Increased expression and function of integrins in enterocytes by endotoxin impairs epithelial restitution
2005; Elsevier BV; Volume: 128; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2005.01.052
ISSN1528-0012
AutoresFaisal Qureshi, Cynthia L. Leaphart, Selma Çetin, Jun Li, Anatoly Grishin, Simon C. Watkins, Henri R. Ford, David J. Hackam,
Tópico(s)Neonatal Respiratory Health Research
ResumoBackground & Aims: Experimental necrotizing enterocolitis (NEC) is characterized by circulating endotoxin (lipopolysaccharide [LPS]) and impaired enterocyte migration. We hypothesized that LPS increases integrin function and cell-matrix adhesion, leading to impaired enterocyte migration in the pathogenesis of NEC. Methods: NEC-like intestinal injury was induced in newborn rats by hypoxia/gavage feedings, and restitution was determined by assessing bromodeoxyuridine-labeled enterocytes along the crypt-villus axis. Newborn mice were injected with 5 mg/kg LPS. IEC-6 cells were treated with LPS ± LY294002 or wortmannin, and β1- and α3-integrins were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunofluorescence. β1-integrin function was determined by adherence of fibronectin beads to IEC-6 monolayers. Migration of IEC-6 cells into a scraped wound was measured by time-lapse microscopy. Results: Newborn intestinal injury was associated with decreased intestinal restitution and increased α3- and β1-integrin expression in the ileal mucosa, which also was observed after LPS injection. In IEC-6 cells, LPS caused an increase in the expression of α3- and β1-integrins, a shift of β1-integrins from the cytoplasm to the plasma membrane and an increase in fibronectin bead adhesion during which β1-integrins accumulated underneath attached beads. These effects could be reversed with LY294002 or wortmannin, suggesting phosphatidylinositol-3-phosphate kinase (PI3K) dependence. The increased integrin-matrix adhesion by LPS led to an inhibition of enterocyte migration, which could be reversed by anti-β1-antibodies. Conclusions: Enterocyte migration is inhibited by LPS through increased expression and function of α3- and β1-integrins. Modulation of enterocyte migration via integrins may provide novel insights into the pathogenesis of NEC, in which intestinal restitution is impaired. Background & Aims: Experimental necrotizing enterocolitis (NEC) is characterized by circulating endotoxin (lipopolysaccharide [LPS]) and impaired enterocyte migration. We hypothesized that LPS increases integrin function and cell-matrix adhesion, leading to impaired enterocyte migration in the pathogenesis of NEC. Methods: NEC-like intestinal injury was induced in newborn rats by hypoxia/gavage feedings, and restitution was determined by assessing bromodeoxyuridine-labeled enterocytes along the crypt-villus axis. Newborn mice were injected with 5 mg/kg LPS. IEC-6 cells were treated with LPS ± LY294002 or wortmannin, and β1- and α3-integrins were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunofluorescence. β1-integrin function was determined by adherence of fibronectin beads to IEC-6 monolayers. Migration of IEC-6 cells into a scraped wound was measured by time-lapse microscopy. Results: Newborn intestinal injury was associated with decreased intestinal restitution and increased α3- and β1-integrin expression in the ileal mucosa, which also was observed after LPS injection. In IEC-6 cells, LPS caused an increase in the expression of α3- and β1-integrins, a shift of β1-integrins from the cytoplasm to the plasma membrane and an increase in fibronectin bead adhesion during which β1-integrins accumulated underneath attached beads. These effects could be reversed with LY294002 or wortmannin, suggesting phosphatidylinositol-3-phosphate kinase (PI3K) dependence. The increased integrin-matrix adhesion by LPS led to an inhibition of enterocyte migration, which could be reversed by anti-β1-antibodies. Conclusions: Enterocyte migration is inhibited by LPS through increased expression and function of α3- and β1-integrins. Modulation of enterocyte migration via integrins may provide novel insights into the pathogenesis of NEC, in which intestinal restitution is impaired. Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in neonates and is a significant cause of long-term morbidity among survivors.1Guthrie S.O. Gordon P.V. Thomas V. Thorp J.A. Peabody J. Clark R.H. Necrotizing enterocolitis among neonates in the United States.J Perinatol. 