Imbalanced Rab3D versus Rab27 increases cathepsin S secretion from lacrimal acini in a mouse model of Sjögren's Syndrome.

The mechanism responsible for the altered spectrum of tear proteins secreted by lacrimal gland acinar cells (LGAC) in patients with Sjögren's Syndrome (SS) remains unknown. We have previously identified increased cathepsin S (CTSS) activity as a unique characteristic of tears of patients with SS. Here, we investigated the role of Rab3D, Rab27a, and Rab27b proteins in the enhanced release of CTSS from LGAC. Similar to patients with SS and to the male nonobese diabetic (NOD) mouse model of SS, CTSS activity was elevated in tears of mice lacking Rab3D. Findings of lower gene expression and altered localization of Rab3D in NOD LGAC reinforce a role for Rab3D in suppressing excess CTSS release under physiological conditions. However, CTSS activity was significantly reduced in tears of mice lacking Rab27 isoforms. The reliance of CTSS secretion on Rab27 activity was supported by in vitro findings that newly synthesized CTSS was detected in and secreted from Rab27-enriched secretory vesicles and that expression of dominant negative Rab27b reduced carbachol-stimulated secretion of CTSS in cultured LGAC. High-resolution 3D-structured illumination microscopy revealed microdomains of Rab3D and Rab27 isoforms on the same secretory vesicles but present in different proportions on different vesicles, suggesting that changes in their relative association with secretory vesicles may tailor the vesicle contents. We propose that a loss of Rab3D from secretory vesicles, leading to disproportionate Rab27-to-Rab3D activity, may contribute to the enhanced release of CTSS in tears of patients with SS.

Role of Matrix Metalloproteinases 2 and 9 in Lacrimal Gland Disease in Animal Models of Sjögren's Syndrome.

PURPOSE: Chronic inflammation of the lacrimal gland results in changes in the composition of the extracellular matrix (ECM), which is believed to compromise tissue repair. We hypothesized that increased production/activity of matrix metalloproteinases (MMPs), especially MMP-2 and -9, in inflamed lacrimal glands modifies the ECM environment, therefore disrupting tissue repair. METHODS: The lacrimal glands from female MRL/lpr and male NOD mice along with their respective control strains were harvested and divided into three pieces and processed for histology, immunohistochemistry, zymography, Western blotting, and RNA analyses. In another study, MRL/lpr mice were treated for 5 weeks with a selective MMP2/9 inhibitor peptide or a control peptide. At the end of treatment, the lacrimal glands were excised and the tissue was processed as described above. RESULTS: There was a 2.5- and 2.7-fold increase in MMP2 gene expression levels in MRL/lpr and NOD mice, respectively. Matrix metalloproteinase 2 and 9 enzymatic activities and protein expression levels were significantly upregulated in the lacrimal glands of MRL/lpr and NOD mice compared to controls. Treatment with the MMP2/9 inhibitor resulted in decreased activity of MMP-2 and -9 both in vitro and in vivo. Importantly, MMP2/9 inhibitor treatment of MRL/lpr mice improved aqueous tear production and resulted in reduced number and size of lymphocytic foci in diseased lacrimal glands. CONCLUSIONS: We conclude that MMP2/9 expression and activity are elevated in lacrimal glands of two murine models of Sjögren's syndrome, suggesting that manipulation of MMP2/9 activity might be a potential therapeutic target in chronically inflamed lacrimal glands.

The effects of estrogen and androgen on tear secretion and matrix metalloproteinase-2 expression in lacrimal glands of ovariectomized rats.

