As the epithelial NF-κB pathway plays a key role in determining the immune outcome in other mucosal sites, we reasoned that it could be involved in the disruptive effect of BAK on conjunctival tolerance that we had previously reported.
9 Therefore, we directly assessed by different approaches the activation status of the NF-κB pathway in epithelial cells on BAK treatment. As primary murine conjunctival epithelial cells could not be obtained in sufficient number by standard culture techniques,
27 we used the Pam212 epithelial cell line, which we had previously used for exploring the immune effects of BAK.
9 To model the instillation of a BAK-containing eye drop onto the ocular surface, we exposed Pam212 monolayers for 15 minutes (the accepted tear film clearance time
28,29 ) to a BAK concentration curve starting at 10
−2% (0.01%, the most frequently used concentration in medical formulations), and extending up to 1000-fold dilution (10
−5%). We have previously shown that 10
−4% and 10
−3% BAK treatment for 15 minutes does not affect Pam212 cell viability on overnight culture, whereas comparable exposure to 10
−2% BAK readily induces cell death.
9 First we quantified the expression of the IκBα protein, an inhibitory cytoplasmic protein that binds the NF-κB complex and prevents its translocation to the nucleus. Benzalkonium chloride decreased IκBα protein levels in epithelial cells, as did lipopolysaccharide (LPS), a well-characterized activator of the NF-κB pathway (
Fig. 1A). These results, obtained by FACS, were confirmed by Western blot under the same conditions (
Fig. 1B). We also determined by immunofluorescence microscopy the actual intracellular location of the NF-κB transcriptional complex in Pam212 cells. Strict cytoplasmic localization of the NF-κB p65 subunit was detected in resting cells, whereas significant translocation to the nucleus was observed 1 h after BAK treatment (
Fig. 1C). Altogether these results show that a brief exposure to BAK is sufficient to trigger the NF-κB pathway in cultured epithelial cells.