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B-cell infection by hepatitis C virus (HCV) has been a controversial topic. To examine whether HCV has a genetically determined lymphotropism through a co-receptor specific for the infection by lymphotropic HCV, we established an infectious clone and chimeric virus of hepatotropic and lymphotropic HCV strains derived from an HCV-positive B-cell lymphoma. The viral envelope and 5′-UTR sequences of the lymphotropic HCV strain were responsible for the lymphotropism. Silencing of the virus sensor, RIGI, or overexpression of microRNA-122 promoted persistent viral replication in B cells. By cDNA library screening, we identified an immune cell-specific, co-stimulatory receptor B7.2 (CD86) as a co-receptor of lymphotropic HCV. Infection of B cells by HCV inhibited the recall reaction to antigen stimulation. Together, a co-receptor B7.2 enabled lymphotropic HCV to infect memory B cells, leading to inhibition of memory B-cell function and persistent HCV infection in HCV-infected hosts., Infection of B cells by hepatitis C virus (HCV) is poorly understood, but is thought to result in lymphoproliferative disorders. Here, Chen et al. identify CD86 as co-receptor for lymphotropic HCV and show that HCV infection inhibits memory B-cell function.

PURPOSE Autologous peripheral blood lymphocytes, activated in a mixed cell reaction when cocultured with purified rabbit lacrimal epithelial cells, are known to induce a severe autoimmune dacryoadenitis when injected directly into the donor animal's remaining inferior lacrimal gland (LG) or subcutaneously at a site remote from the LG. The purpose of the present study was to determine the ability of intravenously (IV) injected autologous stimulated lymphocytes to home to the LG and salivary gland (SG) and induce disease. METHODS One inferior LG was surgically excised from each rabbit. Acinar epithelial cells were purified, cultured for 2 days, gamma-irradiated, and cocultured for 5 days with purified autologous peripheral blood lymphocytes. The activated lymphocytes were used for autoadoptive transfer. RESULTS Tear production was reduced 50% by 4 weeks and tear breakup time was 70% less than normal. Ocular surface defects assessed by rose bengal staining were present but not as pronounced as after direct injection. Four weeks after IV injection, as after direct injection, glands contained large infiltrates composed of predominantly CD4(+) T cells close to interlobular and intralobular ducts; however, they also contained unique areas of streaming lymphocytes. Histopathology at 8 weeks was more severe than at 4 weeks, and SG also showed clusters of abnormal epithelial cells and streaming lymphocytes. CONCLUSIONS Lymphocytes activated against lacrimal antigens and injected IV can home to the LG and SG and initiate autoimmune processes, suggesting that these sites constitutively contain not only antigen-presenting cells displaying potentially pathogenic autoantigen epitopes but also chemokines and homing molecules that recruit CD4(+) T cells. This new rabbit model more closely mimics Sjogren syndrome, in that SG manifestations accompany the LG disease. It should be well suited to elucidating Sjogren pathogenesis and pathophysiology and to evaluating experimental therapies.

PURPOSE To test whether CD4+ T cells proliferate in mixed cell reactions with autologous lacrimal gland (LG) acinar cells and whether these cells can autoadoptively transfer disease. METHODS Purified acinar cells were gamma irradiated and cocultured with peripheral blood lymphocytes. Activated CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS). Unfractionated activated peripheral blood lymphocytes (UF), CD4+-enriched and CD4+-depleted T cells from an autologous mixed cell reaction were injected into the donor rabbit's remaining LG. After 4 weeks, ocular examinations were performed, and the rabbits were euthanized; LGs were removed for histopathology, immunohistochemistry, and real-time reverse transcription-polymerase chain reaction studies. RESULTS CD4 T cells increased in the autologous mixed cell reaction from 20% to 80%. Tear production decreased in the induced disease/UF (ID/UF) group and declined even more in the ID/CD4+-enriched group. Tear breakup times decreased and rose bengal staining increased in all groups. All LGs exhibited significant histopathology and increased messenger RNAs for tumor necrosis factor α. The ID/UF group exhibited the largest increases of CD4+ and rabbit T-lymphocyte antigen-positive cells. The ID/CD4+-enriched group contained fewer infiltrating CD4 cells but more eosinophils, severely altered acinar morphology, and increased fibrosis. LG of the ID/CD4+-depleted group exhibited large increases of CD18, major histocompatibility complex II, and CD4+ cells. Messenger RNAs for interleukin 2, interleukin 4, and CD4+ increased in the ID/CD4+-enriched group compared with the CD4+-depleted group. CONCLUSIONS Autoreactive CD4+ effector cells activated ex vivo and autoadoptively transferred, caused what seems to be a distinct dacryoadenitis. The CD4+-depleted cell fraction also contained pathogenic effector cells capable of inducing disease.

Research into the physiological processes governing both normal and abnormal functions of the lacrimal gland has used animal models to provide insights that might be applied to improving our understanding of human disease and designing of beneficial therapeutic interventions. Animal models most frequently used are mice, rats, and rabbits. As participants in research into normal and abnormal lacrimal gland function, the authors have observed significant differences between the various animal models, and these differences must be considered in investigational studies. This review summarizes a wide range of topics, including structural organization of the lacrimal gland and the immunological, secretomotor and hormonal processes regulating lacrimal gland function in all three animal models. In addition, comparisons with relevant aspects of the human lacrimal gland are included where permitted by available data.

Lacrimal epithelial cells appear to constitutively secrete autoantigens to their underling stroma. The present experiments address the hypothesis that they also secrete soluble factors that regulate immune responses. Epithelial cells, spleen cells and lymphocytes were obtained from rabbits or rats and cultured in various configurations. Monocytes from rat bone marrow were matured to dendritic cells (DC) ex vivo. Proliferation was measured by [3H]-thymidine incorporation; surface MHC Class II and CD86 using flow cytometry; and mRNA relative abundances using real time RT-PCR. Microporous culture inserts containing rat lacrimal cells inhibited proliferation of rabbit lymphocytes co-cultured with autologous lacrimal cells and of rat lymphocytes co-cultured with TNF-alpha-stimulated DC. They inhibited CD86 and MHC Class II surface expression by maturating DC and reversed surface expression of CD86 but not MHC Class II by partially matured DC. Subsequent exposure of partially matured DC to mediators from rat lacrimal cells reversed the ability to stimulate lymphocyte proliferation. TGF-beta(1) and IL-10 mRNAs increased somewhat when rat lacrimal cells were isolated but decreased markedly in rabbit lacrimal cells. Antibodies to TGF-beta prevented soluble factors from rat lacrimal cells from inhibiting proliferation of rabbit lymphocytes co-cultured with rabbit lacrimal cells, but recombinant TGF-beta alone did not mimic the soluble factors. IL-10 immunopositivity was detected in epithelial cells of interlobular ducts and occasional interstitial cells in rabbit lacrimal gland. Rat lacrimal epithelial cells secrete TGF-beta and other factors that synergize to suppress lymphocyte proliferation and regulate DC maturation. Interlobular duct epithelial cells in rabbit lacrimal glands may express similar functions.

To use a rabbit model of induced autoimmune dacryoadenitis to evaluate the efficacy of topical ophthalmic cyclosporine A (CsA).Autoimmune dacryoadenitis was induced by injecting autologous peripheral blood lymphocytes, which had been activated in a mixed cell reaction with acinar cells isolated from one inferior lacrimal gland (LG), back into the donor animal's remaining inferior LG. Schirmer's test, tear breakup time, and rose Bengal staining were assessed. Animals with established disease were treated topically with either CsA or Endura twice daily for 5 months.Without treatment tear production and tear stability were abnormal for 6 months, and clear signs of ocular surface defects were evident. Severe immune cell infiltration was observed in the LG. Long-term CsA treatment increased tear production only slightly, but the severity of LG histopathology decreased noticeably. CD4(+) T-cell infiltration of the LG was decreased and infiltration by MHC class II-expressing cells was also decreased. For the Endura-treated group tear production did not improve, rose Bengal scores remained high, and histopathology showed infiltration comparable to the untreated group, but by the end of the study the tear breakup time did improve.The rabbit model of autoimmune dacryoadenitis had signs of chronic dry eye disease 6 months after induction of disease. Tear production improved slightly with CsA treatment and CD4(+) T-cell infiltration decreased significantly in the LG. This suggests that some Sjögren's patients may benefit from long-term CsA treatment.

