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Induction of nitric oxide synthase and over-production of nitric oxide by interleukin-1β in cultured lacrimal gland acinar cells
Experimental Eye Research 77 (2003) 109–114 www.elsevier.com/locate/yexer
Induction of nitric oxide synthase and over-production of nitric oxide by interleukin-1b in cultured lacrimal gland acinar cells Clay Beauregarda,b, Paul C. Brandtb, George C.Y. Chioua,b,* a
Institute of Ocular Pharmacology, Texas A&M University System Health Science Center, College of Medicine, 304 Reynolds Medical Building, College Station, TX 77843-1114, USA b Department of Medical Pharmacology and Toxicology, Texas A&M University System Health Science Center, College of Medicine, 304 Reynolds Medical Building, College Station, TX 77843-1114, USA Received 25 September 2001; accepted 29 May 2002
Abstract Purpose. Inflammation of the lacrimal gland is one of the major causative factors in aqueous tear-deficient dry eye syndrome. Proinflammatory cytokine production is upregulated in lacrimal gland autoimmune disease (i.e. Sjo¨gren’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-1b (IL-1b), 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-1b, iNOS inhibitor, or IL-1 receptor antagonist 2 (IL-1ra). Colorimetric detection of NO2 2 and NO3 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-1b stimulated a concentration-dependent and time-dependent increase in NO production. IL-1b-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-1b, and was nearly undetectable at 12 hr. IL-1ra greatly reduced IL-1b-induced iNOS expression. Conclusions. Lacrimal gland acinar cells are able to produce iNOS in response to the pro-inflammatory cytokine IL-1b. 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. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: lacrimal gland; dry eye; nitric oxide; iNOS; interleukin-1; cytokines; inflammation; autoimmune
1. Introduction Dry eye syndrome is a chronic condition in which some components of the preocular tear film are dysfunctional, leaving the patient with painful symptoms of dryness. The tear film is composed of three layers of distinct origin: the aqueous layer from the lacrimal glands, the lipid layer from the Meibomian glands, and the mucin layer from the goblet * Corresponding author. Dr George C.Y. Chiou, Department of Medical Pharmacology and Toxicology, Texas A&M University System Health Science Center, College of Medicine, 304 Reynolds Medical Building, College Station, TX 77843-1114, USA. E-mail address: [email protected] (G.C.Y. Chiou).
cells. Aqueous tears provide the ocular surface with water, electrolytes, proteins, and oxygen. Deficiency of aqueous tears, either in quantity or quality, can lead to ocular surface disease, or keratoconjunctivitis sicca (KCS). The factors leading to aqueous tear deficiency are complex and may involve autoimmune disease (i.e. Sjo¨gren’s syndrome), loss of hormonal support, and glandular inflammation (Stern et al., 1998). A diseased lacrimal gland exhibits reduced fluid and protein secretion (Mathers and Daley, 1996). It has also been shown that lacrimal glands from patients with autoimmune dry eye (Sjo¨gren’s syndrome) produce greatly increased levels of pro-inflammatory cytokines, such as interleukin-1 (IL-1), interferon-g (IFN-g), and tumor
0014-4835/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. DOI:10.1016/S0014-4835(03)00058-7
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necrosis factor-a (TNF-a), which are secreted into the tear fluid (Robinson et al., 1998b; Rosenbaum et al., 1998; Pflugfelder et al., 1999). The possible role of nitric oxide (NO) in dry eye syndrome has not received much attention in the literature to date. NO is a diffusible messenger molecule that is constitutively produced from L -arginine in most cell types by two of the three known isoforms of nitric oxide synthase (NOS): endothelial cell NOS (eNOS/ecNOS/NOS-1) and neuronal or brain NOS (nNOS/bNOS/NOS-3). The inducible isoform of NOS (iNOS/NOS-2) is only expressed upon activation of a signal transduction cascade initiated by immunological or other pathophysiological conditions. Constitutive NOS produces low levels of NO for short periods of time and the reaction is calcium/calmodulindependent, whereas iNOS produces relatively high levels of NO for longer periods of time and is calcium/calmodulinindependent (Ignarro, 1996). Over-production of NO can be cytotoxic, both directly by the formation of free radicals and indirectly by initiating signal transduction pathways leading to apoptosis (Brune et al., 1998; Chung et al., 2001). Expression of both eNOS and nNOS has been shown in mouse lacrimal gland slices (Ding et al., 2000), indicating that NO is constitutively produced in the lacrimal gland. The expression of iNOS has been experimentally induced in acinar cells of the exocrine pancreas and in hepatocytes, both of which are secretory epithelial cell types (Eizirik et al., 1996; Smith et al., 1997; Adeghate and Parvez, 2000). Induction of iNOS has also been shown to occur in corneal epithelial cells and anterior segment during experimental uveitis (Becquet et al., 1997; Dighiero et al., 1997). In this study, it will be demonstrated that iNOS expression can be induced in cultured lacrimal gland acinar cells by addition of the pro-inflammatory cytokine, interleukin-1b (IL-1b). The induction of iNOS in these cells leads to a significant increase in nitric oxide production. It will also be demonstrated that the expression of iNOS in lacrimal gland cells is temporally regulated, perhaps by nitric oxide itself. Finally, it will be demonstrated that the induction of iNOS can be inhibited by IL-1 receptor antagonist (IL-1ra).
