Nitric Oxide in the Rabbit Lacrimal Glands with Induced Autoimmune Dacryoadenitis

Abstract

Abstract: : Purpose: Our previous data suggest that neuronal nitric oxide synthase (nNOS) is expressed by mouse and rabbit lacrimal gland epithelial cells (LGEC). The purpose of the present study was to test that: (1) nNOS is present in rabbit LGEC, and (2) lacrimal NO levels are elevated in a rabbit model of induced autoimmune dacryoadenitis that mimics Sjögren’s Syndrome. Methods: Autoimmune dacryoadenitis was induced by activating autologous peripheral blood lymphocytes in mixed cell reactions with isolated epithelial cells from one lacrimal gland and injecting them into the donor’s remaining gland. Lacrimal glands were collected 4 weeks after induction. Purified LGEC were cultured in Matrigel rafts, collected and incubated without agonist or with NO donors, spermine–NONOate, and PAPA NONOate, or L–NAME, the NOS inhibitor. The supernatant culture media and lacrimal gland homogenates were filtered using a 10 kDa cut–off filter. The filtrates were assayed for NO by measuring its metabolic end products, nitrate and nitrite. Total protein concentrations were assayed by Coomassie blue method and Western blot was used to detect the presence of nNOS. Results: nNOS immunoreactivity was detected by Western blot, with the molecular weight of 155 kDa. Rabbit brain used as positive control confirmed these findings. However, Western blot failed to show the presence of endothelial NOS (eNOS) in LGEC. Although significant amounts of NO were released by both NO donors, neither was able to effectively affect protein secretion by cultured LGEC. Likewise, L–NAME also failed to significantly affect protein secretion. Nitrate/nitrite concentrations increased from 225.34±53.78 µMol/kg tissue wet weight in normal glands to 394.8±65.15 µMol/kg in glands with autoimmune dacryoadenitis. Conclusions: Our studies showed that nNOS is expressed by rabbit LGEC, supporting our previous immunocytochemical report of nNOS immunoreactivity in lacrimal non–neuronal cells. While epithelial cells in other tissues have been reported to nNOS or eNOS, this is the first of constitutive NOS expression by LGEC. Neither NO donors nor NOS inhibitor had significant effect on protein secretion from cultured LGEC. These data suggest that NO is not a direct secretagogue in the rabbit lacrimal gland. Rather, it may be a paracrine regulator of the vasculature and of infiltrating lymphocytes. The significant increase of NO production in rabbits with autoimmune dacryoadenitis also suggests that NO may play a role in Sjögren’s Syndrome.

Keywords: lacrimal gland • cornea: tears/tear film/dry eye • nitric oxide