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. 1998 Apr;112(1):34-43.
doi: 10.1046/j.1365-2249.1998.00560.x.

Polarized secretion of IL-6 and IL-8 by human retinal pigment epithelial cells

G M Holtkamp  1 , M Van Rossem, A F de Vos, B Willekens, R Peek, A Kijlstra
Affiliations

Polarized secretion of IL-6 and IL-8 by human retinal pigment epithelial cells

G M Holtkamp et al. Clin Exp Immunol. 1998 Apr.

Abstract

A number of cell types situated along interfaces of various tissues and organs such as the peritoneum and the intestine have been shown to secrete inflammatory cytokines in a polarized fashion. Retinal pigment epithelial (RPE) cells are positioned at the interface between the vascularized choroid and the avascular retina, forming part of the blood-retina barrier. These cells are potent producers of inflammatory cytokines and are therefore considered to play an important role in the pathogenesis of ocular inflammation. Whether cytokine secretion by these cells also follows a vectorial pattern is not yet known, and was therefore the subject of this study. Monolayers of human RPE cells (primary cultures and the ARPE-19 cell line) cultured on transwell filters were stimulated to produce IL-6 and IL-8 by adding IL-1beta (100 U/ml) to either the upper or the lower compartment. After stimulation, the human RPE cell lines showed polarized secretion of IL-6 and IL-8 towards the basal side, irrespective of the side of stimulation. The ARPE- 19 cell line also secreted IL-6 and IL-8 in a polarized fashion towards the basal side after basal stimulation; polarized secretion was, however, not apparent after apical stimulation. The observation that human RPE cells secrete IL-6 and IL-8 in a polarized fashion towards the choroid may represent a mechanism to prevent damage to the adjacent fragile retinal tissue.

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Figures

Fig. 1
Fig. 1
Light micrograph of a section of an ARPE-19 cell monolayer on filter after haematoxylin–eosin staining (× 550).
Fig. 2
Fig. 2
Scanning electron micrograph of an ARPE-19 cell monolayer on filter. (a) Filter with a preconfluent culture of ARPE-19 cells. Arrow indicates pores of the filter. (b) Confluently overgrown filter with ARPE-19 cells. Bar = 50 μm.
Fig. 3
Fig. 3
Transmission electron micrograph of a section of an ARPE-19 cell monolayer on filter. Arrow indicates tight junction complex. Bar = 0.2 μm.
Fig. 4
Fig. 4
Fluorescence micrograph of an ARPE-19 cell monolayer on filter stained with ZO-1 antibody (× 500).
Fig. 5
Fig. 5
Diffusion of IL-8 through a retinal pigment epithelial (RPE) cell monolayer on transwell filter. In the upper or lower compartment of the RPE monolayer, 1 μg/ml of recombinant IL-8 was added. At different time points the amount of IL-8 was measured in the opposite compartment. Data are expressed as the mean of two filters and plotted as percentage of the added amount of IL-8. •, IL-8 added to the upper compartment; ○, IL-8 added to the lower compartment.
Fig. 6
Fig. 6
Secretion of IL-6 by the ARPE-19 cell line after stimulation with IL-1β from the apical side (a) or the basal side (b). ARPE-19 cells were seeded on transwell filters at a concentration of 1.6 × 105 cells/cm2 and cultured for at least 19 days in Dulbecco's modified essential medium (DMEM)/F12 supplemented with 1% fetal calf serum (FCS). Filters were used when the net trans-epithelial resistance (TER) was at least 20 Ω·cm2. Before stimulation monolayers were cultured for 24 h in serum-free medium. Filters were stimulated from either the upper or lower compartment with IL-1β at a final concentration of 100 U/ml. Data are expressed as absolute amounts of IL-6 per filter and are expressed as the means of 10 filters ± s.e.m. Statistical analysis of the log transformed data revealed a significant difference between the upper and the lower secretion after basal stimulation when tested by anova method (*) and a significant difference at the indicated time points (†) by Newman–Keuls method (P < 0.05). Measured in the upper • and lower ○ compartment after IL-1β stimulation, measured in the upper ▪ and lower □ compartment without IL-1β stimulation.
Fig. 7
Fig. 7
Secretion of IL-8 by the ARPE-19 cell line after stimulation with IL-1β from the apical side (a) or the basal side (b). ARPE-19 cells were seeded on transwell filters at a concentration of 1.6 × 105 cells/cm2 and cultured for at least 19 days in Dulbecco's modified essential medium (DMEM)/F12 supplemented with 1% fetal calf serum (FCS). Filters were used when the net trans-epithelial resistance (TER) was at least 20 Ω·cm2. Before stimulation monolayers were cultured for 24 h in serum-free medium. Filters were stimulated from either the upper or lower compartment with IL-1β at a final concentration of 100 U/ml. Data are expressed as absolute amounts of IL-8 per filter. Data are expressed as the means of 10 filters ± s.e.m. Statistical analysis of the log transformed data revealed a significant difference between the upper and the lower secretion after apical and basal stimulation when tested by anova (*) and a significant difference at the indicated time points (†) by Newman–Keuls method (P < 0.05). Measured in the upper • and lower ○ compartment after IL-1β stimulation, measured in the upper ▪ and lower □ compartment without IL-1β stimulation.
Fig. 8
Fig. 8
Secretion of IL-6 by three donor retinal pigment epithelial (RPE) cell cultures (I, II, III) after stimulation with IL-1β from the apical side (a) or the basal side (b). RPE cell lines were seeded on transwell filters at a concentration of 1.6 × 105 cells/cm2 and cultured for at least 19 days in Iscove's modified Dulbecco's medium (IMDM) supplemented with 1% fetal calf serum (FCS) and were used when net trans-epithelial resistance (TER) was at least 20 Ω·cm2. Before stimulation monolayers were cultured for 24 h in serum-free medium. Filters were stimulated from either the upper or lower compartment with IL-1β at a final concentration of 100 U/ml. Data are expressed as absolute amounts of IL-6 per filter and are expressed as the mean of two filters. Statistical analysis of the log transformed data revealed a significant difference between the upper and the lower secretion after both apical and basal stimulation when all donors are accounted for and individual donors as indicated (*) when tested by anova. Measured in the upper • and lower ○ compartment after IL-1β stimulation, measured in the upper ▪ and □ lower compartment without IL-1β stimulation.
Fig. 9
Fig. 9
Secretion of IL-8 by three donor retinal pigment epithelial (RPE) cell cultures (I, II, III) after stimulation with IL-1β from the apical side (a) or the basal side (b). RPE cell lines were seeded on transwell filters at a concentration of 1.6 × 105 cells/cm2 and cultured for at least 19 days in Iscove's modified Dulbecco's medium (IMDM) supplemented with 1% fetal calf serum (FCS) and were used when net trans-epithelial resistance (TER) was at least 20 Ω·cm2. Before stimulation monolayers were cultured for 24 h in serum-free medium. Filters were stimulated from either the upper or lower compartment with IL-1β at a final concentration of 100 U/ml. Data are expressed as absolute amounts of IL-8 per filter and are expressed as the mean of two filters. Statistical analysis of the log transformed data revealed a significant difference between the upper and the lower secretion after both apical and basal stimulation when all donors are accounted for and individual donors as indicated (*) when tested by anova. Measured in the upper • and lower ○ compartment after IL-1β stimulation, measured in the upper ▪ and lower □ compartment without IL-1β stimulation.
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