Cornea Lacritin-Induced Secretion of Tear Proteins From Cultured Monkey Lacrimal Acinar Cells Atsuko Fujii,1,2 Ayumi Morimoto-Tochigi,3 Ryan D. Walkup,1,2 Thomas R. Shearer,2 and Mitsuyoshi Azuma1–3 1Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Beaverton, Oregon 2Department of Integrative Biosciences, Oregon Health and Science University, Portland, Oregon 3Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Kobe, Japan Correspondence: Mitsuyoshi Azuma, PURPOSE. During inflammation of the ocular surface, increased proinflammatory cytokines Senju Pharmaceutical Corporation depress tear protein secretion, suggesting that a decline in lacrimal cell function contributes Limited, 20000 NW Walker Road, to dry eye. Lacritin, a glycoprotein secreted from lacrimal acinar cells, may function as an Suite JM508, Beaverton, OR 97006; autocrine factor to stimulate tear protein secretion. The purpose of the present experiment [email protected]. was to investigate lacritin-induced protein secretion in normal and cytokine-pretreated Submitted: June 14, 2012 (inflammation model) monkey acinar cells. Accepted: March 1, 2013 METHODS. Acinar cells from monkey lacrimal glands were cultured with or without tumor Citation: Fujii A, Morimoto-Tochigi A, necrosis factor alpha (TNF-a) plus interferon gamma (IFN-c). Protein secretion was induced Walkup RD, Shearer TR, Azuma M. by lacritin or carbachol (Cch, positive control). Proteins were detected and identified by Lacritin-induced secretion of tear pro- 2þ teins from cultured monkey lacrimal immunoblotting and immunocytochemistry. Intracellular Ca was measured with the acinar cells. Invest Ophthalmol Vis fluorophore Calcium-4, and cell damage was determined by LDH leakage into the culture Sci. 2013;54:2533–2540. DOI:10. medium. 1167/iovs.12-10394 RESULTS. In cultured monkey acinar cells, lacritin stimulated tear protein secretion in normal cells without elevating intracellular Ca2þ. In contrast, Cch elevated intracellular Ca2þ and release of tear proteins. This contrast suggested an alternate, calcium-independent mechanism for lacritin-induced protein secretion. TNF-a plus IFN-c caused LDH leakage from sensitive human corneal epithelial cells, but even higher doses of TNF-a plus IFN-c did not cause LDH leakage from monkey acinar cells, suggesting a higher tolerance against these cytokines. In cytokine-treated acinar cells, lacritin stimulated protein secretion as much as that in normal cells. In contrast, Cch-induced elevation of Ca2þ and release of proteins were depressed by cytokines. CONCLUSIONS. Lacritin might be a useful biotechnology-based treatment agent against ocular surface diseases where endogenous lacritin is inadequate. Keywords: lacritin, monkey, lacrimal acinar cells, tear protein secretion, cytokine he functions of the tear film include maintaining the nerves. The major neurotransmitters are parasympathetic Tmoisture of the eye surface, supplying oxygen and nutrients acetylcholine, vasoactive intestinal peptide, and sympathetic to the avascular cornea, protecting the ocular surface by norepinephrine. Each neurotransmitter activates a different entrapping and sweeping debris and microorganisms, prevent- signaling pathway.9 For example, in cultured monkey lacrimal ing infection by supplying antibacterial substances and acinar cells, we previously reported that acetylcholine analog antibodies, and maintaining normal osmolarity and tear film carbachol (Cch) increased intracellular Ca2þ, activated the stability.1 Dry eye occurs when there are insufficient tears to protein kinase C (PKC) pathway, and promoted secretion of the provide these functions. tear protein lacritin.10 Inflammation of the ocular surface also plays a key role in Lacritin protein expression has been detected in primates, dry eye syndrome.2 Increased concentrations of proinflamma- and high expression was found in human lacrimal and tory cytokines tumor necrosis factor alpha (TNF-a)and meibomian glands.11 Recently, lacritin expression was also interferon gamma (IFN-c) have been found in the tears of dry found in nonprimate horse tears.12 Monkey lacritin was also eye patients.3–5 In a murine model of human Sj¨ogren syndrome identified in monkey tears, and expressions of lacritin mRNA (MRL/lpr), cytokine expression was upregulated,6,7 and acetyl- and protein were found in monkey lacrimal gland.13 Choliner- choline release was impaired following a decrease in protein gic stimulation induced secretion of lacritin from monkey secretion from the lacrimal gland.8 Proinflammatory cytokines lacrimal acinar cells, suggesting that lacritin is produced in the interleukin-1 alpha, beta, or TNF-a also inhibited peroxidase lacrimal gland and is secreted onto the ocular surface.10 Since a