Studying Permeability in a Commonly Used Epithelial Cell Line: T84 Intestinal Epithelial Cells

2011 ◽  
pp. 115-137 ◽  
Author(s):  
Rino P. Donato ◽  
Adaweyah El-Merhibi ◽  
Batjargal Gundsambuu ◽  
Kai Yan Mak ◽  
Emma R. Formosa ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Line ◽  
Intestinal Epithelial Cells ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial

scholarly journals Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesicular traffic

1992 ◽  
Vol 117 (6) ◽  
pp. 1197-1209 ◽  
Author(s):  
WI Lencer ◽  
C Delp ◽  
MR Neutra ◽  
JL Madara
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cholera Toxin ◽  
Cell Line ◽  
Intestinal Epithelial Cell ◽  
Time Course ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Vesicular Traffic ◽  
Intestinal Epithelial Cell Line

The massive secretion of salt and water in cholera-induced diarrhea involves binding of cholera toxin (CT) to ganglioside GM1 in the apical membrane of intestinal epithelial cells, translocation of the enzymatically active A1-peptide across the membrane, and subsequent activation of adenylate cyclase located on the cytoplasmic surface of the basolateral membrane. Studies on nonpolarized cells show that CT is internalized by receptor-mediated endocytosis, and that the A1-subunit may remain membrane associated. To test the hypothesis that toxin action in polarized cells may involve intracellular movement of toxin-containing membranes, monolayers of the polarized intestinal epithelial cell line T84 were mounted in modified Ussing chambers and the response to CT was examined. Apical CT at 37 degrees C elicited a short circuit current (Isc: 48 +/- 2.1 microA/cm2; half-maximal effective dose, ED50 integral of 0.5 nM) after a lag of 33 +/- 2 min which bidirectional 22Na+ and 36Cl- flux studies showed to be due to electrogenic Cl- secretion. The time course of the CT-induced Isc response paralleled the time course of cAMP generation. The dose response to basolateral toxin at 37 degrees C was identical to that of apical CT but lag times (24 +/- 2 min) and initial rates were significantly less. At 20 degrees C, the Isc response to apical CT was more strongly inhibited (30-50%) than the response to basolateral CT, even though translocation occurred in both cases as evidenced by the formation of A1-peptide. A functional rhodamine-labeled CT-analogue applied apically or basolaterally at 20 degrees C was visualized only within endocytic vesicles close to apical or basolateral membranes, whereas movement into deeper apical structures was detected at 37 degrees C. At 15 degrees C, in contrast, reduction to the A1-peptide was completely inhibited and both apical and basolateral CT failed to stimulate Isc although Isc responses to 1 nM vasoactive intestinal peptide, 10 microM forskolin, and 3 mM 8Br-cAMP were intact. Re-warming above 32 degrees C restored CT-induced Isc. Preincubating monolayers for 30 min at 37 degrees C before cooling to 15 degrees C overcame the temperature block of basolateral CT but the response to apical toxin remained completely inhibited. These results identify a temperature-sensitive step essential to apical toxin action on polarized epithelial cells. We suggest that this event involves vesicular transport of toxin-containing membranes beyond the apical endosomal compartment.

scholarly journals The function of macromolecular complex of CFTR-NHERF2-LPA2 in inflammatory responses of intestinal epithelial cells

2017 ◽  
Author(s):  
Shanshan Kong ◽  
Weiqiang Zhang
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Line ◽  
Human Cell ◽  
Inflammatory Responses ◽  
Intestinal Epithelial Cells ◽  
Human Cell Line ◽  
Macromolecular Complex ◽  
Apical Surface ◽  
Intestinal Epithelial

