1. Volume 132, Issue 7, Pages 2395-2411 (June 2007)
Interferon-γ Inhibits Intestinal Restitution by Preventing Gap Junction Communication Between Enterocytes 
Cynthia L. Leaphart, Faisal Qureshi, Selma Cetin, Jun Li, Theresa Dubowski, Catherine Batey, Donna Beer–Stolz, Fengli Guo, Sandra A. Murray, David J. Hackam 
Gastroenterology 
Volume 132, Issue 7, Pages (June 2007)
DOI: /j.gastro
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2. Figure 1
The gap junction protein Cx43 is expressed between adjacent enterocytes in vivo and in vitro. (A) Lysates were prepared from mouse brain (positive control), the intestinal epithelial cell lines IEC-6, CaCO-2, HT-29, T-84, and the epithelial cell line HeLa (negative control), and mucosal scraping were obtained from the terminal ileum of 2 individual 10-day-old Swiss-Webster mice (“ileum-1” and “ileum-2”), subjected to SDS-PAGE and immunoblotted using antibodies against Cx43. Blots were then stripped and reprobed using antibodies against f-actin to assess for protein loading. (B) Immunofluorescent laser confocal microscopy of Cx43 in IEC-6 enterocytes. Arrows indicate the presence of gap junction plaques (green) on the surface between adjacent cells, identified by the presence of the nuclear marker Draq5 (blue). Bar indicates 10 μm. (C–E) Primary ileal enterocytes were purified from newborn mouse ileum (C shows representative DIC image), confirmed via staining for E-cadherin (D) and stained for Cx43 (E). (F) Confocal micrograph showing the distribution of Cx43 in the terminal ileum of 10-day-old Swiss-Webster mice. (G–H) Images at higher magnification corresponding to the dotted regions in (F), demonstrating the distribution of Cx43 in regions of crypts (D) and villi (E). Arrows indicate the presence of gap junction plaques (green) on the surface between adjacent cells, identified by the presence of the nuclear marker Draq5 (blue). Bar indicates 10 μm. Representative of at least 5 separate experiments. Size bar = 5 μm.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Adjacent IEC-6 enterocytes communicate via gap junctions. (A–F) IEC-6 cells were plated on glass coverslips in the absence (A–C) or presence (D–F) of the gap junction inhibitor oleamide (10 μmol/L, 15 minutes) and microinjected with the gap junction impermeant fluorescent dye Texas red* dextran (“Texas red”) and the gap junction tracer Lucifer yellow (“Lucifer yellow”). Confocal micrographs were obtained 60 seconds later to reveal the differential interference contrast (“DIC”) appearance of the live cells (A), localization of Texas red dextran within the injected cell only (B; asterisk indicates the location of injected cell), and transfer of Lucifer yellow to adjacent cells beyond the injected cell (C). In the presence of oleamide (see DIC image in D), both Texas red dextran (E) and Lucifer yellow remain localized within the injected cell (asterisk). Representative of at least 15 separate experiments. (G–L) IEC-6 cells were plated on glass coverslips, loaded with the gap junction tracer calcein (20 μmol/L, 20 minutes) (G), then bleached in a small area with a laser. The region of bleaching is indicated by an asterisk and dotted lines (H). Cells were then examined by live cell confocal microscopy and allowed to recover fluorescence over the subsequent 3 minutes (I). The experiment was repeated in the presence of the gap junction inhibitor oleamide. (J) Calcein-loaded cells prior to bleaching; (K) micrograph indicating region of bleaching (dotted line). The appearance 3 minutes later indicates that the recovery of fluorescence signal to the bleached area was inhibited (L). (M) Quantification of gap junction communication as determined above in untreated cells (“Control”), or after treatment with the gap junction inhibitors oleamide (10 μmol/L “OLM”) or 18α-glyceric acid 10 μmol/L, 30 minutes (“18α-GA”). Black bars = microinjection, red bars = FRAP. Representative of at least 15 separate experiments. **P < .05 versus control. Size bar = 5 μm.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
siRNA knockdown of Cx43 prevents gap junction communication between enterocytes. (A) SDS-PAGE revealing the expression of Cx43 and f-actin in lysates obtained from IEC-6 cells that had been transfected with either Cx43 siRNA, control siRNA (5 nmol/L), or 2 separate nonoverlapping siRNA constructs that did not affect the expression of Cx43 (5 nmol/L of each, “1” and “2”) or were treated with media alone. (B–H) Quantification of gap junction communication between IEC-6 cells treated as in (A). *P < .05 vs control siRNA and media alone. (C–H) Representative micrographs obtained prior to bleaching (C, F), immediately after bleaching (D, G), or after recovery (E, H) in IEC-6 cells treated with control siRNA (C–E) or Cx43 siRNA (F–H). The region of bleaching is indicated by an asterisk and dotted lines. Representative of at least 5 separate experiments. Size bar = 5 μm.