Rabbit Lacrimal Gland Acinar Cells Research Articles

A thermo-responsive protein treatment for dry eyes

Millions of Americans suffer from dry eye disease, and there are few effective therapies capable of treating these patients. A decade ago, an abundant protein component of human tears was discovered and named lacritin (Lacrt). Lacrt has prosecretory activity in the lacrimal gland and mitogenic activity at the corneal epithelium. Similar to other proteins placed on the ocular surface, the durability of its effect is limited by rapid tear turnover. Motivated by the rationale that a thermo-responsive coacervate containing Lacrt would have better retention upon administration, we have constructed and tested the activity of a thermo-responsive Lacrt fused to an elastin-like polypeptide (ELP). Inspired from the human tropoelastin protein, ELP protein polymers reversibly phase separate into viscous coacervates above a tunable transition temperature. This fusion construct exhibited the prosecretory function of native Lacrt as illustrated by its ability to stimulate β-hexosaminidase secretion from primary rabbit lacrimal gland acinar cells. It also increased tear secretion from non-obese diabetic (NOD) mice, a model of autoimmune dacryoadenitis, when administered via intra-lacrimal injection. Lacrt ELP fusion proteins undergo temperature-mediated assembly to form a depot inside the lacrimal gland. We propose that these Lacrt ELP fusion proteins represent a potential therapy for dry eye disease and the strategy of ELP-mediated phase separation may have applicability to other diseases of the ocular surface.

Polymeric immunoglobulin receptor traffics through two distinct apically targeted pathways in primary lacrimal gland acinar cells

The polymeric immunoglobulin receptor (pIgR) mediates transcytosis of dimeric immunoglobulin A (dIgA) and its release into mucosal secretions. The present study reveals the complexity of the trafficking of pIgR to the apical plasma membrane in epithelial cells with exocrine secretory functions; in rabbit lacrimal gland acinar cells (LGACs), trafficking of pIgR involves both the transcytotic pathway and one arm of the regulated secretory pathway. By specifically tracking pIgR endocytosed from the basolateral membrane, we show here that the Rab11a-regulated transcytotic pathway mediates the basal-to-apical transport of pIgR, and that pIgR sorted into the transcytotic pathway does not access the regulated secretory pathway. However, previous work in LGACs expanded in the present study has shown that some pIgR is localized to Rab3D-enriched mature secretory vesicles (SVs). Myosin Vb and myosin Vc motors modulate release of proteins from the Rab11a-regulated transcytotic pathway and the Rab3D-enriched secretory pathway in LGACs, respectively. Confocal fluorescence microscopy and biochemical assays showed that inhibition of myosin Vb and myosin Vc activity by overexpression of their dominant-negative mutants each significantly but differentially impaired aspects of apically targeted pIgR trafficking and secretory component release, suggesting that these motors function to regulate pIgR trafficking in both the transcytotic and exocytotic pathways. Intriguingly, a second mature SV population enriched in Rab27b was devoid of pIgR cargo, suggesting the specialization of Rab3D-enriched mature SVs to carry a particular subset of cargo proteins from the trans-Golgi network to the apical plasma membrane.

Direct Imaging of RAB27B-Enriched Secretory Vesicle Biogenesis in Lacrimal Acinar Cells Reveals Origins on a Nascent Vesicle Budding Site

This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the “nascent vesicle site,” from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150Glued, a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules.

Use of nucleofection to efficiently transfect primary rabbit lacrimal gland acinar cells

Lacrimal gland acinar cells are an important cell type to study due to their role in production and release of tear proteins, a function essential for ocular surface integrity and normal visual acuity. However, mechanistic studies are often limited by problems with transfection using either plasmid DNA or siRNA. Although various gene delivery methods are available, many have been unproductive due to consistently low transfection efficiencies. We have developed a method using nucleofection that can result in 50% transfection efficiency and 60% knockdown efficiency for plasmid DNA and siRNA, respectively. These results are vastly improved relative to previous studies, demonstrating that nucleofection offers an efficient transfection technique for primary lacrimal gland acinar cells.

Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.