2. Materials and methods 2.1. Materials Immortalized rabbit lacrimal gland (RLG) acinar epithelial cells were obtained by a generous gift from Dr Roger Beuerman (Louisiana State University Eye Center, New Orleans, LA, USA). The protein secretory function, morphology, and cellular protein expression of these cells were found to be identical to primary cultured lacrimal cells (Nguyen et al., 1999). Specific instructions for growth conditions and subcultivation of the immortalized cells were obtained from the Beuerman lab. In the initial study of this
immortalized cell line, the cells were compared to primary lacrimal gland acinar cells for morphology, protein expression, and secretory function (Nguyen et al., 1999). It was determined in the referenced paper that morphologically the cells mainly formed a monolayer on Matrigelcoated plates, with occasional formation of small structures resembling acini, similar to cultured primary cells. The immortalized cells showed positive immunohistochemical reactivity to secretory component (SC), a proteolytic fragment of the IgA receptor produced predominantly in acinar cells, as well as transferrin (Tf) and transferrin receptor (TfR). Total protein secretion in response to carbachol was also measured and found to be similar to primary cell data (Nguyen et al., 1999). In another study examining the similarity of this immortalized RLG acinar cell line to primary cells, expression of cytokeratins, laminin, and integrins in this immortalized line was found to be indistinguishable from expression in primary lacrimal gland cells (Saarloos et al., 1999). RAW 246·7 immortalized mouse macrophages were obtained by a generous gift from Dr Vernon Tesh (Texas A&M University System Health Sciences Center, College Station, TX, USA). RAW 264·7 cells are often used as a positive control for iNOS expression because these immortalized macrophages produce high levels of iNOS protein and NO in vitro (MacMicking et al., 1997). Specific instructions for growth conditions and subcultivation of RAW 264·7 cells were obtained from the Tesh lab. Dulbecco’s modified Eagle’s medium (DMEM), DMEM mixed 1:1 with Ham’s nutrient mixture F-12 (DMEM/F12), fetal bovine serum (FBS), dexamethasone, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), liposolysaccharide (LPS) of Salmonella typhimirum, aprotinin, leupeptin, and pepstatin-A were purchased from Sigma Chemical, St Louis, MO, USA. Matrigel basement membrane matrix and Nu-Serum IV media supplement were purchased from Becton Dickinson, Bedford, MA, USA. Soybean trypsin inhibitor (STI), insulin: transferrin:sodium selenite (ITS-X), murine epidermal growth factor (EGF), gentamicin, penicillin, streptomycin, and 0·25% porcine trypsin-EDTA were purchased from Gibco BRL, Grand Island, NY, USA. Murine recombinant interleukin-1b (IL-1b) and murine recombinant IL-1ra were purchased from R&D Systems, Minneapolis, MN, USA. N-(3-(aminomethyl)benzyl) acetamidine (1400W) was purchased from Calbiochem, San Diego, CA, USA. Nitrate/ nitrite colorimetric assay was purchased from Cayman Chemical, Ann Arbor, MI, USA. Immobilon-P polyvinylidene fluoride (PVDF) transfer membranes were purchased from Millipore, Bedford, MA, USA. Novex NuPAGE 4 – 12% Bis-Tris gradient gels, gel running buffers, and sample buffers were purchased from Invitrogen, Carlsbad, CA, USA. Anti-iNOS monoclonal antibody (961-1144, clone 54) was purchased from Transduction Labs, Lexington, KY, USA. ECL Western blot detection system was purchased from Amersham Pharmacia Biotech, Piscataway,
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NJ, USA. BCA protein assay was purchased from Pierce Chemical, Rockford, IL, USA. All other chemicals used were reagent grade. 2.2. Cell culture RLG cells and murine macrophages (RAW 264·7 cells) were grown in a 378C humidified incubator in 95% air/5% CO2. RLG normal feed culture medium consisted of DMEM/F-12 with or without phenol red supplemented with 5% Nu-Serum IV, 1 mM dexamethasone, 0·1 mg ml21 STI, 10 mg ml21 insulin, 5·5 mg ml21 transferrin, 6·7 ng ml 21 sodium selenite, 10 ng ml 21 EGF, and 10 mg ml21 gentamicin. All RLG culture dishes were coated with 5% Matrigel (v/v) in DMEM/F-12. RLG cells were subcultivated by incubation with trypsin and scraping. RAW 264·7 cells were grown on normal culture dishes in DMEM supplemented with 10% FBS, 500 units ml21 streptomycin, and 500 units ml21 penicillin. RAW 264·7 cells were subcultivated without trypsin. 2.3. Nitric oxide determination Following incubations with drugs, samples of phenol red- and dexamethasone-free culture media were extracted and levels of nitrite and nitrate, the relatively stable end products of NO, were determined using the nitrite/nitrate colorimetric assay system. Eighty microliter aliquots of phenol red-free culture media were incubated with 10 ml each of nitrate reductase preparation and nitrate reductase cofactor preparation (proprietary concentrations), which converts nitrate to nitrite, for 2 hr at room temperature in 96-well microassay plates. One hundred microliters Griess reagent, which converts nitrite to a purple azo compound, were added to each well and incubated at room temperature for 10 min. Absorbance was measured at 540 nm on a Packard SpectraCount plate reader. Final nitrate and nitrite concentrations were determined by comparing sample absorbances to those of nitrate reductase-treated sodium nitrate standards. 2.4. Cell viability and cell number Cell viability was determined using the MTT assay. The MTT assay detects viable cells by measuring the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to an insoluble purple formazan compound by cleavage of the tetrazolium ring by active mitochondrial dehydrogenase enzymes (Mossman, 1983). The formazen compound can be dissolved in an organic solvent such as dimethylsulfoxide (DMSO) and the absorbance at 570 nm directly corresponds to the number of viable cells. Cells in six-well dishes were incubated with 0·5 mg ml21 MTT in phenol red-free media at 378C for exactly 4 hr. The resulting purple formazen compound was dissolved in 1 ml DMSO per well and shaken for 5 min. Absorbance of each well
