HMGB1 B box increases the permeability of Caco-2 enterocytic monolayers and impairs intestinal barrier function in mice

GASTROENTEROLOGY 2002;123:790 – 802

HMGB1 B Box Increases the Permeability of Caco-2 Enterocytic Monolayers and Impairs Intestinal Barrier Function in Mice PENNY L. SAPPINGTON,* RUNKUAN YANG,* HUAN YANG,‡ KEVIN J. TRACEY,‡ RUSSELL L. DELUDE,*,§ and MITCHELL P. FINK*,㛳 Departments of *Critical Care Medicine, §Pathology, and 㛳Surgery, University of Pittsburgh Medical School, Pittsburgh, Pennsylvania; and ‡Laboratory of Biomedical Science, North Shore-Long Island Jewish Research Institute, Manhasset, New York

Background & Aims: High mobility group (HMG) B1 is a nonhistone nuclear protein that was recently identified as a late-acting mediator of lipopolysaccharide-induced lethality in mice. The proinflammatory actions of HMGB1 have been localized to a region of the molecule called the B box. Methods: To determine whether HMGB1 or B box are capable of causing derangements in intestinal barrier function, we incubated cultured Caco-2 human enterocytic monolayers with recombinant human HMGB1 or a 74-residue truncated form of the protein consisting of the B box domain. Results: Both HMGB1 and B box increased the permeability of Caco-2 monolayers to fluorescein isothiocyanate–labeled dextran (FD4) in a time- and dose-dependent fashion. The increase in permeability was reversible following removal of the recombinant protein. Exposure of Caco-2 cells to B box resulted in increased expression of inducible nitric oxide synthase messenger RNA and increased production of NO. When we used various pharmacologic strategies to inhibit NO production or scavenge NO or peroxynitrite (ONOOⴚ), we abrogated B box–induced hyperpermeability. Administration of B box to wild-type mice increased both ileal mucosal permeability to FD4 and bacterial translocation to mesenteric lymph nodes. These effects were not observed in inducible nitric oxide synthase knockout mice. Conclusions: These data support the view that HMGB1 and B box are capable of causing alterations in gut barrier function via a mechanism that depends on the formation of NO and ONOOⴚ.

igh mobility group (HMG) B1 was first described as a nonhistone nuclear protein with high electrophoretic mobility.1 A characteristic feature of the protein is the presence of 2 folded DNA-binding motifs that are termed the “A domain” and the “B domain.”2 Both of these domains contain a characteristic grouping of aromatic and basic amino acids within a block of 75 residues termed the HMG-1 box.3 HMGB1 has several functions within the nucleus, including facilitating DNA repair4 and supporting the transcriptional regulation of genes.5

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Recently, extracellular HMGB1 was identified as a late-acting cytokine-like mediator of lipopolysaccharide (LPS)-induced lethality.6 High circulating concentrations of HMGB1 can be detected in mice 16 –32 hours after the onset of endotoxemia,6 and delayed passive immunization of mice with antibodies against HMGB1 confers significant protection against LPS-induced mortality.6 Administration of highly purified recombinant HMGB1 to mice is lethal,6 and direct application of HMGB1 into the airways of mice initiates an acute inflammatory response and lung injury.7 Exposure of macrophage cultures to low concentrations of HMGB1 induces the release of tumor necrosis factor and other proinflammatory cytokines.8 Rodents challenged with LPS develop manifestations of impaired intestinal barrier function, including increased mucosal permeability to hydrophilic macromolecules9 and increased translocation of viable bacteria to mesenteric lymph nodes (MLN).9,10 Intestinal barrier dysfunction often is not apparent until many hours after the injection of LPS,11 suggesting that a late-acting mediator may be pathophysiologically responsible for this phenomenon. Because HMGB1 is a late-acting mediator of LPS-induced toxicity,6 we sought to determine whether HMGB1 is capable of causing derangements in gut epithelial barrier function in vitro and in vivo. Because recent studies by Yang et al. localized the proinflammatory actions of HMGB1 to the B-box portion of the molecule,12 we used a recombinant 74-residue long B-box construct for most of our studies. Here we present Abbreviations used in this paper: BrdU, 5-bromo-2ⴕ-deoxyuridine; C-PTIO,2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1oxyl-3-oxide; FeTPPS, 5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron [III ]; HMG, high mobility group; iNOS, inducible nitric oxide synthase; L-NIL, L-N(6)-(1-iminoethyl)lysine; LPS, lipopolysaccharide; MLN, mesenteric lymph nodes; NF-␬B, nuclear factor ␬B; PCR, polymerase chain reaction; PDTC, pyrrolidine dithiocarbamate; RAGE, receptor for advanced glycation end products. © 2002 by the American Gastroenterological Association 0016-5085/02/$35.00 doi:10.1053/gast.2002.35391

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results showing that HMGB1 and HMGB1 B box increased the permeability of monolayers of a human enterocytic cell line, Caco-2, growing on permeable supports in bicameral diffusion chambers. We further showed that injecting mice with B box increased ileal mucosal permeability and bacterial translocation to MLN.

Materials and Methods The research protocol complied with the regulations regarding animal care as published by the National Institutes of Health and was approved by the Institutional Animal Use and Care Committee of the University of Pittsburgh Medical School.

Animals Male C57BL/6J (Jackson Laboratories, Bar Harbor, ME) and inducible nitric oxide synthase (iNOS) knockout (iNOS⫺/⫺) mice weighing 20 –25 g were used in this study. iNOS⫺/⫺ mice were generously provided by Dr. T. Billiar (University of Pittsburgh Medical School). These mice were generated as described previously.13 The knockout mice were backcrossed 4 times onto a C57BL/6J background. Polymerase chain reaction (PCR) of genomic DNA was used to confirm the genotype of iNOS⫺/⫺ mice in randomly selected animals (data not shown). All animals were maintained at the University of Pittsburgh Animal Research Center with a 12-hour light-dark cycle and free access to standard laboratory chow and water. Animals were not fasted before the experiments.

