Abstract
Purpose: :
Diabetic Retinopathy has been considered a low–grade and chronic inflammatory disease. It was demonstrated that several genes involved in inflammatory processes are upregulated in diabetic retinas. The production of interleukin–1 beta (IL–1 beta), a pro–inflammatory cytokine, is increased, and it was correlated with an increase in blood–retinal barrier permeability. IL–1 beta elicits its pro–inflammatory action through the activation of IL–1 type I receptor (IL–1RI).The IL–1R accessory protein (IL–1RAcP) is a key element on the activation of the signal transduction cascade. We investigated the effect of high glucose and IL–1 beta on the IL–1RI and IL–1RAcP regulation, in retinal endothelial cells.
Methods: :
Rat retinal capillary endothelial cells (TR–iBRB2 cell line) were exposed to high concentrations of glucose (30 mM) or mannitol (osmotic control), for 7 days. The cells were also exposed, in a time–dependent manner (1, 3, 6, 12 and 24h), to high glucose, mannitol or IL–1 beta (10 ng/ml). IL–1RI and IL–1RAcP immunoreactivity was evaluated by Western Blotting and immunocytochemistry.
Results: :
Long–term exposure of retinal endothelial cells to high glucose or mannitol decreased the protein content of IL–1RI. A time–dependent downregulation of IL–1RI also occurred in cells exposed to high glucose, mannitol or IL–1 beta (1–24h). The protein content of IL–1RAcP did not change. The downregulation of IL–1RI induced by high glucose, mannitol or IL–1 beta was due to activation of IL–1RI by IL–1 beta, since it was prevented by the presence of antibodies against IL–1 beta or IL–1RI. The downregulation of IL–1RI induced by IL–1 beta was prevented by chloroquine (100 µM), a lysosome inhibitor, but not by MG132 (40 µM), a proteasome inhibitor.
Conclusions: :
Retinal endothelial cells express IL–1RI and IL–1RAcP, and, therefore can be affected by IL–1 beta. Our results indicate that high glucose, probably due to osmotic stress, and IL–1 beta downregulate the protein expression of IL–1RI in retinal endothelial cells. The downregulation of IL–1RI is triggered by its activation and it is due, at least partially, to lysosomal degradation.
Keywords: diabetic retinopathy • inflammation • signal transduction