Regulation of soluble interleukin-6 (IL-6) receptor release from corneal epithelial cells and its role in the ocular surface

Abstract

Interleukin (IL)-6 signaling through its soluble receptor (sIL-6R) (IL-6 trans-signaling) plays an important role in various inflammatory states. We investigated production of sIL-6R in the corneal epithelium and examined the role of IL-6 trans-signaling in the cornea. In-vitro experiments were performed using SV40-transformed human corneal epithelial cells (HCEC) and primary human corneal fibroblasts (HCF, keratocytes). Ectodomain shedding in HCEC was stimulated by adding phorbol myristate acetate (PMA, 3 μM: ) both with and without ectodomain shedding inhibition using TNF-α processing inhibitor-1 (TAPI-1, 250 ng/mL), and the concentration of sIL-6R in the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). Expression of differential sIL-6R mRNA splicing (DS-sIL-6R) in HCEC was examined by using reverse transcription (RT)-PCR. The recombinant IL-6 or combination of recombinant IL-6/sIL-6R was added to HCF culture medium and phosphorylation of STAT3 was analyzed by Luminex assay. Tear fluid from patients with Sjögren syndrome was collected and analyzed by ELISA for sIL-6R concentration. In HCEC culture medium, sIL-6R release was increased significantly (P < 0.01) by adding PMA and this increased release of sIL-6R was inhibited significantly by adding TAPI-1, indicating the participation of ectodomain shedding in sIL-6R production. In RT-PCR, DS-sIL-6R expression was noted in HCEC. IL-6/sIL-6R-induced STAT3 phosphorylation was recognized in cultured HCF, suggesting IL-6 trans-signaling induced inflammatory cellular signaling in HCF. In the tear fluid of the patients with Sjögren syndrome, sIL-6R expression was up-regulated (Sjögren syndrome; 2.38 ± 0.98 ng/mL, normal control; 0.16 ± 0.34 ng/mL). Production of sIL-6R was induced by both ectodomain shedding and mRNA splicing in the corneal epithelium. IL-6 trans-signaling can induce an inflammatory response in corneal fibroblasts. The up-regulation of sIL-6R in inflamed ocular surfaces suggests a pivotal role of sIL-6R at the ocular surface.