Joel E. Schechter

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 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.

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 rabbits 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 Sjögrens 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.

Activation of anterior pituitary folliculo-stellate cells in the formation of estrogen-induced prolactin-secreting tumors

The response of folliculo-stellate (FS) cells of the anterior pituitary to estrogen has been studied in two strains of rat which differ in estrogen responsiveness. Fischer 344 (F344) rats are highly estrogen-responsive in comparison to Sprague-Dawley (S-D) rats. Ovariectomized adults were implanted with silastic capsules containing 17 beta-estradiol benzoate. Control and experimental animals were sacrificed 10 and 20 days after implantation of the silastic capsules. Most FS cells of F344 rats revealed dramatic changes, i.e. activation as phagocytes, after 10 and 20 days of estrogen treatment. These activated FS cells separated themselves from adjacent parenchymal cells and frequently contained phagosomes which included portions of granulated cells, mostly somatotrophs and lactotrophs. They also contained a variety of lysosomal dense bodies and dilated cisterns of endoplasmic reticulum. Endfeet processes abutting the parenchymal basal lamina contained large aggregates of dumbbell-shaped granules and tubulo-vesicles. Subjacent to the endfeet processes the basal lamina was often discontinuous, and profiles suggesting uptake of fragments of the basal lamina by FS cells were evident. Most FS cells of estrogen-treated S-D rats either were identical to those of controls or were only minimaly modified. However, a few activated FS cells were present and these contained a variety of lysosomal dense bodies and phagosomes, as well as lipid inclusions and dilated endoplasmic reticulum. Endfeet projections at the parenchymal basal lamina contained very few dumbbell-shaped granules or tubulo-vesicles, and the basal lamina was intact.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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

BackgroundThe 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 Sjögrens 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.MethodsThe 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.ResultsTwo 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.ConclusionWe 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.

Comparison of Gonadotroph, Thyrotroph and Mammotroph Development in situ, in Transplants and in Organ Culture

This study was conducted to examine the role of the hypothalamus in development of gonadotrophs, thyrotrophs, and mammotrophs up to the time of birth in the rat. The development of the in situ newborn pituitary (22 days post-coitus; dpc) was compared with the development of 12 and 15 dpc pituitary anlagen grown under the kidney capsule of an adult host or in organ culture for a time period equivalent to 22 days of gestation. Morphological development was evaluated by light microscopic histology and gonadotrophin immunocytochemistry. Functional development was analyzed by radioimmunoassay determination of luteinizing hormone, thyrotrophin, and prolactin content of the in situ, kidney capsule grafted, and organ-cultured pituitary. The results provide evidence that factors, perhaps blood-borne, play a significant role in pituitary development but that direct hypothalamic influences are of minimal importance in the development of gonadotrophs and mammotrophs. The fetal hypothalamus may play a significant role in stimulating high storage levels of thyrotrophin in the prenatal pituitary.

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 Sjögrens 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.

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.

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.