Autologous peripheral blood lymphocytes (PBL), activated in a mixed cell reaction when co-cultured with purified rabbit lacrimal epithelial cells, are known to induce a Sjőgren’s-like autoimmune dacryoadenitis and keratoconjunctivitis when injected directly back into the donor animal’s inferior lacrimal gland (LG). This study shows that autoreactive lymphocytes injected subcutaneously in a site away from the LG is capable of inducing an autoimmune disease in a rabbit. Induced disease (ID) develops more slowly, taking 4 weeks as compared to 2 weeks in the direct injection model. Initially, both clinical symptoms and histopathology are less pronounced than in the direct injection ID model, but later the immunocytochemistry shows the same CD4+/CD8+ ratio of 4:1 for both injection methods. The finding that lymphocytes activated against lacrimal antigens can travel or home from the injection site back to the inferior and superior LG, as well as the conjunctiva, suggests that these anatomical sites may have common epitopes that induce pathogenic CD4+ T cells that produce a Sjőgren’s-like syndrome.

The rabbit lacrimal gland undergoes an immunophysiological transformation during pregnancy, reminiscent of that of the mammary gland as it prepares to deliver secretory IgA into the nascent fluid product. The contents of TGF-beta and prolactin (PRL) within ductal epithelial cells increase, and their primary localizations shift from the apical to the basal cytoplasm, suggesting a transformation from exocrine to paracrine secretion. Studies with ex vivo acinar cell models demonstrated that elevated PRL suppresses traffic of secretory proteins into the regulated exocrine apparatus and directs them into a novel, induced, regulated paracrine apparatus [Wang, Y., Chiu, C.T., Nakamura, T., Walker, A.M., Petridou, B., Trousdale M.D., Hamm-Alvarez S.F., Schechter J.E., Mircheff A.K., 2007. Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells. Am. J. Physiol. Endocrinol. Metab. 292, E1122-E1134]. However, it was not clear whether PRL itself entered the induced paracrine apparatus. In the present study, confocal immunofluorescence microscopy revealed that natively expressed PRL and over-expressed PRL co-localized with PRL receptors (PRLR); rab11, a marker for the recycling endosome; gamma-adaptin, a marker for the Golgi complex and trans-Golgi network; and rab7, a marker for the autophagic lysosomal apparatus. Natively expressed, over-expressed, and endocytosed PRL also co-localized with rab4 and rab5A, markers for the early endosome, and with rab3D, a marker for regulated exocrine secretory vesicles. Endocytosed PRL was stored in intact form and released in response to stimulation with carbachol. Subcellular fractionation analysis detected relative excesses of PRL over PRLR in fractions that contained fragments of the recycling endosome and fractions that contained both secretory vesicle fragments and prelysosomal and autolysosomal fragments. EM-gold microscopy demonstrated PRL within small vesicles, consistent with endosomes or secondary lysosomes, and in large vesicles, consistent with regulated secretory vesicles. The secretory vesicles were preponderantly localized in the apical cytoplasm of control cells, and in the basal cytoplasm of PRL over-expressing cells. These results indicate that when lacrimal epithelial cells synthesize PRL, and when they endocytose it from their ambient medium, they traffic it both into the endosomes that constitute the constitutive transcytotic paracrine apparatus and also into regulated secretory vesicles, which are associated with the exocrine apparatus at low PRL levels and with the induced paracrine apparatus at high PRL levels.

Aim: To develop a local approach to study rabbit lacrimal secretion in situ by administering specific secretagogues directly onto the lacrimal gland (LG). Methods: After the rabbit has been anesthetized, the inferior bulbar conjunctiva and underlying connective tissue are blunt dissected. A polyethylene tube, for drug delivery, is inserted through the fibrous membrane overlying the inferior surface of the orbital cavity beneath which the LG resides. Lacrimal fluid is collected by cannulating the lacrimal duct. Results: Pilocarpine induced robust lacrimal fluid secretion from the LG that had been bathed with either pilocarpine or phenylephrine, with no detectable effect on the contralateral LG and salivary secretion. By next giving pilocarpine or phenylephrine to the control LG while the experimental gland used before served as control, we duplicated the results exactly. Conclusion: This novel approach avoids the unwanted systemic effects of intravenous injection and is a promising technique to study rabbit LG secretion in situ.

The acinar epithelial cells of the lacrimal gland are responsible for the production, packaging and regulated exocytosis of tear proteins into ocular surface fluid. This review summarizes new findings on the mechanisms of exocytosis in these cells. Participating proteins are discussed within the context of different categories of trafficking effectors including targeting and specificity factors (rabs, SNAREs) and transport factors (microtubules, actin filaments and motor proteins). Recent information describing fundamental changes in basic exocytotic mechanisms in the NOD mouse, an animal model of Sjögren's syndrome, is presented.

Lacrimal glands of male NOD and BALB/c mice have very small, pleomorphic acinar lumens. Acini contain isolated zones of highly complex cell surface interdigitations at the basal surface, sometimes occurring between acinar and myoepithelial cells. In NOD mice, cytological abnormalities, including mitochondrial deterioration, pleomorphic and heterogeneous cytoplasmic vacuoles, and lipid accumulation are evident within acinar cells at 1 month. Accumulation of lipid is further increased as the animal ages, accompanied by lymphocytic infiltration and destruction of acini. These results demonstrate alterations from normal cytology as early as 1 month in NOD mice, well before detection of clinical signs of Sjögren syndrome.

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.

The lacrimal glands of male NOD mice exhibit many of the features of the human lacrimal gland in patients afflicted with the autoimmune disease, Sjögren's syndrome, including loss of secretory functions and lymphocytic infiltration into the lacrimal gland. To elucidate the early changes in the secretory pathway associated with development of Sjögren's syndrome, we investigated the organization of the exocytotic pathway in lacrimal glands of age-matched male BALB/c and NOD mice. Cryosections from lacrimal glands from 1 and 4 month male BALB/c and NOD mice were processed for confocal fluorescence and electron microscopic evaluation of different participants in exocytosis. No changes in apical actin filaments were noted in glands from NOD mice, but these glands exhibited thickening of basolateral actin relative to that seen in the BALB/c mice. Rab3D immunofluorescence associated with mature secretory vesicles was distributed abundantly in a continuous vesicular network concentrated beneath the apical plasma membrane in glands from 1 and 4 month BALB/c mice. In glands from 1 month NOD mice, rab3D immunofluorescence exhibited marked discontinuity and irregularity in the vesicular labeling pattern. While this change was also detected in glands from 4 month NOD mice, many of these glands exhibited an additional extension of rab3D labeling through the cell to the basolateral membrane. Electron microscopic analysis confirmed the formation of irregularly shaped, unusually large secretory vesicles in lacrimal glands from NOD mice. Quantitation of multiple secretory vesicles from electron micrographs revealed a significant (por =0.05) increase in the percentage of secretory vesicles incorporated into multivesicular aggregates in lacrimal glands from 1 and 4 month NOD mice compared to BALB/c mice. The M3 muscarinic receptor, a key signaling effector of exocytosis, was redistributed away from its normally basolateral locale in glands from BALB/c mice, with concomitant enrichment in intracellular aggregates in glands from NOD mice. These findings show that lacrimal glands in NOD mice as young as 1 month contain aberrant secretory vesicles with altered effector composition that undergo premature cytoplasmic fusion, and that changes in the distribution of the M3 muscarinic receptor occur within the same time frame.

