Hepatitis C virus has a genetically determined lymphotropism through co-receptor B7.2.

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.

Potentially pathogenic immune cells and networks in apparently healthy lacrimal glands.

Lacrimal glands of people over 40 years old frequently contain lymphocytic infiltrates. Relationships between histopathological presentation and physiological dysfunction are not straightforward. Data from rabbit studies have suggested that at least two immune cell networks form in healthy lacrimal glands, one responding to environmental dryness, the other to high temperatures. New findings indicate that mRNAs for several chemokines and cytokines are expressed primarily in epithelial cells; certain others are expressed in both epithelial cells and immune cells. Transcript abundances vary substantially across glands from animals that have experienced the same conditions, allowing for correlation analyses, which detect clusters that map to various cell types and to networks of coordinately functioning cells. A core network--expressing mRNAs including IL-1α, IL-6, IL-17A, and IL-10--expands adaptively with exposure to dryness, suppressing IFN-γ, but potentially causing physiological dysfunction. High temperature elicits concurrent increases of mRNAs for prolactin (PRL), CCL21, and IL-18. PRL is associated with crosstalk to IFN-γ, BAFF, and IL-4. The core network reacts to the resulting PRL-BAFF-IL-4 network, creating a profile reminiscent of Sjögren's disease. In a warmer, moderately dry setting, PRL-associated increases of IFN-γ are associated with suppression of IL-10 and augmentations of IL-1α and IL-17, creating a profile reminiscent of severe chronic inflammation.

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

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

Distinct dacryoadenitides autoadoptively transferred to rabbits by different subpopulations of lymphocytes activated ex vivo.

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.

Adeno-associated virus-mediated IL-10 gene transfer suppresses lacrimal gland immunopathology in a rabbit model of autoimmune dacryoadenitis.

PURPOSE: To evaluate the effect of adeno-associated virus (AAV) vector-mediated viral (v)IL-10 gene expression on lacrimal gland (LG) immunopathology and ocular surface disease in a rabbit model of induced autoimmune dacryoadenitis (ID). METHODS: Autologous peripheral blood lymphocytes, activated in a mixed-cell reaction when cocultured with purified rabbit lacrimal epithelial cells, induce a Sjögren's-like autoimmune dacryoadenitis when injected directly back into the donor animal's inferior LG. Four weeks after disease induction, AAV vector expressing the vIL-10 gene under control of a tetracycline-inducible promoter was injected into the inferior LG of the treatment group (ID/Rx), and doxycycline was fed orally to induce transgene expression. The ID group serving as control also received doxycycline. All LGs were removed 16 weeks after disease induction. RESULTS: Clinical symptoms showed overall improvement in the ID/Rx group compared with the ID group. Histopathologic examination of the ID group's LG revealed scattered large lymphocytic foci and areas of altered or distorted acini, whereas the ID/Rx group had scattered small lymphocytic foci. The number of CD18(+) cells was almost fivefold lower in the ID/Rx group than in the ID group. Although the total number of RTLA(+) cells did not differ between the groups, the CD4/CD8 ratio was 16-fold smaller in the ID/Rx group. CONCLUSIONS: Animals with experimentally induced autoimmune dacryoadenitis appeared to benefit from AAV-mediated vIL-10 gene transfer therapy. Quantitative immunohistochemical analysis suggested that the therapy might not have been simply immunosuppressive but rather supported the induction of CD8(+) regulatory cells.

Cytopathology and exocrine dysfunction induced in ex vivo rabbit lacrimal gland acinar cell models by chronic exposure to histamine or serotonin.