AbstractCFTR is a cAMP-regulated chloride channel located in the apical surface of intestinal epithelial cells; where it forms a macromolecular complex with NHERF2 and LPA2. CFTR has been shown to play a role in the pathogenies of several types of secretory diarrheas. Inflammatory bowel disease (IBD) is a chronic condition of intestine characterized by severe inflammation and mucosal destruction, genetic analysis has shown that LPA contribute to IBD and patients of cystic fibrosis also display the phenotype of diarrhea. The purpose of this study is to investigate if this complex plays a role in the inflammatory responses of intestinal epithelium.We then explored the role of this complex in maintaining the integrity of tight junction and inflammatory responses in these cells. In vitro assays show that inhibiting CFTR or LPA2 in the intestinal epithelial cell could disrupt the epithelial cell junction, and reduce the TER of intestinal epithelial cells in both mouse and human cell line. EƯSA assay show that intriguing LPA2 through LPS or LPA can increase the secretion of IL-8, while inhibiting or SiRNA knockdown of LPA2 can decrease the secretion of IL-8 in mouse or human intestinal epithelial cells. The CFTR inhibitor can reduce the IL-8 secretion in both mouse and human cell line, the deletion of CFTR in mouse intestine does not affect the IL-8 level, but the knockdown of CFTR in human cell line reduced the IL-8 protein level. The deletion of CFTR in human also reduced the IL-8 mRNA level. This indicates the CFTR-LPA complex is necessary for the expression of IL-8.

Gene expression of activin, activin receptors, and follistatin in intestinal epithelial cells

2000 ◽  
Vol 278 (1) ◽  
pp. G89-G97 ◽  
Author(s):  
Kei Sonoyama ◽  
Suriya Rutatip ◽  
Takanori Kasai
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Line ◽  
Intestinal Epithelial Cells ◽  
Activin A ◽  
Intestinal Cell ◽  
Rat Jejunum ◽  
Intestinal Epithelial ◽  
Activin Receptors

Gene expression of activin, activin receptors, and follistatin was investigated in vivo and in vitro using semiquantitative RT-PCR in intestinal epithelial cells. Rat jejunum and the intestinal epithelial cell line IEC-6 expressed mRNA encoding the βA-subunit of activin, α-subunit of inhibin, activin receptors IB and IIA, and follistatin. An epithelial cell isolation study focused along the crypt-villus axis in rat jejunum showed that βA mRNA levels were eight- to tenfold higher in villus cells than in crypt cells. Immunohistochemistry revealed the expression of activin A in upper villus cells. The human intestinal cell line Caco-2 was used as a differentiation model of enterocytes. Four- to fivefold induction of βA mRNA was observed in postconfluent Caco-2 cells grown on filter but not in those cells grown on plastic. In contrast, follistatin mRNA was seen to be reduced after reaching confluence. Exogenous activin A dose-dependently suppressed the proliferation and stimulated the expression of apolipoprotein A-IV gene, a differentiation marker, in IEC-6 cells. These results suggest that the activin system is involved in the regulation of such cellular functions as proliferation and differentiation in intestinal epithelial cells.

ADAM-15 inhibits wound healing in human intestinal epithelial cell monolayers

2005 ◽  
Vol 288 (2) ◽  
pp. G346-G353 ◽  
Author(s):  
Laetitia Charrier ◽  
Yutao Yan ◽  
Adel Driss ◽  
Christian L. Laboisse ◽  
Shanthi V. Sitaraman ◽  
...  
Keyword(s):  
Wound Healing ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cell ◽  
Intestinal Epithelial Cells ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Coding Region ◽  
Human Intestinal Epithelial Cell ◽  
New Variant

The disintegrin metalloproteases (or ADAMs) are membrane-anchored glycoproteins that have been implicated in cell-cell or cell-matrix interactions and in proteolysis of molecules on the cell surface. The expression and/or the pathophysiological implications of ADAMs are not known in intestinal epithelial cells. Therefore, our aim was to investigate the expression and the role of ADAMs in intestinal epithelial cells. Expression of ADAMs was assessed by RT-PCR, Western blot analysis, and immunufluorescence experiments. Wound-healing experiments were performed by using the electric cell substrate impedence sensing technology. Our results showed that ADAMs-10, -12, and -15 mRNA are expressed in the colonic human cell lines Caco2-BBE and HT29-Cl.19A. An ADAM-15 complementary DNA cloned from Caco2-BBE poly(A)+ RNA, and encompassing the entire coding region, was found to be shorter and to present a different region encoding the cytoplasmic tail compared with ADAM-15 sequence deposited in the database. In Caco2-BBE cells and colonic epithelial cells, ADAM-15 protein was found in the apical, basolateral, and intracellular compartments. We also showed that the overexpression of ADAM-15 reduced cell migration in a wound-healing assay in Caco2-BBE monolayers. Our data show that 1) ADAM-15 is expressed in human intestinal epithelia, 2) a new variant of ADAM-15 is expressed in a human intestinal epithelial cell line, and 3) ADAM-15 is involved in intestinal epithelial cells wound-healing processes. Together, these results suggest that ADAM-15 may have important pathophysiological roles in intestinal cells.

