UROEPITHELIAL CELLS ARE PART OF A MUCOSAL CYTOKINE NETWORK

This study compared the cytokine production of uroepithelial cell line s in response to gram-negative bacteria and inflammatory cytokines. Hu man kidney (A498) and bladder (J82) epithelial cell lines were stimula ted with either Escherichia coli Hu734, interleukin 1 alpha (IL-1 alph a), or tumor necrosis factor alpha (TNF-alpha). Supernatant samples we re removed, and the RNA was extracted from cells at 0, 2, 6, and 24 h. The secreted cytokine levels were determined by bioassay or immunoass ay; mRNA was examined by reverse transcription-PCR. The two cell lines secreted IL-6 and IL-8 constitutively. IL-6 and IL-8 mRNA were consti tutively produced in both cell lines; IL-1 beta mRNA was detected in J 82 cells. IL-1 alpha induced significantly higher levels of IL-6 secre tion than did E. coli Hu734 or TNF-alpha. IL-1 alpha and TNF-alpha ind uced significantly higher levels of IL-8 secretion than did E. coli Hu 734. Secreted IL-1 beta was not detected; IL-1 alpha and TNF-alpha wer e not detected above the levels used for stimulation. IL-1 alpha, IL-1 beta, IL-6, and IL-8 mRNAs were detected in both cell lines after exp osure to the stimulants. TNF-alpha mRNA was occasionally detected in t he J82 cell line after TNF-alpha stimulation. Cytokine (IL-6 and IL-8) and control (glyceraldehyde 3-phosphate dehydrogenase [G3PDH] and bet a-actin) mRNA concentrations were quantitated with internal PCR standa rds. Cytokine mRNA levels relative to beta-actin mRNA levels were the highest in E. coli-stimulated cells. In comparison, the cytokine mRNA levels relative to G3PDH mRNA levels were the highest in IL-1 alpha-st imulated cells. beta-Actin mRNA levels decreased after bacterial stimu lation but not after cytokine stimulation, while G3PDH mRNA levels inc reased in response to all of the stimulants tested. These results sugg ested that E. coli Hu734 lowered the beta-actin mRNA levels in uroepit helial cells, thus distorting the IL-6 and IL-8 mRNA levels relative t o this control. In summary, E. coli IL-1 alpha and TNF-alpha were foun d to activate the de novo synthesis and secretion of IL-6 and IL-8 in uroepithelial cells. These results emphasize the role of epithelial ce lls in cytokine-mediated responses during the early stages of infectio n.