Cultivation of lacrimal gland acinar cells in a microgravity environment

Background:A rotary cell-culture system (RCCS) allows the creation of a microgravity environment of low shear force, high-mass transfer and three-dimensional cell culture of various cell types. The aim of the...

Transduced viral IL-10 is exocytosed from lacrimal acinar secretory vesicles in a myosin-dependent manner in response to carbachol

The purpose of this study was to determine the intracellular trafficking and release pathways for the therapeutic protein, viral IL-10 (vIL-10), from transduced acinar epithelial cells from rabbit lacrimal gland. Primary cultured rabbit lacrimal gland acinar cells (LGACs) were transduced with adenovirus serotype 5 containing viral interleukin-10 (AdvIL-10). The distribution of vIL-10 was assessed by confocal fluorescence microscopy. Carbachol (CCH)-stimulated release of vIL-10 was quantified by ELISA. vIL-10 localization and exocytosis was probed in response to treatments with agents modulating actin- and myosin-based transport. vIL-10 immunoreactivity was detected in large intracellular vesicles in transduced LGAC. vIL-10 was partially co-localized with biosynthetic but not endosomal compartment markers. vIL-10 release was sensitive to CCH, and the kinetics of release showed an initial burst phase that was similar but not identical to that of the secretory protein, β-hexosaminidase. Disassembly of actin filaments with latrunculin B significantly increased CCH-stimulated vIL-10 secretion, suggesting that vIL-10 was released from stores sequestered beneath the subapical actin barrier. That release required the activity of actin-dependent myosin motors previously implicated in secretory vesicle exocytosis was confirmed by findings that CCH-stimulated vIL-10 release was reduced by inhibition of non-muscle myosin 2 and myosin 5c function, using ML-7 and overexpression of dominant negative myosin 5c, respectively. These results suggest that the majority of vIL-10 transgene product is packaged into a subpopulation of secretory vesicles that utilize actin-dependent myosin motors for aspects of actin coat assembly, compound fusion and exocytosis at the apical plasma membrane in response to CCH stimulation.

Direct interaction between Rab3D and the polymeric immunoglobulin receptor and trafficking through regulated secretory vesicles in lacrimal gland acinar cells

The lacrimal gland is responsible for tear production, and a major protein found in tears is secretory component (SC), the proteolytically cleaved fragment of the extracellular domain of the polymeric Ig receptor (pIgR), which is the receptor mediating the basal-to-apical transcytosis of polymeric immunoglobulins across epithelial cells. Immunofluorescent labeling of rabbit lacrimal gland acinar cells (LGACs) revealed that the small GTPase Rab3D, a regulated secretory vesicle marker, and the pIgR are colocalized in subapical membrane vesicles. In addition, the secretion of SC from primary cultures of LGACs was stimulated by the cholinergic agonist carbachol (CCH), and its release rate was very similar to that of other regulated secretory proteins in LGACs. In pull-down assays from resting LGACs, recombinant wild-type Rab3D (Rab3DWT) or the GDP-locked mutant Rab3DT36N both pulled down pIgR, but the GTP-locked mutant Rab3DQ81L did not. When the pull-down assays were performed in the presence of guanosine-5'-(gamma-thio)-triphosphate, GTP, or guanosine-5'-O-(2-thiodiphosphate), binding of Rab3DWT to pIgR was inhibited. In blot overlays, recombinant Rab3DWT bound to immunoprecipitated pIgR, suggesting that Rab3D and pIgR may interact directly. Adenovirus-mediated overexpression of mutant Rab3DT36N in LGACs inhibited CCH-stimulated SC release, and, in CCH-stimulated LGACs, pull down of pIgR with Rab3DWT and colocalization of pIgR with endogenous Rab3D were decreased relative to resting cells, suggesting that the pIgR-Rab3D interaction may be modulated by secretagogues. These data suggest that the novel localization of pIgR to the regulated secretory pathway of LGACs and its secretion therefrom may be affected by its novel interaction with Rab3D.

