Abstract
Purpose :
Previously we demonstrated that the Secreted Ly-6/uPAR Related Protein-1 (SLURP1), abundantly expressed in the cornea and secreted into the tear film, serves as an anti-angiogenic and anti-inflammatory molecule. Here we have studied the effect of SLURP1 on the inflammatory response of Human Corneal Limbal Epithelial (HCLE) cells to tumor necrosis factor-α (TNF-α).
Methods :
HCLE cells were stably transfected to express SLURP1 and two different clones were selected for the present studies. The effect of SLURP1 on TNF-α-induced expression of (i) IL-8 was quantified by QPCR and ELISA, (ii) CXCL1, CXCL2 and IL-1b by QPCR, in un-transfected control and SLURP1-expressing HCLE cells. The effect of SLURP1 on TNF-α-induced NF-kB nuclear translocation was studied by immunofluorescent staining and immunoblots. The effect of SLURP1 on TNF-α-induced oxidative stress was examined by immunoblots and immunofluorescent staining with anti-4-Hydroxynonenal (4-HNE) antibody.
Results :
QPCR revealed a significant decrease in TNF-α-induced upregulation of (i) IL-8 from 7.4- to 2.9- and 2.1 fold, (ii) IL-1b from 4.9- to 3.9- and 2.9-fold, (iii) CXCL1 from 9- to 3.3- and 5.5-fold, and (iv) CXCL2 from 4.8- to 2.1- and 2.8-fold respectively, in two different SLURP1-expressing HCLE clones compared with the WT cells. ELISAs revealed a concomitant decrease in IL-8 levels in cell culture supernatants from 789 pg/ml in the WT, to 503 and 352 pg/ml in the SLURP1-expressing HCLE clones. Immunoblots and immunofluorescent staining with anti-4-HNE antibodies revealed lower TNF-α-induced oxidative stress in SLURP1-expressing HCLE cells compared with the WT. Furthermore, TNF-α-activated nuclear translocation of NF-kB was suppressed in SLURP1-expressing HCLE cells.
Conclusions :
Collectively, these results demonstrate that SLURP1 (i) suppresses the TNF-α-induced upregulation of IL-8, IL-1b, CXCL-1 and CXCL-2 in HCLE cells by blocking NF-kB nuclear translocation, and (ii) safeguards the HCLE cells from TNF-α-induced oxidative stress.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.