Volume 128, Issue 4, Pages 1012-1022 (April 2005) Increased expression and function of integrins in enterocytes by endotoxin impairs epithelial restitution  Faisal G. Qureshi, Cynthia Leaphart, Selma Cetin, Jun Li, Anatoly Grishin, Simon Watkins, Henri R. Ford, David J. Hackam  Gastroenterology  Volume 128, Issue 4, Pages 1012-1022 (April 2005) DOI: 10.1053/j.gastro.2005.01.052 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Newborn intestinal injury resembling NEC is associated with impaired epithelial restitution and increased expression of α3- and β1-integrins in the intestinal mucosa. Intestinal injury was induced in newborn rats using a combination of gavaged formula and systemic hypoxia. (A) The normal villus architecture of the ileum in control, breastfed animals (inset shows villi at higher magnification). (B) Histopathology of animals with experimental injury resembling NEC showing inflammation of the lamina propria, edema, and necrosis similar to that observed in human NEC (higher magnification shown in inset). (C) The rate of enterocyte migration along the crypt villus axis—a measure of intestinal restitution—in control and rats with intestinal injury (NEC), as determined in the Materials and Methods section. The mean and SEM of over 15 separate experiments are shown. *P < .05 by Student t test. (D) Mucosal scrapings were prepared from the terminal ilea of control newborn rats and rats with intestinal injury (NEC), subjected to SDS-PAGE, immunoblotted with antibodies against β1-integrin, and then stripped and reprobed for actin. Samples from 3 paired experimental groups (labeled 1, 2, and 3) are shown. Representative of 5 separate experiments. (E and F) Mucosal scrapings were purified from the ilea of newborn mice that were injected either with purified LPS (2 mg/mL) or normal saline, and immunoblotted with antibodies against (E) β1- or (F) α3-integrins. Representative of 3 separate experiments. Gastroenterology 2005 128, 1012-1022DOI: (10.1053/j.gastro.2005.01.052) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Endotoxin increases the expression of integrins in IEC-6 enterocytes in an LY294002- or wortmannin-dependent manner. (A) IEC-6 cells were treated with (lps) or without (ctrl) LPS in the presence (lps + ly) or absence of the PI3K inhibitors LY294002 or wortmannin, and immunoblotted against β1- or α3-integrins. Representative of 3 separate experiments. (B) Relative expression of β1- and or α3-integrins in IEC-6 cells. ■, β1-integrin; □, α3-integrin. (C) IEC-6 cells were treated with LPS (lps) or left untreated (ctrl), stained for β1-integrin, and analyzed by flow cytometry. Representative of over 5 separate experiments. (D) IEC-6 cells were either untreated (ctrl) or treated with LPS (lps) in the presence of LY294002 (lps + ly), wortmannin (lps + wm), LY294002 alone (ly), or wortmannin alone (wm), stained for β1-integrin, and analyzed by flow cytometry. The proportion of immunopositive cells is shown. Representative of over 5 experiments. (E) IEC-6 cells were stimulated with epidermal growth factor or left unstimulated (ctrl) in the presence (egf + ly) or absence of LY294002 (egf) or LY294002 alone (ly), and probed for phospho-AKT (pakt). Membranes were stripped and reprobed for total AKT (akt). Representative of 3 separate experiments. (F) IEC-6 cells either were left untreated (ctrl) or treated with LPS (lps), LPS and wortmannin (lps + wm), or wortmannin alone (wm), then immunoblotted for total KT and phospho-AKT as in E. (G) Quantification of results from 3 separate experiments. Comparisons by ANOVA, *P < .05 compared with control cells, **P < .05 compared with LPS-treated cells. Gastroenterology 2005 128, 1012-1022DOI: (10.1053/j.gastro.2005.01.052) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Endotoxin increases the expression of integrins in several enterocyte cell lines in an LY294002- or wortmannin-dependent manner. (A) IEC-18, (B) HT-29, or (C) Caco-2 enterocytes were treated with LPS (lps), in the presence of LY294002 (lps + ly) or wortmannin (lps + wm), or left untreated (ctrl), then probed for β1-integrin as shown. Blots then were stripped and reprobed for α3-integrin then actin. (D and E) Quantification of integrin expression for 3 separate experiments. ■, IEC-18; ▦ HT-29; □, Caco-2. Comparisons by ANOVA, *P < .05 compared with control cells, **P < .05 compared with LPS-treated cells. Gastroenterology 2005 128, 1012-1022DOI: (10.1053/j.gastro.2005.01.052) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Endotoxin increases the surface delivery of β1-integrin in IEC-6 cells in an LY294002-dependent manner. IEC-6 cells either were treated or not treated with LPS or LPS + LY294002 or LY294002 alone, immunostained for β1-integrin, and examined by fluorescence confocal microscopy. Arrowheads indicate intracellular integrins; arrows indicate surface integrins. Images are representative of 4 separate experiments. Gastroenterology 2005 128, 1012-1022DOI: (10.1053/j.gastro.2005.01.052) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Endotoxin increases the binding of fibronectin-coated beads to IEC-6 cells in an LY294002- or wortmannin-dependent manner. (A and B) Fibronectin-coated latex beads were incubated with (A) IEC-6 or (B) IEC-18 cells in the presence of either β1-integrin, LPS, LPS + β1, LPS + LY294002, LY294002, LPS + wortmannin (lps + wm), or wortmannin alone (wm) for 12 hours. Cells were washed, and the number of attached beads was counted. Representative of 5 separate experiments. *P < .05 compared with control, **,†,‡P < .05 compared with LPS, by ANOVA. (C and D) Cells either were (C) untreated or (D) treated with LPS, immunostained against β1-integrin, and examined by immunofluorescent confocal microscopy by obtaining slices at the level of the cover slip (bottom) or through the beads (top). A representative differential interference contrast (dic) image and fluorescent image is displayed. Representative of 5 separate experiments. Gastroenterology 2005 128, 1012-1022DOI: (10.1053/j.gastro.2005.01.052) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 LPS impairs enterocyte migration through increased β1-integrin function. Confluent IEC-6 or IEC-18 cells were plated on glass cover slips, scraped with a cell scraper, treated with 50 μg/mL LPS in the presence or absence of anti-β1-integrin antibody, and allowed to migrate at 37°C. Shown are images from a typical experiment of the migration of (A and B) control, (C and D) LPS, and (E and F) LPS + β1-treated IEC-6 cells. The dotted line indicates the position of the cells at the edge of the scraped wound, at t = 0. Quantification of the migration rate for (G) IEC-6 and (H) IEC-18 cells, calculated as the mean distance traveled by 50 individual cells in an orientation perpendicular to the axis of the scrape over the time course of the experiment. Representative of 5 experiments. *P < .05 compared with control, **P < .05 compared with LPS, by ANOVA. Gastroenterology 2005 128, 1012-1022DOI: (10.1053/j.gastro.2005.01.052) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Increased surface localization of β1-integrins in LPS-treated compared with untreated enterocytes during migration. Confluent IEC-6 cells were induced to migrate across a scraped wound and the distribution of β1-integrins in cells at the leading edge (insets) was determined 12 hours after scraping. Confocal images showing the expression of β1-integrins in (A) untreated migrating cells compared with (B) LPS (50 μg/mL) treated nonmigrating cells. The insets show representative wound edges, arrowheads indicate surface integrin expression, arrows indicate direction of migration, Ø denotes blocked migration. Representative of 5 experiments. Gastroenterology 2005 128, 1012-1022DOI: (10.1053/j.gastro.2005.01.052) Copyright © 2005 American Gastroenterological Association Terms and Conditions