Tissue‐engineered tear secretory system: Functional lacrimal gland acinar cells cultured on matrix protein‐coated substrata

Dry eye is a general term that refers to a myriad of ophthalmic disorders resulting in the inadequate wetting of the corneal surface by the tear film. Dry eyes are typically treated by the application of artificial tears. However, patients with lacrimal insufficiencies such as Stevens-Johnson syndrome, chemical and thermal injuries, or ocular cicatricial pemphigoid have very limited options because of the short duration and action of lubricating agents. As a therapeutic strategy, we are working to develop a bioengineered tear secretory system for such patients. This article describes the growth and physiological properties of purified rabbit lacrimal gland acinar cells (pLGACs) on several matrix protein-coated polymers such as silicone, collagen I, copolymers of poly-D,L-lactide-co-glycolide (PLGA; 85:15 and 50:50), poly-L-lactic acid (PLLA), and Thermanox plastic cell culture coverslips. Monolayers of acinar cells were established on all of the polymeric substrata. An assay of beta-hexosaminidase activity in the supernatant medium showed significant increases in protein secretion, following stimulation with 100 microM carbachol on matrix protein-coated and uncoated polymers such as silicone, PLGA 85:15, and PLLA. Our study demonstrates that PLLA supported the morphological and physiological properties of purified rabbit lacrimal gland epithelial cells more successfully than the others.

Characterization of β-hexosaminidase secretion in rabbit lacrimal gland

The present study was aimed at validating the use of the lysosomal enzyme β-hexosaminidase as a marker of secretory function in cultured rabbit lacrimal gland acinar cells. The secretory response and morphological characteristics of isolated acinar cells cultured in a serum-free medium supplemented with an extracellular matrix extract were monitored over time as part of optimization of our culturing protocol. Secreted β-hexosaminidase activity was analyzed and compared with that of another lysosomal enzyme, cathepsin B, as well as protein secreted into the media, w or w/o the presence of secretagogues or protein kinase C activators and inhibitors. Lacrimal gland fluid was obtained from pilocarpine stimulated rabbits, and the activities of β-hexosaminidase and cathepsin B were measured. A membrane fraction and a soluble fraction were obtained from isolated acinar cells and used for kinetic studies of β-hexosaminidase in comparison with that released from cultured cells, in the lacrimal gland fluid and in serum. Optimal secretory response was obtained when the cells had been in culture for 2–3 days, coinciding with the formation of acinus-like structures. Stimulation of the cultured cells by carbachol or phorbol esters resulted in a more than 3-fold increase of β-hexosaminidase release over basal, whereas no effect on cathepsin B release could be detected. Treatment with the protein kinase C inhibitor, chelerythrine chloride, significantly decreased the carbachol and phorbol ester-stimulated secretion. Cathepsin B could not be detected in rabbit lacrimal fluid, but β-hexosaminidase was easily measured in quantities corresponding to as low as 0.4 μl of tear fluid. Using 4-methylumbelliferyl N-acetyl-β- d-glucosaminide as a substrate for β-hexosaminidase, the K m in lacrimal gland fluid (1.22 ± 0.15 mM) was not significantly different from that of the membrane-associated fraction, the soluble fraction, rabbit serum or activity secreted from cultured cells. β-hexosaminidase is secreted by rabbit lacrimal gland, in vivo, and by acinar cells in primary culture, whereas cathepsin B is not secreted under the conditions described. β-Hexosaminidase therefore provides a versatile marker for secretion in studies of tear production utilizing the rabbit as a model. Our results also indicate that PKC is an important regulator of rabbit lacrimal gland secretion.