Materials All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) unless otherwise noted. Dulbecco’s modified Eagle medium and phosphate-buffered saline (PBS) were from BioWhittaker (Walkersville, MD). Fetal bovine serum was from Hyclone (Logan, UT). L-N(6)-(1-iminoethyl)lysine (L-NIL) was from A.G. Scientific (San Diego, CA). The ONOO⫺ decomposition catalyst, 5,10,15,20-tetrakis-[4sulfonatophenyl]-porphyrinato-iron [III ] (FeTPPS), was from Calbiochem (San Diego, CA). An antibody against the extracellular domain of the receptor for advanced glycation end products (RAGE) was from Santa Cruz Biotechnology (Santa Cruz, CA). Caco-2 human intestinal epithelial cells were obtained from American Type Culture Collection (Manassas, VA).

Recombinant Human HMGB1 and HMGB1 B Box Recombinant human HMGB1 was cloned by PCR amplification from human brain Quick-Clone cDNA (Clontech, Palo Alto, CA) using the following primers: 5⬘-GATGGGCAAAGGAGATCCTAAG-3⬘ and 5⬘- GCGGCCGCTTATTCATCATCATCATCTTC-3⬘. The product length was 651 base pairs. A truncated mutant, HMGB1 B box, was also cloned by PCR amplification from human brain Quick-Clone

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cDNA. The primers used were 5⬘-AAGTTCAAGGATCCCAATGCAAAG-3⬘ and 5⬘-GCGGCCGCTCAATATGCAGCTATATCCTTTTC-3⬘. The product length was 233 base pairs. A stop codon was added to the mutant to ensure the accuracy of protein size. The PCR products were cloned into the pCRII-TOPO vector between the EcoRI sites using the TA cloning method according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). The complementary DNA (cDNA) inserts were excised with EcoRI and subcloned into the expression vector, pGEX, with a GST tag (Pharmacia, Piscataway, NJ); correct orientation of positive clones and PCR fidelity were confirmed by DNA sequencing of both strands. The recombinant plasmids were transformed into the proteasedeficient Escherichia coli strain BL21 (Novagen, Madison, WI), and fusion protein expression was induced by incubation with 1 mmol/L isopropyl-D-thiogalactopyranoside for 3 hours at 25°C. Recombinant proteins were obtained using affinity purification with a glutathione sepharose resin column (Pharmacia). A GST vector lacking HMGB1 protein (M7) was included as a control. The protein eluates were passed over a polymyxin B column (Pierce, Rockford, IL) to remove contaminating LPS and dialyzed extensively against PBS to remove excess amounts of reduced glutathione. The proteins were lyophilized and redissolved in sterile water before use. LPS levels were about 19 pg/␮g protein for B box and 300 – 400 pg/␮g for wild-type HMGB1 as measured by Limulus amebocyte lysate assay (BioWhittaker). The integrity of the protein was verified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis with Coomassie blue staining. The recombinant human HMGB1 B box used for the studies described herein contained 74 amino acids (residues 88 –162 from the holoprotein) and had a molecular mass of 10,000 daltons.

Anti–B Box Antibody Polyclonal antibodies against HMGB1 B box were raised in rabbits (Cocalico Biologicals, Inc., Reamstown, PA) and assayed for titer by immunoblotting. Immunoglobulin was purified from anti-HMGB1 antiserum using Protein A agarose (Pierce) according to the manufacturer’s instructions.

Cell Culture Caco-2 human intestinal epithelial cells were routinely maintained on collagen I– coated Biocoat tissue culture dishes (Becton-Dickinson, Bedford, MA) at 37°C in a 5% CO2 humidified atmosphere in Dulbecco’s modified Eagle medium 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.

Monolayer Permeability Assays Caco-2 human enterocytes (105 cells/well) were plated on permeable filters in 12-well Transwell bicameral chambers (COSTAR, Corning, NY) and fed biweekly. Permeability studies were performed using confluent monolayers between 21 and 28 days after seeding. The permeability probe was fluorescein isothiocyanate–labeled dextran (4000 daltons;

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FD4). A sterile stock solution of FD4 (25 mg/mL) was prepared by dissolving the compound in HEPES-buffered Dulbecco’s modified Eagle medium complete medium (pH 6.8) and passing it through a filter (0.45-␮m pore size). For permeability studies, the medium was aspirated from the apical and basolateral sides of the Transwell chambers. FD4 solution (200 ␮L) was added to the apical compartments. The medium on the basolateral side of the Transwell chambers was replaced with 500 ␮L of control medium or medium containing recombinant HMGB1, recombinant B box, or medium plus B box plus one of several different pharmacologic agents. After 24 and 48 hours of incubation, 30 ␮L of medium was aspirated from the basolateral compartments for spectrofluorometric determination of FD4 concentration as previously described.14 The permeability of monolayers was expressed as an average clearance (C), which was calculated as previously described.14

Assessment of Cell Viability Caco-2 cells (100,000 cells/well) were grown on collagen I– coated 8-well culture slides (Becton Dickinson) and fed biweekly. The cells were incubated for 48 hours with control medium or medium containing M7 vector or medium containing recombinant HMGB1 B box. Preliminary studies to determine cell viability were performed by assessing exclusion of the dye, trypan blue, by light microscopy. A more rigorous assessment of cell viability was performed using the live/dead viability/cytotoxicity kit from Molecular Probes (Eugene, OR). After washing the cells twice with PBS, 100 ␮L of a solution containing 2 ␮mol/L calcein AM and 4 ␮mol/L ethidium homodimer 1 was added to each well and incubated for 45 minutes. Cells were imaged using a model DM HC inverted fluorescent microscope (Leica, Germany) equipped with a Diagnostic Instruments SPOT-II Model 1.4 Digital Microscope Imaging System (Burroughs, MI). Captured images were minimally manipulated for publication using Adobe Photoshop software (San Jose, CA).