PURPOSE: Previous studies have shown that ovariectomy (OVX) induces lacrimal gland regression and that androgens are implicated. This study explored the effects of estrogen and androgen on tear secretion and matrix metalloproteinase 2 (MMP-2) expression in lacrimal glands of ovariectomized rats. METHODS: Sixty-four adult female Wistar rats were randomly divided into three groups (control, sham operated, and OVX). Bilateral OVX was performed in the OVX group. After 5 months, the OVX group was further divided into six subgroups receiving topical ophthalmic or systemic treatment with corn oil vehicle, estradiol, or testosterone for 6 weeks. Schirmer test (SIT), assessment of tear film breakup time (BUT), corneal fluorescein staining, and measurement of estradiol and testosterone levels were performed before OVX and 1, 2, 3, 4, and 5 months after OVX, as well as after 6 weeks of treatment. Lacrimal glands were assessed for MMP-2 mRNA and protein expression. RESULTS: The mean (SD) tear film BUT decreased from 10.53 (0.79) to 9.98 (1.00) seconds (P < 0.01) in the first month after OVX, and the mean (SD) SIT result decreased by 50% from 7.32 (1.61) to 3.39 (1.15) mm (P < 0.01) in the third month after OVX. The mean (SD) corneal fluorescein staining score increased from 0.35 (0.11) to 6.02 (1.34) (P < 0.05) in the fourth month after OVX. The values increased or decreased in parallel with the time course (P < 0.01). In serum, ovariectomy resulted in a mean (SD) decline in estradiol levels from 44.38 (9.78) to 23.00 (3.78) pg/mL (P < 0.01), and the mean (SD) testosterone levels decreased from 2.42 (0.26) to 1.87 (0.15) ng/mL (P < 0.05). The mean (SD) estradiol level was elevated to 35.38 (3.34) pg/mL by systemic estradiol administration for 6 weeks, which also led to further mean (SD) decreases in tear film BUT from 5.28 (0.81) to 3.65 (0.55) seconds (P < 0.01) and in SIT result from 2.19 (1.01) to 1.47 (0.85) mm (P < 0.05), as well as a higher mean (SD) corneal fluorescein staining score from 7.39 (1.34) to 9.89 (1.27) (P < 0.05). However, the mean (SD) testosterone level was increased to 3.53 (0.67) ng/mL by systemic testosterone administration for 6 weeks. As a result, the mean (SD) tear film BUT increased from 5.08 (0.40) to 6.03 (1.48) seconds (P < 0.05), and the mean (SD) SIT result increased from 2.38 (1.20) to 3.66 (1.90) mm (P < 0.05). The mean (SD) corneal fluorescein staining score declined from 7.45 (0.73) to 4.56 (1.21) (P < 0.05). In the nontreated OVX group, the mean (SD) MMP-2 mRNA (0.66 [0.10]) and protein (0.55 [0.13]) expression in lacrimal glands was significantly increased compared with that in the sham-operated group (0.50 [0.09] and 0.40 [0.07], respectively) (P < 0.05). Systemic estradiol administration further increased the mean (SD) MMP-2 mRNA (0.83 [0.10]) and protein (0.69 [0.12]) expression (P < 0.05), while systemic testosterone administration decreased the mean (SD) MMP-2 mRNA (0.12 [0.04]) and protein (0.27 [0.07]) expression (P < 0.01). Topical ophthalmic administration of two sex hormones had no effect on the mean (SD) MMP-2 mRNA (0.59 [0.12] for estradiol and 0.57 [0.14] for testosterone) or protein (0.49 [0.11] for estradiol and 0.46 [0.13] for testosterone) expression (P > 0.05). CONCLUSIONS: Ovariectomy-induced ocular surface impairment may be associated with androgen deficiency. A pathogenetic role for estrogen in dry eye may involve upregulation of MMP-2 expression, while androgen suppresses MMP-2 expression.

Contributions of ocular surface components to matrix-metalloproteinases (MMP)-2 and MMP-9 in feline tears following corneal epithelial wounding.

PURPOSE: This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding. METHODS: Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs. RESULTS: The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining. CONCLUSIONS: Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.

Enzymes of urea synthesis are expressed at the ocular surface, and decreased urea in the tear fluid is associated with dry-eye syndrome.