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was measured at 570 nm. Exact cell number was determined using Trypan Blue exclusion and a hemacytometer. Counting cells using trypan blue exclusion and simultaneously performing the MTT assay on cells seeded at identical densities established a standard curve of A570 vs. cell number. 2.5. Western blotting To identify iNOS protein expression, cells were washed twice in ice-cold PBS, scraped, added to ice-cold lysis buffer (20 mM Tris HCl, pH 7·4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mg ml21 leupeptin, 1 mg ml21 pepstatin-A, and 1 mg ml21 aprotinin), and kept on ice for 30 min to lyse cells. Lysates were centrifuged at 10 000g for 15 min and total protein concentration of supernatants were determined using the BCA assay. Sixty micrograms of cell protein was separated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS– PAGE) on 4– 12% Bis-Tris gradient gels. Proteins were transferred to Immobilon-P membranes at 1 A constant current in transfer buffer 25 mM Tris HCl, pH 7·4, 190 mM glycine, 0·05% SDS, and 20% methanol) for 90 min. Transfer membranes were blocked by incubating in 10% non-fat dry milk in TBS-T (20 mM Tris HCl, pH 7·4, 150 mM NaCl, and 0·1% Tween-20) for 60 min at room temperature with agitation. Membranes were then transferred to a solution of monoclonal anti-iNOS diluted 1:1000 in blocking solution and incubated for 60 min at room temperature with agitation, followed by three 5 min washes in TBS-T. Membranes were then incubated in anti-mouse HRP-conjugate diluted 1:1000 in blocking solution for 60 min at room temperature with agitation, followed by six 5 min washes in TBS-T. Antibodies were detected using the ECL detection system and visualized by exposure to autoradiography film. 2.6. Statistical analysis Results for nitric oxide measurements are expressed as mM nitrite and nitrate per 106 cells. Data are expressed as mean ^ standard error of the mean (s.e.m.). Means were compared using Student’s pooled t-test. Differences between means were considered significant at p # 0·05:
3. Results 3.1. Nitric oxide production by interleukin-1b Incubation of lacrimal gland acinar cells with recombinant murine IL-1b for 24 hr produced a concentrationdependent increase in NO production (as determined by accumulation of nitrate and nitrite in culture media) above vehicle-treated cells (Fig. 1). Maximum NO production occurred after 24 hr incubation with 100 ng ml21 IL-1b
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Fig. 1. Nitric oxide production in lacrimal gland acinar cells following 24 hr incubation with IL-1b alone (X), IL-1b plus 100 ng ml21 IL-1ra (B), or 0.01% BSA vehicle (P). Conditioned media was assayed for total nitrate and nitrite concentration using a combination of nitrate reductase and Griess reagent. Values were normalized to cell number. IL-1b stimulated a concentration-dependent increase in NO production. A single concentration of IL-1ra significantly inhibited nitric oxide production at all concentrations of IL-1b. Vehicle for control was added at equivalent concentrations to vehicle used with IL-1b. Error bars represent standard error (s.e.m.). * Indicates significant difference from IL-1b alone at corresponding time point ðp , 0:05Þ.
(12·28 ^ 0·96 mM /106 cells; n ¼ 6), however, this was not significantly different from NO produced by 10 ng ml21 IL1b (10·49 ^ 0·87 mM /106 cells; n ¼ 6; p # 0·05). Nitric oxide production at all concentrations of IL-1b was significantly higher than that by vehicle-treated cells ðp # 0·05Þ. Co-incubation of IL-1b with a single concentration of 100 ng ml21 IL-1ra significantly reduced NO production by all concentrations of IL-1b ðp # 0·05Þ. Cells co-incubated with 100 ng ml 21 IL-1ra and 10 and 100 ng ml21 IL-1b produced NO levels that were significantly higher than vehicle-treated cells, yet significantly lower than IL-1b-treated cells (3·62 ^ 0·1 mM /106 cells; 4·78 ^ 0·13 mM /106 cells, respectively). Co-incubation of lacrimal gland cells for 24 hr with IL1b and the iNOS-specific inhibitor, 1400W, significantly lowered NO production compared to 1, 10 and 100 ng ml21 IL-1b alone (Fig. 2). Ten micromolar 1400W significantly reduced 100 ng ml21 IL-1b-stimulated NO production from 12·28 ^ 0·96 mM /106 cells ðn ¼ 6Þ to 1·35 ^ 0·07 mM /106 cells ðn ¼ 6Þ.
Fig. 2. Nitric oxide production (as determined by nitrate and nitrite in media) in lacrimal gland acinar cells by IL-1b alone (X), IL-1b plus 1 mM (B) or 10 mM (S) 1400 W, or vehicle alone (P). Conditioned media was assayed for total nitrate and nitrite concentration using a combination of nitrate reductase and Griess reagent. Values were normalized to cell number. Both 1 and 10 mM 1400 W significantly inhibited nitric oxide production stimulated by 1, 10, and 100 ng ml21 IL-1b after 24 hr incubation. Nitric oxide produced by IL-1b- and 1400 W-treated cells was significantly higher than vehicle alone at all concentrations. Error bars represent standard error of the mean (s.e.m.). * Indicates significant difference from IL-1b alone at corresponding time point ðp , 0:05Þ.
to SDS – PAGE and Western blotting. Fig. 3 reveals that iNOS expression was greatest when cells were exposed to IL-1b for 4 hr and that high levels of expression remained at 6 and 8 hr. However, iNOS expression was nearly completely diminished after 12 hr exposure to IL-1b. Cells incubated with IL-1b for 12– 24 hr had no iNOS expression (data not shown). To correlate this phenomenon with NO production, cells were exposed to IL-1b for 0– 24 hr and NO production was measured every 3 hr (Fig. 4). NO production was greatly increased from 0 to 3 hr and maintained at near steady state from 3 to 6 hr. Nitric oxide production increased slightly thereafter from 6 to 24 hr, yet remained greatly increased above control levels. To confirm that the inhibitory effect of IL-1 receptor antagonist on iNOS expression was due to blockade of
3.2. Induction of iNOS expression Previous studies on the induction of iNOS by cytokines in other cell types have reported iNOS expression at different time points after drug addition (Balligand et al., 1994; Adeghate and Parvez, 2000; Tabatabaie et al., 2000). To determine the time course of iNOS expression, lacrimal cells were incubated with 10 ng ml21 IL-1b for 0 –24 hr and cells were harvested every 2 hr and subjected
Fig. 3. Western blot of iNOS (130 kDa) expression in lacrimal gland acinar cells following incubation with 10 ng ml21 IL-1b for 0 –12 hr. Sixty micrograms total protein from cells were separated by SDS– PAGE and probed for iNOS expression with monoclonal antibody. Blots were visualized on autoradiography film by ECL. Position of iNOS band was determined using molecular weight markers and iNOS positive control. This blot is representative of three separate experiments.
