Long-term topical cyclosporine treatment improves tear production and reduces keratoconjunctivitis in rabbits with induced autoimmune dacryoadenitis.

PURPOSE: To use a rabbit model of induced autoimmune dacryoadenitis to evaluate the efficacy of topical ophthalmic cyclosporine A (CsA). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Technique for preparation of the corneal endothelium-Descemet membrane complex for transplantation.

PURPOSE: Replacing diseased corneal endothelium with a preparation of Descemet membrane carrying functional endothelium and no stroma may be a feasible method for treating corneal endothelial decompensation. To obtain a viable donor of a Descemet membrane endothelium disc, we modified the Descemet membrane stripping technique and monitored the percentage of endothelial damage to the donor tissue preparation. METHODS: Forty-eight human corneas were used. Cornea buttons were mounted on an artificial anterior chamber, endothelial side up. Endothelia were stained with alizarin red, examined under the microscope, and photographed at 5 different sites (microscope, x100; digital magnification, x2.83). A 6 x 7-mm rectangular piece of endothelium-Descemet membrane complex was obtained using a Grieshaber microsurgical knife and Kelman-McPherson forceps. Digital photographs of endothelia were analyzed with a computer, and the percentage of endothelial damage was calculated. Specimens were processed for hematoxylin-eosin staining. RESULTS: Forty of 48 endothelium-Descemet membrane preparations (83.3%) were complete peels with minimal endothelial damage. Endothelial damage before and after the surgery was 1.57 +/- 2.11% and 2.61 +/- 1.77%, respectively. Eight preparations (16.7%) failed because of tearing. Multiple hematoxylin-eosin-stained sections showed the presence of endothelium with intact Descemet membrane and no stromal tissue. CONCLUSION: We modified the technique of Melles and obtained a sheet of Descemet membrane and endothelium with minimal endothelial damage and with no remaining stroma observed. This simple technique can be used to obtain the endothelium-Descemet membrane complex in minutes. It may be useful for corneal endothelium transplantation.

Cellular redox state predicts in vitro corneal endothelial cell proliferation capacity.

Cellular redox state using the non-invasive mitochondrial autofluorescence technique of redox fluorometry was evaluated as a predictor for corneal endothelial proliferative capacity in vitro. Human corneal endothelial cells (HCEC) harvested from eye bank corneas were cultured in plates with two different coating substrates; type I collagen and poly-D-lysine. Cellular autofluorescence was measured with both DAPI (excitation: G365, emission: bandpass 445/50) and FITC (excitation: bandpass 450-490, emission bandpass 515-565) filter sets on days 3, 5, 7, and 14. The redox fluorometric ratio was calculated as net "DAPI" signal intensity divided by net "FITC" signal intensity. Normalized redox ratio was calculated as redox ratio divided by individual cell size. Cellular proliferation was analyzed by live cell count on days 2, 7, and 14. Mitochondrial staining was performed on days 4 and 14. The poly-d-lysine substrate decreased the proliferation capacity of HCEC in comparison to type I collagen out to 2 weeks (p=0.045). The cellular redox fluorometric ratio decreased significantly as the cells proliferated (p<0.001). The cells cultured on type I collagen coated plates exhibited significantly lower redox fluorometric ratios than cells cultured on poly-D-lysine coated plates at day 7 (p=0.015). Normalized redox ratio showed significantly lower value in type I collagen coated plates at days 7 (p=0.015) and 14 (p=0.039). Correlated cell proliferation capacity was significantly higher on type I collagen coating at days 7 and 14 (p=0.045 and p=0.049 respectively). HCECs showed different growth potential in vitro on different culture surface coating agents. This difference was well correlated with cellular redox ratios determined using redox fluorometry. Cellular redox ratio can be a potential predictor of cellular proliferation capacity.

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.

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.

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.

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.