Objective. Salivary gland epithelial cells in patients with Sjögren's syndrome (SS) and in NOD andNOD.scid mice express Fas and Fas ligand, and these cells die from apoptosis. To elucidate the intracellular molecular mechanisms responsible for this salivary gland epithelial cell apoptosis, expression of the Bcl-2 family of proteins (Bcl-2, Bcl-x L , Bax) and caspase (caspases 3 and 8) was studied in young (ages 8-10 weeks) and old (ages 17-28 weeks) NOD and NOD.scid mice.Methods. Sections of frozen salivary gland tissue were obtained from NOD and NOD.scid mice and from the lip biopsy material of SS patients. Immunohistochemistry or Western blot analysis was performed to assess the apoptotic-associated proteins.Results. Levels of Bax and caspase 3 were elevated in the epithelial cells of glands from old NOD mice, but not in those from young NOD mice. In contrast, epithelial cells from both young and old NOD.scid mice exhibited strong expression of Bax and caspase 3. Western blot analysis showed that the activated form of caspase 3 was increased 2-5-fold in the glands from old NOD, old NOD.scid, and young NOD.scid mice compared with those from young NOD mice. Caspase 3 was also significantly elevated (P < 0.01) in SS patients whose focus scores were grade 3 or 4. In the SS patients' biopsy tissue and in the mouse glands, cells with fragmented DNA were positive for caspase 3.Conclusion. These results demonstrate that salivary gland epithelial cells in NOD and NOD.scid mice overexpress the proapoptotic molecules Bax and caspase 3. Bax could be the gene responsible for initiation of caspase activation, epithelial cell destruction, and lymphocyte glandular localization in SS.
Objective Salivary gland epithelial cells in patients with Sjögren's syndrome (SS) and in NOD and NOD.scid mice express Fas and Fas ligand, and these cells die from apoptosis. To elucidate the intracellular molecular mechanisms responsible for this salivary gland epithelial cell apoptosis, expression of the Bcl‐2 family of proteins (Bcl‐2, Bcl‐xL, Bax) and caspase (caspases 3 and 8) was studied in young (ages 8–10 weeks) and old (ages 17–28 weeks) NOD and NOD.scid mice. Methods Sections of frozen salivary gland tissue were obtained from NOD and NOD.scid mice and from the lip biopsy material of SS patients. Immunohistochemistry or Western blot analysis was performed to assess the apoptotic‐associated proteins. Results Levels of Bax and caspase 3 were elevated in the epithelial cells of glands from old NOD mice, but not in those from young NOD mice. In contrast, epithelial cells from both young and old NOD.scid mice exhibited strong expression of Bax and caspase 3. Western blot analysis showed that the activated form of caspase 3 was increased 2–5‐fold in the glands from old NOD, old NOD.scid, and young NOD.scid mice compared with those from young NOD mice. Caspase 3 was also significantly elevated (P < 0.01) in SS patients whose focus scores were grade 3 or 4. In the SS patients' biopsy tissue and in the mouse glands, cells with fragmented DNA were positive for caspase 3. Conclusion These results demonstrate that salivary gland epithelial cells in NOD and NOD.scid mice overexpress the proapoptotic molecules Bax and caspase 3. Bax could be the gene responsible for initiation of caspase activation, epithelial cell destruction, and lymphocyte glandular localization in SS.
Salivary epithelial cells from patients with primary Sjo È gren's syndrome (SS) undergo Fas-mediated apoptosis. Bcl-2 and Bcl-xL are apoptosis suppressing oncogenes. Very little is known about the role of these oncogene molecules in salivary epithelial cells. To investigate the possible prevention of salivary glandular destruction in SS by Bcl-2 and Bcl-xL, stable transfectants expressing these molecules were made from HSY cells, a human salivary epithelial cell line. HSY cells were transfected with an expression vector for human Bcl-2 or BclxL. Stable transfectants were selected and apoptosis was induced by anti-Fas antibody. Apoptosis was quantified by propidium iodide staining followed by flow cytometry. Caspase activity was detected by immunohistochemical analysis and enzyme cleavage of DEVD-AMC, a fluorescent substrate. Response to carbachol, a muscarinic receptor agonist, and EGF was measured by Ca 2+ mobilization and influx. Fas-mediated apoptosis was significantly inhibited in Bcl-2 and Bcl-xL transfectants compared to wild-type and control transfectants (empty vector). Surprisingly, caspase activity was not inhibited in Bcl-2 and Bcl-xL transfectants. Activation of the Fas pathway in the Bcl-2 and Bcl-xL transfectants by antibody also inhibited carbachol and EGF responsiveness (i.e., Ca 2+ mobilization and/or influx) by 50 ± 60%. This Fas-mediated inhibition of cell activation was partially or completely restored by specific peptide interference of caspase enzyme activity. The prevention of Fasmediated apoptosis by the overexpression of Bcl-2 and Bcl-xL in salivary gland epithelial cells results in injured cells expressing caspase activity and unable to respond normally to receptor agonists. Such damaged cells may exist in SS patients and could explain the severe dryness out of proportion to the actual number of apoptotic cells seen on salivary gland biopsy.
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