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4. Discussion
Fig. 4. Nitric oxide production in lacrimal gland acinar cells by 10 ng ml21 IL-1b (X) or vehicle alone (P) at different incubation times from 0 to 24 hr. Conditioned media was assayed for total nitrate and nitrite concentration using a combination of nitrate reductase and Griess reagent. Values were normalized to cell number. Nitric oxide production by IL-1b-stimulated cells was significantly higher than vehicle at all time points except 0 hr. Error bars represent standard error of the mean (s.e.m.). * Indicates significant difference from vehicle alone at corresponding time point ðp , 0:05Þ.
iNOS induction, lacrimal cells were incubated with 10 ng ml21 IL-1b with and without 100 ng ml21 IL-1ra for 6 hr (Fig. 5). Western blot of these cells indicates that iNOS expression is greatly reduced, but not fully diminished, after 6 hr of co-incubation with IL-1b and IL-1ra. To compare the levels of iNOS protein expression between lacrimal gland acinar cells and macrophages, macrophage lysate (from RAW 264·7 cells stimulated with 1 mg ml21 LPS for 6 hr) was run on the same gel as the lacrimal cell lysate (Fig. 5). Macrophage iNOS production was seen to be greater than lacrimal cell lysate when incubated for equal time periods.
Fig. 5. Western blot of iNOS expression in both RAW 264.7 macrophages and lacrimal gland acinar cells. Sixty micrograms total protein from lacrimal cells and 5 mg protein from RAW 264.7 cells were separated by SDS–PAGE and probed for iNOS expression with monoclonal antibody. Blots were visualized on autoradiography film by ECL. Position of iNOS band was determined using molecular weight markers. Lane 1 represents RAW 264.7 cells stimulated with 1 mg ml21 LPS for 6 hr. Lanes 2–4 represent lacrimal cells incubated for 6 hr with vehicle alone (2), 10 ng ml21 IL-1b alone (3), or 10 ng ml21 IL-1b plus 100 ng ml21 IL1ra (4). This blot is representative of three separate experiments.
Dry eye syndrome, or KCS, is a chronic condition that causes ocular pain and discomfort for millions of people in the USA (Foulks, 1998). The etiology of this condition is multi-faceted and not clearly understood. One of the leading causes of dry eye is the autoimmune disease, Sjo¨gren’s syndrome, in which autoantibodies are produced against the lacrimal and salivary glands. In both Sjo¨gren’s and nonSjo¨gren’s dry eye, inflammation of the lacrimal gland occurs, often accompanied by significant lymphocytic infiltration of the gland. Lymphocytic infiltration is accompanied by increases in local levels of inflammatory mediators, such as cytokines and chemokines (Robinson et al., 1998a,b). At the present time there is no pharmacological agent on the market approved for dry eye and patients must rely on artificial tear solutions, surgery, or other treatments that replenish moisture. The anti-inflammatory immune modulator cyclosprorin A is currently in clinical testing for topical treatment of KCS. However, the pathway connecting lacrimal gland inflammation to KCS is not yet fully understood. The role of nitric oxide in lacrimal gland cells has not been studied extensively. It is well known that the induction of nitric oxide synthase (iNOS) in many cell types occurs by stimulation with a complex milieu of pro-inflammatory cytokines and bacterial endotoxins during periods of inflammation or infection (Eizirik et al., 1996; Kroncke et al., 1997). Expression of iNOS catalyzes production of NO from L -arginine in concentrations much greater then those produced by constitutive isoforms of NOS. High levels of NO can be cytotoxic, which may be beneficial in terms of microbial infection or tumor suppression, but which may also be harmful to the host cells (Brune et al., 1998; Chung et al., 2001). Induction of iNOS has been observed in other inflammatory diseases of the eye, including experimental autoimmune uveitis (Jacquemin et al., 1996) and corneal endothelial inflammation (Dighiero et al., 1997). It has been demonstrated in this study that iNOS expression, not constitutively seen in lacrimal gland acinar cells, can be induced in these cells by addition of proinflammatory cytokines, such as IL-1b and that iNOS catalyzes a significant increase in NO production in these cells. Induction of iNOS and concomitant increase in NO production are partially inhibited by IL-1ra. Induction of iNOS was evidenced both by Western blot analysis and by inhibition of IL-1b-stimulated NO production by the iNOSspecific inhibitor 1400W. During the course of demonstrating that iNOS is induced in lacrimal gland cells, it was revealed that expression of iNOS is temporally autoregulated within the cells in a manner that is somewhat different than in previous work involving other cell types. In cultured corneal epithelial, stromal, and endothelial cells, for example, iNOS protein was found to be expressed at peak levels up to 24 hr poststimulus (O’Brien et al., 2001). While it is dogmatic that
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iNOS expression can produce high levels of NO for periods of days (compared to low levels of NO produced by constitutive NOS for periods of hours) (Ignarro, 1996; Gorren and Mayer, 1997), it was found in the current study that iNOS in lacrimal gland acinar cells is upregulated early after IL-1b stimulation and is inhibited soon thereafter (Figs. 3 and 4). It has been demonstrated previously that NO itself can inhibit the induction of iNOS in cardiac myocytes (Balligand et al., 1994). While the data presented in this study do not clearly prove that NO is responsible for this temporal inhibition of iNOS expression, this remains a possibility. An autofeedback mechanism would be important to prevent prolonged overproduction of NO by iNOS and possible cytotoxicity by NO in lacrimal gland cells during short-term inflammation. In conclusion, the data from this study indicate that lacrimal gland inflammation might be caused by induction of iNOS protein expression in acinar cells by proinflammatory cytokines, such as IL-1b, and that iNOSinduced inflammation might play a role in dry eye syndrome. Based on our data that IL-1ra inhibits iNOS expression and NO production and that 1400W also inhibits NO production, clinically useful drugs that inhibit the induction of iNOS or prevent overproduction of NO might be useful as therapeutic agents for controlling autoimmune lacrimal gland inflammation. The effects of upregulated iNOS on lacrimal gland cell function have yet to be determined. Further studies examining cell death, protein secretion, and gene expression need to be conducted to fully understand the effects of increased NO on lacrimal gland acinar cells in relation to dry eye syndrome.