PURPOSE: The present study aims at determining whether enzymes of urea synthesis are expressed in the human lacrimal gland and in tissues of ocular surface (conjunctiva, cornea), to give evidence for the hypothesis that urea can be locally formed from ocular tissues and is important for the composition of the tear fluid. METHODS: The presences of enzymes (arginase 1, 2 and agmatinase) that directly contribute to the formation of urea were investigated in the lacrimal gland and tissues of ocular surface by RT-PCR and immunohistochemistry. We collected tear fluid, aqueous humour, and blood samples from a total of 38 subjects, and tear fluid samples from a total of 78 subjects, with and without dry-eye syndrome (DES, keratoconjunctivitis sicca), and determined the urea concentration. RESULTS: The enzymes arginase 1, 2 and agmatinase were expressed in all tissues examined except for arginase 1, which was not expressed in the cornea. There was no correlation of urea concentration in tear fluid with aqueous humour and blood plasma (r = 0.13, p = 0.58 and r = 0.45, p = 0.05 respectively). However, correlation of urea concentration between aqueous humour and blood plasma was highly significant (r = 0.7, p = 0.0001). The concentration of urea in the tear fluid of patients with DES compared to healthy control group was significantly reduced (p < 0.0001). CONCLUSION: Enzymes that are directly involved in the formation of urea are expressed in ocular tissues. This may imply that in the ocular surface is a well-coordinated system of enzymes that can produce urea which might be independent of external urea supply.

Isoforms of secretory group two phospholipase A (sPLA2) in mouse ocular surface epithelia and lacrimal glands.

PURPOSE: To compare and contrast the distribution patterns of select secretory group two phospholipase A (sPLA2) isoforms in corneal epithelia (CN), conjunctival epithelia (CNJ), and lacrimal glands (LG) of BALB/c and C57BL/6 mice. METHODS: Gene expression of select sPLA2 isoforms was quantified via real-time reverse-transcription PCR (qRT(2)-PCR). Immunofluorescence assay (IFA) of the sPLA2-IIa, -V, and -X isoforms were used to confirm qRT(2)-PCR results. sPLA2-IIa function was confirmed via in vitro CN and CNJ culturing. RESULTS: qRT(2)-PCR revealed that sPLA2 isoforms (pla2g5, 12a, and 12b), cPLA2 isoform (pla2g4a), iPLA2 isoform (pla2g6), and PLA2-receptor (pla2r1) were present in all tissues of both strains, whereas sPLA2 isoforms (pla2g1b, 2e, and 3) were absent. sPLA2 isoforms (pla2g2a, 2d, 2f, and 10) showed tissue- and strain-specific expression: 2a in BALB/c CNJ only; 2d at higher levels in CNJ than LG; and 2f and 10 in CN and CNJ, but absent in LG. Upon dry eye (DE) induction, pla2g2a, 2d, and 2f were upregulated in BALB/c CNJ, and 10 was absent from CN. Furthermore, BALB/c DE mice showed upregulation of pla2r1 in CN and CNJ and downregulation of 12a and 12b in LG. IFA of sPLA2-IIa, -V, and -X in DE CNJ confirmed the upregulation of pla2g2a, 5, and 10. Last, in vitro CN and CNJ culturing confirmed that sPLA2-IIa amplifies ocular surface inflammation in CNJ but not in CN. CONCLUSIONS: sPLA2 isoforms exhibit differential expression patterns when comparing BALB/c with C57BL/6 mice; and DE with control BALB/c mice. These findings suggest that at least some sPLA2 isoforms must have significant roles in ocular surface physiology and inflammation.

p38 Mitogen-activated protein kinase modulates exocrine secretion in rabbit lacrimal gland.

The lacrimal gland (LG) is an exocrine gland important for secretion of the tear film. The kinase p38 has important signal transduction functions, e.g. in gene transcription, but has previously not been known to modulate exocrine secretion. The aim of the current study was to investigate the role of p38 in carbachol (Cch)-induced LG secretion in LG acinar cells in vitro. Western blotting was used to determine the phosphorylation status of p38 and p42/44 and determine expression of p38 isoforms. To determine the effect of p38 inhibition on LG secretion, PD 169316, a general p38 inhibitor, and SB 239063, an inhibitor of p38α and ß, were added to the cells prior to secretion measurements. The results revealed activation of p38 mediated by Cch stimulation and inhibition of Cch-induced secretion as a result of p38 inhibition. The inhibition was observed with PD 169316 isoforms, but not with SB 239063. The p38δ isoform was shown to have robust expression both by Western blotting of acinar cells and immunofluorescence of the whole gland. In conclusion, p38 activation mediates secretion in cholinergic stimulation of rabbit LG cells.

