Inset: Higher power magnification of CD45+ cells. and strand breaks in epithelial cells, which could be reversed by antioxidant treatment. These findings support the importance of immune cellCmediated esophageal injury in esophagitis and confirms the power of the OTC platform to characterize the underlying molecular events in esophagitis. Inflammatory conditions of the gastrointestinal tract, such?as reflux esophagitis, studies under controlled conditions. In past studies, we have used OTC to model early molecular events that promote the development of Barrett esophagus (BE) and esophageal adenocarcinoma. We exhibited that expression of the proinflammatory enzyme cyclooxygenase 2 in human telomerase reverse transcriptaseCimmortalized human esophageal epithelial (STR) cells led to prostaglandin E2 production and the induction of a Rabbit Polyclonal to DDX3Y mucin-secreting metaplasia with features of BE but only under OTC conditions.10 In addition, with the use of OTCs we decided that this onset of BE metaplasia is a multistep course of action Exemestane that requires increased proliferation, senescence inhibition, and epigenetic Exemestane alterations.11 Moreover, when evaluated under OTC conditions, the Barrett cell lines CP-A, CP-B, CP-C, and CP-D demonstrate distinct phenotypes.12 These new phenotypes emerged only as a result of the 3D OTC system. Here, we change an established 3D organotypic culture system to model esophageal inflammation.13 Under 3D OTC conditions, human esophageal keratinocytes undergo a complete differentiation and stratification to produce a fully mature epithelium.9 To this culture we added human peripheral blood mononuclear cells (PBMCs) and stimulated them to induce an Exemestane acute inflammatory response. After the addition and appropriate stimulation of immune cells in OTCs, we analyzed the associated morphologic changes and the effect of the inflammatory environment on epithelial differentiation, proliferation, and apoptosis. Finally, we sought to identify the contribution and potential mechanism of immune response to DNA damage. This physiologically relevant human cell culture system thus provides a novel platform for the study of human esophagitis and the effects of inflammation on esophageal epithelial responses. Materials and Methods Cell Lines Immortalized human primary normal esophageal epithelial/human telomerase reverse transcriptase9 cells designated as STR herein were managed in keratinocyte serum-free medium (Invitrogen, Carlsbad, CA) supplemented with epidermal growth factor, pituitary bovine extract, and 1% penicillin/streptomycin. FEF3 human esophageal fibroblasts9 were managed in Dulbecco’s altered Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Immune Cells and Cytokine Treatment PBMCs were collected from de-identified healthy volunteers and isolated freshly by the Human Immunology Core at the University or college of Pennsylvania under an Institutional Review BoardCapproved protocol. Proinflammatory cytokines IL-7 (10 Exemestane ng/mL; Cell Signaling Technologies, Danvers, MA) and IL-15 (20 ng/mL; ProSpec, Rehovot, Israel) were added to the cell culture media; IL-2 (10 U/mL; BD Biosciences, San Jose, CA) was also added to support PBMC viability. The cytokines were replenished during each media exchange. 3D Organotypic Culture Organotypic culture was performed as explained previously.9 The fibroblast feeder layer and 6.75??105 PBMCs were embedded within a collagen/Matrigel matrix and was allowed to mature for 7 days, after which time 5??105 epithelial cells were seeded on top and allowed to grow to confluence for an additional 4 days. Then the culture media level was reduced, exposing the keratinocytes to the airCliquid interface, which Exemestane stimulates epithelial differentiation into a multilayer epithelium common for the esophagus. On day 15 OTCs were harvested for histologic examination, RNA, and/or protein isolation. N-acetyl cysteine (5 mmol/L; Sigma-Aldrich, St. Louis, MO) was added to the culture media as an antioxidant in some experiments as indicated, based on a prior published study.14 RNA Isolation, Reverse Transcription, and Real-Time Quantitative PCR The epithelial layer was peeled off the collagen base, and total RNA was isolated (Nucleospin RNA II kit; Macherey-Nagel, Dren, Germany), according to the manufacturer’s instructions. The quantity of isolated RNA samples was checked by spectrophotometry (NanoDrop 3.1.0; NanoDrop Technologies, Rockland, DE). Total RNA (1 g) was utilized for cDNA synthesis using the SuperScript VILO cDNA Synthesis Kit (Invitrogen). All real-time PCR reactions were performed using the StepOne Plus instrument, and the amplifications were performed using the SYBR Green PCR Grasp Mix (Applied Biosystems, Foster City, CA). Glyceraldehyde-3-phosphate dehydrogenase was used as the normalization control. The relative changes in gene expression were decided using the CT method as explained in Applied Biosystems User Bulletin No. 2 (P/N 4303859). Western Blot Analysis Denatured protein.
Categories