Flagellin–Induced Calcium Signaling in Human Corneal Epithelial Cells

Abstract

Abstract: : Purpose: Flagellin, a toll–like receptor (TLR) 5 agonist, in human corneal epithelial cells stimulates MAPK and NFΚB activity, and up–regulates interleukin (IL)–6 and –8 expression. We determined whether calcium transients are a component of signaling pathways eliciting these responses to TLR5 stimulation. Methods: Single cell fluorescence imaging was performed on fura2–AM loaded serum–starved human corneal epithelial cells (HCEC) on the stage of an inverted microscope. Intracellular calcium levels were evaluated based on F₃₄₀/F₃₈₀nm. Values are expressed as mean±SD. Protein microarrays were used to detect cytokine production. Results: Flagellin, between 25 and 125 ng/ml, induced dose–dependent transient increases in F₃₄₀/F₃₈₀nm. Flagellin (75 ng/ml) caused an immediate increase in F₃₄₀/F₃₈₀nm of 0.99±0.25 units (n=75 cells). Other flagellin concentrations (25, 50 and 100 ng/ml) (n=75 cells for each concentration) also induced a transient increase in F₃₄₀/F₃₈₀nm, but with reduced magnitudes ranging from 0.08 to 0.69 units, which were delayed for 20–40 s. After reaching peak stimulation, the ratios plateau at levels above their baseline values. Pre–incubation with an anti–TLR5 antibody completely abolished the effect of flagellin on F₃₄₀/F₃₈₀nm. Furthermore, protein microarray studies showed that flagellin induced an up–regulation of TNFα production. Conclusions: Flagellin–induces calcium signaling through stimulation of TLR5. This occurs over a concentration range known to induce IL–6 and –8 up–regulation. Such correspondence suggests that calcium signaling may be necessary for increases in MAPK and NFΚB activity.

Keywords: calcium • cornea: epithelium • signal transduction: pharmacology/physiology