Purpose
To evaluate the molecular and celular mechanisms involved in the disruptive immune effect of benzalkonium chloride (BAK) on the ocular surface.
Methods
A previously validated in vivo murine model was used (Mucosal Immunol. 2012 Jun 13. doi: 10.1038/mi.2012.44). In brief, Balb/c mice were given ovalbumin (OVA) alone or in combination with 0.01% BAK (B+O) daily for 5 days in both eyes, with and without nuclear factor κB (NFκB) pathway inhibitors: pyrrolidine dithiocarbamate (PDTC) and sulfasalazine (SSZ). On day 7, mice were immunized intraperitoneally with OVA in alum to evaluate the systemic response on day 14 as OVA-induced splenocyte proliferation by thymidine incorporation. To model in vitro the ocular surface epithelium, Pam212 cells were exposed for 15 min to different BAK concentrations (0.00001%-0.01%), then cultured for 96 h with syngeneic splenocytes and finally cell viability and expression of major histocompatibility complex class II (MHC-II) as activation marker were assayed.
Results
In the in vivo model, OVA instillation induced conjunctival tolerance (43±9% of control systemic response, p<0.05), in contrast to B+O (90±8%), which did not affect the subsequent cellular response. Addition of NFκB inhibitors restored tolerance in B+O mice (PDTC 39±3%, SSZ 54±8%, p<0.05) but did not significantly affect it in OVA mice (PDTC 31±5%, SSZ 69±5%, p<0.05). In the epithelial cultures, exposure to BAK decreased cell viability after 96 h but did not modify the MHC-II+ fraction (5% control, <8% BAK). Both γ interferon and splenocyte coculture increased MHC-II expression (15% and 19%, respectively, p<0.05). In BAK-exposed Pam212 cells, coculture with splenocytes increased even further the MHC-II+ fraction (24%-46% with increasing BAK concentrations, p<0.05). Epithelial pretreatment with PDTC or SSZ did not affect MHC-II expression in any case.
Conclusions
In the in vivo model, BAK-induced conjunctival tolerance breakdown could be reverted by the instillation of two known inhibitors of NFκB activation. Regarding the increased MHC II expression observed in conjunctival biopsies from preservative-exposed patients, BAK indirectly induced a similar change in cultured epithelial cells that involved their interaction with lymphocytes. These results suggest that NFκB modulation could have a therapeutic role in ocular surface immunology.
Keywords: 621 ocular irritants •
555 immunomodulation/immunoregulation •
553 immune tolerance/privilege