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Induction of nitric oxide synthase and over-production of nitric oxide by interleukin-1b in cultured lacrimal gland acinar cells Clay Beauregarda,b, Paul C. Brandtb, George C.Y. Chioua,b,* a
Institute of Ocular Pharmacology, Texas A&M University System Health Science Center, College of Medicine, 304 Reynolds Medical Building, College Station, TX 77843-1114, USA b Department of Medical Pharmacology and Toxicology, Texas A&M University System Health Science Center, College of Medicine, 304 Reynolds Medical Building, College Station, TX 77843-1114, USA Received 25 September 2001; accepted 29 May 2002
Abstract Purpose. Inflammation of the lacrimal gland is one of the major causative factors in aqueous tear-deficient dry eye syndrome. Proinflammatory cytokine production is upregulated in lacrimal gland autoimmune disease (i.e. Sjo¨gren’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-1b (IL-1b), 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-1b, iNOS inhibitor, or IL-1 receptor antagonist 2 (IL-1ra). Colorimetric detection of NO2 2 and NO3 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-1b stimulated a concentration-dependent and time-dependent increase in NO production. IL-1b-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-1b, and was nearly undetectable at 12 hr. IL-1ra greatly reduced IL-1b-induced iNOS expression. Conclusions. Lacrimal gland acinar cells are able to produce iNOS in response to the pro-inflammatory cytokine IL-1b. 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. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: lacrimal gland; dry eye; nitric oxide; iNOS; interleukin-1; cytokines; inflammation; autoimmune
1. Introduction Dry eye syndrome is a chronic condition in which some components of the preocular tear film are dysfunctional, leaving the patient with painful symptoms of dryness. The tear film is composed of three layers of distinct origin: the aqueous layer from the lacrimal glands, the lipid layer from the Meibomian glands, and the mucin layer from the goblet * Corresponding author. Dr George C.Y. Chiou, Department of Medical Pharmacology and Toxicology, Texas A&M University System Health Science Center, College of Medicine, 304 Reynolds Medical Building, College Station, TX 77843-1114, USA. E-mail address: [email protected] (G.C.Y. Chiou).
cells. Aqueous tears provide the ocular surface with water, electrolytes, proteins, and oxygen. Deficiency of aqueous tears, either in quantity or quality, can lead to ocular surface disease, or keratoconjunctivitis sicca (KCS). The factors leading to aqueous tear deficiency are complex and may involve autoimmune disease (i.e. Sjo¨gren’s syndrome), loss of hormonal support, and glandular inflammation (Stern et al., 1998). A diseased lacrimal gland exhibits reduced fluid and protein secretion (Mathers and Daley, 1996). It has also been shown that lacrimal glands from patients with autoimmune dry eye (Sjo¨gren’s syndrome) produce greatly increased levels of pro-inflammatory cytokines, such as interleukin-1 (IL-1), interferon-g (IFN-g), and tumor
0014-4835/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. DOI:10.1016/S0014-4835(03)00058-7
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necrosis factor-a (TNF-a), which are secreted into the tear fluid (Robinson et al., 1998b; Rosenbaum et al., 1998; Pflugfelder et al., 1999). The possible role of nitric oxide (NO) in dry eye syndrome has not received much attention in the literature to date. NO is a diffusible messenger molecule that is constitutively produced from L -arginine in most cell types by two of the three known isoforms of nitric oxide synthase (NOS): endothelial cell NOS (eNOS/ecNOS/NOS-1) and neuronal or brain NOS (nNOS/bNOS/NOS-3). The inducible isoform of NOS (iNOS/NOS-2) is only expressed upon activation of a signal transduction cascade initiated by immunological or other pathophysiological conditions. Constitutive NOS produces low levels of NO for short periods of time and the reaction is calcium/calmodulindependent, whereas iNOS produces relatively high levels of NO for longer periods of time and is calcium/calmodulinindependent (Ignarro, 1996). Over-production of NO can be cytotoxic, both directly by the formation of free radicals and indirectly by initiating signal transduction pathways leading to apoptosis (Brune et al., 1998; Chung et al., 2001). Expression of both eNOS and nNOS has been shown in mouse lacrimal gland slices (Ding et al., 2000), indicating that NO is constitutively produced in the lacrimal gland. The expression of iNOS has been experimentally induced in acinar cells of the exocrine pancreas and in hepatocytes, both of which are secretory epithelial cell types (Eizirik et al., 1996; Smith et al., 1997; Adeghate and Parvez, 2000). Induction of iNOS has also been shown to occur in corneal epithelial cells and anterior segment during experimental uveitis (Becquet et al., 1997; Dighiero et al., 1997). In this study, it will be demonstrated that iNOS expression can be induced in cultured lacrimal gland acinar cells by addition of the pro-inflammatory cytokine, interleukin-1b (IL-1b). The induction of iNOS in these cells leads to a significant increase in nitric oxide production. It will also be demonstrated that the expression of iNOS in lacrimal gland cells is temporally regulated, perhaps by nitric oxide itself. Finally, it will be demonstrated that the induction of iNOS can be inhibited by IL-1 receptor antagonist (IL-1ra).