Integrin adhesion in regulation of lacrimal gland acinar cell secretion

The extracellular microenvironment regulates lacrimal gland acinar cell secretion. Culturing isolated rabbit lacrimal gland acinar cells on different extracellular matrix proteins revealed that laminin enhances carbachol-stimulated secretion to a greater extent than other extracellular matrix proteins investigated. Furthermore, immunofluorescence indicated that integrin subunits, potentially functioning as laminin receptors are present in acinar cells. Among these, the integrin α6 and β1 subunit mRNA expression was also confirmed by RT–PCR and sequence analysis. Secretion assays, which measured β-hexosaminidase activity released in the culture media, demonstrated that function-blocking integrin α6 and β1 monoclonal antibodies (mAbs) induce a rapid, transient and dose-dependent secretory response in cultured cells. To determine the intracellular pathways by which integrin α6 and β1 mAbs could induce secretion, selected second messenger molecules were inhibited. Although inhibitors of protein kinase C and IP 3-induced Ca 2+ mobilization attenuated carbachol-stimulated secretion, no effect on integrin mAb-induced release was observed. In addition, protein tyrosine kinases do not appear to have a role in transducing signals arising from mAb interactions. Our data clearly demonstrate, though, that cell adhesion through integrins regulates secretion from lacrimal gland acinar cells. The fact that the integrin mAbs affect the cholinergic response differently and that the integrin β1 mAb secretion, but not the α6, was attenuated by the phosphatase inhibitor, sodium orthovanadate, suggests that each subunit utilizes separate intracellular signaling pathways to induce exocytosis. The results also indicate that the secretory response triggered by the β1 integrin mAb is generated through dephosphorylation events.

211. Novel Fiber-Mediated Adenovirus Interactions in Primary Rabbit Lacrimal Acini

Top of pageAbstract Purpose: Cellular infection by adenovirus serotype 5 (Ad5) is thought to be mediated by fiber capsid and coxsackie-adenovirus receptor (CAR) binding followed by the interaction of penton capsid with |[alpha]|v integrins, which triggers clathrin-mediated endocytosis. The purpose of this study was to identify the capsid proteins responsible for mediating Ad5 entry in cultured lacrimal acinar epithelial cells, the major source of tear proteins released into ocular surface fluid. Methods: Rabbit lacrimal gland acinar cells were isolated and cultured for 2 - 3 d. To examine the mechanism of entry and the intracellular trafficking of Ad5 and its capsids, reconstituted acini were exposed to Ad-LacZ (MOI=5-20), recombinant penton (20 |[mu]|g/ml) or knob protein (20 |[mu]|g/ml) at 37|[deg]| for short time intervals (0-120 m) and for 16-18 h. Their distributions were evaluated by confocal fluorescence microscopy or a trypsinization-based biochemical assay. To determine the identity of capsids and their binding partners responsible for mediating Ad5 entry, acinar cells were preincubated with various doses of penton, fiber, knob, or penton-blocking peptides, RGD or LDV, for 2 h at 4|[deg]|. Ad-GFP (MOI=2) was then added and incubated for 1 h at 4|[deg]| before incubating cells for 10-12 h at 37|[deg]|. Transduction efficiency was assayed by GFP positivity, determined by FACS analysis. To determine the involvement of heparin sulfate glycosaminoglycan (HS-GAG), in some experiments Ad-GFP was preincubated with heparin at 37|[deg]| for 1 h before being added to the cells. Results: Ad5 showed pronounced intracellular punctate staining after 30 m incubation and retained an intracellular labeling pattern by 2 h that was comparable to that seen with overnight exposure. Penton remained associated with the plasma membrane after acute and chronic exposure, while sustained knob exposure resulted in a comparable intracellular labeling pattern to that seen for Ad5. Fiber significantly (p|[le]|0.05) and substantially inhibited Ad5 infectivity (17.42|[plusmn]|3.53% of control, n=4) while penton had no effect (102.68|[plusmn]|9.63% of control, n=7). Knob also induced a comparable inhibitory effect on Ad5 infectivity (17.23|[plusmn]|3.39% of control, n=8) relative to fiber while RGD and LDV had no effect. Lower doses of fiber and knob also exhibited an inhibitory effect which was not as potent. However, CAR expression appeared to be minimal in acinar cells compared to other cells. Heparin had a modest but significant inhibitory effect on Ad5 infectivity which was additive with that of knob. Conclusions:These studies suggest that Ad5 internalization into lacrimal acini is through a unique fiber-dependent pathway rather than the consensus penton-mediated mechanism seen in simple cell models. HS-GAGs may serve as cooperative receptors for Ad5 by increasing the efficiency of the binding and infection mediated by CAR or another fiber receptor. Further characterization of the nature of virus-cell interactions will be important in consideration of the toxicity and efficacy of Ad5 capsids for non-viral gene delivery.

