IFN{gamma} regulates retinal pigment epithelial fluid transport

Abstract

The present experiments show that IFNgamma receptors are mainly localized to the basolateral membrane of human retinal pigment epithelium (RPE). Activation of these receptors in primary cultures of human fetal RPE inhibited cell proliferation and migration, decreased RPE mitochondrial membrane potential, altered transepithelial potential and resistance, and significantly increased transepithelial fluid absorption. These effects are mediated through JAK-STAT and p38 MAPK signaling pathways. Second messenger signaling through cAMP-PKA pathway- and interferon regulatory factor-1-dependent production of nitric oxide/cGMP stimulated the CFTR at the basolateral membrane and increased transepithelial fluid absorption. In vivo experiments using a rat model of retinal reattachment showed that IFNgamma applied to the anterior surface of the eye can remove extra fluid deposited in the extracellular or subretinal space between the retinal photoreceptors and RPE. Removal of this extra fluid was blocked by a combination of PKA and JAK-STAT pathway inhibitors injected into the subretinal space. These results demonstrate a protective role for IFNgamma in regulating retinal hydration across the outer blood-retinal barrier in inflammatory disease processes and provide the basis for possible therapeutic interventions.

Figures

Fig. 1.

Expression and localization of IFNγ receptors. A: immunoblots of IFNγ receptor subunits 1 and 2 (IFNGR1 and IFNGR2) in human fetal and adult retinal pigment epithelium (RPE) and fetal choroidal (hfCHC) cells. M, molecular weight marker lanes; lane 1, primary hfRPE cell culture; lane 2, native human adult RPE; lane 3, primary hfCHC cells. B: immunofluorescence localization of IFNγ receptor in hfRPE cells. For IFNGR1 and IFNGR2, cross section through the z plane is shown at top. In each case, x-y plane is shown as an en face view of the apical membrane (maximum-intensity projection through the z-axis). ZO-1 (green) stains tight junctions; 4,6-diamidino-2-phenylindole (DAPI, blue) labels nuclei. Inset at higher gain shows a z-section above each panel; note that IFNGR1 and IFNGR2 are mainly located on the basolateral membrane (Ba). Ap, apical membrane.

Fig. 2.

IFNγ activates JAK-STAT signaling pathway in hfRPE cells. A: IFNγ stimulated tyrosine phosphorylation of STAT1 in hfRPE cells (15 min) and can be blocked by anti-IFNGR1 blocking antibody. GAPDH was used as loading control. B: regulation of interferon regulatory factor (IRF) gene expression in hfRPE cells. Cells were treated with serum-free medium [SFM (Ctrl)], IFNγ, or cycloheximide (CHX) + IFNγ for 4 h. ICSBP, IFN consensus sequence-binding protein.

Fig. 3.

Proinflammatory cytokines regulate hfRPE and human fetal choroidal (hfCHC) cell proliferation and migration. A: modulation of hfRPE cell proliferation by combinations of IFNγ, TNFα, and IL-1β (n = 4). *P < 0.05 vs. control in 0% FBS. #P < 0.05 vs. control in 5% FBS. ^P < 0.05, IFNγ vs. IFNγ + TNFα or IL-1β. B: modulation of hfRPE cell migration by combinations of IFNγ, TNFα, and IL-1β (n = 3). C: modulation of growth factor-induced hfRPE cell proliferation by IFNγ. *P < 0.05 vs. SFM control. ^P < 0.05, growth factor alone vs. growth factor + IFNγ. D: effects of JAK inhibitors on IFNγ-induced inhibition of hfRPE cell proliferation (n = 4). *P < 0.05 vs. control. ^P < 0.05, IFNγ alone vs. IFNγ + different JAK inhibitors. E: modulation of hfCHC proliferation by combinations of IFNγ, TNFα, and IL-1β (n = 4). *P < 0.05 vs. control in 0% FBS. #P < 0.05 vs. control in 5% FBS. ^P < 0.05, IFNγ vs. IFNγ + TNFα or all 3 components. F: modulation of PDGF-BB-induced hfCHC proliferation by IFNγ or proinflammatory cytokine mixture (ICM, i.e., TNFα, IL-1β, and IFNγ) (n = 4). *P < 0.05 vs. SFM control. ^P < 0.05, PDGF-BB + IFNγ or ICM-induced proliferation vs. single components.

Fig. 4.

