Human retinal endothelial cells express functional interleukin-6 receptor

Abstract

Background: Interleukin (IL)-6 is an inflammatory cytokine present in the eye during non-infectious uveitis, where it contributes to the progression of inflammation. There are two major IL-6 signaling pathways: classic signaling and trans-signaling. Classic signaling requires cellular expression of the IL-6 receptor (IL-6R), which exists in membrane-bound (mIL-6R) and soluble (sIL-6R) forms. Prevailing dogma is that vascular endothelial cells do not produce IL-6R, relying on trans-signaling during inflammation. However, the literature is inconsistent, including with respect to human retinal endothelial cells.

Findings: We examined IL-6R transcript and protein expression in multiple primary human retinal endothelial cell isolates, and assessed the effect of IL-6 on the transcellular electrical resistance of monolayers. Using reverse transcription-polymerase chain reaction, IL-6R, mIL-6R and sIL-6R transcripts were amplified in 6 primary human retinal endothelial isolates. Flow cytometry on 5 primary human retinal endothelial cell isolates under non-permeabilizing conditions and following permeabilization demonstrated intracellular stores of IL-6R and the presence of mIL-6R. When measured in real-time, transcellular electrical resistance of an expanded human retinal endothelial cell isolate, also shown to express IL-6R, decreased significantly on treatment with recombinant IL-6 in comparison to non-treated cells across 5 independent experiments.

Conclusions: Our findings indicate that human retinal endothelial cells produce IL-6R transcript and functional IL-6R protein. The potential for classic signaling in human retinal endothelial cells has implications for the development of therapeutics targeted against IL-6-mediated pathology in non-infectious uveitis.

Keywords: Endothelial cell; Human; Interleukin-6; Receptor; Retina; Uveitis.

Fig. 1

IL-6R transcript expression in human retinal endothelial cells. A Images showing IL-6R amplicons run on 2% agarose gel. L: DNA ladder (500 base pairs indicated by red cross); 1–7: primary retinal endothelial cell isolates from individual donors; 8: expanded retinal endothelial cell isolate; NT: no cDNA template control. Expected product sizes (indicated by red arrows): IL-6R: 240 bp; mIL-6R: 202 bp; sIL-6R: 195 bp. B Graphs showing relative normalized expression of corresponding IL-6R transcripts in the same cell isolates showed in (A). Reference genes were RPLP0 and PPIA

Fig. 2

IL-6R protein expression in human retinal endothelial cells. A-C Representative flow cytometry plots from one cell isolate: (A) Debris and doublets were excluded based on forward scatter (FSC) and side scatter (SSC) properties. B CD31-positive were gated in each sample relative to unstained controls. C IL-6R expression was assessed in non-permeabilized and permeabilized CD31-positive cells. Blue and red traces indicate the fluorescence in unstained and IL-6R antibody-stained of CD31-positive cells, respectively. D Histograms showing percentage of CD31-positive cells expressing IL-6R for individual primary cell isolates (n = 5 donors) and the expanded cell isolate (n = 4 experiments). Bars indicate mean. Non-permeabilized and permeabilized groups were compared by donor-paired (primary cell isolates) and experiment-paired (expanded cell isolate) 2-tailed Student’s t-test: p > 0.05

Fig. 3

Effect of IL-6 on permeability of human retinal endothelial cell monolayers. Results were generated in 5 independent experiments using the expanded cell isolate. A Plots of transcellular electrical resistance across IL-6-treated (red) versus untreated control (blue) cell monolayers, measured as cell index each hour. Arrowheads mark time of IL-6 treatment. Dots represent mean, with error bars indicating standard deviation. n = 3–4 monolayers per condition. B Graphs showing cell index at specified time intervals following IL-6 treatment for corresponding experiments. Bars represent mean, with error bars showing standard deviation. n = 3–4 monolayers per condition. Groups were compared by 2-tailed Student’s t-test: * = p < 0.05; ** = p ≤ 0.01