This paper articulates a new working hypothesis that explains many of the pathophysiological conditions described under the common rubric "dry eye" as altered states of mucosal immune regulation. A central principle of mucosal immune physiology is that the parenchymal tissues at the effector sites, i.e., the sites at which secretory antibodies are produced, maintain local signaling milieus that support differentiation of IgA+ plasmablasts and survival of IgA+ plasmacytes. These local signaling milieus also support robust regulatory networks that maintain tolerance to commensual microbes, benign antigens, and parenchymal autoantigens. The regulatory networks are mediated by cycles of interactions between successive generations of dendritic cells, which normally mature with tolerogenic functions, and regulatory T cells, which normally reinforce the system's ability to generate new tolerogenic dendritic cells. The systemic endocrine environment controls expression of the local signaling milieu in the mammary gland and in the prostate and male urethral glands. Emerging evidence indicates that the local signaling milieu in the lacrimal gland also is determined, in part, by the systemic endocrine environment. This working hypothesis suggests explanations for the excess incidence of Sjogren syndrome among women and for the mechanisms of several different immunophysiological states in addition to Sjogren syndrome that, like Sjogren syndrome, are associated with the classical symptoms and signs of dry eye. It also comprises a promising rationale for specific new approaches to therapy.

PURPOSE. To evaluate the effect of viral IL-10 on the lacrimal gland immunopathologic response in the ocular surface disease, induced autoimmune dacryoadenitis. METHODS. Disease was induced in rabbits by injecting inferior lacrimal glands with peripheral blood lymphocytes activated by 5 days of coculture with autologous acinar cells in a mixed-cell reaction. In the treated group, an adenoviral vector carrying the vIL-10 gene was concurrently injected with activated lymphocytes. Tears were collected periodically for quantitation of IL-10 by ELISA. Two weeks after disease induction, tear production, tear film breakup time, and rose bengal staining score were determined. Sectioned glands were immunostained for expression of CD4, CD8, rabbit thymic lymphocyte antigen (RTLA), CD18 and major histocompatibility complex class II. RESULTS. The titer of vIL-10 in tears was at its maximum on day 3, started to decline by day 7, and was undetectable by day 14. In the diseased group, the tear production rate and tear film breakup time were significantly decreased, and rose bengal staining was significantly increased. Diseased glands had immune cell infiltrates containing CD4 + , RTLA + , and CD18 + cells, and major histocompatibility complex class II expression was increased. These changes were significantly ameliorated by expression of vIL-10. CONCLUSIONS. In vivo transduction of the lacrimal gland with AdvIL-10 resulted in the transient appearance of VIL-10 in tears. The presence of vIL-10 partially suppressed the appearance of Sjogren-syndrome-like features of reduced tear production, accelerated tear breakup, ocular surface disease, and immunopathologic response. Anti-inflammatory cytokine gene expression may offer a therapeutic modality for the treatment of autoimmune dacryoadenitis, once suitable vectors become available.

Purpose. To evaluate the effect of tumor necrosis factor (TNF) inhibitor protein on lacrimal gland immunopathology and ocular surface disease resulting from induced dacryoadenitis. Methods. Autoimmune dacryoadenitis was induced in rabbits by injecting the lacrimal glands with peripheral blood lymphocytes (PBLs) activated by 5 days of coculture with autologous acinar cells in a mixed cell reaction. In the treated group, an adenoviral vector carrying the TNF inhibitor gene (AdTNFRp55-Ig) was concurrently injected with AMCR-PBL. Tear production was monitored by Schirmer test, and tears were collected for detection of TNF-inhibitor protein. Frozen sections of the glands were immunostained for expression of CD4, CD8, rabbit thymic lymphocyte antigen (RTLA), and CD18. Histological sections of lacrimal glands were examined using the TUNEL technique to monitor apoptosis. Results. Soluble TNF-inhibitor protein was detected by ELISA in tears, with titers at a maximum on day 3, declining by day 7, and undetectable by day 14. Tear production declined in the induced dacryoadenitis group but did not change when glands had been treated with AdTNFRp55-Ig simultaneously with disease induction. Tear break-up time and rose bengal staining properties were not altered by treatment. Fourteen days after the glands were injected with activated PBLs, focal mononuclear cell infiltrates were observed around ducts and venules, some of which assumed the high endothelial phenotype, and between acini. Immune cells in the infiltrates stained positive for CD4, RTLA, and CD18. Glands that received AdTNFRp55-Ig concurrently with activated PBLs had decreased numbers of CD4 cells, CD18 cells, RTLA, and apoptotic cells. Conclusions. In vivo transduction of the lacrimal gland with AdTNFRIp55-Ig resulted in transient expression in the gland and the appearance of TNF-inhibitor protein in tears. The presence of soluble TNF-inhibitor protein partially suppressed the appearance of Sjogren's syndrome-like features of reduced tear production and the immunohistopathology associated with induced autoimmune dacryoadenitis but not tear break-up time and ocular surface disease. This may reflect immunoregulation in the lacrimal gland but not in the conjunctiva.

We have undertaken a series of studies to elucidate the roles of growth factors (FGF-2, EGF, TGF-beta1) and prolactin (PRL) in lacrimal gland function during pregnancy and lactation, and to better understand the status of the immune system within the lacrimal gland during those physiological states. In this initial study, lacrimal glands of pregnant (d15, d29), lactating (9d, 22d), and adult female control rabbits, were evaluated by immunohistochemistry, Western blotting and image analysis. In control rabbits EGF, TGF-beta1, and PRL, were immunolocalized primarily in the apical cytoplasm of intralobular ductal epithelial cells, and acini demonstrated a basement membrane-associated immunopositivity for TGF-beta1. FGF-2 immunolocalized in myoepithelial cells in the basal ductal epithelium and complexed to the basement membrane enclosing ducts and acini. Cells immunopositive for immune cell markers (RTLA and CD18) were apparent primarily around interlobular ducts. In d29 pregnant rabbits immunopositivity for EGF and TGF-beta1 was increased within intralobular ducts, both apically and basally, and within some interlobular ductal epithelial cells. Immunopositivity for PRL was strongest in d29 pregnant rabbits within the apical and basal cytoplasm of intralobular ductal epithelial cells. Immunopositivity for FGF-2 in myoepithelial cells was strong in d15 and d29 pregnant rabbits, although basement membrane-associated immunopositivity around acini was often decreased. Immunostaining for EGF and TGF-beta1 in lactating rabbits was similar to that in d29 pregnant rabbits, although basement membrane-associated immunopositivity around acini was more comparable to controls. By 22d lactation immunopositivity for FGF-2 closely resembled that in controls. Image analysis of pregnant and lactating rabbits demonstrated that cells immunopositive for RTLA and CD18 were less abundant around ducts and more abundant between acini, although in 22d lactating rabbits the size of periductal foci was increased to nearly that of controls. Western blots correlated well with the immunohistochemistry. Our findings demonstrate that pregnancy and lactation are accompanied by a shift in the distributions of growth factors and PRL, suggestive of increased release both apically into the lacrimal fluid and basally into the interstitium. Additional shifts in the distributions of cells of the immune system from periductal foci to interacinar sites suggest that there is a recruitment of immune cells away from ducts and toward the connective tissue interstitium surrounding the acini, possibly as part of a heightened state of immune readiness during pregnancy and lactation.

The purpose of this study was to determine the distribution of FGF-2 within rabbit lacrimal glands and to determine whether corneal insult affects that distribution. The scarified corneas of experimental animals were inoculated either with adenovirus type 5 or buffer. Control animals were either untreated, or animals whose corneas were scarified. Twenty-one days later all animals were killed and the lacrimal glands were studied by immunocytochemistry and Western blotting to detect FGF-2. In untreated control animals, FGF-2 was immunolocalized predominantly within a population of elongated cells in the basal epithelium of ducts, and to a lesser degree in the basal epithelium of the acini. The elongated immunopositive cells appear to be myoepithelial cells known to be present at these sites. Interstitial cells around ducts and acini, and the basement membranes of the ducts and acini, were also immunopositive for FGF-2. Twenty-one days after adenovirus inoculation and scarification of the cornea, immunopositivity for FGF-2 was dramatically decreased in basement membranes, but increased within myoepithelial cells of the duct epithelium. These myoepithelial cells were frequently enlarged, bulging toward the duct lumen. In animals whose corneas were inoculated with buffer and scarified, or animals whose corneas were simply scarified, the changes in the lacrimal gland were similar, but somewhat less pronounced, to those of adenovirus-inoculated animals. Western blots confirmed the presence of FGF-2 immunoreactivity in all groups. The major band in untreated controls was at 24 kD, whereas all animals with corneal scarification had major bands at 38 kD. Densitometry of Western blots demonstrated that the amount of 24 kD FGF-2 present within the lacrimal gland after corneal scarification was at least 50% less than in untreated controls, whereas 38 kD FGF-2 was at least ten-fold greater. Our findings indicate that corneal scarification results in an altered distribution of FGF-2 within the lacrimal gland, which involves a decrease in low molecular weight FGF-2 and a dramatic increase in a higher molecular weight isoform of FGF-2. FGF-2 may be released from myoepithelial cells apically (exocrine) into the tear fluid and basally (autocrine/paracrine) into the connective tissue, as well as from extracellular complexes within basal laminae.