Apoptosis Assay Caco-2 cells (100,000 cells/well) were grown on 6-well collagen I– coated polystyrene tissue culture dishes for 7 days and fed biweekly. After a 48-hour incubation in control medium, medium containing 1 ␮g/mL M7 vector, or medium containing 1 ␮g/mL B box, the cells were fixed with 1% paraformaldehyde and incubated in 70% ethanol for 18 hours. Apoptotic cells were detected using the APO-BrdU TUNEL assay kit (Molecular Probes) according to the manufacturer’s instructions. Briefly, the cells were washed twice with 1 mL of the wash buffer provided with the kit and centrifuged at 300g for 5 minutes between washes. The cells were then incubated for 60 minutes with 50 ␮L of DNA labeling solution containing 10 ␮L of reaction buffer, 0.75 ␮L of terminal deoxynucleotidyl transferase solution, 8.0 ␮L of 5-bromo-2⬘-deoxyuridine (BrdU) solution, and 31.25 ␮L of distilled H2O. The cells were then rinsed twice with 1 mL of rinse buffer and centrifuged at 300g for 5 minutes between rinses. BrdU-labeled DNA was detected by adding 100 ␮L of a solution containing

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5.0 ␮L of Alexa Fluor 488 dye-labeled anti-BrdU antibody and 95 ␮L of rinse buffer to each sample and incubating for 30 minutes. Lastly, each sample was incubated for 30 minutes with 0.5 mL of propidium iodide/ribonuclease A staining buffer. A FACS Vantage SE flow cytometer (Becton Dickinson, Santa Cruz, CA) was used to measure the red (propidium iodide) and green (Alexa Fluor 488 – conjugated anti-BrdU antibody) fluorescence of the labeled nuclei. Data from 104 events were collected by doublet discrimination, and the results were displayed as bivariate dot plots of red (y-axis) versus green (x-axis) fluorescence. Positive and negative control cells (supplied with the kit) were also assayed.

Measurement of NO Production To determine NO3⫺/NO2⫺ in culture supernatants, Caco-2 enterocytes were plated in 6-well dishes and incubated for 21 days. Confluent monolayers were incubated for 48 hours with control medium, medium plus B box, or medium plus B box plus one of several different pharmacologic agents. To first reduce NO3⫺ to NO2⫺, cadmium filings (0.4 – 0.7 g/tube; Fluka, Milwaukee, WI) were loaded into 1.5-mL microfuge tubes. The filings were washed twice with 1.0 mL of deionized water, twice with 1.0 mL of 0.1 mol/L HCl, and twice with 1.0 mL of 0.1 mol/L NH4OH. A total of 10 ␮L of 30% ZnSO4 was added to 200 ␮L of culture supernatant, vortexed, incubated at room temperature for 15 minutes, and centrifuged at 14,000g for 5 minutes. The resulting supernatant was added to a cadmium-containing microcentrifuge tube and incubated at room temperature overnight with constant mixing. The samples were transferred to fresh microcentrifuge tubes and centrifuged again. The supernatants were subsequently assayed for NO2⫺ using the modified Griess assay as previously described.9

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Nuclear Extract Preparations Caco-2 enterocytes were plated at 106 cells per well in 6-well dishes. Forty-eight hours after seeding, the cells were incubated with control medium or medium plus B box. After various intervals of stimulation, the cells were removed from the incubator and immediately placed on ice. Cells were washed once with PBS and then harvested in 1 mL of PBS containing 2% fetal bovine serum using a rubber policeman. The cells were transferred to a 1.5-mL microfuge tube and centrifuged at 14,000g for 10 seconds. The cell pellet was resuspended in 600 ␮L of buffer I (10 mmol/L KCl, 1.5 mmol/L MgCl2 , 0.3 mol/L sucrose, 500 ␮mol/L phenylmethylsulfonyl fluoride, 1.0 mmol/L sodium orthovanadate, 1 mmol/L dithiothreitol, 10 mmol/L Tris-HCl, pH 7.8) and incubated for 15 minutes. A total of 38.3 ␮L of 10% NP40 was added, and the tubes were vortexed at full speed for 10 seconds. The nuclei were isolated by centrifugation at 310g for 3 minutes and the supernatants aspirated. The nuclear pellets were gently resuspended in 80 ␮L of buffer II (500 ␮mol/L phenylmethylsulfonyl fluoride, 1.0 mmol/L sodium orthovanadate, 1 mmol/L dithiothreitol, 420 mmol/L KCl, 1.5 mmol/L MgCl2 , 20% glycerol, 10 mmol/L Tris-HCl, pH 7.8). Following a 15-minute incubation, nuclear extracts were cleared by centrifugation at

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14,000g for 10 minutes. The supernatants were transferred to new tubes, and protein concentration was determined using a commercially available Bradford assay (Bio-Rad Protein Assay, Hercules, CA). Nuclear extracts were frozen at ⫺80°C.

Electrophoretic Mobility Shift Assay The sequence of the double-stranded nuclear factor ␬B (NF-␬B) oligonucleotide was as follows: sense, 5⬘-AGT TGA GGG GAC TTT CCC AGG C-3⬘; antisense: 3⬘-TCA ACT CCC CTG AAA GGG TCC G-5⬘ (Promega, Madison, WI). The oligonucleotides were end-labeled with ␥-[32P] adenosine triphosphate (New England Nuclear, Boston, MA) using T4 polynucleotide kinase (Promega). A total of 3 ␮g of nuclear protein was incubated with ␥-32P–labeled NF-␬B probe solution (1 ␮L) in 20 ␮L of 1⫻ bandshift buffer (325 mmol/L NaCl, 5 mmol/L dithiothreitol, 0.7 mmol/L ethylenediaminetetraacetic acid, 40% vol/ vol glycerol, 65 mmol/L HEPES, pH 8.0) in the presence of 2 ␮g of poly[d(I 䡠 C)] (Boehringer Mannheim, Indianapolis, IN) for 20 minutes at room temperature. For competition reactions, a 100fold molar excess of cold oligonucleotide was added simultaneously with labeled probe. Supershift assays were performed by incubating nuclear extracts with 2 ␮L of anti-p65 and anti-p50 (Santa Cruz Biotechnology) for 1 hour before the addition of the radiolabeled probe. The binding reaction mixture was electrophoresed on 4% nondenaturing polyacrylamide gel electrophoresis gels containing 5% glycerol and 1⁄4 ⫻ Tris-borate-ethylenediaminetetraacetic acid. After polyacrylamide gel electrophoresis, the gels were dried and exposed to Biomax-5 film (Kodak, Rochester, NY) at ⫺80°C overnight using an intensifying screen.

In Vivo Experiments In the first experiment, 4 groups of mice were studied. All agents were injected intraperitoneally. Controls were injected with either 1.0 mL PBS or M7 protein dissolved in PBS (50 ␮g/mL per animal). One group was injected with 1.0 mL of a well-sonicated suspension of E. coli serotype O111:B4 LPS (0.1 mg/mL) in PBS, and one group was injected with 1.0 mL of a solution of B box in PBS (50 ␮g/mL). Six, 12, and 18 hours later, subgroups of mice (n ⫽ 5) were anesthetized with intramuscular injections of sodium pentobarbital (90 mg/kg) and segments of ileum were excised for determination of mucosal permeability. The MLN complex was harvested to measure bacterial translocation. Blood was aspirated from the heart to measure the plasma concentration of alanine aminotransferase. In the second experiment, 2 groups (n ⫽ 2) of iNOS⫺/⫺ mice were studied. Each animal was injected with 1.0 mL of either M7 protein (50 ␮g/mL) or B box (50 ␮g/mL). Eighteen hours later, the mice were anesthetized as previously described for determinations of ileal mucosal permeability, bacterial translocation, and plasma alanine aminotransferase concentration.

Intestinal Barrier Function Intestinal mucosal permeability to FD4 was determined using an everted gut sac method as previously described by our laboratory with minor modifications necessitated by the use of mice instead of rats.15 Permeability was expressed as the mucosal-

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to-serosal clearance of FD4 calculated as previously described.15 To assess the extent of bacterial translocation, the abdominal cavity was opened and the viscera exposed using sterile technique. The MLN complex was removed, weighed, and disrupted with 1 mL PBS with 13 strokes of Dounce homogenizer fitted with the B pestle. Aliquots (200 ␮L) of the homogenate were coated onto plates containing brain-heart and MacConkey’s agar. The plates were examined 24 hours later after being aerobically incubated at 37°C. Visible colonies were counted and expressed as colonyforming units per gram of tissue.

Reverse-Transcription PCR Total RNA was extracted from harvested cells with chloroform and TRI Reagent (Molecular Research Center, Cincinnati, OH) exactly as directed by the manufacturer. The total RNA was treated with DNAFree (Ambion, Houston, TX) as instructed by the manufacturer using 10 U deoxyribonuclease I/10 ␮g RNA. Two micrograms of total RNA was reverse transcribed in a 40-␮L reaction volume containing 0.5 ␮g of oligo(dT)15 (Promega), 1 mmol/L of each deoxynucleoside triphosphate, 15 U AMV reverse transcriptase (Promega), and 1 U/␮L of recombinant RNasin ribonuclease inhibitor (Promega) in 5 mmol/L MgCl2 , 50 mmol/L KCl, 0.1% Triton X-100, and 10 mmol/L Tris-HCl (pH 8.0). The reaction mixture was preincubated at 21°C for 10 minutes before DNA synthesis. The reverse-transcription reaction was performed for 50 minutes at 42°C and was heated to 95°C for 5 minutes to terminate the reaction. Reaction mixtures (50 ␮L) for PCR were assembled using 5 ␮L of cDNA template, 10 U AdvanTaq Plus DNA Polymerase (Clontech), 200 ␮mol/L of each deoxynucleoside triphosphate, 1.5 mmol/L MgCl2 , and 1.0 ␮mol/L of each primer in 1⫻ AdvanTaq Plus PCR buffer. PCR reactions were performed in a Perkin Elmer Model 9700 thermocycler (Norwalk, CT). Amplification was initiated with 5 minutes of denaturation at 94°C. The PCR conditions for amplifying cDNA for iNOS was performed by denaturing at 94°C for 45 seconds, annealing at 61°C for 45 seconds, and polymerizing at 68°C for 1:30 minutes for 25 cycles. After the last cycle of amplification, the samples were incubated at 72°C for 7 minutes and then held at 4°C. The 5⬘ and 3⬘ primers for iNOS were 5⬘-GCG CCT GGA GGA CCT GGA TGA GA -3⬘ and 5⬘-CCC GGG AGG AGC TGA TGG AGT AGA-3⬘, respectively (Invitrogen); the expected product length was 341 base pairs. 18S ribosomal RNA was amplified to verify equal loading. For this reaction, the 5⬘ and 3⬘ primers were 5⬘-CCC GGG GAG GTA GTG ACG AAA AAT-3⬘ and 5⬘-CGC CCG CTC CCA AGA TCC AAC TAC-3⬘, respectively; the expected product length was 200 base pairs. The PCR conditions for amplifying cDNA for 18S amplification were performed by denaturing at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerizing at 68°C for 1 minute for 20 cycles. After the last cycle of amplification, the samples were incubated at 68°C for 3 minutes and then held at 4°C. Ten microliters of each PCR reaction was electrophoresed on a 2% agarose gel in 1⫻ TAE buffer, scanned in NucleoVision im-

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aging workstation (NucleoTech, San Mateo, CA), and quantified using GelExpert release 3.5.

Statistical Methods Results are presented as means ⫾ SEM. Data were analyzed using analysis of variance followed by least significant difference test or the Mann–Whitney U test. P ⬍ 0.05 was considered significant.

Results HMGB1 and B Box Increase the Permeability of Caco-2 Monolayers Confluent Caco-2 monolayers were incubated for 24 and 48 hours under control conditions or with graded concentrations of recombinant human HMGB1 or the truncated protein, B box. HMGB1 increased the permeability of Caco-2 monolayers to FD4 in a concentrationand time-dependent fashion (Figure 1A). A similar pattern was observed when monolayers were incubated with B box instead of HMGB1 (Figure 1B). When a more detailed time course study was performed comparing control medium and medium containing 1 ␮g/mL B box, 6 hours was the shortest period of incubation that resulted in a statistically significant increase in average FD4 clearance (12.5 ⫾ 2.8 vs. 25.4 ⫾ 1.3 nL 䡠 cm⫺2 䡠 h⫺1; n ⫽ 4 per condition; P ⬍ 0.05). All subsequent in vitro studies were performed using 1 ␮g/mL B box. B Box–Induced Hyperpermeability Is Not Secondary to Cell Death Preliminary studies showed no evidence of increased uptake of trypan blue when Caco-2 monolayers were incubated for 24 or 48 hours with B box (data not shown). To obtain additional information regarding the effect of B box on the viability of Caco-2 cells, we incubated cells growing on culture slides with control medium, 1 ␮g/mL B box, or 1 ␮g/mL M7 control protein for 48 hours. Other cells were incubated with 10 mmol/L KCN for 60 minutes. The cells were then stained with calcein AM and ethidium homodimer 1. The ester linkage of the former compound is cleaved by esterases present in the cytosol, and the green fluorescent product (calcein) is retained only by viable cells. The nuclear membrane of dead (but not living) cells is permeable to ethidium homodimer 1. Only scattered cells incubated with the control medium were stained by the red fluorescent ethidium homodimer 1 (Figure 2). The number of dead cells was similarly low following incubation with either B box or the M7 control protein. As expected, incubation with KCN killed all of the cells. Additional evidence that B box is not lethal to cells was obtained by using flow cytometry to detect apoptotic

