Clay Beauregard

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

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. Sjögrens 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-1beta (IL-1beta), 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-1beta, 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-1beta stimulated a concentration-dependent and time-dependent increase in NO production. IL-1beta-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 4hr after addition of IL-1beta, and was nearly undetectable at 12hr. IL-1ra greatly reduced IL-1beta-induced iNOS expression. CONCLUSIONS Lacrimal gland acinar cells are able to produce iNOS in response to the pro-inflammatory cytokine IL-1beta. 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.

Down regulation of interleukin-1β-induced nitric oxide production in lacrimal gland acinar cells by sex steroids

Purpose. Because the ocular surface is constantly exposed to allergens and irritants, it was reasoned that one cause of dry eye might be damage from inflammatory responses normally regulated by sex steroids. To test this hypothesis, we determined if sex steroids could down regulate nitric oxide (NO) production induced by interleukin-1β (IL-1β) in cultured rabbit lacrimal gland acinar cells. Methods. Cultured rabbit lacrimal gland acinar cells were exposed to IL-1β to stimulate NO production. Stimulated cells were treated with different sex steroids and expression of iNOS protein determined by Western blotting and NO production by a nitrate/nitrite colorimetric assay. Results. It was found that the androgens testosterone, dihydrotestosterone, dehydroepiandrosterone and dehydroepiandrosterone-sulfate and 17β-estradiol were able to inhibit interleukin-1β-induced NO production in rabbit lacrimal gland acinar cells at physiological concentrations, while progesterone was not able to inhibit NO production. Sex steroid inhibition of NO production was not due to down regulation of iNOS protein production nor was it due to down regulation of GTP cyclohydrolase I with consequent loss of tetrahydrobiopterin production. Conclusions. The results reported here show that androgens and estrogens can down regulate cytokine-mediated responses in cells that are part of the ocular surface protection system and thereby may have an important role in regulating inflammatory responses in the eye. Deficiencies in these steroids, as occurs in postmenopausal women, may lead to damage of the cells responsible for producing the fluids that protect the ocular surface and subsequently to dry eye disease.

Peroxisome Proliferator-Activated Receptor Agonists Inhibit Interleukin-1β-Mediated Nitric Oxide Production in Cultured Lacrimal Gland Acinar Cells

Development of dry eye disease often occurs in individuals with autoimmune disorders such as Sjögrens syndrome. The cause of dry eye in these patients is thought to be due, at least in part, to lymphocytic infiltration of the lacrimal glands, with subsequent loss of secretion of the aqueous component of tear film. How this lymphocytic infiltration leads to loss of secretion is not fully understood. We have previously shown that the proinflammatory cytokine, interleukin-1beta (IL-1beta), can stimulate the production of nitric oxide (NO) in cultured lacrimal gland acinar cells. It is possible that IL-1beta, produced by the infiltrating macrophages, stimulates production of inducible nitric oxide synthase (iNOS), and subsequently excessive production of NO. Peroxynitrate and other radical byproducts associated with excessive synthesis of NO may be detrimental to normal function of the lacrimal gland. Here we show that the peroxisome proliferator-activated receptor (PPAR)alpha and gamma agonists can inhibit NO production in cultured lacrimal gland acinar cells. Further, this is accomplished without loss of iNOS expression or tetrahydrobiopterin. These data suggest that the use of ointments or eye drops containing these PPAR agonists may provide an effective therapeutic intervention for the prevention of dry eye in Sjögrens syndrome patients.

Effects of nitric oxide donors and nitric oxide synthase substrates on ciliary muscle contracted by carbachol and endothelin for possible use in myopia prevention.

Research has suggested that the development of myopia may possibly be prevented by the use of drugs which facilitate relaxation of the intraocular ciliary muscle. We examined the effects of five nitric oxide-producing agents--two nitric oxide donors, hydralazine and sodium nitrite, and three nitric oxide synthase substrates, L-arginine, L-canavanine, and N-benzoyl-L-arginine ethyl ester--on isolated bovine ciliary muscle maximally contracted with either carbachol or endothelin-1. Of these agents, hydralazine and L-canavanine produced a relaxing effect on endothelin-1-contracted muscle that was significantly greater than relaxing effect on carbachol-contracted muscle. These results indicate that hydralazine and L-canavanine could possibly be used for the prevention of myopia by relaxing the ciliary muscle with few anticholinergic and cycloplegic side effects.

Nitric oxide and cyclic GMP-mediated protein secretion from cultured lacrimal gland acinar cells

PURPOSE Nitric oxide (NO) donors and NO synthase (NOS) substrates were tested for their use to stimulate protein secretion from cultured lacrimal gland acinar cells, through activation of guanylate cyclase. METHOD Rabbit lacrimal gland epithelial cells (RLG cells) were incubated with NO donors and/or NOS substrates and the protein released into culture medium was determined with bicinchoninic acid assay. Guanylate cyclase activation by NO precursors was determined by measurement of c-GMP produced. RESULTS Both NO donors and NOS substrates were able to stimulate protein release from RLG cells. Among 6 compounds studied, sodium nitroprusside, isosorbide dinitrate and N(a)-benzoyl L-arginine ethyl ester (BAEE) were most potent to release protein over 100% of the basal release. The guanylate cyclase activity was stimulated by these NO precursors and was inhibited by guanylate cyclase inhibitor, [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). CONCLUSION NO donors and NOS substrates were able to stimulate protein release from RLG cells via activation of guanylate cyclase and c-GMP release, which was blocked by guanylate cyclase inhibitor, ODQ. It indicates that NO donors and NOS substrates could be used for the treatment of dry eye syndrome if the same holds true in dry eye animal models.