Background Basic fibroblast growth factor (FGF-2), a potent angiogenic peptide, is known to be present in gonadotropes of the anterior pituitary parenchyma of rats and mice, and has been isolated from endothelial cells of many organs. Its localization within endothelial cells has not been determined, nor the mechanisms by which it might be released from endothelial cells during normal organogenesis. Methods Localization of FGF within endothelial cells of the anterior pituitary was accomplished by immunocytochemistry and studied by light-and electron microscopy. Capillaries within the anterior pituitary were studied in fetal rats from day 15 to term, and in adult rats. Results At the onset stages of vascularization (15–18 days fetal), the cytoplasm of the endothelial cells of many of the invading, immature capillaries (thick-walled with few or no fenestrations) was intensely immunopositive for FGF. Immunoprecipitate-filled blebs and slender cytoplasmic processes projected from the endothelial cells into the presumptive pericapillary space and toward the parenchymal cells. As gestation progressed (19–20 day fetal), and an increasing number of capillaries acquired the features characteristic of capillaries in the anterior pituitary of adult animals, i.e., thin-walled and fenestrated, there were fewer capillaries demonstrating immunopositivity for FGF. Foci of released FGF, i.e., extracellular, were occasionally evident within the presumptive pericapillary spaces throughout gestation. By comparison, capillaries of the anterior pituitary of adult rats did not contain immunostainable FGF in their cytoplasm, nor were any blebs and/or processes filled with immunoprecipitate evident. However capillaries did reveal an immunopositive enhancement of their lumenal and ablumenal surfaces. Conclusions During vascularization of the anterior pituitary, FGF within the cytoplasm of endothelial cells is released from blebs and/or processes of endothelial cells, and after the capillary bed is stabilized postnatally, these characteristics of vascularization are absent. © 1996 Wiley-Liss, Inc.

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 Rab27ash/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.

Normal tear film is crucial to the integrity and function of the human cornea and conjunctiva. Disturbance of the homogeneity of the tear film gives rise to a chronic condition known as keratoconjunctivitis sicca, or dry eye. One of the most severe forms of dry eye is found in patients with Sjogren's syndrome, an inflammatory autoimmune disorder characterized by lymphocytic infiltration of the lacrimal gland (LG) and affecting approximately 2 to 4 million of the population in the United States. The lymphocytic infiltrates produce immune mediators that act as toxic factors, resulting in reduced secretory function caused by secretory tissue atrophy and dysfunction of the surviving tissue. Although the pathogenesis of the disease is yet to be defined, increasing evidence shows that immunologic, genetic, hormonal, and environmental factors play crucial roles. Sjogren infiltrates consist primarily of CD4+ and CD8+ T cells at a ratio of 4:1, plus IgG+ B cells and plasmacytes. The proinflammatory cytokines infiltrating lymphocytes produce include interleukins (IL)-1β, -6, -12, and -18, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. In addition to these proinflammatory cytokines, anti-inflammatory cytokines, including IL-10, transforming growth factor-β, and IL-4 also have been detected. To some extent these are produced by glandular epithelial cells rather than by lymphocytes.1 Numerous studies have shown that administering or increasing expression of specific inhibitors of proinflammatory cytokines (sTNFR, IL-ra, anti–TNF-α) or anti-inflammatory cytokines (IL-10 and IL-4) can moderate inflammatory signals and suppress harmful effector functions in autoimmune disorders.2 Among these approaches, vector-mediated transduction of IL-10 has received increased attention for its promising therapeutic potential. IL-10 is produced by many activated immune cell types, including type 2 helper T cells, T regulatory cells, NKT cells, B cells, monocytes, and macrophages. IL-10 is pleiotropic, but it is considered an immunoregulatory cytokine because of its inhibitory effects on the expression of a large spectrum of proinflammatory cytokines and other inflammatory mediators, such as chemokines, major histocompatibility complex (MHC-II) molecules, and costimulatory molecules. IL-10 has been shown to attenuate inflammation in various experimental models, including arthritis, diabetes, uveitis, systemic lupus erythematosus, chronic renal disease, orchitis, and uveoretinitis.3–8 Injected IL-10 is short-lived and requires repeated administration, whereas viral vector–mediated IL-10 gene transfer therapy provides an extended source of therapeutic IL-10. Previous publications have demonstrated that rabbit peripheral blood lymphocytes (PBLs) proliferate when cocultured with autologous LG epithelial cells. These activated lymphocytes induce dacryoadenitis within 2 weeks after having been injected into the donor rabbit's remaining, contralateral LG, thus providing an induced in vivo model of autoimmune dacryoadenitis.9,10 The histopathology of the induced disease is characterized by lymphocytic foci containing large proportions of CD4+ T cells. The inflammatory process is accompanied by LG dysfunction, characterized by reduced tear production, and by signs of ocular surface inflammation, characterized by reduced tear film stability and increased Rose Bengal staining. These histopathologic features and clinical manifestations mimic those associated with Sjogren syndrome. The severity of the induced disease shows no signs of abatement but, rather, increases with time over a period of 6 months after induction.11 We have reported that the transfer of viral (v)IL-10 with an adenoviral vector (AdIL-10) produces prophylaxis against LG immunopathology and ocular surface disease induced by subsequent autoadoptive transfer of activated lymphocytes.7 However, as in other tissues, adenovirus-mediated gene transfer results only in transient transgene expression, with vIL-10 detectable in tears for

Paracrine mediators orchestrate the lacrimal gland's organization into ductal and acinar epithelial elements and its immunophysiology, that is, its maintenance of an optimal niche for dimeric immunoglobulin A (IgA)-secreting plasmacytes, secretion of secretory IgA, and prevention of inflammatory autoimmune responses. Transforming growth factor-beta (TGF-β) conveys important differentiation signals, but it may also be antiproliferative and pro-apoptotic. Prolactin, epidermal growth factor (EGF), and fibroblast growth factor (FGF) are suggested to convey important survival signals that abrogate TGF-β's deleterious influences. However, prolactin also exerts mitogenic and proinflammatory influences; TGF-β likely abrogates these. Pronounced cytophysiological and immunoarchitectural changes that the lacrimal gland undergoes during pregnancy suggest that reproductive hormones influence expression of both.