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Gap junctions are required for enterocyte migration in vitro and in vivo. (A–D, G) IEC-6 cells were plated to confluence and scraped to create a wound in the absence (A, B) or presence of the gap junction inhibitors oleamide (10 μmol/L, 15 minutes) (C–D), 18αG-A (G) (10 μmol/L, 30 minutes), control siRNA (“si-ctl,” G), or Cx43 siRNA (“si-cx,” G), and allowed to migrate at 37°C. Shown are images from a typical experiment at the beginning (“t = 0 hours”) of the experiment and 14 hours later (“t = 14 hours”), where the dotted line indicates the position of the cells at the edge of the scraped wound, at t = 0. Quantification of migration rate (G). Representative of 5 experiments. **P < .05, compared to control, by analysis of variance. Representative of 10 separate experiments. (E–F, H) Ten-day-old Swiss-Webster mice were injected intraperitoneally with either saline or oleamide (5 mg/kg) daily for 4 days, then injected intraperitoneally with BrdU 14 hours prior to sacrifice and immunostained for BrdU (arrows indicate position of peroxidase staining). In saline-treated animals, most BrdU has accumulated at varying positions along the villi (E, arrows). In oleamide-treated animals, the majority of the BrdU uptake remains in the crypts (F), demonstrating a significant impairment in migration. The rate of migration is significantly decreased in oleamide-treated animals compared to controls (H). Data are means ± SE of 4 separate experiments. A minimum of 100 cells were counted per experiment.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Experimental NEC is associated with impaired expression of Cx43. (A) NEC (“NEC”) was induced in 10-day-old Swiss-Webster mice using a combination of gavaged formula and hypoxia, while control animals were allowed to breast feed (see the Materials and Methods section). The expression of Cx43 by SDS-PAGE in mucosal scrapings obtained from the terminal ileum is shown, along with the reprobed immunoblot against actin to assess for protein loading. (B–C) Representative gross images of the small intestine obtained from freshly sacrificed control (B) and NEC (C) animals are shown. (D) Serum IL-6 concentration in control animals (“Ctl”), animals with NEC (“NEC”), animals that had been injected with oleamide (5 mg/kg) for each of the 4 days of the NEC induction (“NEC + Olm”), and animals injected with oleamide alone (5 mg/kg/day for 4 days). *P < .05 vs control; **P < .05 vs NEC. (E–J) Samples from the terminal ileum from control mice (E, G, H) and those with NEC (F, I, J) were immunostained with antibodies against Cx43 (green) and the nuclear marker Draq-5 (red). Representative confocal micrographs are shown at low magnification (E, F), in which dotted lines reflect regions at higher of representative villi and crypts (G–J). Representative of 5 separate experiments. Arrows indicate location of gap junctions. Size bar = 5 μm.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
The expression of Cx43 is reduced in the ileum of patients with IBD. (A) Expression of Cx43 by SDS-PAGE in lysates from the ileum of 2 specimens of bowel affected by inflammatory bowel disease (“IBD”), and 2 specimens from unaffected bowel (“control”); f-actin expression after stripping of the blot is shown. Representative of 3 separate experiments. (B–G) Confocal micrograph showing the distribution of Cx43 in human ileum in unaffected bowel (B–D) and bowel affected by inflammatory bowel disease (E–G), revealing the distribution of E-cadherin (B, E), Cx43 (C, F), and the codistribution of these proteins represented by orange staining on the merged image (D, G). Size bar = 5 μm. Arrows indicate presence of gap junctions between enterocytes.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Interferon gamma, whose mucosal expression is increased in experimental NEC, impairs enterocyte migration and gap junction communication. (A) SDS-PAGE showing the expression of interferon gamma (“IFN”) in mucosal scrapings obtained from the terminal ileum of control animals (lanes 1 and 2) and animals with NEC (lanes 3 and 4). The blot was reprobed for f-actin to assess protein loading as shown. Relative band density of 3 separate experiments is shown, *P < .05 versus control. (B) Rate of migration into a scraped wound of IEC-6 cells in the presence or absence of IFN (1000 IU/mL, 14 hours). *P < .05 versus control, n = 5 separate experiments. (D–F) Micrographs showing the migration of BrdU-labeled enterocytes in newborn animals that had been injected with either saline (D) or IFN (1000 IU/g/day, 4 days) (E). (F) Quantification of migration, P < .05 versus saline. Representative of 3 separate experiments. (G) Gap junction communication as measured using fluorescent recovery after photobleaching, of IEC-6 cells in the presence or absence of IFN (1000 IU/mL, 14 hours), indicating impaired gap junction communication by IFN. *P < .05 versus control, n = 5 separate experiments.