Figure 1. The effect of (A) recombinant HMGB1 or a (B) truncated form of the protein, the B box, on the permeability of Caco-2 enterocytic monolayers. The cells were incubated for 24 (䊐) or 48 (■) hours under control conditions or with graded doses of either HMGB1 or B box. Permeability was assessed by measuring the transepithelial flux of FD4 added to the apical compartments. n ⫽ 6 –12 per condition. *P ⬍ 0.05 vs. control.

cells. The percentages of doubly stained (i.e., apoptotic) cells following 48 hours of incubation with control medium, medium containing 1 ␮mol/L M7 control protein, or 1 ␮mol/L B box were 1.2%, 0.43%, and 0.33%, respectively (Figure 3). To further confirm that B box–induced hyperpermeability was not secondary to cell death, monolayers were incubated with either control medium or B box for 48 hours. At the end of this period, the monolayers were washed extensively and incubated with fresh medium without B box. Permeability was reassessed every 12 hours over the subsequent 48 hours. As expected, incubation with B box significantly increased permeability to FD4 (Figure 4). However, following washout of the protein, barrier function progressively improved, suggesting that the cells were still viable.

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Figure 2. Effect of B box on the viability of Caco-2 cells. Caco-2 cells growing on collagen I– coated 8-well culture slides were incubated for 48 hours with (A) control medium or (B) medium containing 1 ␮g/mL M7 control protein or (C) with medium containing 1 ␮g/mL recombinant B box. Other cells were incubated for 60 minutes with 10 mmol/L KCN as a positive control for cell death (D). Viability of cells was assessed using a commercially available kit that uses uptake and retention of calcein (green fluorescence) as a marker for viability and nuclear staining, with ethidium homodimer 1 (red fluorescence) as a marker for cell death.

B Box–Induced Hyperpermeability Is Not Caused by Stable Soluble Factors Secreted by Caco-2 Cells We considered the possibility that stimulation by B box caused Caco-2 cells to release one or more soluble factors (e.g., cytokines) into the medium. We hypothesized that these factors, acting in an autocrine fashion, are responsible for B box–induced hyperpermeability. To test this hypothesis, we measured the permeability of Caco-2 monolayers after 48 hours of incubation under control conditions or with B box (1 ␮g/mL). We also measured the permeability of monolayers that were incubated for 48 hours with a 50:50 mixture of fresh medium and medium conditioned by Caco-2 monolayers incubated for 48 hours with B box (1 ␮g/mL). Because the conditioned medium presumably contained residual B box, we measured the

permeability of monolayers incubated for 48 hours with a 50:50 mixture of fresh and conditioned medium in the presence of a polyclonal anti–B box antibody (10 ␮g/mL). Finally, to verify the neutralizing activity of the anti–B box antibody, we measured the permeability of monolayers incubated for 48 hours with B box (1 ␮g/mL) plus anti–B box antibody (10 ␮g/mL). As expected, incubation with B box increased the permeability of Caco-2 monolayers (Figure 5). Incubation with a 50:50 mixture of fresh and conditioned media also significantly increased the permeability of Caco-2 monolayers. However, this effect was most likely due to the presence of residual B box in the conditioned medium, because the 50:50 mixture of fresh and conditioned media failed to increase permeability when a neutralizing anti–B box antibody was added.

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Figure 4. Reversibility of B box–induced hyperpermeability. The permeability of Caco-2 enterocytic monolayers was measured after 48 hours of exposure to either control medium (C) or medium containing 1 ␮g/mL B box (B). In additional experiments, monolayers that had been incubated with B box for 48 hours were washed extensively and then incubated in B box–free medium. Permeability was measured at 24, 36, and 48 hours after incubation in B box–free medium (R24, R36, and R48, respectively). n ⫽ 6 per condition. †P ⬍ 0.05 vs. control; *P ⬍ 0.05 vs. B box.

cubated with B box for 48 hours (Figure 6). However, when Caco-2 cells were exposed to B box in the presence of 10 ␮g/mL of anti-RAGE antibody, the increase in permeability was only about 1⁄3 as great as the increase caused by incubation with B box alone. When monolayers were incubated with B box and an irrelevant antibody, 10 ␮g/mL of anti-myosin light chain, the clearance of FD4 was similar to that observed for monolayers incubated with just B box. Figure 3. Flow cytometric assessment of apoptosis of Caco-2 cells using the APO-BrdU TUNEL assay kit (Molecular Probes). Caco-2 cells were incubated for 48 hours under (A ) control conditions, (B) with 1 ␮g/mL M7 control protein, or (C ) with 1 ␮g/mL B box. To verify the performance of the assay, results are also shown for (D) positive and (E ) negative control cells supplied with the kit. DNA was nick end labeled with BrdU, and incorporated BrdU was detected with green fluorescent Alexa Fluor 488 dye-labeled anti-BrdU antibody according to the instructions from the manufacturer. Nuclei were also stained with the red fluorescent compound propidium iodide. Results are displayed as bivariate dot plots of red (y-axis) vs. green (x-axis) fluorescence. Cells in the upper right quadrant were scored as being apoptotic.

