulated Caco-2 cells.
Enterocyte-like cells, including Caco-2 cells, are known to secrete a number of cytokines. In some cases, secretion of these mediators is polarized. For example, the secretion of CC and CXC chemokines by stimulated HT-29, T84, or Caco-2 enterocyte-like cells is predominantly basolateral (10, 15, 20). In this study, we observed that the secretion of HMGB1 by stimulated Caco-2 cells was also polarized, favoring release into the apical rather than the basolateral environment. Because key receptors for HMGB1, such as Tlr2 and Tlr4, are localized to the apical surface of enteroyctes (8, 25), our observation that HMGB1 was secreted apically supports the idea that release of this protein might serve an autocrine role to amplify the activation of enterocytes by other factors. This notion is supported by our previously reported (30) observation that HMGB1 is promotes activation of the proinflammatory transcription factor, NK-
B, in Caco-2 cells and also increases the permeability of Caco-2 monolayers. To specifically test this hypothesis, we stimulated Caco-2 monolayers in the absence or presence of a polyclonal neutralizing anti-HMGB1 antibody added to the apical compartment of Transwell chambers. Although failing to completely block the increase in epithelial permeability, the anti-HMGB1 antibody significantly blunted the cytomix-induced development of hyperpermeability. Thus secretion of HMGB1 may be an important positive feedback phenomenon that promotes the development of intestinal epithelial barrier dysfunction due to inflammation. HMGB1 appears to modulate intestinal epithelial barrier function by decreasing the expression of key tight junction proteins, including zonula occludens-1 and occludin (Liu S, Sappington PL, and Fink MP, unpublished observations).
To our knowledge, the data presented here are the first to indicate that exposing (both transformed human and primary murine) enterocyte-like epithelial cells to a variety of proinflammatory stimuli leads to active secretion of HMGB1. Our data suggest that the release of HMGB1 associated with exosomes is a key mechanism for the secretion of this protein by Caco-2 cells. Finally, our data support the view that HMGB1 secretion by IECs may have functional significance, serving to amplify the derangements in barrier function induced by other proinflammatory stimuli.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* S. Liu and D. B. Stolz contributed equally to this work. 
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P < 0.05 vs. incubation with cytomix in the absence of antibody.