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Isabella Venza, Maria Cucinotta, Silvana Caristi, Giuseppe Mancuso, Diana Teti; Transcriptional Regulation of IL-8 by Staphylococcus aureus in Human Conjunctival Cells Involves Activation of AP-1. Invest. Ophthalmol. Vis. Sci. 2007;48(1):270-276. doi: 10.1167/iovs.06-0081.
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purpose. To identify signal transduction pathways involved in interleukin (IL)-8 expression by human conjunctival cells challenged with Staphylococcus aureus.
methods. Conjunctival cells were cultured in the presence of live or heat-killed S. aureus. IL-8 protein and mRNA were determined by ELISA and RT-PCR, respectively. Activation of mitogen-activated protein kinases (MAPKs) and NF-κB was analyzed by Western blot analysis with phosphospecific antibodies. Conjunctival cells were transfected with wild-type (wt) or mutated IL-8 promoters (IL-8-97, lacking the AP-1 site; IL-8-97 mutant C/EBP; IL-8-97 mutant NF-κB; IL-8/AP-1 double mutant for C/EBP and NF-κB) or c-Jun-NH2-terminal kinase (JNK)–responsive GAL-c-Jun. In further experiments, cells were cotransfected with wt IL-8 promoter and expression plasmids for p38MAPK-responsive C/EBP homologous protein (CHOP) or wt or dominant negative transactivation domain mutant (TAM-67) c-Jun. A protein–DNA binding study was performed by electrophoretic mobility shift assay (EMSA), to identify the transcription factors bound to the IL-8 promoter.
results. S. aureus induced significant IL-8 expression and synthesis in human conjunctival epithelial cells by activating c-Jun phosphorylation and transactivation potential via JNK. The IL-8 promoter activation was NF-κB- and p38MAPK-independent. Transfection and EMSA experiments suggested that only AP-1 transcription factors were necessary for optimal IL-8 expression.
conclusions. Human conjunctival epithelial cells possess the ability to respond to Gram-positive S. aureus and to activate the innate immune response by the IL-8 gene expression. These results are the first to delineate the transcription factors involved in S. aureus–induced IL-8 release by conjunctival epithelium.
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