Localization of CFTR in hfRPE cells. A: CFTR was detected in membrane-enriched extracts of hfRPE cells. M, molecular weight marker lane; lane 1, major mature (band C) and immature (bands A and B) bands of CFTR in primary hfRPE cell culture. B: immunofluorescence localization of CFTR (green label). Top: cross section through the z plane and below the x-y plane showing an en face view of the apical membrane (maximum-intensity projection through the z-axis). ZO-1 (labeled as red) serves as a tight junction marker delineating apical (Ap) and basolateral (Ba) sides of the cell, and DAPI (blue) labels the nuclei located close to the basement membrane. ZO-1 is seen as yellow/orange, since its red label overlaps green fluorescence from CFTR. In the inset at higher gain, DAPI (blue) channel was removed for more clear visualization of the basolateral localization of CFTR (green).

Fig. 5.

IFNγ-induced changes in hfRPE fluid transport. A: basal bath addition of IFNγ increased transepithelial fluid transport (JV) across monolayer of hfRPE cells. JV is plotted as a function of time in the top trace and net fluid absorption (apical to basal bath) is indicated by positive values; transepithelial potential (TEP, −) and total tissue resistance (RT, ▵) are plotted as a function of time in bottom traces. B: pretreatment with JAK inhibitor I (5 μM) in apical/basal baths inhibited IFNγ-stimulated JV increase. C: summary of JV and RT measurements after 24-h treatment with IFNγ. A statistically significant increase in JV and decrease in RT were observed in 24-h IFNγ-treated filters vs. control: *P < 0.05, **P < 0.01. NS, nonsignificant. D: IFNγ-stimulated JV increase was inhibited by addition of 5 μM CFTRinh-172 to basal bath. E and F: addition of CHX (62 μM) for ∼30 min and 4 h, respectively. Subsequent addition of IFNγ (Ap/Ba) increased JV acutely (E), whereas 4 h of incubation with CHX significantly inhibited IFNγ-stimulated JV increase (F).

Fig. 6.

Role of second messengers in IFNγ-induced JV changes. A: PKA inhibitor H-89 inhibited IFNγ-stimulated JV across the hfRPE monolayer. A similar effect was produced by CFTRinh-172. B: pretreatment with Rp-8-Br-cAMPS blocked IFNγ-stimulated JV. C: NOC-5 [a nitric oxide (NO) donor] stimulated JV in hfRPE cells. D: pretreatment with 5 μM CFTRinh-172 blocked NOC-5-stimulated JV. E: IFNγ-induced JV increase was inhibited by 100 μM aminoguanidine hydrochloride, an inducible NO synthase (iNOS) inhibitor.

Fig. 7.

Role of p38 in IFNγ-induced JV increase. A: IFNγ stimulates phosphorylation of p38 MAPK. pY/pS-STAT1 antibodies were used to detect tyrosine or serine phosphorylation of STAT1. B: p38 MAPK-specific inhibitor blocks IFNγ-stimulated JV increase. Subsequent addition of CFTRinh-172 further decreased JV.

Fig. 8.

A: intracellular recording of IFNγ-induced changes in TEP and apical and basolateral membrane potential (VA and VB, respectively, −) in a modified Ussing chamber. ▵, RT; ◊, ratio of apical to basolateral resistance (RA/RB). B: intracellular recordings of forskolin- and 8-Br-cAMP-induced changes in TEP, RT, VA, VB, and RA/RB. Data represent results from 5 similar recordings for A and B.

Fig. 9.

Time course of volume change of retinal detachment measured by optical coherence tomography (OCT). MPBS, modified PBS. A–D: OCT images from 4 different experiments showing difference in detachment size before addition of IFNγ to the anterior surface (left) and after 40–70 min of IFNγ addition (right), in the absence or presence of JAK-STAT pathway and PKA inhibitors (C and D). A and B: 2 examples of a decrease in detachment size/volume after IFNγ treatment. C: IFNγ was added in the presence of JAK inhibitor I (40 μM) and Rp-8-Br-cAMPS (80 μM). D: IFNγ was added in the presence of Rp-8-Br-cAMPS alone. Arrows indicate boundary of the bleb for comparison with area enclosed in dashed line, which indicates starting volume. E: 3-dimensional sections of experiment summarized in B. Pseudocolor in blue indicates spatial extent of detachment at 0 and 40 min. Middle panel in E is an en face view. F: summary of all in vivo experiments (n = 3). *P < 0.05; **P < 0.01.

Fig. 10.

Schematic diagram of IFNγ signaling pathway in retinal pigment epithelium. Major plasma membrane transporters and channels mediate fluid absorption across the RPE (Ref. 1) after activation of the canonical JAK-STAT pathway. GAS, γ-interferon activation site.