secretion induced by neurotransmitters from mouse lacrimal decrease of tear lacritin was reported in patients with glands.7 blepharitis14 and in contact lens–related dry eye,15 lacritin Secretion of tear proteins from lacrimal glands responds to might play an important role in maintaining tear fluid and the neurotransmitters from parasympathetic and sympathetic ocular surface. Indeed, lacritin promoted protein secretion Copyright 2013 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783 2533 Downloaded from iovs.arvojournals.org on 09/30/2021 Lacritin-Induced Secretion of Tear Proteins IOVS j April 2013 j Vol. 54 j No. 4 j 2534 from cultured rat acinar cells16 and stimulated proliferation in ma-Aldrich), 25 lg L-ascorbic acid/mL (Sigma-Aldrich), 1 mM cultured human corneal cells and salivary ductal cells.17,18 putrescine (Sigma-Aldrich), and 50 lg gentamicin/mL (Life Furthermore, in vivo instillation of recombinant human lacritin Technologies). After culture for approximately 16 hours at stimulated basal tear flow in rabbits.19 However, no data are 378C to allow adhesion and formation of epithelial-like available on lacritin-induced secretion of tear proteins in the morphology, the acinar cells were rinsed and pretreated with more relevant monkey model. Thus, the purpose of the supplement-free medium for 30 minutes. Acinar cells were present study was to determine if lacritin induces protein stimulated for 10 minutes with monkey lacritin or carbachol secretion from monkey lacrimal acinar cells cultured under (Cch; Sigma-Aldrich) at the concentrations indicated in the physiologic and inflammatory conditions. figures. The cultured medium was then collected, and detached cells were removed by centrifugation. The collected media were concentrated and subjected to immunoblotting. To MATERIALS AND METHODS determine calcium requirements for protein secretion, calci- um-free medium (EpiLife Medium; Life Technologies) was used Experimental Animals during pretreatment and stimulation in replace of medium containing calcium. Lacrimal glands from rhesus monkeys ( ), Macaca mulatta Since the amount of tear proteins secreted into the culture ranging in age from 1 to 10 years, were obtained at necropsy medium was less than 0.1% of total tear proteins within the from procedures unrelated to the present studies from the cultured acinar cells, and variability of cell numbers had a Oregon National Primate Research Center (Beaverton, OR). negligible influence on results, protein secretion was ex- Tear fluids were collected from adult monkeys (Macaca pressed as the amount in the medium not as a ratio to cellular fascicularis; Evebioscience Co., Ltd., Wakayama, Japan). number. Experimental animals were handled in accordance with the ARVO Statement for the use of Animals in Ophthalmic and Vision Research and the Guiding Principles in the Care and Use Establishment of Inflammatory Conditions in of Animals (DHEW Publication, NIH 80-23). Lacrimal Acinar Cells To mimic inflammatory conditions, monkey lacrimal acinar Purification of Native Lacritin From Monkey Tears cells were cultured with TNF-a (R&D Systems, Minneapolis, Adult monkeys were secured in a monkey chair, and 50 lL MN) and IFN-c (R&D Systems) for an additional 1, 2, or 3 days. saline (Otsuka Pharmaceutical, Tokyo, Japan) was instilled in To compare with damage due to inflammation on the ocular surface, human corneal epithelial cells (HCE-T; a gift from the nasal side of one eye by a micropipette. After blinking, Kaoru Sasaki, Ideta Eye Hospital, Kumamoto, Japan) were also diluted tear fluids were collected from the temporal side of the treated with TNF-a and/or IFN-c at indicated concentrations, palpebral fissure. after plating at 3 3 104 cells/cm2 and preculturing for Diluted tear fluids (20 mL) were mixed with 80 mL 50 mM approximately 16 hours. sodium phosphate solution (pH 7.0) and applied onto a After incubation, the medium was collected, and leakage of cationic exchange column (HiTrap SP Sepharose; GE Health- LDH was measured as a marker for cell damage (LDH care Life Sciences, Buckinghamshire, UK) and eluted with 50 Cytotoxicity Detection Kit; Takara Bio Inc., Shiga, Japan). mM sodium phosphate solution containing 10 mM NaCl (pH The cells were harvested into buffer containing 20 mM Tris-HCl 7.0). Flow-through fractions containing lacritin were collected (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsul- and concentrated using disposable ultrafiltration spin columns fonyl fluoride, 1 mM sodium orthovanadate, 1 mM dithiothre- (Vivaspin 20; Sartorius Stedim Biotech S.A., Aubagne Cedex, itol, protease inhibitor (complete Mini-EDTA free; Roche France). The concentrated sample was then purified on a gel Diagnostics Corp., Indianapolis,