2. Materials and methods 2.1. Materials Immortalized rabbit lacrimal gland (RLG) acinar epithelial cells were obtained by a generous gift from Dr Roger Beuerman (Louisiana State University Eye Center, New Orleans, LA, USA). The protein secretory function, morphology, and cellular protein expression of these cells were found to be identical to primary cultured lacrimal cells (Nguyen et al., 1999). Specific instructions for growth conditions and subcultivation of the immortalized cells were obtained from the Beuerman lab. In the initial study of this
immortalized cell line, the cells were compared to primary lacrimal gland acinar cells for morphology, protein expression, and secretory function (Nguyen et al., 1999). It was determined in the referenced paper that morphologically the cells mainly formed a monolayer on Matrigelcoated plates, with occasional formation of small structures resembling acini, similar to cultured primary cells. The immortalized cells showed positive immunohistochemical reactivity to secretory component (SC), a proteolytic fragment of the IgA receptor produced predominantly in acinar cells, as well as transferrin (Tf) and transferrin receptor (TfR). Total protein secretion in response to carbachol was also measured and found to be similar to primary cell data (Nguyen et al., 1999). In another study examining the similarity of this immortalized RLG acinar cell line to primary cells, expression of cytokeratins, laminin, and integrins in this immortalized line was found to be indistinguishable from expression in primary lacrimal gland cells (Saarloos et al., 1999). RAW 246·7 immortalized mouse macrophages were obtained by a generous gift from Dr Vernon Tesh (Texas A&M University System Health Sciences Center, College Station, TX, USA). RAW 264·7 cells are often used as a positive control for iNOS expression because these immortalized macrophages produce high levels of iNOS protein and NO in vitro (MacMicking et al., 1997). Specific instructions for growth conditions and subcultivation of RAW 264·7 cells were obtained from the Tesh lab. Dulbecco’s modified Eagle’s medium (DMEM), DMEM mixed 1:1 with Ham’s nutrient mixture F-12 (DMEM/F12), fetal bovine serum (FBS), dexamethasone, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), liposolysaccharide (LPS) of Salmonella typhimirum, aprotinin, leupeptin, and pepstatin-A were purchased from Sigma Chemical, St Louis, MO, USA. Matrigel basement membrane matrix and Nu-Serum IV media supplement were purchased from Becton Dickinson, Bedford, MA, USA. Soybean trypsin inhibitor (STI), insulin: transferrin:sodium selenite (ITS-X), murine epidermal growth factor (EGF), gentamicin, penicillin, streptomycin, and 0·25% porcine trypsin-EDTA were purchased from Gibco BRL, Grand Island, NY, USA. Murine recombinant interleukin-1b (IL-1b) and murine recombinant IL-1ra were purchased from R&D Systems, Minneapolis, MN, USA. N-(3-(aminomethyl)benzyl) acetamidine (1400W) was purchased from Calbiochem, San Diego, CA, USA. Nitrate/ nitrite colorimetric assay was purchased from Cayman Chemical, Ann Arbor, MI, USA. Immobilon-P polyvinylidene fluoride (PVDF) transfer membranes were purchased from Millipore, Bedford, MA, USA. Novex NuPAGE 4 – 12% Bis-Tris gradient gels, gel running buffers, and sample buffers were purchased from Invitrogen, Carlsbad, CA, USA. Anti-iNOS monoclonal antibody (961-1144, clone 54) was purchased from Transduction Labs, Lexington, KY, USA. ECL Western blot detection system was purchased from Amersham Pharmacia Biotech, Piscataway,
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NJ, USA. BCA protein assay was purchased from Pierce Chemical, Rockford, IL, USA. All other chemicals used were reagent grade. 2.2. Cell culture RLG cells and murine macrophages (RAW 264·7 cells) were grown in a 378C humidified incubator in 95% air/5% CO2. RLG normal feed culture medium consisted of DMEM/F-12 with or without phenol red supplemented with 5% Nu-Serum IV, 1 mM dexamethasone, 0·1 mg ml21 STI, 10 mg ml21 insulin, 5·5 mg ml21 transferrin, 6·7 ng ml 21 sodium selenite, 10 ng ml 21 EGF, and 10 mg ml21 gentamicin. All RLG culture dishes were coated with 5% Matrigel (v/v) in DMEM/F-12. RLG cells were subcultivated by incubation with trypsin and scraping. RAW 264·7 cells were grown on normal culture dishes in DMEM supplemented with 10% FBS, 500 units ml21 streptomycin, and 500 units ml21 penicillin. RAW 264·7 cells were subcultivated without trypsin. 2.3. Nitric oxide determination Following incubations with drugs, samples of phenol red- and dexamethasone-free culture media were extracted and levels of nitrite and nitrate, the relatively stable end products of NO, were determined using the nitrite/nitrate colorimetric assay system. Eighty microliter aliquots of phenol red-free culture media were incubated with 10 ml each of nitrate reductase preparation and nitrate reductase cofactor preparation (proprietary concentrations), which converts nitrate to nitrite, for 2 hr at room temperature in 96-well microassay plates. One hundred microliters Griess reagent, which converts nitrite to a purple azo compound, were added to each well and incubated at room temperature for 10 min. Absorbance was measured at 540 nm on a Packard SpectraCount plate reader. Final nitrate and nitrite concentrations were determined by comparing sample absorbances to those of nitrate reductase-treated sodium nitrate standards. 2.4. Cell viability and cell number Cell viability was determined using the MTT assay. The MTT assay detects viable cells by measuring the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to an insoluble purple formazan compound by cleavage of the tetrazolium ring by active mitochondrial dehydrogenase enzymes (Mossman, 1983). The formazen compound can be dissolved in an organic solvent such as dimethylsulfoxide (DMSO) and the absorbance at 570 nm directly corresponds to the number of viable cells. Cells in six-well dishes were incubated with 0·5 mg ml21 MTT in phenol red-free media at 378C for exactly 4 hr. The resulting purple formazen compound was dissolved in 1 ml DMSO per well and shaken for 5 min. Absorbance of each well