Phospholipase D1, but not D2, regulates protein secretion via Rho/ROCK in a Ras/Raf-independent, MEK-dependent manner in rat lacrimal gland.

PURPOSE: A prior study showed that cholinergic agonists activate phospholipase D (PLD). The purpose of this study was to determine whether cholinergic agonists use the PLD pathway to alter protein secretion and to identify the molecular signaling components of this pathway in rat lacrimal gland acini. METHODS: Rat lacrimal gland acini were isolated by collagenase digestion. Presence and localization of PLD1 and -2 were determined by immunofluorescence and Western blot experiments. Acini were incubated with adenoviruses overnight or the inhibitors 1-butanol, Y-27632, or C3 exotoxin before stimulation with the cholinergic agonist carbachol (Cch, 10(-4) M) for 5 minutes. Western blot analysis was performed for 20 minutes, and protein secretion was measured spectrophotometrically. Activation of ERK, MEK, Pyk2, Ras, and Raf was determined by Western blot analysis. RESULTS: 1-Butanol increased Cch-stimulated protein secretion and decreased ERK activity. Incubation with catalytically inactive PLD1, but not catalytically inactive mutant PLD2 adenovirus, also increased Cch-stimulated protein secretion and decreased ERK activity. Inhibition of Rho with C3 exotoxin and a dominant negative Rho adenovirus and inhibition of ROCK with Y-27632 inhibited Cch-stimulated PLD1 activity, increased protein secretion, and decreased ERK activity. The association of PLD1 and ROCK increased with Cch stimulation, as determined by immunoprecipitation. PMA-stimulated ERK activity was also inhibited by 1-butanol. 1-Butanol had no effect on Cch-stimulated Pyk2, Ras, and Raf activity, but decreased MEK activity. CONCLUSIONS: Cholinergic agonists activate PLD1 through Rho and ROCK, which in turn activate MEK and ERK, which attenuate protein secretion in freshly isolated epithelial cells.

Identification of the Raf-1 signaling pathway used by cAMP to inhibit p42/p44 MAPK in rat lacrimal gland acini: role in potentiation of protein secretion.

PURPOSE: The lacrimal gland is primarily responsible for the aqueous portion of the tear film. Simultaneous addition of cholinergic agonists or growth factors with cAMP-dependent agonists potentiates secretion. Recent investigations revealed that cAMP decreases p44/p42 mitogen-activated protein kinase (MAPK) activity stimulated by cholinergic agonists and growth factors that could account for this potentiation. In this study the authors identify the signal transduction pathway used by cAMP to inhibit MAPK activity. METHODS: Rat lacrimal gland acini were incubated with H89, an inhibitor of protein kinase A, before the addition of dibutyryl cAMP (dbcAMP, 10(-3) M) for 30 minutes. Basal MAPK and CREB activity and MAPK activity after stimulation with the cholinergic agonist carbachol (Cch) or epidermal growth factor (EGF) for 5 minutes was determined. The effect of dbcAMP on EGF receptor activity and basal and stimulated Ras, Raf-1, mitogen-activated protein kinase kinase (MEK), and MAPK activity was determined. The effect of a Rap-1 inhibitor, GGTI-298, on MAPK activity after the addition of dbcAMP was also determined. RESULTS: H89 relieved the inhibition of cAMP on MAPK activity and inhibited CREB activity. Incubation with dbcAMP did not have any effect either on the EGF receptor or on Ras but significantly inhibited both basal and Raf-1 and MEK activity stimulated with Cch or EGF. GGTI-298 did not have any effect on cAMP-dependent decrease in MAPK activity. CONCLUSIONS: The authors conclude that cAMP mediates the inhibition of MAPK by PKA in a Raf-1-dependent manner.

Altered morphology and function of the lacrimal functional unit in protein kinase C{alpha} knockout mice.

