Abstract
Purpose :
The Secreted Ly-6/uPAR Related Protein-1 (SLURP1), abundantly expressed by the cornea, serves as an immunomodulatory molecule that prevents neutrophil influx while promoting angiogenic privilege. Here, we evaluate the effect of SLURP1 on neutrophil-endothelial cell interaction, an essential step in inflammation.
Methods :
The effect of SLURP1 (expressed in Pichia pastoris and partially purified) on tumor necrosis factor-α (TNF-α)-stimulated (i) interaction between differentiated neutrophil-like HL-60 (dHL-60) and human umbilical vein endothelial cells (HUVEC), (ii) expression of HUVEC surface adhesion molecules ICAM, VCAM, E-selectin, and L-selectin , (iii) dHL-60 chemotaxis towards chemoattractant fMLP, (iv) HUVEC monolayer permeability, and (v) cytokine expression in dHL-60 was studied by (a) fluorimetry of calcein-labeled dHL-60 retained on HUVEC monolayer, (b) fluorescence-assisted cell sorting (FACS), (c) transwell migration assays with 5mm pore size filters, (d) assaying 40 kDa FITC-dextran permeability through confluent HUVEC monolayer, and (e) QPCR, respectively.
Results :
SLURP1 treatment of TNF-α-activated HUVEC resulted in decreased binding of dHL-60 cells (59% bound, compared with 68% in albumin-treated TNF-α-activated HUVEC). The fraction of VCAM+, E-selectin+, and L-selectin+ cells decreased to 39, 34, and 13% in SLURP1-treated TNF-α-activated HUVEC, from 48, 46, and 17%, respectively, in albumin-treated TNF-α-activated cells. SLURP1 suppressed the disruptive effect of TNF-a on confluent HUVEC barrier function by 15% in permeability assays. SLURP1 also suppressed the TNF-a stimulated IL-1A, IL-1B and MMP9 production by 41, 34, and 45% respectively, in dHL-60 cells, and their chemotaxis towards fMLP by 18.2%.
Conclusions :
Together, these results suggest that SLURP1 suppresses neutrophil docking on endothelial cells and extravasation- key steps in inflammation- by suppressing TNF-α-stimulated cytokine production in dHL-60, cell adhesion molecules in HUVEC, and promoting HUVEC barrier function.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.