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was measured at 570 nm. Exact cell number was determined using Trypan Blue exclusion and a hemacytometer. Counting cells using trypan blue exclusion and simultaneously performing the MTT assay on cells seeded at identical densities established a standard curve of A570 vs. cell number. 2.5. Western blotting To identify iNOS protein expression, cells were washed twice in ice-cold PBS, scraped, added to ice-cold lysis buffer (20 mM Tris HCl, pH 7·4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mg ml21 leupeptin, 1 mg ml21 pepstatin-A, and 1 mg ml21 aprotinin), and kept on ice for 30 min to lyse cells. Lysates were centrifuged at 10 000g for 15 min and total protein concentration of supernatants were determined using the BCA assay. Sixty micrograms of cell protein was separated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS– PAGE) on 4– 12% Bis-Tris gradient gels. Proteins were transferred to Immobilon-P membranes at 1 A constant current in transfer buffer 25 mM Tris HCl, pH 7·4, 190 mM glycine, 0·05% SDS, and 20% methanol) for 90 min. Transfer membranes were blocked by incubating in 10% non-fat dry milk in TBS-T (20 mM Tris HCl, pH 7·4, 150 mM NaCl, and 0·1% Tween-20) for 60 min at room temperature with agitation. Membranes were then transferred to a solution of monoclonal anti-iNOS diluted 1:1000 in blocking solution and incubated for 60 min at room temperature with agitation, followed by three 5 min washes in TBS-T. Membranes were then incubated in anti-mouse HRP-conjugate diluted 1:1000 in blocking solution for 60 min at room temperature with agitation, followed by six 5 min washes in TBS-T. Antibodies were detected using the ECL detection system and visualized by exposure to autoradiography film. 2.6. Statistical analysis Results for nitric oxide measurements are expressed as mM nitrite and nitrate per 106 cells. Data are expressed as mean ^ standard error of the mean (s.e.m.). Means were compared using Student’s pooled t-test. Differences between means were considered significant at p # 0·05:
3. Results 3.1. Nitric oxide production by interleukin-1b Incubation of lacrimal gland acinar cells with recombinant murine IL-1b for 24 hr produced a concentrationdependent increase in NO production (as determined by accumulation of nitrate and nitrite in culture media) above vehicle-treated cells (Fig. 1). Maximum NO production occurred after 24 hr incubation with 100 ng ml21 IL-1b
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Fig. 1. Nitric oxide production in lacrimal gland acinar cells following 24 hr incubation with IL-1b alone (X), IL-1b plus 100 ng ml21 IL-1ra (B), or 0.01% BSA vehicle (P). Conditioned media was assayed for total nitrate and nitrite concentration using a combination of nitrate reductase and Griess reagent. Values were normalized to cell number. IL-1b stimulated a concentration-dependent increase in NO production. A single concentration of IL-1ra significantly inhibited nitric oxide production at all concentrations of IL-1b. Vehicle for control was added at equivalent concentrations to vehicle used with IL-1b. Error bars represent standard error (s.e.m.). * Indicates significant difference from IL-1b alone at corresponding time point ðp , 0:05Þ.
(12·28 ^ 0·96 mM /106 cells; n ¼ 6), however, this was not significantly different from NO produced by 10 ng ml21 IL1b (10·49 ^ 0·87 mM /106 cells; n ¼ 6; p # 0·05). Nitric oxide production at all concentrations of IL-1b was significantly higher than that by vehicle-treated cells ðp # 0·05Þ. Co-incubation of IL-1b with a single concentration of 100 ng ml21 IL-1ra significantly reduced NO production by all concentrations of IL-1b ðp # 0·05Þ. Cells co-incubated with 100 ng ml 21 IL-1ra and 10 and 100 ng ml21 IL-1b produced NO levels that were significantly higher than vehicle-treated cells, yet significantly lower than IL-1b-treated cells (3·62 ^ 0·1 mM /106 cells; 4·78 ^ 0·13 mM /106 cells, respectively). Co-incubation of lacrimal gland cells for 24 hr with IL1b and the iNOS-specific inhibitor, 1400W, significantly lowered NO production compared to 1, 10 and 100 ng ml21 IL-1b alone (Fig. 2). Ten micromolar 1400W significantly reduced 100 ng ml21 IL-1b-stimulated NO production from 12·28 ^ 0·96 mM /106 cells ðn ¼ 6Þ to 1·35 ^ 0·07 mM /106 cells ðn ¼ 6Þ.
Fig. 2. Nitric oxide production (as determined by nitrate and nitrite in media) in lacrimal gland acinar cells by IL-1b alone (X), IL-1b plus 1 mM (B) or 10 mM (S) 1400 W, or vehicle alone (P). Conditioned media was assayed for total nitrate and nitrite concentration using a combination of nitrate reductase and Griess reagent. Values were normalized to cell number. Both 1 and 10 mM 1400 W significantly inhibited nitric oxide production stimulated by 1, 10, and 100 ng ml21 IL-1b after 24 hr incubation. Nitric oxide produced by IL-1b- and 1400 W-treated cells was significantly higher than vehicle alone at all concentrations. Error bars represent standard error of the mean (s.e.m.). * Indicates significant difference from IL-1b alone at corresponding time point ðp , 0:05Þ.
to SDS – PAGE and Western blotting. Fig. 3 reveals that iNOS expression was greatest when cells were exposed to IL-1b for 4 hr and that high levels of expression remained at 6 and 8 hr. However, iNOS expression was nearly completely diminished after 12 hr exposure to IL-1b. Cells incubated with IL-1b for 12– 24 hr had no iNOS expression (data not shown). To correlate this phenomenon with NO production, cells were exposed to IL-1b for 0– 24 hr and NO production was measured every 3 hr (Fig. 4). NO production was greatly increased from 0 to 3 hr and maintained at near steady state from 3 to 6 hr. Nitric oxide production increased slightly thereafter from 6 to 24 hr, yet remained greatly increased above control levels. To confirm that the inhibitory effect of IL-1 receptor antagonist on iNOS expression was due to blockade of
3.2. Induction of iNOS expression Previous studies on the induction of iNOS by cytokines in other cell types have reported iNOS expression at different time points after drug addition (Balligand et al., 1994; Adeghate and Parvez, 2000; Tabatabaie et al., 2000). To determine the time course of iNOS expression, lacrimal cells were incubated with 10 ng ml21 IL-1b for 0 –24 hr and cells were harvested every 2 hr and subjected
Fig. 3. Western blot of iNOS (130 kDa) expression in lacrimal gland acinar cells following incubation with 10 ng ml21 IL-1b for 0 –12 hr. Sixty micrograms total protein from cells were separated by SDS– PAGE and probed for iNOS expression with monoclonal antibody. Blots were visualized on autoradiography film by ECL. Position of iNOS band was determined using molecular weight markers and iNOS positive control. This blot is representative of three separate experiments.
