FIGURE 4.

IL-6 concentration reported in the amniotic fluid of infection associated pPROM does not induce cellular aging, cell death, or cellular transitions in AECs. (A) Flow cytometry analysis of IL-6 induced senescence in AECs. Physiologic and pathologic concentrations of IL-6 did not increase the population of senescence-associated-β-galactosidase (SA-β-Gal) positive cells compared to controls. N = 5; mean ± SEM. (B) Flow cytometry analysis of necrosis and apoptosis of cells. Physiologic (3,300 pg/mL) and pathologic (16,000 pg/mL) concentrations of IL-6 did not induce necrosis, as indicated by a lack of changes in annexin-V + propidium iodide (PI) positive cells (top right box), nor apoptosis, as indicated by no changes in Annexin-V (bottom right box) expression between IL-6 treated and untreated cells. A protein kinase panel further confirmed a lack of apoptosis by showing that phospho-p53, a pro-apoptotic marker, did not change after treatment with IL-6. Fluorescence intensity units (FIU). N = 5; mean ± SEM. (C) Immunocytochemistry of intermediate filaments in AEC to assess cell transition. AECs were stained with both cytokeratin-18 (CK-18; epithelial marker) and vimentin (mesenchymal marker) to determine transition after physiologic and pathologic IL-6 treatment. Uniform intensity analysis was conducted on vimentin and CK-18 levels to derive the vimentin/CK-18 ratio. A high vimentin/CK-18 ratio indicates a mesenchymal phenotype, while a low ratio indicates an epithelial phenotype. IL-6 treatment did not change the vimentin/CK-18 ratio compared to control cells. Fluorescent images were captured at 20×. Red: cytokeratin-18 (CK-18), green: vimentin, blue: DAPI. N = 5; mean ± SEM. Scale bar 50 μm.