PURPOSE: Protein kinase C (PKC) α plays a major role in the parasympathetic neural stimulation of lacrimal gland (LG) secretion. It also has been reported to have antiapoptotic properties and to promote cell survival. Therefore, the hypothesis for the present study was that PKCα knockout ((-/-)) mice have impaired ocular surface-lacrimal gland signaling, rendering them susceptible to desiccating stress and impaired corneal epithelial wound healing. In this study, the lacrimal function unit (LFU) and the stressed wound-healing response were examined in PKCα(-/-) mice. METHODS: In PKCα(+/+) control mice and PKCα(-/-) mice, tear production, osmolarity, and clearance rate were evaluated before and after experimental desiccating stress. Histology and immunofluorescent staining of PKC and epidermal growth factor were performed in tissues of the LFU. Cornified envelope (CE) precursor protein expression and cell proliferation were evaluated. The time course of healing and degree of neutrophil infiltration was evaluated after corneal epithelial wounding. RESULTS: Compared with the PKCα(+/+) mice, the PKCα(-/-) mice were noted to have significantly increased lacrimal gland weight, with enlarged, carbohydrate-rich, PAS-positive acinar cells; increased corneal epithelia permeability, with reduced CE expression; and larger conjunctival epithelial goblet cells. The PKCα(-/-) mice showed more rapid corneal epithelial healing, with less neutrophil infiltration and fewer proliferating cells than did the PKCα(+/+) mice. CONCLUSIONS: The PKCα(-/-) mice showed lower tear production, which appeared to be caused by impaired secretion by the LG and conjunctival goblet cells. Despite their altered tear dynamics, the PKCα(-/-) mice demonstrated more rapid corneal epithelial wound healing, perhaps due to decreased neutrophil infiltration.

Immunohistolocalization and gene expression of the carbonic anhydrase isoenzymes (CA-II and CA-VI) in glands associated with the canine lacrimal apparatus.

Cytosolic and secretory carbonic anhydrase isoenzymes (CA-II and CA-VI, respectively) were detected by immunohistolocalization using specific canine CA-II and CA-VI antisera. CA-II and CA-VI were identified in glands associated with the canine lacrimal apparatus, such as lacrimal gland, superficial gland of the third eyelid (third eyelid gland) and tarsal gland. CA-II and CA-VI mRNA signals were also detected by reverse-transcriptase polymerase chain reaction in the same tissues. Some serous acinar cells and duct segments in the lacrimal gland and serous acinar cells in the third eyelid gland were immunopositive for anti-CA-II and CA-VI antisera. In particular, some immunopositive acini to CA-II and CA-VI on the edge of the third eyelid gland are histologically similar to sebaceous gland cells. Sebaceous gland cells in the tarsal and ciliary glands also showed immunopositivity to both CA antisera. CA-II and CA-VI gene transcripts were detected in the same regions. These results suggest that secreted CA-VI may form together with cytosolic CA-II, a high-activity isozyme mostly considered as a bicarbonate producer, in a mutually complementary system for the maintenance of bicarbonate levels to regulate pH in tear fluid and protect the corneal epithelia against injuries. In sebaceous gland cells in the lacrimal apparatus, CA-VI may be related to lipogenesis in an unknown function.

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 study was to evaluate the growth pattern and the secretory function of rabbit lacrimal gland acinar cells in a microgravity environment using an RCCS. METHODS: Lacrimal gland acinar cells from male New Zealand White rabbits were isolated and cultured in an RCCS up to 28 days. Cells were analysed by light and electron microscopy, and apoptosis was assessed by the TUNEL assay at days 7, 14, 21 and 28. Secretory function was tested by measuring the beta-hexosaminidase activity. RESULTS: After 7 days of culture, spheroidal aggregates were found inside the RCCS. The spheroids consisted of acinus-like cell conglomerates. Apoptotic centres inside the spheroids were observed at all time points by means of the TUNEL assay. Evaluation of the secretory function revealed beta-hexosaminidase release after carbachol stimulation which decreased over the culture period. CONCLUSION: A simulated microgravity environment promotes the development of three-dimensional cell spheroids containing viable acinar cells up to 28 days. Due to the evolving central apoptosis, it is unlikely that such simple three-dimensional cell communities can serve as tissue equivalents for clinical transplantation, but they promise opportunities for further applications in basic and applied cell research on lacrimal gland cells.

