Control of chemokine gradients by the retinal pigment epithelium

Abstract

Purpose: Proinflammatory cytokines in degenerative diseases can lead to the loss of normal physiology and the destruction of surrounding tissues. In the present study, the physiological responses of human fetal retinal pigment epithelia (hfRPE) were examined in vitro after polarized activation of proinflammatory cytokine receptors.

Methods: Primary cultures of hfRPE were stimulated with an inflammatory cytokine mixture (ICM): interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. Western blot analysis and immunofluorescence were used to determine the expression/localization of the cytokine receptors on hfRPE. Polarized secretion of cytokines was measured. A capacitance probe technique was used to measure transepithelial fluid flow (J(V)) and resistance (R(T)).

Results: IL-1R1 was mainly localized to the apical membrane and TNFR1 to the basal membrane, whereas IFN-gammaR1 was detected on both membranes. Activation by apical ICM induced a significant secretion of angiogenic and angiostatic chemokines, mainly across the hfRPE apical membrane. Addition of the ICM to the basal but not the apical bath significantly increased net fluid absorption (J(V)) across the hfRPE within 20 minutes. Similar increases in J(V) were produced by a 24-hour exposure to ICM, which significantly decreased total R(T).

Conclusions: Chemokine gradients across the RPE can be altered (1) through an ICM-induced change in polarized chemokine secretion and (2) through an increase in ICM-induced net fluid absorption. In vivo, both of these factors could contribute to the development of chemokine gradients that help mediate the progression of inflammation/angiogenesis at the retina/RPE/choroid complex.

Figure 1

Western blot analysis to identify IFN-γR1, IL-1R1, and TNFR1. IFN-γR1 displayed two bands, close to 100 and 55 kDa. TNFR1, and IFN-γR1 were detected at approximately 55 and 80 kDa, respectively.

Figure 2

Immunofluorescence staining of hfRPE cultures grown on transwells. In each part of the figure, the left panel is an en face view of the apical membrane shown as a set of superimposed sections (MIP image). For each of the three en face views, the x- and y-axis side views are also shown as MIP images. In each case, the right panel shows a part of the y-axis side view image (labeled area) at higher magnification. ZO-1 highlights cell circumferences in the en face images. In the side view, ZO-1 also marks the boundary between the apical and basolateral membranes. (A) IL-1R1 (green) was mainly localized apically (ZO-1, yellow). (B) TNF-R1 (green) was mainly localized to the basal side (basal ZO-1, red). (C) IFN-γR1 (red) was localized to both the apical and basolateral membranes (ZO-1, green). In all three panels, nuclei of the cells were stained with DAPI (blue).

Figure 3

Summary of mean chemokine secretion levels as a heat map. The heat map variations in color intensity correspond to the measured secretion levels for different combinations of inflammatory mediators added to the apical or basal baths. The data were log₁₀ transformed; the lightest shade of yellow corresponds to secretion levels between 0 and 10 pg/mL and each increment represents a log unit increase in secretion level.

Figure 4

hfRPE monolayers were stimulated by addition of ICM to both the apical and basal baths for 24 hours (n = 3). Cytokine and chemokine activities were measured in the apical (top) and basal (bottom) baths after the reduction of basal bath volume in three steps from 1500 to 500 μL. The basal volumes were adjusted with O-rings.

Figure 5

ICM induced changes in hfRPE fluid transport. Top traces: J_V plotted as a function of time, with net fluid absorption is indicated by positive values; bottom traces: TEP and R_T are plotted as function of time. (A) Addition of ICM to the basal bath increased J_V ∼10 μL · cm^-2 · h^-1 with no significant changes in TEP and R_T. (B) Concomitant addition of ICM to apical and basal baths increased J_V ∼5 μL · cm^-2 · h^-1 with no changes in TEP and a slight increase in resistance that was not consistent from experiment to experiment. In four experiments, the mean change in R_T was not statistically significant. We used the Student’s t-test to analyze these acute responses to ICM (paired, two tailed).

Figure 6

ICM-induced changes in hfRPE fluid transport, formatted as in Figure 5. (A) Addition of IL-1β to the apical and IFN-γ+TNF-α to the basal baths consistent with their primary receptor locations increased J_V ∼13 μL · cm^-2 · h^-1. (B) Addition of these component cytokines to the bath opposite their primary receptor location did not appreciably alter J_V, R_T, or TEP (n = 4). Student’s t-test used to analyze acute responses to ICM components (paired, two-tailed).

Figure 7

Twenty-four-hour ICM-induced changes in hfRPE fluid transport and R_T. R_T and J_V summary for hfRPE monolayers treated with ICM in both apical and basal bath for 24 hours and hfRPE control monolayers. R_T decrease was statistically significant for ICM-treated filters at t = 0 versus t = 24 hours (*P < 0.05). A statistically significant increase in J_V was observed in control versus 24-hour ICM-treated filters (**P < 0.01). NS, nonsignificant.