scholarly journals Shiga Toxin 2e-Producing Escherichia coli Isolates from Humans and Pigs Differ in Their Virulence Profiles and Interactions with Intestinal Epithelial Cells

2005 ◽  
Vol 71 (12) ◽  
pp. 8855-8863 ◽  
Author(s):  
Anne-Katharina Sonntag ◽  
Martina Bielaszewska ◽  
Alexander Mellmann ◽  
Nadine Dierksen ◽  
Peter Schierack ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Shiga Toxin ◽  
Intestinal Epithelial Cell ◽  
Intestinal Epithelial Cells ◽  
Epithelial Cell Line ◽  
Uptake System ◽  
Intestinal Epithelial ◽  
E Coli

ABSTRACT Thirteen Escherichia coli strains harboring stx 2e were isolated from 11,056 human stools. This frequency corresponded to the presence of the stx 2e allele in 1.7% of all Shiga toxin-producing E. coli (STEC) strains. The strains harboring stx 2e were associated with mild diarrhea (n = 9) or asymptomatic infections (n= 4). Because STEC isolates possessing stx 2e are porcine pathogens, we compared the human STEC isolates with stx 2e-harboring E. coli isolated from piglets with edema disease and postweaning diarrhea. All pig isolates possessed the gene encoding the F18 adhesin, and the majority possessed adhesin involved in diffuse adherence; these adhesins were absent from all the human STEC isolates. In contrast, the high-pathogenicity island encoding an iron uptake system was found only in human isolates. Host-specific patterns of interaction with intestinal epithelial cells were observed. All human isolates adhered to human intestinal epithelial cell lines T84 and HCT-8 but not to pig intestinal epithelial cell line IPEC-J2. In contrast, the pig isolates completely lysed human epithelial cells but not IPEC-J2 cells, to which most of them adhered. Our data demonstrate that E. coli isolates producing Shiga toxin 2e have imported specific virulence and fitness determinants which allow them to adapt to the specific hosts in which they cause various forms of disease.

scholarly journals Interleukin (IL)-22 from IL-20 Subfamily of Cytokines Induces Colonic Epithelial Cell Proliferation Predominantly through ERK1/2 Pathway

2019 ◽  
Vol 20 (14) ◽  
pp. 3468 ◽  
Author(s):  
Md. Moniruzzaman ◽  
Ran Wang ◽  
Varinder Jeet ◽  
Michael A. McGuckin ◽  
Sumaira Z. Hasnain
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Imaging System ◽  
Intestinal Epithelial Cells ◽  
Human Colon ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Human Colon Cancer ◽  
Transcriptional Changes

The interleukin (IL)-20 subfamily of cytokines consists of IL-19, IL-20, IL-22, IL-24, and IL-26, and the expression of IL-20, IL-22, and IL-24 is reported to be higher in the colon of patients with ulcerative colitis. Although the receptors for these cytokines are highly expressed in the colon epithelium, their effects on epithelial renewal are not clearly understood. This study evaluated the effects of IL-20, IL-22, and IL-24 in epithelial renewal using the LS174T human colon cancer epithelial cell line. LS174T cells were treated with IL-20, IL-22, and IL-24 (25, 50, and 100 ng/mL) and a live-cell imaging system was used to evaluate the effects on cell proliferation. Following treatment, the signaling pathways contributing to cell proliferation were investigated through Western blotting in LS174T cells and downstream transcriptional changes through qRT-PCR in LS174T cells, and RNA-Seq in primary murine intestinal epithelial cells. Our results demonstrated that only IL-22 promoted LS174T cell proliferation, mediated via extracellular-signal-regulated kinase (ERK)1/2-mediated downstream regulation of p90RSK, c-Jun, and transcriptional changes of TRIM15 and STOM. IL-22 also promoted expression of ERK1/2-independent genes such as DDR2, LCN2, and LRG1, which are known to be involved in cell proliferation and migration. This study suggests that IL-22 induces cell proliferation in highly proliferative cells such as intestinal epithelial cells.

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