Anti-RAGE Antibody Partially Prevents B Box–Induced Hyperpermeability Previous studies have shown that amphoterin (another name for HMGB1) binds to RAGE and can trigger cellular responses following ligation of this receptor.16,17 Accordingly, we sought to determine whether coincubation with an anti-RAGE antibody would attenuate the increase in permeability caused by exposing Caco-2 monolayers to B box. Permeability was significantly increased when monolayers were in-

Figure 5. Effect of conditioned medium on permeability of Caco-2 monolayers. The permeability of Caco-2 enterocytic monolayers was measured after 48 hours of exposure to either control medium (C) or medium containing 1 ␮g/mL B box (B). In additional experiments, conditioned medium was collected from monolayers that had been incubated with B box for 48 hours. Monolayers were incubated for 48 hours with a 50:50 mixture of fresh (control) medium and conditioned medium (CM). Some monolayers were incubated with either a 50:50 mixture of fresh (control) medium and conditioned medium plus anti–B box antibody (10 ␮g/mL) or B box and anti–B box antibody (CM ⫹ a-B and C ⫹ a-B, respectively). n ⫽ 6 per condition. *P ⬍ 0.05 vs. C; †P ⬍ 0.05 vs. B; ‡P ⬍ 0.05 vs. CM.

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der control conditions or with B box for varying periods of time. DNA binding of NF-␬B was increased at 15 minutes and 3 hours after cells were exposed to B box (Figure 8A ). To confirm the identity of the activated protein-DNA complex, binding assays were performed with samples that were preincubated with specific antibodies directed against p50 and p65. We observed both a supershifted band and decreased intensity of the NF-␬B band with the p65 antibody (Figure 8B). Moreover, binding of the protein to labeled NF-␬B binding element was completely inhibited by a 100-fold excess of unlabeled NF-␬B duplex oligonucleotide but not by a similar molar excess of unlabeled HIF-1 duplex oligonucleotide. Figure 6. Effect on permeability of coincubating Caco-2 enterocytic monolayers with B box and anti-RAGE antibody. Monolayers were coincubated for 48 hours with control medium (C), medium containing 10 ␮g/mL of anti-RAGE antibody (a-RAGE), medium containing 10 ␮g/mL of antibody against myosin light chain (a-Myo), medium containing 1 ␮g/mL B box (B), medium containing B box and anti-RAGE antibody (B ⫹ a-RAGE), or medium containing B box and anti-myosin light chain antibody (B ⫹ a-Myo). n ⫽ 6 per condition. *P ⬍ 0.05 vs. control; †P ⬍ 0.05 vs. B box alone.

Incubation of Caco-2 Cells With B Box Induces iNOS Expression and Increases NO Production Because we previously showed that exposing Caco-2 monolayers to interferon gamma, either alone18 or in combination with tumor necrosis factor and interleukin 1␤,19,20 induces iNOS expression and increases the release of NO by these cells, we hypothesized that exposing Caco-2 cells to B box might also lead to induction of iNOS messenger RNA expression and increased NO production. Incubation of Caco-2 cells with B box for 48 hours increased steady-state iNOS messenger RNA levels as assessed by semiquantitative reverse-transcription PCR (Figure 7A). In addition, the concentration of nitrite plus nitrate in culture supernatants was significantly increased after 48 hours of incubation with B box (Figure 7B). Coincubation with anti-RAGE antibody significantly blunted the increase in NO production induced by B box, whereas with an irrelevant antibody had no effect on B box–induced NO production. Incubation of Caco-2 Cells With B Box Activates Nuclear Binding of NF-␬B Increased expression of the iNOS gene in human enterocytes is partially dependent on activation of NF-␬B.21 Accordingly, we sought to determine whether B box increases NF-␬B DNA binding. Nuclear extracts were prepared from cells incubated un-

Figure 7. Effect of B box on (A) iNOS messenger RNA expression in Caco-2 cells and (B) nitrite/nitrate concentrations in Caco-2 culture supernatants. For determinations of iNOS expression, Caco-2 cells growing as monolayers in collagen-coated 6-well dishes were incubated for 48 hours with control medium, medium containing 1 ␮g/mL of a recombinant polypeptide (M7) generated by transforming E. coli with a GST vector lacking sequences coding for HMGB1 or B box, or medium containing 1 ␮g/mL of B box. Reverse-transcription PCR was performed as described in Materials and Methods. For determination of NO2⫺/NO3⫺ concentrations in supernatants, Caco-2 cells growing as monolayers in collagen-coated 6-well dishes were incubated for 48 hours with control medium (C), medium containing 10 ␮g/mL of anti-RAGE antibody (a-RAGE), medium containing 10 ␮g/mL of antibody against myosin light chain (a-Myo), medium containing 1 ␮g/mL B box (B), medium containing B box and anti-RAGE antibody (B ⫹ a-RAGE), or medium containing B box and anti-myosin light chain antibody (B ⫹ a-Myo). n ⫽ 4 per condition. *P ⬍ 0.05 vs. control; †P ⬍ 0.05 vs. B box.

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B Box Increases Ileal Mucosal Permeability and Promotes Bacterial Translocation and Hepatocellular Injury in Mice

Figure 8. Effect of B box on DNA binding of NF-␬B. Caco-2 cells growing as monolayers in collagen-coated 6-well dishes were incubated for 15 or 180 minutes with control medium or medium containing 1 ␮g/mL of B box. After these incubation periods, nuclear extracts were prepared and electrophoretic mobility shift assay performed. (A) Incubation with B box increased NF-␬B DNA binding. (B) Supershift and cold competition assays were performed to verify the identity of the putative NF-␬B band. Results shown are from a representative experiment, which was repeated twice.

With the anti-p50 antibody, the density of the NF-␬B band was somewhat diminished.