2003; 23: 278-285Crossref PubMed Scopus (318) Google Scholar Clinical manifestations of NEC include abdominal distention, feeding intolerance, and multisystem organ failure, which develop as a result of diffuse intestinal necrosis.2Noerr B. Current controversies in the understanding of necrotizing enterocolitis.Adv Neonatal Care. 2003; 3: 107-120Crossref PubMed Scopus (65) Google Scholar The pathogenesis of NEC remains incompletely understood, although evidence suggests that it develops after a breakdown in the intestinal mucosal barrier in the preterm infant.3Chan K.L. Hui C.W. Chan K.W. Fung P.C. Wo J.Y. Tipoe G. Tam P.K. 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To this end, we recently showed that intestinal restitution is impaired in an experimental model of newborn intestinal injury resembling human NEC.8Cetin S. Ford H.R. Sysko L.R. Agarwal C. Wang J. Neal M.D. Baty C. Apodaca G. Hackam D.J. Endotoxin inhibits intestinal epithelial restitution through activation of rho-GTPase and increased focal adhesions.J Biol Chem. 2004; 279: 24592-24600Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar The current study focuses specifically on the molecular mechanisms governing impaired enterocyte migration during the pathogenesis of NEC. Although a variety of cytokines may be important in the initiation and propagation of the intestinal injury in NEC, the gram-negative bacterial cell wall component endotoxin (lipopolysaccharide, LPS) is likely to act at one of the earliest time points in this process. LPS is one of the most abundant proinflammatory stimuli in the gastrointestinal tract. 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Endotoxin inhibits intestinal epithelial restitution through activation of rho-GTPase and increased focal adhesions.J Biol Chem. 2004; 279: 24592-24600Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar This leads to an increase in the degree of adhesiveness with which the enterocyte binds to the extracellular matrix. By sticking too tightly to the matrix, enterocytes that have been exposed to endotoxin are unable to move.8Cetin S. Ford H.R. Sysko L.R. Agarwal C. Wang J. Neal M.D. Baty C. Apodaca G. Hackam D.J. Endotoxin inhibits intestinal epithelial restitution through activation of rho-GTPase and increased focal adhesions.J Biol Chem. 2004; 279: 24592-24600Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar Importantly, the receptors that govern enterocyte-matrix adhesion and the mechanisms by which they may be affected during either experimental NEC or after endotoxin treatment remain incompletely understood. 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Characterization by morphologic and immunologic criteria.J Cell Biol. 1979; 80: 248-265Crossref PubMed Scopus (699) Google Scholar Antibodies were obtained as follows: β1- and α3-integrin were from BD Transduction Laboratories (Palo Alto, CA), AKT and phospho-AKT were from Cell Signaling Technology (Beverly, MA), Cy3 and Alexa-488 secondary antibodies were from Jackson Immunoresearch Laboratories (West Grove, PA) and Molecular Probes (Eugene, OR), respectively. Contaminant free, purified LPS was purchased from Sigma (St. Louis, MO). The phosphatidylinositol-3-phosphate kinase (PI3K) inhibitors LY294002 and Wortmannin were from Biomol (Plymouth Meeting, PA) and Sigma, respectively. Epidermal growth factor was from Collaborative Research (Bedford, MA). Where indicated, cells were treated with LPS (50 μg/mL) with or without LY294002 (10 μmol/L) or wortmannin (150 nmol/L) for 12 hours. 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Cells then were washed twice with PBS/1% bovine serum albumin and resuspended in 500 μL PBS and analyzed immediately by FACS Calibur (Becton-Dickinson, San Jose, CA). Fluorescence signals from 10,000 gated cells from each of the treatment groups were collected, and differences between the groups were calculated using 1-way analysis of variance (ANOVA). For immunofluorescence, cells were processed as described24Hackam D. Rotstein O. Schreiber A. Zhang W. Grinstein S. Rho is required for the initiation of calcium signaling and phagocytosis by Fcgamma receptors in macrophages.J Exp Med. 1997; 186: 955-966Crossref PubMed Scopus (149) Google Scholar and viewed on an Olympus Fluoview 500 confocal microscope (Melville, NY) using standard filter sets. Studies of cell migration were performed as described.8Cetin S. Ford H.R. Sysko L.R. Agarwal C. Wang J. Neal M.D. Baty C. Apodaca G. Hackam D.J. Endotoxin inhibits intestinal epithelial restitution through activation of rho-GTPase and increased focal adhesions.J Biol Chem. 2004; 279: 24592-24600Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar Briefly, IEC-6 or IEC-18 cells were grown on glass cover slips to 100% confluence, serum-starved