(WHS-P89) FIBROBLASTS SHOW DIFFERENTIAL EXPRESSION OF INTERLEUKINS ACROSS MONOLAYERS INJURED IN VITRO
Friday, May 17, 2024
7:30 AM – 5:00 PM East Coast USA Time
Expression of pro-inflammatory cytokines by human dermal fibroblasts occurs when the cells are activated by cellular injury. We have reported previously, using an in vitro wound model, that within 8-12 hours of wounding a fibroblast monolayer, cells express and secrete a number of cytokines including interleukin (IL) -1, 6 and 8. We had hypothesized that this expression arose from cells immediately adjacent to the area of wounding, however we now report that cells at several cell distances from the wound grid express these cytokines suggesting that a compound released from injured cells may trigger this cell expression. Non-transformed human dermal fibroblasts (GM23973 and GM1872, Coriell Institute) were obtained from the NIGMS cell bank and cultured under standard conditions. The cells were seeded onto acid-stripped glass coverslips grown in 6-well plates using DMEM medium containing 10% FBS and penicillin/streptomycin. Upon confluence, the coverslips had cells scraped in a grid pattern and IL-8 localized by immunofluorescence using FITC-labelled primary antibody (BD Bioscience) at various times post scraping. Total immunoreactive IL-8 secretion from confluent cell monolayers following scraping was also performed in large cell culture dishes by ELISA (Raybiotech) and Western blotting. Findings of note were that cells immediately adjacent to the scraped area showed variable expression of IL-8 with many cells not showing any expression. In contrast, cells away from the scraped edge showed the brightest immunofluorescence. This suggested that cells at the immediate edge, which are either migrating or entering for cell division, may not also express pro-inflammatory genes. These cells may release soluble mediators to stimulate other cells in the wound area to express inflammatory mediators, or factors released from the scraped cells are able to act as paracrine mediators. Based on cell injury in epithelial cells, we also examined the potential that uridine 5’-diphosphate (UDP) released from damaged fibroblasts could serve as a soluble mediator to activate the distant cells. Preliminary studies with LC/MSMS failed to detect significant levels of this nucleotide in culture media after scraping. Addition of exogenous UDP also failed to significantly enhance fibroblast monolayer expression of these ILs. Suggesting that in fibroblasts this compound is not involved with the injury response. These findings suggest that fibroblast IL expression is dynamically regulated with cells triggered to move and/or proliferate showing less IL synthesis whereas cells that are not directly impacted by injury show significant expression. Understanding the biochemical triggers for this induced production of ILs may lead to pharmacologic approaches to reduce excess inflammation in problematic wounds.