Method: Snapin expression in synovial tissue was examined by histology, and single and two-color immunohistochemistry. Snapin knock down in macrophages were performed using siRNA technique. Snapin, Lamp1, 2 and Lac3 protein levels were checked by Western blot analysis. The localization and co-localization of Snapin with Rab7 in macrophages were studied with confocal immunofluorescence microscopy.
Result: Snapin was significantly increased in RA synovial tissue compared to control synovial tissues from arthritis-free controls. The percentage of Snapin positive cells (lining: 21 vs 60%, sublining: 18 vs 54%; p < 0.01) and the Snapin expression score (lining: 18 vs 124, sublining: 10 vs 144; p < 0.05), reflecting the percent positive and intensity of staining, were both significantly increased in the synovial lining and in the sublining of the RA synovial tissues compared to the controls. Snapin expression level was correlated with inflammation. Two color immunohistochemistry showed that CD68 positive macrophages, especially those in the sublining region, strongly expressed Snapin in RA synovial tissue. Additionally, within lymphoid aggregates some CD3 positive T cells also expressed Snapin. Therefore, studies were performed to determine the homeostatic function of Snapin in macrophages. Employing in-vitro differentiated macrophages, Snapin was present in a diffuse granular pattern and it co-localized with Rab7, a marker for late endosomes. The forced reduction of Snapin by siRNA in macrophages resulted in increased Lamp-1, Lamp-2, two lysosomal markers, and LC3-II, a marker of autophagosomes, suggesting that Snapin might be functionally involved in endosome to lysosome fusion in macrophages. Studies are underway to determine the potential role of Snapin in the autophagy-lysosomal pathway, which may contribute to the pathogenesis of RA.
Conclusion: Snapin was highly expressed in RA synovial tissue, especially in sublining macrophages. Snapin was localized in the late endosomes and may play an important role in endosome to lysosome fusion in macrophages.
Disclosure: B. Shi, None; Q. Q. Huang, None; A. Dorfleutner, None; C. Stehlik, None; P. P. Tak, Arthrogen B.V. , 3 ; R. M. Pope, None.