When injected into mice, B box increased ileal mucosal permeability in a time-dependent fashion (Figure 10A). B box also promoted bacterial translocation as evidenced by greater numbers of viable colony-forming units compared with control mice when MLN homogenates were cultured for total aerobic and facultative organisms on brain-heart infusion agar (Figure 10B) or only Gram-negative organisms on MacConkey’s agar (data not shown). Six hours after injection of B box, ileal permeability remained normal and bacterial translocation was no greater than in controls injected with M7. However, by 12 hours, both ileal mucosal permeability and the degree of bacterial translocation were significantly greater in B box– challenged mice compared with animals treated with M7. Whereas evidence of gut barrier dysfunction was not evident until 12 hours after injecting B box, biochemical evidence of hepatocellular injury was evident at 6 hours and remained more or less constant over the ensuing 12-hour period of observation (Figure 10C). iNOSⴚ/ⴚ Mice Are Protected From B Box– Induced Gut Barrier Dysfunction We measured ileal mucosal permeability to FD4 in 2 iNOS⫺/⫺ mice after injection of B box (50 ␮g per mouse). For comparison, we also studied 2 iNOS⫺/⫺ mice injected with the M7 control protein. FD4 clearances 18 hours after injection of M7 were 0.08 and 0.09

Pharmacologic Studies Suggest That B Box–Induced Hyperpermeability Is Mediated by ONOOⴚ The permeability of Caco-2 monolayers was assessed after being incubated for 48 hours under control conditions or with B box in the absence or presence of various pharmacologic agents. Coincubating monolayers with B box and pyrrolidine diothiocarbamate (PDTC), an inhibitor of NF-␬B activation, completely prevented the development of increased permeability (Figure 9). Both L-NIL, an isoform selective iNOS inhibitor, and 2-(4-carboxyphenyl)4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), an NO scavenger,22 also prevented B box– induced hyperpermeability. Tiron, which scavenges O2⫺,23 partially prevented B box–induced hyperpermeability, whereas FeTPPS, which is an ONOO⫺ decomposition catalyst,24 completely blocked it.

Figure 9. Effect of pharmacologic agents on B box–induced hyperpermeability. Caco-2 enterocytic monolayers were incubated for 48 hours with one of the following solutions: control medium (C), medium containing 1 ␮g/mL B box, or medium containing B box plus 100 ␮mol/L PDTC, 20 ␮mol/L L-NIL, 100 ␮mol/L C-PTIO, 10 mmol/L Tiron, or 50 ␮mol/L FeTPPS. n ⫽ 12 per condition. *P ⬍ 0.05 vs. control; †P ⬍ 0.05 vs. B box.

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necessary for B box–induced intestinal barrier dysfunction. Interestingly, circulating alanine aminotransferase concentrations at 18 hours were similar for iNOS⫺/⫺ mice injected with B box (23 and 64 U/L) or M7 (25 and 27 U/L), suggesting that iNOS induction also may be important for B box–induced hepatocellular injury.

Discussion

Figure 10. Effect of B box on (A) ileal mucosal permeability, (B) bacterial translocation to MLN, and (C) plasma alanine aminotransferase concentration in mice. Groups of male C57BL/6 mice were injected intraperitoneally with 1 mL of PBS, a suspension of E. coli serotype O111:B4 LPS in PBS (0.1 mg/mL), M7 protein dissolved in PBS (50 ␮g/mL), or B box dissolved in PBS (50 ␮g/mL). Groups were killed 6, 12, or 18 hours later. n ⫽ 5 per time point and condition. *P ⬍ 0.05 vs. time-matched value in M7 or PBS groups.

pL 䡠 min⫺1 䡠 cm⫺1. FD4 clearances 18 hours after injection of B box were 0.11 and 0.11 pL 䡠 min⫺1 䡠 cm⫺1. Thus, there was at most a 30% increase in permeability in the B box–treated iNOS⫺/⫺ mice relative to the M7-treated iNOS⫺/⫺ mice. In contrast, when wild-type C57Bl/6J mice were injected with the same dose of B box, we measured an ⬃300% increase in ileal mucosal permeability relative to the permeability measured in animals injected with M7. Furthermore, in contrast to the results obtained using wild-type mice, there was no bacterial translocation to MLN in any of the iNOS⫺/⫺ mice, whether injected with B box or M7. Collectively, these data support the view that iNOS induction is

The intestinal epithelium normally functions as a selective barrier, permitting the absorption of nutrients, electrolytes, and water but restricting passage from the lumen into the systemic compartment of larger, potentially toxic compounds or microbes. In both experimental animals and humans, a number of conditions that are associated with either local (i.e., mucosal) or systemic inflammation are known to cause alterations in intestinal barrier function. These conditions include inflammatory bowel disease,25,26 thermal injury,27,28 multisystem trauma,29 endotoxemia,30 and sepsis.31 In these conditions, derangements in gut barrier function have been detected by measuring increased permeation of a hydrophilic macromolecular probe across mucosa or by quantifying increased translocation of viable enteric bacteria to mesenteric lymph nodes or other organs. The mechanisms responsible for altered barrier function in these conditions are incompletely understood, but it is well established that certain proinflammatory cytokines, such as interferon gamma and tumor necrosis factor, are capable of increasing the permeability of cultured enterocytic monolayers.18,32–34 The data presented here support the view that HMGB1 is another cytokine that is capable of inducing alterations in intestinal barrier function both in vitro and in vivo. We showed that both recombinant human HMGB1 and a 74-residue mutant form of the protein, B box, increased the permeability of Caco-2 monolayers to the hydrophilic macromolecular probe, FD4. This effect was concentration dependent and increased progressively over a 48-hour period of observation. We used the B box truncated protein for all of our subsequent experiments, because Yang et al. have localized the proinflammatory activity of HMGB1 to this region of the molecule.12 The increase in permeability induced by B box was probably not caused by cell death, because we saw no evidence that incubation of Caco-2 cultures with B box increased the number of dead cells as assessed by nuclear staining with the fluorescent probe, ethidium homodimer 1. Furthermore, we found no evidence from studies using TUNEL staining and flow cytometry that exposure of Caco-2 cultures to B box increased the number of apoptotic cells. Finally, the increase in permeability induced by B box