Roles of caspase 1 and extracellular signal-regulated kinase in inflammation-induced inhibition of lacrimal gland protein secretion.

PURPOSE: The purpose of the present study was to investigate the roles of caspase 1 and extracellular signal-regulated kinase (ERK) in inflammation-induced inhibition of lacrimal gland secretion. METHODS: Lacrimal gland inflammation was induced by injection of lipopolysaccharide (LPS; to study the role of caspase 1) or IL-1beta (to study the role of ERK). Lacrimal gland protein secretion was measured using a spectrofluorometric assay. Caspase 1 and ERK activities in the lacrimal gland were measured by immunohistochemistry, Western blotting, or both. Aqueous tear production was measured using phenol red-impregnated cotton threads. RESULTS: Injection of LPS into the lacrimal gland inhibited neurally and adrenergic agonist-induced protein secretion by 77% and 54%, respectively, and activated caspase 1. The degree of inhibition achieved by LPS was similar to that obtained with injection of IL-1beta. Inhibition of caspase 1 alleviated the inhibitory effect of LPS on lacrimal gland secretion. IL-1beta activated ERK in the lacrimal gland in vitro and in vivo, and this effect was blocked by UO126, an inhibitor of MEK, the ERK-activating enzyme. IL-1beta injection into the lacrimal gland inhibited aqueous tear production by 52% and inhibited neurally and adrenergic agonist-induced protein secretion by 80% and 55%, respectively. UO126 alleviated the inhibitory effect of IL-1beta on aqueous tear production and lacrimal gland protein secretion. CONCLUSIONS: LPS inhibits lacrimal gland secretion by activating caspase 1, and IL-1beta activates the ERK pathway to inhibit lacrimal gland protein secretion and aqueous tear production.

Clonal relationship between infiltrating immunoglobulin G4 (IgG4)-positive plasma cells in lacrimal glands and circulating IgG4-positive lymphocytes in Mikulicz's disease.

Mikulicz's disease (MD) is gaining acceptance as an immunoglobulin G4 (IgG4)-related disease characterized by bilateral lacrimal and salivary gland swelling. The aetiology of MD and other IgG4-related diseases is still unclear. The present work was performed to study the clonality of infiltrating IgG4-positive plasma cells in lacrimal glands and circulating peripheral blood cells in patients with MD, and compare the clonal relationship between infiltrating and circulating IgG4 positive cells. Total cellular RNA was extracted from the lacrimal glands and peripheral blood in five MD patients. Reverse transcription polymerase chain reaction was performed with primers specific for activation-induced cytidine deaminase (AID) and for Ig VH and IgG4. Sequences of Ig VH were compared with the structure of Ig VH of the lacrimal glands and the peripheral blood cells. AID was expressed to varying degrees in lacrimal glands of all MD patients. Most IgG4-positive cells infiltrating lacrimal glands and in peripheral blood were polyclonal, although several clonally related pairs were detected. In one patient, two of the circulating IgG4 VH4-59 clones shared identical CDR3 sequences with the clones within the lacrimal glands. In conclusion, while most tissue-infiltrating and circulating IgG4-positive cells in MD are polyclonal, some clonally related IgG4 positive cells exist between lacrimal gland and peripheral blood, accounting for the clinical features of MD as an IgG4-related disease involving multiple organs.

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.

Human chitotriosidase is expressed in the eye and lacrimal gland and has an antimicrobial spectrum different from lysozyme.

