Cytokines (TNF-α, IFN-γ, and IL-1β)
19 20 21 and chemokines (MIP-1α, MCP-1, and RANTES)
22 23 play critical roles during the pathogenesis of EAU. Although some growth factors and oxidative stress are reported to affect RPE TJs.
24 25 Whether cytokines and chemokines can directly affect TJ protein expression during EAU is unknown. Retinas and choroids (including RPE) from normal B10.RIII and C57BL/6 mice were cultured in vitro with various cytokines (TNF-α, IFN-γ, and IL-1β) and chemokines (MIP-1α, MCP-1, and RANTES) for 24 hours, and expression of TJ proteins was evaluated by Western blot analysis. With rabbit anti-claudin-1/3, two bands were detected in both retinal and RPE samples in Western blot analysis. In the retinal tissue, the top band was stronger than the bottom band, whereas in the RPE tissue, the bottom band was stronger then the top band
(Fig. 6) . Further comparisons conducted with the more specific anti-claudin-1 antibody and anti-claudin-3 antibodies showed that the top band was claudin-3 (22 kDa) and the bottom band was claudin-1 (20 kDa; data not shown), suggesting that, in the retina, claudin-3 is predominant, whereas in RPE cells, claudin-1 is predominant, consistent with differential distribution of claudin proteins in separate tissues.
Table 1shows that retinal endothelial cell TJ proteins were not downregulated by any of the cytokines and chemokines tested. In contrast, in RPE cells, both IL-1β and MCP-1 significantly reduced ZO-1 (
P < 0.02) protein expression. However, TJ regulatory proteins occludin-1 and claudin-1/3 expression remained unaffected by cytokine and chemokine treatment.