PURPOSE: Lacrimal immunohistopathology has diverse clinical presentations, suggesting that inflammatory mediators exert diverse influences. Chronic exposure to agonistic acetylcholine receptor autoantibodies has been studied previously; the present work addressed mediators that signal through other G protein-coupled receptors. METHODS: Acinus-like structures and reconstituted acinar epithelial monolayers from rabbit lacrimal glands were exposed to varying concentrations of histamine or 5-hydroxytryptamine (5-HT) for 20 hours. Net and vectorial beta-hexosaminidase secretion, cytosolic Ca(2+) (Ca(i)) elevation, apical recruitment of p150(Glued), actin microfilament meshwork organization, and ultrastructure were assessed. RESULTS: Histamine and 5-HT acutely stimulated beta-hexosaminidase secretion at lower, but not higher, concentrations. Neither of them acutely elevated Ca(i) levels. Both recruited p150(Glued) at concentrations that failed to induce secretion. Chronic exposure to 10 mM histamine inhibited carbachol (CCh)-induced beta-hexosaminidase secretion and prevented the formation of continuous monolayers; 1 mM 5-HT partially inhibited secretion at the apical medium. Neither altered secretion to the basal medium. Chronic exposure to histamine or 5-HT partially decreased CCh induced Ca(i) elevations and p150(Glued) recruitment, even at concentrations that did not inhibit secretion. Both expanded acinar lumina and thickened microfilament meshworks, and both caused homotypic fusion of secretory vesicles and formation of aqueous vacuoles in the apical and basal cytoplasm. Chronic exposure to forskolin, which activates adenylyl cyclase, induced similar cytopathologic changes but impaired secretion modestly and only at the highest concentration tested. CONCLUSIONS: Inflammatory mediators that signal through G protein-coupled receptors cause acinar cell cytopathology and dose-dependent reductions of CCh-induced beta-hexosaminidase secretion. Although agonistic acetylcholine receptor autoantibodies may cause pervasive functional quiescence, inflammatory mediators may cause varying degrees of exocrine dysfunction.

The class V myosin motor, myosin 5c, localizes to mature secretory vesicles and facilitates exocytosis in lacrimal acini.

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 < or = 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.

A novel, local technique for studying rabbit lacrimal gland secretion in situ.

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.

Traffic of endogenous, transduced, and endocytosed prolactin in rabbit lacrimal acinar cells.

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.

Transepithelial bioelectrical properties of rabbit acinar cell monolayers on polyester membrane scaffolds.

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 (I(sc)) with a Na(+),K(+)-ATPase inhibitor, ouabain (100 microM; basal-lateral, BL), and under Cl(-)-free buffer conditions after carbachol stimulation (CCh; 100 microM). 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 beta-hexosaminidase catalytic activity in the AP culture medium in response to 100 microM basal CCh. In summary, rabbit lacrimal acinar cell monolayers generate a Cl(-)-dependent, ouabain-sensitive AP --> BL I(sc) in response to CCh, consistent with current models for Na(+)-dependent Cl(-) secretion.

Tissue-engineered tear secretory system: functional lacrimal gland acinar cells cultured on matrix protein-coated substrata.

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

Elevated prolactin redirects secretory vesicle traffic in rabbit lacrimal acinar cells.

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 beta-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 beta-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.

Current status of gene delivery and gene therapy in lacrimal gland using viral vectors.

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 Sjögren'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.

Molecular mechanisms of lacrimal acinar secretory vesicle exocytosis.

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.

Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells.

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 microM). 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< or =0.05) increases and decreases, respectively, in mobile fraction (Mf) and turnover times (t1/2) 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 microM, 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.

Expression of TNF inhibitor gene in the lacrimal gland promotes recovery of tear production and tear stability and reduced immunopathology in rabbits with induced autoimmune dacryoadenitis.

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)-alpha 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.

Prophylactic effect of IL-10 gene transfer on induced autoimmune dacryoadenitis.

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 Sjögren-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.

Impairing actin filament or syndapin functions promotes accumulation of clathrin-coated vesicles at the apical plasma membrane of acinar epithelial cells.

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, alpha-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

Estrogen prevention of lacrimal gland cell death and lymphocytic infiltration.

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.

Tumor necrosis factor inhibitor gene expression suppresses lacrimal gland immunopathology in a rabbit model of autoimmune dacryoadenitis.

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 Sjögren'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.