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4. Discussion
Fig. 4. Nitric oxide production in lacrimal gland acinar cells by 10 ng ml21 IL-1b (X) or vehicle alone (P) at different incubation times from 0 to 24 hr. Conditioned media was assayed for total nitrate and nitrite concentration using a combination of nitrate reductase and Griess reagent. Values were normalized to cell number. Nitric oxide production by IL-1b-stimulated cells was significantly higher than vehicle at all time points except 0 hr. Error bars represent standard error of the mean (s.e.m.). * Indicates significant difference from vehicle alone at corresponding time point ðp , 0:05Þ.
iNOS induction, lacrimal cells were incubated with 10 ng ml21 IL-1b with and without 100 ng ml21 IL-1ra for 6 hr (Fig. 5). Western blot of these cells indicates that iNOS expression is greatly reduced, but not fully diminished, after 6 hr of co-incubation with IL-1b and IL-1ra. To compare the levels of iNOS protein expression between lacrimal gland acinar cells and macrophages, macrophage lysate (from RAW 264·7 cells stimulated with 1 mg ml21 LPS for 6 hr) was run on the same gel as the lacrimal cell lysate (Fig. 5). Macrophage iNOS production was seen to be greater than lacrimal cell lysate when incubated for equal time periods.
Fig. 5. Western blot of iNOS expression in both RAW 264.7 macrophages and lacrimal gland acinar cells. Sixty micrograms total protein from lacrimal cells and 5 mg protein from RAW 264.7 cells were separated by SDS–PAGE and probed for iNOS expression with monoclonal antibody. Blots were visualized on autoradiography film by ECL. Position of iNOS band was determined using molecular weight markers. Lane 1 represents RAW 264.7 cells stimulated with 1 mg ml21 LPS for 6 hr. Lanes 2–4 represent lacrimal cells incubated for 6 hr with vehicle alone (2), 10 ng ml21 IL-1b alone (3), or 10 ng ml21 IL-1b plus 100 ng ml21 IL1ra (4). This blot is representative of three separate experiments.
Dry eye syndrome, or KCS, is a chronic condition that causes ocular pain and discomfort for millions of people in the USA (Foulks, 1998). The etiology of this condition is multi-faceted and not clearly understood. One of the leading causes of dry eye is the autoimmune disease, Sjo¨gren’s syndrome, in which autoantibodies are produced against the lacrimal and salivary glands. In both Sjo¨gren’s and nonSjo¨gren’s dry eye, inflammation of the lacrimal gland occurs, often accompanied by significant lymphocytic infiltration of the gland. Lymphocytic infiltration is accompanied by increases in local levels of inflammatory mediators, such as cytokines and chemokines (Robinson et al., 1998a,b). At the present time there is no pharmacological agent on the market approved for dry eye and patients must rely on artificial tear solutions, surgery, or other treatments that replenish moisture. The anti-inflammatory immune modulator cyclosprorin A is currently in clinical testing for topical treatment of KCS. However, the pathway connecting lacrimal gland inflammation to KCS is not yet fully understood. The role of nitric oxide in lacrimal gland cells has not been studied extensively. It is well known that the induction of nitric oxide synthase (iNOS) in many cell types occurs by stimulation with a complex milieu of pro-inflammatory cytokines and bacterial endotoxins during periods of inflammation or infection (Eizirik et al., 1996; Kroncke et al., 1997). Expression of iNOS catalyzes production of NO from L -arginine in concentrations much greater then those produced by constitutive isoforms of NOS. High levels of NO can be cytotoxic, which may be beneficial in terms of microbial infection or tumor suppression, but which may also be harmful to the host cells (Brune et al., 1998; Chung et al., 2001). Induction of iNOS has been observed in other inflammatory diseases of the eye, including experimental autoimmune uveitis (Jacquemin et al., 1996) and corneal endothelial inflammation (Dighiero et al., 1997). It has been demonstrated in this study that iNOS expression, not constitutively seen in lacrimal gland acinar cells, can be induced in these cells by addition of proinflammatory cytokines, such as IL-1b and that iNOS catalyzes a significant increase in NO production in these cells. Induction of iNOS and concomitant increase in NO production are partially inhibited by IL-1ra. Induction of iNOS was evidenced both by Western blot analysis and by inhibition of IL-1b-stimulated NO production by the iNOSspecific inhibitor 1400W. During the course of demonstrating that iNOS is induced in lacrimal gland cells, it was revealed that expression of iNOS is temporally autoregulated within the cells in a manner that is somewhat different than in previous work involving other cell types. In cultured corneal epithelial, stromal, and endothelial cells, for example, iNOS protein was found to be expressed at peak levels up to 24 hr poststimulus (O’Brien et al., 2001). While it is dogmatic that
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iNOS expression can produce high levels of NO for periods of days (compared to low levels of NO produced by constitutive NOS for periods of hours) (Ignarro, 1996; Gorren and Mayer, 1997), it was found in the current study that iNOS in lacrimal gland acinar cells is upregulated early after IL-1b stimulation and is inhibited soon thereafter (Figs. 3 and 4). It has been demonstrated previously that NO itself can inhibit the induction of iNOS in cardiac myocytes (Balligand et al., 1994). While the data presented in this study do not clearly prove that NO is responsible for this temporal inhibition of iNOS expression, this remains a possibility. An autofeedback mechanism would be important to prevent prolonged overproduction of NO by iNOS and possible cytotoxicity by NO in lacrimal gland cells during short-term inflammation. In conclusion, the data from this study indicate that lacrimal gland inflammation might be caused by induction of iNOS protein expression in acinar cells by proinflammatory cytokines, such as IL-1b, and that iNOSinduced inflammation might play a role in dry eye syndrome. Based on our data that IL-1ra inhibits iNOS expression and NO production and that 1400W also inhibits NO production, clinically useful drugs that inhibit the induction of iNOS or prevent overproduction of NO might be useful as therapeutic agents for controlling autoimmune lacrimal gland inflammation. The effects of upregulated iNOS on lacrimal gland cell function have yet to be determined. Further studies examining cell death, protein secretion, and gene expression need to be conducted to fully understand the effects of increased NO on lacrimal gland acinar cells in relation to dry eye syndrome.
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