Abstract
Purpose:
Inflammation mediated by interleukin-1β (IL-1β)/IL-1 receptor plays a key role in the progression of diabetic retinopathy. Müller cells are the main source of IL-1β contributing to retinal inflammation in diabetes. Whereas much is known about the activation of IL-1β signaling, less is known about interleukin-1α (IL-1α), which activates the same IL-1 receptor and possesses the same inflammatory properties as IL 1β. IL-1α can translocate to the nucleus and participates as a transcription factor for pro-inflammatory genes. With cell death, the IL-1α precursor is released and is biologically active. To date there are no reports of hyperglycemia influencing IL-1α localization and function in retinal cells. Thus, the focus of this study was to identify whether hyperglycemia influences IL-1α cellular localization, IL-1β mediated inflammatory signaling, and cell viability in Müller cells.
Methods:
Müller cells were treated with normal (5mM) or high (25mM) glucose containing medium for 48 or 96 hours. Nuclear localization of IL-1α was determined using immunohistochemistry (IHC) or Western Blot analysis of nuclear fractions. Caspase-1 activity was measured in Müller treated with normal and high glucose medium in the presence or absence of a specific human monoclonal anti-human anti-IL-1α (10μg/ml) after 96 hours of treatment. Cell death was measured using Trypan Blue exclusion method.
Results:
Hyperglycemia led to a significant decrease of 24±6% in active IL-1α in the cytosol of Müller. At 48 hours of high glucose treatment, IL-1α was predominantly cytosolic. At 96 hours of high glucose treatment, there was a significant, 3-fold increase in IL-1α levels in the nucleus. Nuclear IL-1α seemed clustered. Western Blot analysis of nuclear fractions confirmed increased nuclear localization of IL-1α at 96 hours. Anti-IL-1α treatment led to an 87±13.5% decrease in caspase activity and 76±14% decrease in cell death in Müller treated with high glucose.
Conclusions:
Hyperglycemia alters cellular localization of IL-1α in Müller cells. For the first time, we have shown that under hyperglycemic conditions IL-1α influences activation of caspase-1 and thus the IL-1β pathway indicating that these members of the IL-1 family strongly regulate each other. Identifying the interaction between IL-1 family members is crucial for the understanding of the development of diabetic retinopathy.
Keywords: 557 inflammation •
490 cytokines/chemokines •
603 Muller cells