Adult female Fischer 344 (F344) and Sprague-Dawley (SD) rats, intact and ovariectomized (10–30d), have been used for immunolocalization of basic fibroblast growth factor (FGF). Tissues were selected from three specific sites (postero-lateral, lateral wing, and anterior wedge) of the periphery of the anterior pituitary (AP) carefully maintaining the association between the gland and the highly vascular meningeal connective tissue which envelops it. In both rat strains, most of the periphery of the AP was characterized by intact parenchymal cells delimited from the meningeal connective tissue by an intact basal lamina. However, foci also were evident in which parenchymal cells projected directly into the connective tissue without a basal lamina intervening. These zones, designated the Non-Delimited Peripheral Parenchyma (NDPP), were present minimally in control rats, but were more numerous in ovariectomized rats. Profiles of focally disrupted gonadotropes were evident within the NDPP of 20–30d ovariectomized rats juxtaposed against intact, granulated parenchymal cells. Partially disrupted gonadotropes also were evident within the peripheral parenchyma within approximately 100μ of the edge, and occasionally the disruptions resulted in an association of neighboring gonadotropes as a syncytium. FGF was localized only within the cytosol of gonadotropes, i.e., cells immunopositive for LH-β and FSH-β subunits. Gonadotropes nearer to the edges of the AP, especially the postero-lateral edge, were the most intensely stained. Electron microscopy and immunostaining for S-100 protein, a marker for folliculostellate cells (FSC), demonstrated that in intact and ovariectomized SD rats FSC were present in all peripheral zones of the AP, whereas portions of the postero-lateral periphery of the AP of intact and ovariectomized F344 rats often lacked FSC. We propose that FGF may be released from the cytosol of gonadotropes by a mechanism of cellular disruption. FGF released at peripheral sites of the AP would be well-positioned to stimulate angiogenesis from systemic blood vessels within the meninges. Since FSC are known phagocytes within the AP, their consistent presence in the periphery of the AP of SD rats may help regulate the effects of the released FGF, and by contrast, their absence in F344 rats may intensify or prolong the effects of released FGF. Such differences may underlie the higher incidence of pituitary tumors in F344 rats.

Pituitary tumorigenesis occurs in a transgenic line of mice, alpha-T7, which carries a hybrid transgene composed of the 5' flanking region of the human glycoprotein hormone alpha-subunit gene (1.8 kb) linked to the coding region of the SV40 T-antigen gene (alpha-Tag). Tumor foci were identified within the anterior pituitary of both male and female transgenic mice. In addition to a parenchyma with hypertrophied endocrine cells, mostly of the gonadotrope lineage, we here report the unexpected presence of neural tissue within the anterior pituitary, either as foci as large as 1.0 mm in diameter or greater, or in delicate bundles ramifying amongst the granulated parenchymal cells. Areas richest in neural tissue frequently were associated with tumor tissue composed of giant cells of three varieties, all with electron-lucent cytoplasm and similar organellar distribution including small secretory granules (80-160 nm diameter). In type I cells, the secretory granules were aligned at the plasma membrane; in type II cells, the secretory granules were distributed throughout the cytoplasm; type III cells formed colloid-filled follicles and their secretory granules rarely exceeded 100 nm diameter. These giant cells frequently had bizarre pleomorphic nuclei intensely immunopositive for T-antigen and cytoplasm which was lightly immunopositive for alpha-subunit, and immunopositive either for the LH-beta or TSH-beta subunits. Neural tissue contacted the normal granulated parenchymal cells directly, i.e., without a basal lamina or any connective tissue intervening, but only rarely formed synaptoid junctions with these granulated cells. Synaptoid junctions containing round, smooth vesicles, as well as dense core vesicles, were numerous between the neural processes themselves and between the neural tissue and the giant cells of the tumor tissue. These data suggest that in alpha-T7 transgenic mice the giant cells represent highly transformed gonadotropes or thyrotropes, and that a neurotrophic factor may be expressed by these transformed pituitary parenchymal cells.

With the eventual goal of developing a tissue-engineered tear secretory system, we found that primary lacrimal gland acinar cells grown on solid poly(L-lactic acid) (PLLA) supports expressed the best histiotypic morphology. However, to be able to perform vectorial transport functions, epithelia must be supported by a permeable substratum. In the present study, we describe the use of a solvent-cast/particulate leaching technique to fabricate microporous PLLA membranes (mpPLLAm) from PLLA/polyethylene glycol blends. Scanning electron microscopy revealed pores on both the air-cured ( approximately 4 microm) and glass-cured sides (2 microm) of the mpPLLAm. Diffusion studies were performed with mpPLLAm fabricated from 57.1% PLLA/42.9% polyethylene glycol blends to confirm the presence of channelized pores. The data reveal that glucose, L-tryptophan, and dextran (a high molecular weight glucose polymer) readily permeate mpPLLAm. Diffusion of the immunoglobulin G through the mpPLLAm decreased with time, suggesting the possible adsorption and occlusion of the pores. Cells cultured on the mpPLLAm (57.1/42.9 wt%) grew to subconfluent monolayers but retained histiotypic morphological and physiological characteristics of lacrimal acinar cells in vivo. Our results suggest that mpPLLAm fabricated using this technique may be useful as a scaffold for a bioartificial lacrimal gland device.

We investigated the role of the actin-based myosin motor, myosin 5c (Myo5c) in vesicle transport in exocrine secretion. Lacrimal gland acinar cells (LGAC) are the major source for the regulated secretion of proteins from the lacrimal gland into the tear film. Confocal fluorescence and immunogold electron microscopy revealed that Myo5c was associated with secretory vesicles in primary rabbit LGAC. Upon stimulation of secretion with the muscarinic agonist, carbachol, Myo5c was also detected in association with actin-coated fusion intermediates. Adenovirus-mediated expression of green fluorescent protein (GFP) fused to the tail domain of Myo5c (Ad-GFP-Myo5c-tail) showed that this protein was localized to secretory vesicles. Furthermore, its expression induced a significant ( P ≤ 0.05) decrease in carbachol-stimulated release of two secretory vesicle content markers, secretory component and syncollin-GFP. Adenovirus-mediated expression of GFP appended to the full-length Myo5c (Ad-GFP-Myo5c-full) was used in parallel with adenovirus-mediated expression of GFP-Myo5c-tail in LGAC to compare various parameters of secretory vesicles labeled with either GFP-labeled protein in resting and stimulated LGAC. These studies revealed that the carbachol-stimulated increase in secretory vesicle diameter associated with compound fusion of secretory vesicles that was also exhibited by vesicles labeled with GFP-Myo5c-full was impaired in vesicles labeled with GFP-Myo5c-tail. A significant decrease in GFP labeling of actin-coated fusion intermediates was also seen in carbachol-stimulated LGAC transduced with GFP-Myo5c-tail relative to LGAC transduced with GFP-Myo5c-full. These results suggest that Myo5c participates in apical exocytosis of secretory vesicles.

In our quest to develop a tissue-engineered tear secretory system, we have tried to demonstrate active transepithelial ion fluxes across rabbit lacrimal acinar cell monolayers on polyester membrane scaffolds to evaluate the bioelectrical properties of the cultured cells. Purified lacrimal gland acinar cells were seeded onto polyester membrane inserts and cultured to confluency. Morphological properties of the cell monolayers were evaluated by transmission electron microscopy and immunofluorescence staining for Na+,K+-ATPase and the tight junction-associated protein occludin. Sections revealed cell monolayers with well-maintained epithelial cell polarity, i.e., presence of apical (AP) secretory granules, microvilli, and junctional complexes. Na+,K+-ATPase was localized on both the basal-lateral and apical plasma membranes. The presence of tight cell junctions was demonstrated by a positive circumferential stain for occludin. Bioelectrical properties of the cell monolayers were studied in Ussing chambers under short-circuit conditions. Active ion fluxes were evaluated by inhibiting the short-circuit current ( Isc) with a Na+,K+-ATPase inhibitor, ouabain (100 μM; basal-lateral, BL), and under Cl−-free buffer conditions after carbachol stimulation (CCh; 100 μM). The directional apical secretion of Cl−was demonstrated through pharmacological analysis, using amiloride (1 mM; BL) and bumetanide (0.1 mM; BL), respectively. Regulated protein secretion was evaluated by measuring the β-hexosaminidase catalytic activity in the AP culture medium in response to 100 μM basal CCh. In summary, rabbit lacrimal acinar cell monolayers generate a Cl−-dependent, ouabain-sensitive AP → BL Iscin response to CCh, consistent with current models for Na+-dependent Cl−secretion.

