Soluble hyaluronan receptor CD44 levels secreted by human conjunctival cells change in inflammatory conditions

Abstract

Purpose: Our group has reported the presence of soluble CD44 (sCD44) in human tears and measured sCD44 levels in corneal and conjunctival epithelial cell lines under in vitro inflammatory conditions. We also reported that those cell lines are not a good model to perform hyaluronan-related studies. For that reason our purpose is to determine if cultured human conjunctival primary cells release sCD44 when exposed to different in vitro simulated inflammatory conditions.

Methods: Human primary fibroblasts and epithelial cells were grown from conjunctival tissue obtained from cadaveric donors (n=7) and cultured in supplemented DMEM/F12 medium. This study was in strict accordance with the tenets of the Declaration of Helsinki and Spanish Regulations concerning the use of human tissue for biomedical research. When confluence was reached cells were treated for 24 h with inflammatory cytokines: TNF-α (25 ng/ml), IFN-y (10 ng/ml), and IL-13 (20 ng/ml). Then, supernatants were collected and levels of sCD44 and IL-6 were measured by ELISA. In addition, proliferation was studied with Alamar Blue™ reagent in cytokine-exposed cells. Data (mean ± SEM) were analyzed by the Student t test.

Results: Conjunctival fibroblasts basally released sCD44 (0.32 ± 0.041 ng/ml); when exposed to TNF-α or IL-13, a significant increase to 0.46 ± 0.038 ng/ml (p=0.022) and 0.59 ± 0.084 ng/ml (p=0.012), respectively, was observed. Similar results were obtained for IL-6 production. Untreated fibroblasts secreted 274.98 ± 63.80 pg/ml IL-6, and TNF-α and IL-13 increased secreted IL-6 levels to 7920.33 ± 2530.18 (p=0.006) and 1386.92 ± 444.39 (p=0.023) pg/ml, respectively. In epithelial cells, a tendency to decrease IL-6 levels was observed with IFN-y treatment, from 21.19 ± 1.41 ng/ml to 18.19 ± 1.18 ng/ml (p=0.061). Fibroblast proliferation rate was reduced (29% decrease, p=0.007) by IL-13 exposure whereas no changes were observed in epithelial cells.

Conclusions: We demonstrated that basal release of sCD44 occurred in both conjunctival fibroblasts and epithelial cells. Increased sCD44 levels measured under simulated inflammatory conditions would suggest an involvement of sCD44 in conjunctival inflammation. These results are consistent with our previous studies with hyaluronan receptors, where we reported an increase in CD44 and RHAMM levels in patients with immune-atopic diseases.