Characterization of Soluble Nsf Attachment Protein Receptors (Snares) in Rabbit Lacrimal Gland Acinar Cells

The soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein (SNAP) receptor (SNARE) hypothesis has been applied to exocytosis in salivary glands. The expression of SNARE proteins has not been well investigated in the parotid gland. In this study, the mRNA expression of SNAREs and membrane-fusion-related proteins were investigated in the rat parotid by reverse transcriptase-polymerase chain reaction (RT-PCR). All syntaxins except syntaxin 1, and the vesicle-associated membrane proteins (VAMP) except VAMP-7, NSF, SNAP-23 and α-SNAP, were expressed for the SNAREs in rat parotid. Synaptotagmins 3, 4 and 11, Munc18s (1, 2 and 3), syncollin, prenylated Rab acceptor (PRA1), zygin 1, pantophysin and cellugyrin, which are the other membrane-fusion-related proteins, were also detected, but neither Rim nor rabphilin 3A, which have high specificity of binding to Rab 3A, were found. mRNA expressions of many SNAREs and of the membrane-fusion-proteins suggest novel interactions for the regulation of salivary exocytosis.

Adenosine Receptor Signalling in the Rabbit Lacrimal Gland Acinar Cells is Ca2+ Dependent

Adenosine Receptor Signalling in the Rabbit Lacrimal Gland Acinar Cells is Ca2+ Dependent

Altered traffic to the lysosome in an ex vivo lacrimal acinar cell model for chronic muscarinic receptor stimulation

Evidence suggests that lacrimal and salivary epithelial cells constitutively expose potentially pathogenic autoantigens, but that active regulatory networks normally suppress pathological autoimmune responses [Mircheff, A.K., 2003. Sjögren's syndrome as failed local immunohomeostasis: prospects for cell-based therapy. Ocular Surface 1, 160–179; Mircheff, A.K., Hamm-Alvarez, S.F., Kaslow, H.R., Schechter, J.E., Trousdale, M.D., 2004. Epithelial cells in lymphocyte regulation in the lacrimal gland. In: Zierhut, M. (Ed.), Immunology of the Ocular Surface and the Lacrimal Glands. Swets & Zeitlinger, Amsterdam; in press]. Events that potentially disrupt the regulatory networks include increased exposure of constitutive autoantigens and induced exposure of previously cryptic autoantigen epitopes. Chronic muscarinic receptor (MAChR) stimulation in an ex vivo rabbit lacrimal acinar cell model induces functional and biochemical alterations reminiscent of the functional quiescence associated with Sjögren's syndrome [Qian, L., Wang, Y., Xie, J., Rose, C.M., Yang, T., Nakamura, T., Sandberg, M., Zeng, H., Schechter, J.E., Chow, R.H., Hamm-Alvarez, S.F., Mircheff, A.K., 2003. Biochemical changes contributing to functional quiescence in lacrimal gland acinar cells after chronic ex vivo exposure to a muscarinic agonist. Scand. J. Immunol. 58, 550–565]. Chronic MAChR stimulation also elicits changes in the compartmental distribution of β-hexosaminidase, a product that normally is dually targeted into the lysosomal pathway and the regulated apical secretory pathway. Here, we use subcellular fractionation analyses to further explore the nature of the stimulation-induced traffic changes and to identify effectors that might mediate this change. Overnight stimulation of primary cultured rabbit lacrimal gland acinar cells with 10 μ m carbachol (CCh) significantly decreased the abundance of mature cathepsin B in the pre-lysosome and lysosome; decreased the abundance of preprocathepsin B in fractions containing the TGN and late endosome; increased the abundance of procathepsin B in fractions containing the basal-lateral membrane; and increased the accumulation of endocytosed [ 125I]-EGF in the recycling endosome. Alterations in distribution or abundance of traffic effectors included: increased abundances of rab5A and rab6 in the TGN; decreased overall abundance of γ-adaptin; remarkably increased relative abundance of membrane phase-associated actin; redistribution of cytoplasmic dynein from biosynthetic and proximal endocytic compartments to the lysosome; and redistribution of p150 Glued from the lysosome to biosynthetic or proximal endocytic compartments. We conclude that chronic MAChR stimulation blocks traffic from the early endosome and the TGN to the lysosome, causing lysosomal proteins to reflux to the TGN, endosomes, and basal-lateral membrane. These traffic alterations may be mediated through action on one or more of the effectors noted.