Chitotriosidase is a chitinolytic enzyme expressed by maturing macrophages and preformed in neutrophil granules, suggesting a role in antimicrobial defence. Although available evidence supports a role in anti-fungal immunity, there is a lack of an obvious phenotype in humans homozygous for a mutation which renders chitotriosidase inactive. This may be explained by compensatory effects of enzymes co-expressed with chitotriosidase, such as lysozyme. We have found that chitinase is highly expressed in mouse and human eye, particularly in lacrimal glands. Chitotriosidase is the only member of the chitinase/chilectin gene cluster expressed in the murine eye. As lacrimal glands also produce lysozyme, we have asked whether chitotriosidase, in addition to its documented anti-fungal effects, has synergistic anti-bacterial properties with lysozyme. The effect of recombinant chitotriosidase on the growth of five Gram-positive (Bacillus cereus, Bacillus subtilis, Listeria innocua, Staphylococcus aureus and Staphylococcus aureus OatA(+/-)) and two Gram-negative strains (Escherichia coli and Pseudomonas aeruginosa), were tested in a luminometric assay. Recombinant chitotriosidase did not inhibit bacterial growth and did not synergize with lysozyme. Though the expression of chitotriosidase in the eye supports a role in innate immunity, the antimicrobial spectrum appears to be complementary to lysozyme and may indeed be limited to fungi.

Bud specific N-sulfation of heparan sulfate regulates Shp2-dependent FGF signaling during lacrimal gland induction.

Preferential outgrowth of the bud cells forms the basis of branching morphogenesis. Here, we show that lacrimal gland development requires specific modification of heparan sulfates by Ndst genes at the tip of the lacrimal gland bud. Systemic and conditional knockout experiments demonstrate the tissue specific requirement of Ndst1 and Ndst2 in the lacrimal gland epithelial, but not mesenchymal, cells, and the functional importance of Ndst1 in Fgf10-Fgfr2b, but not of Fgf1-Fgfr2b, complex formation. Consistent with this, Fgf10-induced ectopic lacrimal gland budding in explant cultures is dependent upon Ndst gene dose, and epithelial deletion of Fgfr2 abolishes lacrimal gland budding, its specific modification of heparan sulfate and its phosphorylation of Shp2 (Ptpn11 - Mouse Genome Informatics). Finally, we show that genetic ablation of Ndst1, Fgfr2 or Shp2 disrupts ERK signaling in lacrimal gland budding. Given the evolutionarily conserved roles of these genes, the localized activation of the Ndst-Fgfr-Shp2 genetic cascade is probably a general regulatory mechanism of FGF signaling in branching morphogenesis.

Role of cAMP inhibition of p44/p42 mitogen-activated protein kinase in potentiation of protein secretion in rat lacrimal gland.

We previously found that addition of cAMP and a Ca(2+)/PKC-dependent agonist causes synergism or potentiation of protein secretion from rat lacrimal gland acini. In the present study we determined whether cAMP decreases p44/p42 mitogen-activated protein kinase (MAPK) activity in the lacrimal gland. Since we know that activation of MAPK attenuates protein secretion stimulated by Ca(2+)- and PKC-dependent agonists, we also determined whether this activation causes potentiation of secretion. Freshly prepared rat lacrimal gland acinar cells were incubated with dibutyryl cAMP (DBcAMP), carbachol (a cholinergic agonist), phenylephrine (an alpha(1)-adrenergic agonist), or epidermal growth factor (EGF). The latter three agonists are known to activate p44/p42 MAPK. p44/p42 MAPK activity and protein secretion were measured. As measured by Western blot analysis, DBcAMP inhibited both basal and agonist-stimulated p44/p42 MAPK activity. Cellular cAMP levels were increased by 1) using two different cell-permeant cAMP analogs, 2) activating adenylyl cyclase (L-858051), or 3) activation of G(s)-coupled receptors (VIP). The cell-permeant cAMP analogs, L-858051, and VIP inhibited basal p44/p42 MAPK activity by 50, 40, and 40%, respectively. DBcAMP and VIP inhibited carbachol- and EGF-stimulated MAPK activity. cAMP, but not VIP, inhibited phenylephrine-stimulated MAPK activity. Potentiation of secretion was detected when carbachol, phenylephrine, or EGF was simultaneously added with DBcAMP. We conclude that increasing cellular cAMP levels inhibits p44/p42 MAPK activity and that this could account for potentiation of secretion obtained when cAMP was elevated and Ca(2+) and PKC were increased by agonists.