During pregnancy, lymphocytes infiltrating the rabbit lacrimal gland disperse to the interacinar space from their normal focal concentrations, basal fluid secretion decreases, pilocarpine-induced fluid secretion increases, and stimulated fluid protein concentration decreases. Ductal epithelial cell prolactin (PRL) content increases and redistributes from the apical to the basal-lateral cytoplasm. A replication-incompetent adenovirus vector for rabbit PRL (AdPRL) was used to test the hypothesis that increased intracrine/autocrine PRL signaling alters secretory protein traffic in an ex vivo lacrimal acinar cell model. AdPRL had no discernable influence on microtubules or actin microfilaments or their responses to carbachol (CCh). Endogenous and transduced PRLs exhibited similar, nonpolarized, punctate distributions. Cells secreted PRL consititutively and at increased rates in response to CCh. In contrast, constitutive secretion of β-hexosaminidase was negligible, suggesting that the constitutive pathway for PRL is relatively inaccessible to typical secretory proteins. AdPRL had no significant effect on total secretion of β-hexosaminidase or syncollin-green fluorescent protein (GFP), a chimeric secretory protein construct. However, it reversed the polarized distributions of vesicles containing rab3D and syncollin-GFP. Live-cell imaging indicated that AdPRL redirected CCh-dependent syncollin-GFP exocytosis from the apical plasma membrane to the basal-lateral membrane. Elevated concentrations of exogenous rabbit PRL in the ambient medium elicited similar changes. These observations suggest that elevated PRL, as occurs in the physiological hyperprolactinemia of pregnancy, induces lacrimal epithelial cells to express a mixed exocrine/endocrine phenotype that secretes fluid to the acinus-duct lumen but secretes proteins to the underlying tissue space. This phenotype may contribute to the pregnancy-associated immunoarchitecture.

Gene delivery is one of the biggest challenges in the field of gene therapy. It involves the efficient transfer of transgenes into somatic cells for therapeutic purposes. A few major drawbacks in gene delivery include inefficient gene transfer and lack of sustained transgene expression. However, the classical method of using viral vectors for gene transfer has circumvented some of these issues. Several kinds of viruses, including retrovirus, adenovirus, adeno-associated virus, and herpes simplex virus, have been manipulated for use in gene transfer and gene therapy applications. The transfer of genetic material into lacrimal epithelial cells and tissues, both in vitro and in vivo, has been critical for the study of tear secretory mechanisms and autoimmunity of the lacrimal gland. These studies will help in the development of therapeutic interventions for autoimmune disorders such as Sjogren's syndrome and dry eye syndromes which are associated with lacrimal dysfunction. These studies are also critical for future endeavors which utilize the lacrimal gland as a reservoir for the production of therapeutic factors which can be released in tears, providing treatment for diseases of the cornea and posterior segment. This review will discuss the developments related to gene delivery and gene therapy in the lacrimal gland using several viral vector systems.

The acinar epithelial cells of the lacrimal gland exocytose the contents of mature secretory vesicles containing tear proteins at their apical membranes in response to secretagogues. Here we use time-lapse confocal fluorescence microscopy and fluorescence recovery after photobleaching to investigate the changes in actin filaments located beneath the apical membrane during exocytosis evoked by the muscarinic agonist, carbachol (100 μM). Time-lapse confocal fluorescence microscopy of apical actin filaments in reconstituted rabbit lacrimal acini transduced with replication-deficient adenovirus containing GFP-actin revealed a relatively quiescent apical actin array in resting acini. Carbachol markedly increased apical actin filament turnover and also promoted transient actin assembly around apparent fusion intermediates. Fluorescence recovery after photobleaching measurements revealed significant (P≤0.05) increases and decreases, respectively, in mobile fraction (Mf) and turnover times (t½) for apical actin filaments in carbachol-stimulated acini relative to untreated acini. The myosin inhibitors, 2,3-butanedione monoxime (BDM, 10 mM, 15 minutes) and ML-7 (40 μM, 15 minutes), significantly decreased carbachol-stimulated secretion of bulk protein and the exogenous secretory vesicle marker, syncollin-GFP; these agents also promoted accumulation of actin-coated structures which were enriched, in transduced acini, in syncollin-GFP, confirming their identity as fusion intermediates. Actin-coated fusion intermediates were sized consistent with incorporation of multiple rather than single secretory vesicles; moreover, BDM and ML-7 caused a shift towards formation of multiple secretory vesicle aggregates while significantly increasing the diameter of actin-coated fusion intermediates. Our findings suggest that the increased turnover of apical actin filaments and the interaction of actin with non-muscle myosin II assembled around aggregates of secretory vesicles facilitate exocytosis in lacrimal acinar epithelial cells.

Background The most common cause of ocular morbidity in developed countries is dry eye, many cases of which are due to lacrimal insufficiency. Dry eye affects approximately 10 million in the United States., most of whom are women. In the U.S. alone, an estimated 2 million Sjögren's syndrome patients have dysfunctional lacrimal glands and severe dry eye, and there is no satisfactory treatment. These patients would benefit if their lacrimal tissue function could be restored. Methods The effect of adenovirus-mediated transfer of tumor necrosis factor (TNF)-α inhibitor gene on induced autoimmune dacryoadenitis was evaluated in a rabbit model. Soluble transgene protein was detected in tears by ELISA for 7 days following transduction. Results Two weeks after induction of disease with activated lymphocytes, tear production, as determined by Schirmer testing, was reduced by about 40%, while tear film stability, as measured by tear breakup time (BUT), declined by 43%. Adenovirus-mediated gene therapy using AdTNFRp55-Ig given 2 weeks after disease induction, resulted in the return of tear production to normal levels by week 4. In the treated disease group, tear BUT improved significantly by week 4. Rose bengal scores, an indicator of corneal surface defects, increased after disease induction and declined after gene therapy. In the lacrimal gland, the CD4 to CD8 T cell ratio was 4:1 in the disease group compared to 1:2 in the treated group. Infiltration of T cells and CD18+ cells was reduced approximately 50% after gene therapy. Conclusion We concluded that therapeutic levels of soluble TNF inhibitor were achieved in the lacrimal gland and on the corneal surface. Anti-inflammatory cytokine gene expression might offer a potential therapeutic modality for the treatment of autoimmune dacryoadenitis, once suitable vectors become available.

In this article, we investigate the contributions of actin filaments and accessory proteins to apical clathrin-mediated endocytosis in primary rabbit lacrimal acini. Confocal fluorescence and electron microscopy revealed that cytochalasin D promoted apical accumulation of clathrin, α-adaptin, dynamin, and F-actin and increased the amounts of coated pits and vesicles at the apical plasma membrane. Sorbitol density gradient analysis of membrane compartments showed that cytochalasin D increased [14C]dextran association with apical membranes from stimulated acini, consistent with functional inhibition of apical endocytosis. Recombinant syndapin SH3 domains interacted with lacrimal acinar dynamin, neuronal Wiskott-Aldrich Syndrome protein (N-WASP), and synaptojanin; their introduction by electroporation elicited remarkable accumulation of clathrin, accessory proteins, and coated pits at the apical plasma membrane. These SH3 domains also significantly (p ≤ 0.05) increased F-actin, with substantial colocalization of dynamin and N-WASP with the additional filaments. Coelectroporation with the VCA domain of N-WASP blocked the increase in F-actin and reversed the morphological changes indicative of impaired apical endocytosis. We suggest that transient modulation of actin polymerization by syndapins through activation of the Arp2/3 complex via N-WASP coordinates dynamin-mediated vesicle fission at the apical plasma membrane of acinar epithelia. Trapping of assembled F-actin intermediates during this process by cytochalasin D or syndapin SH3 domains impairs endocytosis.