Sequencing, expression, and enzymatic characterization of beta-hexosaminidase in rabbit lacrimal gland and primary cultured acinar cells.

The lysosomal enzyme, beta-hexosaminidase, exists as two major isoforms; HexA and HexB. HexA is an alpha beta-subunit heterodimer and HexB a beta-subunit homodimer. Both isoforms can remove nonreducing beta-N-acetyl-D-glucosamine residues, whereas HexA hydrolyzes charged substrates as G(M2) gangliosides as well. beta-Hexosaminidase is present in both human and rabbit tear fluid and is secreted from rabbit lacrimal gland acinar cells in primary culture on stimulation with secretagogs. To further characterize the enzyme, the alpha- and beta-subunit mRNA expression was explored in rabbit lacrimal gland tissue as well as in cultured cells. Possible correlation between mRNA expression and HexA specific enzymatic activity was also investigated. Because existing beta-hexosaminidase antibodies are unable to recognize the rabbit enzyme, cloning and sequencing of the alpha- and beta-subunits were performed. Sequencing of the these subunits indicate that both are highly conserved between human, mouse, and rabbit. In contrast to the beta-subunit, showing an even mRNA expression between tissue and cultured cells, the level of alpha-subunit expression was higher in cultured acinar cells compared to tissue, with no alteration after cell stimulation. A minor but significant increase in total beta-hexosaminidase as well as HexA activity was observed in cultured cells compared to tissue. Enzymatic activity assays also revealed that HexA is the dominating isoform of beta-hexosaminidase in lacrimal gland and cultured acinar cells.

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.

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. 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.

Presence of alpha-and beta-integrin subunits in rabbit lacrimal gland acinar cells cultured on a laminin-rich matrix.

Integrins play critical roles in the interactions between the extracellular matrix and the cell by directing a number of functions, e.g. cell adhesion, cytoskeletal organization, and signal transduction. It is our hypothesis that they are also involved in modulating the regulation of secretion by lacrimal gland acinar cells, either by direct interaction with intracellular signaling cascades, or through modulation of cytoskeletal organization. Relatively little is known about the integrins in the lacrimal gland, but our laboratory is presently surveying for potential candidates capable of modulating secretion by lacrimal acinar cell. We have previously reported the presence of α2 αv, β1 and β4 integrin subunits in cells obtained by a cytospin technique.1, 2 The present study is an expanded survey for integrin subunits using rabbit lacrimal gland acinar cells in primary culture on laminin coated multi-well slides.

Cloning, partial sequencing and expression of rabbit lacrimal gland beta-hexosaminidase.

Several proteins actively secreted by the lacrimal gland have now been identified, including lactoferrin, lysozyme, tear specific pre-albumin and secretory IgA in human tear fluid.1 In addition, some lysosomal hydrolases have also been detected in tears and lacrimal gland fluid.2,3 In our ongoing studies of rabbit lacrimal gland acinar cells in primary culture, we noted that β-hexosaminidase catalytic activity, which we had expected to serve as a marker for lysosomes, diverged from another potential lysosomal marker, cathepsin B. Both enzymes were distributed primarily to the soluble phase, consistent with release from disrupted vesicular and cisternal structures. However, the membrane-associated component of hexosaminidase activity, when compared to cathepsin B, showed a relatively greater steady-state content in membranes of the endoplasmic reticulum and Golgi complex, suggestive of a product undergoing significant bulk flow through the synthesis and assembly pathway.4,5 Since β-hexosaminidase has been reported to be present in human tears,6,7 we examined the possibility that β-hexosaminidase might serve as a marker of regulated protein secretion by rabbit lacrimal gland acinar cells (Gierow et al., submitted).8,9 KeywordsOcular SurfaceLacrimal GlandKeratoconjunctivitis SiccaConjunctival Epithelial CellOcular Surface InflammationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.