Amniotic membrane as a carrier for lacrimal gland acinar cells.

BACKGROUND: The secretion of the lacrimal gland provides 95% of the aqueous tears, which are essential for lubrication, nutrition and protection of the ocular surface. Long-term studies of acinar lacrimal gland cells in vitro are complicated by low proliferation rate and fast loss of cell function on plastic. Aim of this study was to evaluate the growth pattern and the secretory function of lacrimal gland acinar cells on amniotic membrane (AM) in a rabbit model. METHODS: Lacrimal gland acinar cells from Chinchilla Bastard and New Zealand White rabbits of both sexes were isolated and cultured on denuded amniotic membrane. Cells were analysed by light and electron microscopy. Secretory function was tested by measuring the beta-hexosaminidase activity. RESULTS: Three days after seeding to the amniotic membrane, the acinar cells had attached to each other and formed small cluster. Cell clusters consisted of 2-5 cell layers, and the cells showed fine granulation in their cytoplasm, typical for secreting cells. Between days 7 and 14 cell clusters increased in size, and acini-like structures with a central lumen were found. Cells showed polarity, with a basal nucleus and apical secretory granules. Between days 21 and 28 acini-like structures were still found inside the cell clusters. Accumulation of secretory material in the central lumen and desmosome formation connecting the apical cell structures was frequently evident. However, the number of cytoplasmatic granules decreased, and on parts of the AM, cell morphology changed to flat, spindle-shaped cells with a small nucleus. Stimulation with carbachol showed a strong beta-hexosaminidase release until day 7, with a decreasing secretory function detectable until day 21. CONCLUSION: Acinar lacrimal gland cells can be successfully cultured on amniotic membrane up to 28 days, with a secretory response to carbachol up to 21 days. This model may be used for further experimental work, to elucidate cellular mechanisms in normal and diseased lacrimal tissue.

Estrogen up-regulation of metalloproteinase-2 and -9 expression in rabbit lacrimal glands.

Increased levels of the matrix metalloproteinases (MMPs)-2 and -9 have been found in tear fluids of patients with dry eye disease, suggesting that these MMPs may be implicated in the pathogenesis of this disease. One of the main causes of dry eye disease is lacrimal gland insufficiency. However, the contribution of the lacrimal gland (LG) to the expression and production of MMP-2 and MMP-9 in tears is not known. Since dry eye disease occurs more frequently in women, sex hormones, especially estrogens, have also been implicated in the pathogenesis of this disease. Estrogens have been shown to regulate the synthesis levels of MMP-2 and MMP-9 in several tissues, Thus, the purpose of these studies was to determine if: (1) rabbit lacrimal glands secrete MMP-2 and MMP-9; (2) MMP-2 and MMP-9 are produced by lacrimal epithelial cells and/or lacrimal lymphocytes; and (3) the expression, activity and level of these enzymes are regulated by sex hormones. Lacrimal epithelial cells (LEC) and lacrimal lymphocytes (LL) from sexually mature New Zealand White female rabbits were isolated, purified and cultured with and without 10(-6)M dihydrotestosterone (DHT) or 10(-6), 10(-8), 10(-9) and 10(-10)M 17beta-estradiol (E2). The culture supernatants were analyzed by zymography and western blotting (WB) using polyclonal anti-human MMP-2 and MMP-9 antibodies. LGs were also collected from rabbits 7 days after being sham-operated, ovariectomized (OVX), OVX treated with 4 mg/kg DHT, and OVX treated with 0.5 mg/kg of E2. LGs were collected and processed for RNA extraction as well as protein determination using WB and immunocytochemistry. The pro-forms of MMP-2 and MMP-9 were detected in primary LEC and LL culture medium by zymography and WB. Pro-MMP-2 and pro-MMP-9 were also detected at the gene and protein levels in the lacrimal glands of all four treatment groups, with the highest levels and gene expression found in the estrogen-treated group. These results suggest that both pro-MMP-2 and pro-MMP-9 are secreted by the lacrimal gland and appear to be up-regulated by estrogen. The role of the lacrimal MMPs in the pathogenesis of dry eye disease needs to be further investigated.