Previous studies have shown that ovariectomy causes necrosis of lacrimal acinar cells, apoptosis of plasma cells and gland lymphocytic infiltration. Both, lacrimal gland cell death and lymphocytic infiltration were prevented by androgen treatment. Since estrogens are removed by ovariectomy, and the synthetic estrogen diethylstilbestrol has been shown to affect some biochemical correlates of lacrimal secretion, the purpose of this study was to determine the effect of 17-beta-estradiol treatment on ovariectomy-induced cell death and lymphocytic infiltration. Sexually mature female New Zealand white rabbits (4-4.5 kg) were ovariectomized and divided into two groups. One group was treated with 0.5 mg kg(-1) per day 17-beta-estradiol, and the other group with vehicle alone. A third group of sham operated rabbits was used as controls and they also were treated with vehicle alone. Six days after surgery, the animals were euthanized, the lacrimal glands removed and processed for analysis of apoptosis as assessed by DNA fragmentation, and for morphological examination. DNA fragmentation was determined using the TUNEL assay and agarose gel electrophoresis. Sections were also stained for rabbit thymic lymphocyte antigen (RTLA), and rabbit CD18. Labelled nuclei and stained areas were quantified by automated densitometry. Ovariectomized rabbits showed a significant increase in the values for degraded DNA as a percent of total nuclear area (2.90+/-0.40%) compared to sham operated rabbits (0.73+/-0.22%). 17-beta-estradiol treatment in ovariectomized rabbits prevented the increase in DNA degradation. Examination of TUNEL assay at higher magnification (40x) confirmed previous studies showing that ovariectomy caused apoptosis of interstitial cells. Significant numbers of bulging cells of very pale appearance under light microscopy, also confirm previously identified necrotic cells in acinar regions. Treatment with 17-beta-estradiol prevented this necrosis. Increased numbers of RTLA(+) and CD18(+) interstitial cells were also evident after ovariectomy. 17-beta-estradiol treatment prevented the increase in the number of lymphoid cells. We confirmed previous observations that suggest that glandular atrophy observed after ovariectomy is likely to proceed by necrosis of acinar cells rather than apoptosis, and that ovariectomy triggers an inflammatory response in the gland. These results suggest that in addition to androgens, estrogens also seem to play a role to maintain lacrimal gland structure and function. A decrease in available estrogen levels could trigger both lacrimal gland apoptosis and necrosis, as well as lymphocytic infiltration. Although, the effect of estrogens in these experiments seems to be direct and not through androgens, the possibility of the role of an autocrine and/or paracrine factors, promoted by estrogen on lacrimal gland cells still needs to be investigated.

Purpose To study the effects of induced autoimmune dacryoadenitis on lacrimal gland function, histopathology, and ocular surface disease in a rabbit model. Methods One lacrimal gland was surgically excised from each experimental rabbit, and epithelial cells were purified, cultured, irradiated, and then cocultured with autologous peripheral blood lymphocytes (PBLs) for 5 days. Autoimmune dacryoadenitis was induced by injecting the autologous mixed cell reactions (AMCRs) into the rabbit's remaining lacrimal gland. Normal rabbits and rabbits with both lacrimal glands injected with nonstimulated PBLs were examined as controls. Eyes were evaluated biweekly for 8 weeks by slit-lamp biomicroscopy, Schirmer testing, tear break-up time measurement, and rose bengal examination. Sections of lacrimal glands removed at 8 weeks post-operation were immunostained using antibodies against rabbit class II major histocompatibility complex molecule (MHC-II), CD4, CD8, CD18, and rabbit thymic lymphocyte antigen (RTLA). Relative numbers of positively stained cells were quantified with a ChromaVision image analysis system. Results During an 8-week period, a continuous decrease in tear production and stability, accompanied by a continuous increase in rose bengal staining, occurred in eyes in which AMCR-PBL had been injected into the ipsilateral lacrimal glands. Similar, though generally less severe, changes occurred in eyes contralateral to the AMCR-PBL-injected eyes. No obvious changes by 8 weeks in these parameters were found in eyes in which the lacrimal glands had been injected with nonstimulated PBLs or in the lacrimal gland-excised eyes contralateral to normal eyes. Interstitial cells in normal lacrimal glands expressed CD18 and RTLA antigens, but few expressed CD4, CD8, or MHC-II. Focal mononuclear cell infiltrates were only found in lacrimal glands from animals with induced autoimmune dacryoadenitis. These cells were predominantly positive for CD4 (7.3-fold increase), RTLA (7.8-fold increase), or CD18 (42-fold increase). MHC-II expression in interstitial and ductal epithelial cells was also significantly greater in these animals than in control animals. The mononuclear cell infiltrates were frequently found enveloping venules, some of which appeared to be high endothelial cell venules. The ductal epithelium also contained CD4 and CD8 immunopositivity, within the epithelium, at the lumenal surface, or surrounding the ducts. Occasionally CD4 and CD8 immunopositive cells could be identified within the acinar lumens. Conclusions Injection of activated PBLs (i.e., AMCR-PBLs) in the lacrimal gland induces autoimmune dacryoadenitis with immunopathologic features similar to those of Sjogren's syndrome. The lacrimal immunopathology is accompanied by typical clinical manifestations of dry eye syndrome. The persistent significant dry eye does not appear to result just from failure of the diseased gland but from a more general dysfunction of the surface secretory tissues.

The objective of this study was to develop a tissue culture system which closely mimics the in situ lacrimal gland for improved study of lacrimal acinar cell physiology. Highly purified preparations of lacrimal acinar cells from adult female New Zealand White rabbits were isolated and grown in suspension culture in the form of Matrigel 'rafts', i.e., aggregates of acinar cells enclosed within a Matrigel coating. The rafts were seeded onto Matrigel-coated culture plates and their growth was followed for up to 28 days. Immunohistochemistry was used to demonstrate the cellular sites of prolactin (PRL), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), secretory component (SC) and major histocompatibility complex class-II molecules (MHC-II) within the acinar cells. By 3 days the cultures contained numerous, well-formed acini enclosed within the Matrigel. The acinar epithelial cells demonstrated histotypic polarity, with large, pale-staining, secretory granules aggregated adjacent to the lumen, and exocytotic release of secretory material into the lumen. From 5-10 days the pale-staining secretory granules decreased in number, while the lumenal contents of the acini increased in staining density. Throughout the culturing period as the pale-staining, secretory granules decreased in number, smaller more densely stained, secretory granules increased in number. The number of cells and size of acinar clusters increased steadily throughout the culturing period, and acini frequently achieved dimensions in excess of 0.5 mm. Increases in the size of acinar clusters were often accompanied by an increase in the size of the lumen. Frequently the lumen and its contents bulged asymmetrically towards one edge of the acinus. Immunhistochemistry demonstrated PRL and EGF within the lumens and within the apical cytoplasm of the acinar cells. Acini were strongly immunopositive for SC throughout the 28 day culture period, whereas immunopositivity for MHC-II molecules was strong initially, but diminished dramatically by 21 days. Immunostaining for FGF-2 was most intense on days 1 and 3, with staining throughout the cytoplasm, but became progressively more localized to the periphery of the acini as the culture period lengthened. In cultures of 1-28 days duration, Western blots of cell lysates demonstrated a major band (approximately 40 kDa) for PRL in 3-28 day preparations; a major band (approximately 80 kDa) for SC in 3 day and 7 day preparations that decreased in intensity in 14-28 day preparations; and a major band (approximately 23 kDa) for MHC-II protein in 1-21 day preparations that decreased in intensity in 28 day preparations. Lysosomes increased in number with time in culture, becoming a dominant cytoplasmic feature in 21 and 28 day cultures. Carbachol stimulation of 4 day rafts resulted in increased release of beta-hexosaminidase and SC from the rafts. The authors conclude that Matrigel rafts containing purified lacrimal gland acinar cells offer a highly advantageous system for study of lacrimal acinar cell function and one that correlates well with the in situ gland.

Purpose. To determine whether the expression of either interleukin-10 (IL-10) or tumor necrosis factor (TNF) inhibitor genes in transduced rabbit lacrimal gland epithelial cells suppresses lymphocyte proliferation in an autolog us mixed cell reaction, an apparent in vitro model of autoimmune dacryoadenitis. Methods. Purified lacrimal gland epithelial cells, transduced with an adenovirus vector carrying either viral IL-10 or TNF-inhibitor genes, were used to study their effects on the proliferation of autologous lymphocytes as monitored by 3 H-thymidine incorporation in a mixed cell reaction. After transduction, both epithelial cells and lymphocytes were cultured separately for 2 days and then epithelial cells were irradiated. Equal numbers of both cell types were then cocultured together for 5 days. Cocultures were pulsed with 3 H-thymidine and isotope incorporation was determined. Gene expression was detected by enzyme-linked immunosorbent assay and Western blots. Results. Lymphocyte proliferation was stimulated by epithelial cells and 3 H-thymidine incorporation was significantly greater in these cocultures than in controls. The proliferation was significantly diminished in the presence of transduced cells producing either IL-10 or TNF inhibitor. Conclusions. Transduction of lacrimal gland epithelial cells with adenovirus vectors encoding for either IL-10 or TNF-inhibitor proteins leads to expression of functional proteins capable of suppressing lymphocyte proliferation. Thus, lacrimal gland epithelial cells are a plausible target for gene therapy methods meant to produce immunoregulatory proteins.