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Interferon gamma (IFN) causes an internalization of gap junction plaques between enterocytes. (A–B) Representative confocal micrographs of IEC-6 cells that were either untreated (A, “Control”) or treated with IFN (1000 IU/mL, 14 hours) (B, “IFN”) then immunostained with antibodies against Cx43 (green) and the nuclear marker Draq-5 (red). Bar = 10 μm. Arrows indicate the location of gap junction Cx43 plaques. (C–D) Immunoelectron micrographs of IEC-6 cells that were either untreated (C) or treated with IFN (1000 IU/mL, 14 hours, D), and probed with antibodies against Cx43 as described in the Materials and Methods section. Inset reveals region of dotted lines at higher magnification. Arrows indicate the location of gap junction Cx43 plaques. Representative of at least 5 experiments. (E–H) The lack of effect of IFN on the kinetics of Cx43 transcription and translation is shown as IEC-6 cells were either untreated (E) or treated with IFN (F, 1000 IU/mL, 14 hours) and then treated with cycloheximide (10 μmol/L) or actinomycinD (ActD, 5 μmol/L) as indicated, and the expression of Cx43 and f-actin over time is shown. (G) Quantification of Cx43/f-actin ratio, showing no differences in Cx43 expression at each time point between control (closed bars) and IFN (open bars)-treated cells. Representative of 3 separate experiments. (H) The expression of 35S-Cx43 over time in IEC-6 cells in the presence (open bars) or absence (closed bars) of IFN (1000 IU/mL,14 hours). Representative of 3 separate experiments. Size bar = 5 μm.
Gastroenterology  , DOI: ( /j.gastro )
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10. Figure 9
IFN decreases the phosphorylation of Cx43 on enterocytes in vitro and in vivo, while treatment with phosphatase inhibitors reverses the inhibitory effects of IFN on Cx43 surface expression and communication, and restores enterocyte migration. (A) SDS-PAGE showing the expression of phosphorylated Cx43 (“pCx43”) in mucosal scrapings obtained from the terminal ileum of newborn mice injected either with saline (lanes 2 and 3) or IFN (lanes 4 and 5). Blots were then reprobed for total Cx43 (“tCX43”). Lane 1 was loaded with mouse brain (positive control). Representative of 4 separate experiments. (B) SDS-PAGE showing pCx43 in IEC-6 cell lysates that were either untreated (“Ctl”) or treated with IFN (“IFN”), IFN plus calyculin (IFN Ca), or calyculin alone (Ca). Blots were then reprobed for total Cx43 (“tCX43”). (C–E) Confocal micrographs showing the expression of total Cx43 (green) and the nuclear marker Draq-5 (red) in IEC-6 cells. Cx43 is found at the cell surface that was either untreated (C, “Control”), treated with IFN (1000 IU/mL, 14 hours) (D, “IFN”) or treated with IFN in the presence of calyculin (2 μmol/L, 30 minutes) (E, “IFN + Ca”). Arrows indicate the location of Cx43 plaques. (F–G) Gap junction communication as measured using fluorescent recovery after photobleaching (F) and rate of migration into a scraped wound (G) of IEC-6 cells treated under the conditions above. **P < .05 versus control, *P < .05 versus IFN-treated cells; n = 5 separate experiments. Size bar = 5 μm.
Gastroenterology  , DOI: ( /j.gastro )
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11. Figure 10
Interferon gamma knockout mice are protected from the development of NEC, and show enhanced intestinal restitution compared to wild-type counterparts. (A–D) H&E sections of the terminal ileum obtained from wild-type (A, B) and IFN−/− (C, D) mice that were either breast fed (“control,” A, C) or subjected to formula gavage/hypoxia for 4 days (“NEC,” B, D). (E–H) Micrographs showing the migration of BrdU-labeled enterocytes in the terminal ileum from wild-type (E, F) or IFN−/− mice (G, H) that were either breast fed (“control,” E, G) or induced to develop NEC (“NEC,” F, H). (I) SDS-PAGE showing the expression of Cx43 in mucosal scrapings from the terminal ilea of wild-type and IFN−/− mice that were either breast fed or induced to develop NEC as above. Blots were stripped and reprobed for actin, and the quantification of the expression ratio of Cx43/actin is shown. (J) Quantification of IL-6 release in animals corresponding to panels A–D. (K) Quantification of enterocyte migration in animals corresponding to panels E–H. *P < .05 versus ctl; **P < .05 versus NEC in wild-type animals. Representative of 3 separate experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions