Abstract
Purpose :
Early Diabetic Retinopathy (DR) involves chronic low-level inflammation resulting in increased retinal levels of TNFα, IL1β, IL8, and IL6. Vitreous accumulation of these inflammatory mediators is associated with DR progression. We aim to characterize how leukocyte adhesion and vascular permeability are altered in human retinal microvascular endothelial cells (HRMEC) in response to these inflammatory mediators.
Methods :
Primary HRMEC were treated with 1 ng/mL of TNFα, IL1β, IL8, IL6 or vehicle control, respectively. To assess expression, HRMEC were treated for 4hrs, mRNA isolated from lysed cells, and transcripts measured using qRT-PCR. Primers tested were TNFα, IL1β, CXCL8, IL6, ICAM, VCAM, SELE. To measure adhesion, cells were treated for 6hrs whereupon peripheral blood mononuclear cells (PBMCs) were allowed to adhere to HRMEC monolayers for 30 minutes. Unadhered PBMCs were washed off, images taken using phase contrast microscopy, and adhered PBMCs counted using Adobe Photoshop®. Permeability of confluent HRMEC monolayers was measured by quantifying trans-endothelial electrical resistance (TEER) using the ECIS ZTheta.
Results :
Transcript changes (as fold-change of TNFα, IL1β, CXCL8, and IL6 transcripts vs vehicle, respectively) were as follows after 4hr stimulation with: TNFα (42, 69, 6, 2); IL1β (177, 184, 11, 34); IL8 (1, 1, 1, 1); IL6 (2, 1, 1, 1). Similarly, fold changes for ICAM, VCAM, and SELE transcripts, respectively, were as follows: TNFα (26, 1063, 3691); IL1β (20, 1758, 874); IL8 (1, 1, 1); IL6 (3, 2, 3). Both TNFα and IL1β increased PBMC adhesion 10-fold (p<0.0001), while IL8 and IL6 had no significant effect. Similarly, TNFα and IL1β significantly reduced TEER in HRMEC by at least 15% in comparison to vehicle while IL8 and IL6 had no significant changes.
Conclusions :
These data suggest that TNFα and IL1β have autocrine effects on inflammatory amplification in HRMEC, induce transcription of cell adhesion molecules resulting in significantly increased leukocyte adhesion in vitro, and reduce the barrier capacity of HRMEC, suggesting they may be targets for early stages of DR. Conversely, IL8 and IL6 do not significantly affect HRMEC in these experiments. However, they may promote later DR stages unrelated to inflammation. More work is needed to better understand the role of these cytokines on HRMEC and DR.
This is a 2020 ARVO Annual Meeting abstract.