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was reversible following removal of the recombinant protein. Molecular cloning studies have shown that the coding sequence of HMGB1 is identical to the sequence for amphoterin, a compound previously identified as a cytoplasmic and extracellular protein involved in neurite outgrowth.35 A number of studies have shown that amphoterin binds to RAGE,16,17,36 a member of the immunoglobulin superfamily of cell surface molecules whose repertoire of ligands also includes advanced glycation end products, amyloid fibrils, and S100/calgranulins.16 Caco-2 cells are known to express RAGE.37 In our studies, we found that coincubation of Caco-2 cells with anti-RAGE antibody partially inhibited both B box– induced hyperpermeability and B box–induced NO production. These findings support the notion that activation of RAGE by the B box of HMGB1 is important for promoting the activation of enterocytes, leading to deranged barrier function on this basis. The permeability of Caco-2 monolayers is increased when the cells are incubated with various NO donors, such as S-nitroso-N-acetylpenicillamine or sodium nitroprusside.38,39 This effect does not seem to be mediated by NO directly but rather by ONOO⫺, a potent oxidizing and nitrosating agent that is formed when NO reacts with O⫺ 2. Support for this view comes from studies showing that S-nitroso-N-acetylpenicillamine–induced hyperpermeability is augmented by addition of diethyldithiocarbamate, a superoxide dismutase inhibitor, or pyrogallol, an 39 Furthermore, S-nitroso-N-acetylpenicilO⫺ 2 generator. lamine–induced hyperpermeability is blocked by Tiron, ⫺ an agent that scavenges O⫺ 2 , as well as various ONOO scavengers, such as urate and deferoxamine.39 Hyperpermeability of Caco-2 monolayers induced by interferon gamma or a combination of interferon gamma, tumor necrosis factor, and interleukin 1␤ is associated with iNOS induction and can be blocked by inhibiting NO production or scavenging ONOO⫺.18,19 These findings support the view that cytokine-induced intestinal epithelial hyperpermeability is mediated, at least in part, by the formation ONOO⫺. This view is further supported by studies showing that pharmacologic inhibition of iNOS ameliorates LPS-induced intestinal mucosal hyperpermeability in vivo.9 Herein, we showed that B box induces iNOS messenger RNA expression in Caco-2 cells and promotes the release of NO2⫺/NO3⫺ into culture supernatants. B box– induced NO synthesis was partially inhibited when the cells were coincubated with an anti-RAGE antibody, suggesting that binding of B box to the RAGE receptor initiates a signaling cascade that ultimately leads to

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induction of iNOS expression. Activation of RAGE by various ligands has been shown to activate NF-␬B.40,41 Moreover, activation of NF-␬B is known to be an important step leading to iNOS induction in enterocytes,21 and in our studies, B box was shown to promote nuclear localization of this transcription factor. In other studies, we showed that B box–induced hyperpermeability was blocked by pharmacologic inhibition of multiple steps along the pathway leading to ONOO⫺ formation and starting with NF-␬B activation. Thus, we blocked B box–induced hyperpermeability with PDTC (a commonly used inhibitor of NF-␬B activation), L-NIL (an iNOS inhibitor), C-PTIO (an NO scavenger), Tiron (an O2 䡠 ⫺ scavenger), and FeTPPS (an ONOO⫺ decomposition catalyst). Collectively, these data support the view that B box increased the permeability of Caco-2 monolayers through a mechanism involving iNOS induction and ONOO⫺ formation. In addition to performing studies using Caco-2 monolayers as a model of the intestinal epithelium, we also evaluated the effects of B box on gut barrier function in vivo. When mice were challenged with a sublethal dose of B box, we observed evidence of both increased mucosal permeability to FD4 and increased bacterial translocation to MLN. These alterations in barrier function developed gradually over time. These findings support the notion that HMGB1 B box is capable of causing derangements in gut barrier function. We did not perform an extensive series of experiments to determine whether the iNOS/ NO/ONOO⫺ pathway is important in the development of B box–induced intestinal barrier damage in vivo. Nevertheless, the results from a limited experiment using a small number of iNOS⫺/⫺ mice were consistent with our in vitro observations, because the iNOS-deficient animals seemed to be protected from B box–induced mucosal hyperpermeability and bacterial translocation. It seems unlikely that the small amount of LPS present in our B box preparation could have accounted for the observed effects of the recombinant protein when injected into mice. The B box preparation contained 19 pg/␮g LPS, which is equivalent to about 1 ng LPS per mouse. Mice are relatively insensitive to the toxic effects of LPS; in our studies, a much larger dose of LPS (100 ␮g per mouse or approximately 100,000 times the contaminating dose in the B box– challenged animals) was necessary to observe a comparable degree of gut barrier dysfunction. Along these same lines, it is important to note that LPS contamination was almost certainly not responsible for B box–induced hyperpermeability in our

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in vitro studies, because Caco-2 cells fail to respond to LPS.42 We considered the possibility that B box–induced hyperpermeability was not caused by the protein directly but rather was mediated by one or more soluble factors (e.g., cytokines) secreted into the medium when Caco-2 cells are stimulated by HMGB1 or B box. However, when we performed experiments using medium conditioned by B box–stimulated Caco-2 cells, we observed that all of the permeability-enhancing activity present was neutralized by anti–B box antibody. In view of these findings, we think it is unlikely that B box–induced hyperpermeability is mediated by the release of one or more stable soluble factors released by Caco-2 cells. Nevertheless, our results are insufficient to rule out the importance of labile factors as secondary mediators of HMGB1-induced barrier dysfunction. HMGB1 is released by monocytes, macrophages, and pituicytes stimulated with LPS, interleukin 1, or tumor necrosis factor.6,43 Murine erythroleukemia cells also release large quantities of HMGB1 into the extracellular medium following stimulation with hexamethylene bisacetamide, an agent that promotes terminal differentiation of these cells.44 Thus, it is likely that numerous cell types can release HMGB1 following appropriate stimulation. It is unknown at present whether immunostimulated intestinal epithelial cells secrete HMGB1, but this notion seems plausible and would create the possibility that HMGB1 can act in an autocrine fashion to promote intestinal barrier dysfunction. Further studies to evaluate this concept are clearly warranted. In summary, the studies presented here indicate that HMGB1 and its B-box domain are capable of causing alterations in intestinal barrier function both in vitro and in vivo. Our in vitro studies suggest that both binding of HMGB1 to RAGE and iNOS induction are important in this phenomenon. Taken together with other recent studies regarding the role of HMGB1 as a late-acting cytokine mediator of endotoxemia and sepsis,6,7,12 the findings presented here suggest that HMGB1 (or its B-box domain) are attractive targets for the development of new therapeutic agents for the treatment or prevention of organ system dysfunction associated with systemic inflammation.

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Received December 18, 2001. Accepted May 31, 2002. Address requests for reprints to: Mitchell P. Fink, M.D., Department of Critical Care Medicine, University of Pittsburgh Medical School, 616 Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. e-mail: fi[email protected]; fax: (412) 647-5258. Supported by grants GM58484, GM37631, and GM62508 from the National Institutes of Health.