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.

A lacrimal gland is a lacrimal gland, but rodent's and rabbit's are not human.

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.

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.

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.

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

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.

Interacting influences of pregnancy and corneal injury on rabbit lacrimal gland immunoarchitecture and function.

PURPOSE: Previous reports indicated that pregnancy and corneal injury (CI) trigger alterations of lacrimal gland (LG) growth factor expression and redistributions of lymphocytes from periductal foci to acini. The purpose of this study was to test our hypothesis that pregnancy would exacerbate the changes induced by CI. METHODS: Corneas were injured with scalpel blades, and, 2 weeks later, LGs were collected for immunocytochemistry and Western blot analysis. Lacrimal fluid was collected under basal- and pilocarpine-stimulated conditions for protein determination and Western blot analyses. RESULTS: There were significant increases of immunoreactivity for prolactin, TGF-beta1, and EGF in duct cells during pregnancy and after CI, most prominent in pregnant animals with CI. Pregnancy decreased baseline lacrimal fluid secretion, whereas CI did not have a noticeable effect; pregnancy and CI combined resulted in increased fluid production. Pregnancy and CI each increased pilocarpine-induced lacrimal fluid production, whereas protein concentrations were decreased. Prolactin, TGF-beta1, and EGF were detected in LG by Western blot analysis but were minimally detectable in lacrimal fluid. RTLA+ and CD18+ cells were redistributed from periductal to interacinar sites during pregnancy and after CI, most prominent in pregnant animals with CI. CONCLUSIONS: Like pregnancy, CI is associated with redistribution of immune cells from periductal to interacinar sites and enhanced immunoreactivity of prolactin, TGF-beta1, and EGF in ductal cells. Although baseline lacrimal fluid secretion varied, the glands of all three experimental groups produced significant amounts of fluid in response to pilocarpine, but protein concentrations were decreased.

Unique ultrastructure of exorbital lacrimal glands in male NOD and BALB/c mice.

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.

Multiple Natural and Experimental Inflammatory Rabbit Lacrimal Gland Phenotypes.

PURPOSE: To investigate lacrimal gland (LG) immunophysiological and immune-mediated inflammatory process (IMIP) phenotype diversity. METHODS: Ex vivo matured dendritic cells (mDC) were loaded with acinar cell microparticles (MP). Peripheral blood lymphocytes (PBL) were activated in mixed cell reactions with mDC and injected directly into autologous, unilateral LG (1°ATD-LG) of two rabbit cohorts, one naïve, one immunized with a LG lysate membrane fraction (Pi). Autoimmune IgG titers were assayed by ELISA, MCR PBL stimulation indices (SI) by [3H]-thymidine incorporation. Schirmer tests without and with topical anesthetic (STT-I, STT-IA) and rose Bengal (RB) staining tests were performed. H&E and immunohistochemically stained sections were examined. RNA yields and selected transcript abundances were measured. Immune cell number and transcript abundance data were submitted to Principal Component Analysis (PCA). RESULTS: Immunizing Pi dose influenced SI but not IgG titers. STT scores were decreased, and rose Bengal scores increased, by day 118 after immunization. Previous immunization exacerbated scores in 1°ATD-eyes and exacerbated 1°ATD-LG atrophy. IMIP were evident in 2°ATD-LG as well as 1°ATD-LG. PCA described diverse immunophysiological phenotypes in control LG and diverse IMIP phenotypes in ATD-LG. IgG titers and SI pre-adoptive transfer were significantly associated with certain post-adoptive transfer IMIP phenotype features, and certain LG IMIP features were significantly associated with RB and STT IA scores. CONCLUSIONS: The underlying variability of normal states may contribute to the diversity of experimental IMIP phenotypes. The ability to generate and characterize diverse phenotypes may lead to phenotype-specific diagnostic and therapeutic paradigms.

Mucosal immunity and self-tolerance in the ocular surface system.

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.

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.

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.

Lacrimal histopathology and ocular surface disease in a rabbit model of autoimmune dacryoadenitis.

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 Sjögren'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.

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.

Expression of IL-10 and TNF-inhibitor genes in lacrimal gland epithelial cells suppresses their ability to activate lymphocytes.

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 autologous 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 3H-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 3H-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 3H-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.

Autoimmune dacryoadenitis and sialadenitis induced in rabbits by intravenous injection of autologous lymphocytes activated ex vivo against lacrimal antigens.

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 Sjögren syndrome, in that SG manifestations accompany the LG disease. It should be well suited to elucidating Sjögren pathogenesis and pathophysiology and to evaluating experimental therapies.

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.