Volume 123, Issue 3, Pages 790-802 (September 2002) 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, Mitchell P. Fink  Gastroenterology  Volume 123, Issue 3, Pages 790-802 (September 2002) DOI: 10.1053/gast.2002.35391 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 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 (□) 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. Gastroenterology 2002 123, 790-802DOI: (10.1053/gast.2002.35391) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 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 μg/mL M7 control protein or (C) with medium containing 1 μg/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. Gastroenterology 2002 123, 790-802DOI: (10.1053/gast.2002.35391) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 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 μg/mL M7 control protein, or (C) with 1 μg/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. Gastroenterology 2002 123, 790-802DOI: (10.1053/gast.2002.35391) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 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 μg/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. Gastroenterology 2002 123, 790-802DOI: (10.1053/gast.2002.35391) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 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 μg/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 μg/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. Gastroenterology 2002 123, 790-802DOI: (10.1053/gast.2002.35391) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 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 μg/mL of anti-RAGE antibody (a-RAGE), medium containing 10 μg/mL of antibody against myosin light chain (a-Myo), medium containing 1 μg/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. Gastroenterology 2002 123, 790-802DOI: (10.1053/gast.2002.35391) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 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 μg/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 μg/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 μg/mL of anti-RAGE antibody (a-RAGE), medium containing 10 μg/mL of antibody against myosin light chain (a-Myo), medium containing 1 μg/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. Gastroenterology 2002 123, 790-802DOI: (10.1053/gast.2002.35391) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Effect of B box on DNA binding of NF-κB. 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 μg/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-κB DNA binding. (B) Supershift and cold competition assays were performed to verify the identity of the putative NF-κB band. Results shown are from a representative experiment, which was repeated twice. Gastroenterology 2002 123, 790-802DOI: (10.1053/gast.2002.35391) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 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 μg/mL B box, or medium containing B box plus 100 μmol/L PDTC, 20 μmol/L L-NIL, 100 μmol/L C-PTIO, 10 mmol/L Tiron, or 50 μmol/L FeTPPS. n = 12 per condition. *P < 0.05 vs. control; †P < 0.05 vs. B box. Gastroenterology 2002 123, 790-802DOI: (10.1053/gast.2002.35391) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 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 μg/mL), or B box dissolved in PBS (50 μg/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. Gastroenterology 2002 123, 790-802DOI: (10.1053/gast.2002.35391) Copyright © 2002 American Gastroenterological Association Terms and Conditions