CoHP - SoP - Student Works

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Browsing CoHP - SoP - Student Works by Author "Kennard, Emma"

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    EXAMINATION OF THE POTENTIAL ROLE OF UDP IN INTERLEUKIN-8 EXPRESSION BY FIBROBLASTS DURING CELL INJURY
    (2023-04-20) Kennard, Emma; Lindblad, William J.
    Prior studies from our lab show scraping a monolayer of fibroblasts, an in vitro wound model, results in cell migration and enhanced expression of inflammatory mediators including interleukin-8 (IL-8) by residual cells. This effect is more pronounced in fibroblasts from the skin of diabetic people than non-affected people. This expression is seen in the cells adjacent to the scraped area but also in cells 2 and 3 cells removed from the scraped edge. Studies in epithelial cells suggest that nucleotides, specifically uridine 5’-diphosphate (UDP) released from cells upon wounding in the same model bind to P2Y6 receptors on cells inducing cell migration. We hypothesized that UDP may serve as a wound mediator for fibroblasts and that it may induce the expression of IL-8. Human dermal fibroblasts (GM 1872 cells, Coriell Institute) were cultured in 100 mm dishes and grown to confluence in DMEM high glucose media containing 10% fetal calf serum and penicillin-streptomycin. Cell monolayers were scraped in a grid pattern and cultured for 24 hrs. with/out additives. UDP at 100 and 10 µM was added to non-scraped and scraped cultures of fibroblasts to determine the potential involvement of UDP that we have seen in IL-8 expression. Following 24 hr. incubation, total RNA was extracted and quantitation of IL-8 mRNA content was assessed by qPCR. Modified culture media used for the 24 hr. culturing was analyzed for UDP content by ion-pairing HPLC. UDP, immediately released from scraped cell monolayers, was determined by HPLC following a 10X concentration of PBS used to maintain cell viability during scraping. Despite a lower limit of detection of 20 ng, no UDP could be detected and further studies with LC/MS are continuing to ascertain if any quantifiable UDP is released from the scraped cells. Cell levels of mRNA for IL-8 normalized to GAPDH are being analyzed to determine if exogenous UDP enhances the expression of this cytokine. These studies will increase our understanding of what factors during fibroblast injury promote migration and the expression of pro-inflammatory mediators.