Lymphocytic infiltration of the lacrimal gland decreases the gland’s ability to produce the fluid needed to keep the ocular surface healthy and comfortable. This immune-related lacrimal insufficiency is responsible for some severe forms of dry eye. Increasing anti-inflammatory cytokines in a dysfunctional lacrimal gland may suppress the inflammatory process and restore secretory function.

Sjogren’s syndrome is an autoimmune disease primarily involving the lacrimal and salivary glands. Recent studies on the initiation of autoimmune dacryoadenitis suggest that when acinar cells from rabbit lacrimal glands are induced to express MHC Class II molecules, the ability to present processed autoantigen peptides to CD4 T cells is also induced (Guo et al., 2000). However, it is likely that the outcome of MHC Class II molecule-mediated autoantigen presentation depends on the accessory signals present in the interstitial milieu. One potential accessory factor is interleukin 2 (IL-2), which is classically produced by Thl CD4 cells.

Recently, dry eye disease has been linked with hormonal changes in several patient populations. For instance, there is a high incidence of dry eye disease that accompanies aging in postmenopausal women (Molina et al., 1986), and there appears to be an increased incidence of disease during pregnancy (unpublished data). Furthermore, increased addition infiltration of lymphocytes into the lacrimal glands has also been associated with aging (Murray et al., 1981; Damato et al., 1984). Serum levels of androgens have been shown to be important in maintenance of normal lacrimal gland function (Mircheff et al., 1996).

We have recently established procedures for the isolation of highly purified lacrimal gland acinar cells (pLGAC) (Guo et al., 2000) and a culture method that promotes proliferation of the cells (Schonthal et al., 2000). Many studies have shown the benefits of seeding lacrimal cells onto Matrigel coated plates (Kelleher et al., 1991; Menerey et al., 1994; Millar, et al., 1996; Chen et al., 1998). Our procedure utilizes a method which coats the pLGAC with Matrigel to more closely mimic the in vivo condition where acinar cells are enveloped by matrix molecules on their basal surfaces. This has been achieved by trapping pLGAC in Matrigel rafts. These free-floating raft cultures have been maintained in vitro for up to 28 days for morphological and physiological studies.

Autoimmune dacryoadenitis, such as occurs in Sjogren's syndrome, is a frequent cause of lacrimal insufficiency, which in turn can cause dry eye. Rabbits are used frequently to test ocular therapies. Our goal is to develop a rabbit model of autoimmune dacryoadenitis to identify and test candidate therapies. Our approach arises from the observations that lacrimal gland epithelial cells stimulate proliferation in cultured autologous lymphocyte preparations and that an anti-MHC II antibody blocks this proliferation. The purpose of this study was to determine if injecting this proliferating autologous mixed cell reaction could induce dacryoadenitis in rabbits. After establishing that irradiated lacrimal gland epithelial cells stimulate proliferation in autologous peripheral blood lymphocytes, irradiated cells from a single lacrimal gland were co-cultured with autologous lymphocytes and after 5 days the mixed cell reaction, or components of the reaction, were injected into the contralateral lacrimal gland of the donor rabbit. After 2 weeks, the injected glands were removed and lymphocytic infiltration quantitated using digital image analysis of immunostained histological sections. Injecting an autologous mixed cell reaction of co-cultured irradiated lacrimal gland epithelial cells and lymphocytes reliably induced abundant periductal foci of >200 lymphocytes expressing CD18 and/or a rabbit thymic lymphocyte antigen (RTLA). Injection of medium or autologous lymphocytes alone elicited little response; injections of lymphocytes cultured with lysates of lacrimal gland epithelial cells elicited variable, modest responses. These lysates did not stimulate proliferation in the mixed cell reaction and proliferation was not observed if a porous membrane separated co-cultured lacrimal gland cells and lymphocytes. The results demonstrate that injecting an autologous mixed cell reaction of lacrimal gland epithelial cells and lymphocytes reliably creates a model of autoimmune dacryoadenitis. The relative ineffectiveness of components of the reaction to do the same supports the hypothesis that lacrimal gland epithelial cells trigger or exacerbate lacrimal autoimmune disease by presentation of autoantigens via MHC II. This experimental system can aid efforts to further understand mechanisms of diseases, and to identify and test candidate therapies.

Autoimmune dacryoadenitis is a frequent cause of lacrimal insufficiency. In order to test hypotheses regarding mechanisms that can trigger this syndrome, we developed a method to obtain a preparation of rabbit lacrimal gland epithelial cells essentially free of immune-system cells. The method relies on controlled digestion to disperse lacrimal acini, and recovers acini by filtration through various sizes of nylon mesh. Purity and integrity of the preparation were established qualitatively using light and electron microscopy. Contamination by immune-system cells was quantitated by immunohistochemistry using anti-CD18, and -RTLA (rabbit thymic lymphocyte antigen) antibodies. The novel method produced preparations of highly-purified lacrimal gland epithelial cells (pLGEC) with expected morphological characteristics with less than 1.5% of the cells staining for CD18 or RTLA. The method also yielded preparations of lacrimal gland interstitial cells (LGIC) enriched for lymphocytes; in these preparations either CD18 or RTLA were detected on nearly 10% of the cells. pLGEC promoted proliferation in preparations of autologous splenic lymphocytes (SPL) that was blocked by anti-MHC class II but not anti-MHC class I antibodies. This observation, combined with the apparent requirement that pLGEC must contact the autologous lymphocyte preparation to promote proliferation, supports the hypothesis the proliferation arises from antigen-presentation via MHC class II by pLGEC.

Light and electron microscopic immunocytochemistry, in situ hybridization and Dot Blot analysis revealed intracellular localization of prolactin-like molecules and prolactin mRNA in epithelial cells of the lacrimal glands of rabbits. There was also positive immunostaining for prolactin receptors on acinar cells and some interstitial cells. On Western blots of homogenates of whole lacrimal gland, isolated lacrimal acinar cells, isolated lacrimal interstitial cells and peripheral blood lymphocytes, prolactin antibody consistently labeled protein bands migrating at approximately 36 and 50 kD. These data confirm that lacrimal gland acinar cells produce endogenous prolactin-like molecules, but also express prolactin receptors. Since prolactin immunoreactivity has been detected in tear fluid and we found no accumulations of immunogold label in endocytic or transport vesicles, we hypothesize that the prolactin-like molecules in tear fluid originate primarily from synthesis within the acinar cells. We hypothesize further that prolactin from pituitary and other non-acinar cell origin has a modulating influence on acinar cell activity as well as immune function in the lacrimal gland, and that some of the prolactin-like molecules produced by the acinar cells contribute to these functions by autocrine/paracrine mechanisms.

Growth factors have diverse functions and are distributed throughout the body. A number have been detected in the lacrimal gland, eg, EGF,1,2 FGF-2,3 TGF-α,4 TGF-β,5 and HGF.6 In many tissues, FGF-2 and TGF-β have an inverse functional relationship. FGF-2, released in response to tissue damage, activates proteases that function in degradation of extracellular matrix molecules. TGF-β, released during later stages of tissue repair, inhibits proteases, thereby stabilizing extracellular matrix molecules. Although both FGF-2 and TGF-β are present within the lacrimal gland, their sites of synthesis and localization have not been documented. FGF-2 and TGF-β also have been shown to exert inverse functional effects on the synthesis and release of PRL, an immunomodulating hormone that we have localized within the lacrimal gland.7 The present study was undertaken to determine the sites of localization of FGF-2 and TGF-β within the lacrimal gland, as a basis for elucidating their functional roles in the lacrimal gland’s response to corneal injury or other initiators of lacrimal gland inflammation.