overnight for 12 hours, then preincubated for 12 hours with LPS (50 μg/mL) ± anti-β1 antibody (1:10 dilution) or nonspecific IgG (1:10 dilution; Baxter Healthcare Corp, Deerfield, IL). Enterocyte migration was induced by creating a wound within the confluent monolayer with a cell scraper. Cells then were observed as they moved into the wound on the stage of an Olympus 1 × 71 inverted microscope perfused with Dulbecco's modified Eagle medium + 10 mmol/L HEPES pH 7.4 at .5 mL/h at 37°C. Images were recorded every 5 minutes for 20 hours and analyzed using Metamorph software (Universal Imaging Corporation, Downingtown, PA). In parallel, cells were returned to the incubator during migration then imaged at 6-hour intervals using Metamorph software on an Axiovert 200 microscope (Carl Zeiss Incorporated, Thornwood, NY). The migration rate was 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. The following experimental protocols were approved by the Animal Research and Care Committee of the Children's Hospital of Pittsburgh. Pregnant Sprague-Dawley rats were purchased from Harlan Sprague Dawley (Indianapolis, IN). Immediately after birth, control animals were breastfed under normoxic conditions; intestinal injury resembling NEC was induced by the administration of .2 mL enteral formula (15 g Similac PM 60/40; Ross Metabolics, Columbus, OH; and 75 mL Esbilac; PetAg Inc, Hampshire, IL), and treatment with 5% oxygen for 10 minutes before each feeding.25Nadler E.P. Dickinson E. Knisely A. Zhang X.R. 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Sysko L.R. Agarwal C. Wang J. Neal M.D. Baty C. Apodaca G. Hackam D.J. Endotoxin inhibits intestinal epithelial restitution through activation of rho-GTPase and increased focal adhesions.J Biol Chem. 2004; 279: 24592-24600Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar To determine integrin expression in the terminal ilea mucosa, mucosal scrapings were prepared after 4 days of treatment and placed in cold lysis buffer (10% glycerol, 1% SDS, 62.5 mmol/L Tris pH 6.6, .5 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 2 μg/mL aprotinin), homogenized on ice, centrifuged at 9000 × g for 30 minutes, then subjected to SDS-PAGE. In parallel, 2-week-old C57BL/6J mice (Jackson Laboratory) were injected with either normal saline (100 μL) or LPS (5 mg/kg) intraperitoneally. After 8 hours, animals were killed and mucosal scrapings were prepared from freshly isolated segments of the terminal ileum and subjected to SDS-PAGE as described earlier. 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To do so, intestinal injury resembling NEC was induced in newborn rats using a combination of formula gavage and hypoxia. As compared with the healthy mucosa of the control animals (Figure 1A), rats with experimental NEC showed morphologic changes of intestinal inflammation, including edema of the lamina propria, loss of the villus architecture, and the accumulation of an inflammatory infiltrate (Figure 1B, see inset for further mucosal detail). These changes are similar to those observed in human NEC28Hopkins G.B. Gould V.E. Stevenson J.K. Oliver T.K.J. Necrotizing enterocolitis in premature infants. A clinical and pathologic evaluation of autopsy material.Am J Dis Child. 1970; 120: 229-232Crossref PubMed Scopus (66) Google Scholar in that the treatment results in patchy intestinal necrosis as opposed to a global event. Importantly, this model has been validated as being similar to clinical NEC in several ways, including similarities in proinflammatory gene expression,25Nadler E.P. 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Endotoxin inhibits intestinal epithelial restitution through activation of rho-GTPase and increased focal adhesions.J Biol Chem. 2004; 279: 24592-24600Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar enterocyte migration—a measure of intestinal restitution—was impaired significantly in rats with NEC compared with control animals (Figure 1C). Importantly, the expression of β1-integrin was increased significantly in the ileal mucosa of newborn animals with intestinal inflammation compared with controls (Figure 1D). These results suggest that experimental NEC is associated with impaired intestinal restitution and increased integrin expression in ileal enterocytes. Based on the foregoing observations, we hypothesized that the translocation of endotoxin through an impaired barrier may be one of the early events in the pathogenesis of NEC that leads to activation of enterocyte integrins and impaired intestinal restitution. 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