| Objectives:Our study purposes are to develop a rabbit model of autoimmune dacryoadenitis, and 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 dry eye model.Methods:1. Combined with filtration through nylon mesh and the density gradient centrifugation, we developed a method to obtain a preparation of purified rabbit lacrimal gland epithelial cells(pLGEC). pLGEC and autologous peripheral blood lymphocytes(PBL) were co-cultured for 5 days. [3H]-thymidine incorporation was tested to monitor PBL proliferation. Cell surface staining was done with monoclonal antibody of rabbit T cell, B cell, CD4, and CD8 antibodies and analyzed by FACS.2. Activated PBL were injected into the ear veins of the rabbits from which they had been obtained. One set of induced disease (ID/IV) rabbits were sacrificed after 4 weeks(ID/IV4 group, n=9), and a second group of rabbits were sacrificed after 8 weeks(ID/IV8 group, n=6). A control group of animals also had their left iLGs removed, but were injected with non-stimulated lymphocytes (n=8). Clinical assessment were performed on day 0 for baseline and every 2 weeks after injection. Lacrimal gland, conjunctiva and salivary gland were collected for histopathology H&E staining and immunohistochmical staining.3. 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 LG were removed for analysis 16 weeks after disease induction.Results:1. Visual inspection of pLGEC using light microscopy indicated that the cells were primarily acinar cell. The magnitude of [3H]-thymidine incorporation in PBL-pLGEC co-cultures was approximately 5-to 8-fold great than that of either PBL or irradiated pLGEC. FACS analysis revealed that the T-cell population increased approximately twofold in the mixed cell reaction when compared to unstimulated PBL. CD4 T cells constituted 20%of the total T cells counted in PBL samples before activation in the AMCR and 80%after activation.2.Tear production was reduced 50%by 4 weeks and BUT was 70% less than normal. Ocular surface defects were present. Four weeks after IV injection, glands contained large infiltrates composed of predominantly CD4" T cells close to interlobular and intralobar ducts. Histopathology at 8 weeks was more severe than at 4 weeks, and SG also showed clusters of abnormal epithelial cells and streaming lymphocytes.3. Clinical symptoms showed overall improvement in the ID/Rx group compared to ID group. Histopathologic examination of the ID group's LG revealed scattered large lymphocytic foci, whereas the ID/Rx group had scattered small lymphocytic foci. The number of CD18+cells was almost fivefold less in the ID/Rx group than in the ID group. The CD4:CD8 ratio was 16-fold smaller in the ID/Rx group.Conclusion:1. The method obtained highly purified lacrimal gland epithelial cells, and the pLGEC preparations were sufficient to promote proliferation of autologous PBL in the AMCR.2.Lymphocytes activated against lacrimal antigens and injected intravenously can home to the LG and SG and initiate autoimmune processes, suggesting that these sites constitutively contain not only antigen presenting cells displaying potentially pathogenic autoantigen epitopes. but also chemokines and homing molecules that recruit CD4+T cells. This new rabbit model more closely mimics Sjogren's syndrome in that salivary gland manifestations accompany the lacrimal gland disease.3. Animals with experimentally induced autoimmune dacryoadenitis appeared to benefit from AAV-mediated vIL-10 gene transfer therapy. The quantitative immunohistochemical analysis suggested that the therapy may not have been simply immunosuppressive, but rather, that it supported induction of CD8+regulatory cells. |
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