Induction of nitric oxide synthase and over-production of nitric oxide by interleukin-1β in cultured lacrimal gland acinar cells

Abstract

Purpose. Inflammation of the lacrimal gland is one of the major causative factors in aqueous tear-deficient dry eye syndrome. Pro-inflammatory cytokine production is upregulated in lacrimal gland autoimmune disease (i.e. Sjögren's syndrome) and is associated with cell death. The expression of inducible nitric oxide synthase (iNOS/NOS-2) is known to be induced in the presence of pro-inflammatory cytokines in several secretory epithelial cell types. We hypothesize that pro-inflammatory cytokines, such as interleukin-1β (IL-1β), cause a marked increase in nitric oxide (NO) production via induction of iNOS in lacrimal gland epithelial cells and that this may be a significant pathophysiological pathway of dry eye syndrome. Methods. Cultured immortalized rabbit lacrimal gland acinar cells were incubated with IL-1β, iNOS inhibitor, or IL-1 receptor antagonist (IL-1ra). Colorimetric detection of NO 2 − and NO 3 − in the media, measured by the Griess reaction, was used as an index of NO production. Expression of iNOS was determined by SDS–PAGE and Western blot. Results. IL-1β stimulated a concentration-dependent and time-dependent increase in NO production. IL-1β-induced NO production was significantly antagonized by co-incubation with IL-1ra or the iNOS-specific inhibitor, 1400W. Expression of iNOS protein was greatest at 4 hr after addition of IL-1β, and was nearly undetectable at 12 hr. IL-1ra greatly reduced IL-1β-induced iNOS expression. Conclusions. Lacrimal gland acinar cells are able to produce iNOS in response to the pro-inflammatory cytokine IL-1β. The amount of iNOS expressed and the subsequent levels of NO that are produced by lacrimal cells are far lower than those seen in macrophages, but are consistent with those reported for other cell types in the literature. This pathway of iNOS induction and overproduction of NO may be a factor in lacrimal gland cell death in dry eye syndrome. Inhibitors of iNOS or IL-1 receptor may be beneficial for controlling lacrimal gland inflammation.