The effects of Th17 cytokines on the inflammatory mediator production and barrier function of ARPE-19 cells

Abstract

Th17 cells have emerged as a key factor in the pathogenesis of uveitis as well as other autoimmune disorders. They secrete a number of cytokines including IL-17A, IL-17F and IL-22 and until now the effects of these cytokines on resident cells of the eye were not yet clear. The purpose of this study was to investigate the effects of Interleukin (IL)-17A, IL-17F and IL-22 on the production of inflammatory mediators and barrier function of retinal pigment epithelium cells. We showed that ARPE-19 cells, a spontaneously arisen cell line of retinal pigment epithelium (RPE), constitutively expressed IL-17RC and IL-22R, but not IL-17RA. IL-17A significantly enhanced the production of CXCL8, CCL2, CCL20 and IL-6 by these cells. IL-17F had a similar effect on the production of CXCL8, CCL2 and IL-6 by ARPE-19 cells, but did not influence the expression of CCL20. Both IL-17A and IL-17F significantly decreased the transepithelial electrical resistance (TER) of the ARPE-19 monolayer and increased the diffusion rate of fluorescein isothiocyanate (FITC)-dextran. They also disrupted the distribution of the junction proteins zonula occludens (ZO)-1 and occludin at the interface of adjacent cells. IL-22 did not have a detectable effect on the production of the tested inflammatory mediators by ARPE-19 cells, TER of the ARPE-19 monolayer, the diffusion rate of FITC-dextran or the distribution of ZO-1 and occludin. This study demonstrates that IL-17A and IL-17F, but not IL-22, significantly promoted ARPE-19 cells to secrete inflammatory mediators and compromised the ARPE-19 monolayer barrier function in association with a disrupted distribution of ZO-1 and occludin. These results suggest that both IL-17A and IL-17F may play a role in posterior segment inflammation of the eye.

Figure 1. Expression of IL-22R and IL-17RC in ARPE-19 cells.

(A) Coexpression of IL-22R and IL-17RC in ARPE-19 cells was shown by immunocytochemistry. Scale bar  = 75 µm. (B) Western blot revealed that ARPE-19 cells expressed only IL-17RC. However, PBMCs expressed both IL-17RA and IL-17RC.

Figure 2. IL-17A and IL-17F but not IL-22 promoted chemokines and IL-6 production in ARPE-19 cells.

Confluent ARPE-19 cells were stimulated with different concentrations of IL-17A, IL-17F or IL-22 as indicated. After 24 hours of incubation, protein concentrations of CXCL8, CCL2, CCL20 and IL-6 released into the supernatants were measured by ELISA. Data were shown as the means±SEM of four independent experiments. **p<0.01 versus the control group.

Figure 3. Effect of IL-17A, IL-17F or IL-22 on TER of cultured ARPE-19 monolayer.

Monolayers were cultured for 21 days, where after the various stimuli were added. Incubation of ARPE-19 monolayers with 50 ng/ml IL-17A or IL-17F induced a gradual decrease of TER, and a significant effect occurred 5 days (p = 0.019, p = 0.045) after stimulation. The continuous decreases were also observed 6 days (p = 0.01, p = 0.016) and 7 days (p = 0.023, p = 0.008) after stimulation. IL-22 had no effect on TER. Data were shown as the means±SEM of four independent experiments. *p<0.05 versus the control group.

Figure 4. Effect of IL-17A, IL-17F or IL-22 on transepithelial diffusion rate of FITC-dextran in ARPE-19 monolayer.

Stimulation of ARPE-19 monolayer with 50 ng/ml IL-17A, IL-17F or IL-22 for 6 days induced a higher FITC-dextran diffusion rate at 24 hours compared with the control group. A diffusion percentage approaching 100% indicates that the amount of dextran-FITC in the upper and lower chamber approaches the same values. IL-22 had no effect on diffusion rate. Data were shown as the means±SEM of four independent experiments. *p<0.05 versus the control group.

Figure 5. Effect of IL-17A, IL-17F or IL-22 on the distribution of junction proteins in ARPE-19 monolayer.

Cells were incubated with or without 50 ng/ml IL-17A, IL-17F or IL-22 for 6 days, then fixed and immunolabeled with ZO-1 or occludin. Immunostaining for ZO-1 and occludin in untreated or IL-22-treated ARPE-19 monolayer showed a continuous labeling in the region of cell-cell contact. Incubation with IL-17A or IL-17F caused a marked disruption of ZO-1 and occludin staining. The immunostaining shown is representative of three independent experiments. Scale bar  = 15 µm.