Figure 1.
Schematic diagram of CNTF, CT1 and OsM cytokine receptor complexes.
The cytokines are depicted schematically as grey circles. Signal transducing-receptor subunits are light blue (LIFRβ and OsMRβ) or dark blue (gp130). α-receptor subunits are shown in light grey.
Figure 2.
Schematic diagram of fluid transport chamber.
Top part of figure shows internal chamber design consisting of two identical hemichambers. Each hemichamber has a voltage-sensing electrode, three current-passing electrodes, and a thermistor. Hemichamber A contains media bathing the apical or retinal surface; hemichamber B contains solution bathing the basal or choroidal surface. Surface of each bathing solution is covered with hexadecane to eliminate evaporation. A capacitance probe is immersed in the hexadecane in one hemichamber. Volume change is detected by changes of distance between probe and bathing media surface. Solutions are exchanged by gravity flow from reservoirs through inflow valves at the bottom and suction from the surface on top. A thin Kel-F disc (dark horizontal line) is used to isolate probe tip from media and avoid short circuit. RPE monolayer (E) is mounted between two circular Kel-F discs with O-rings (shown in lower left corner of the figure). This chip is mounted between two water-jacketed Kel-F blocks in main chamber located in a modified CO2 incubator (5% CO2, 36.5°C) for fluid transport (Jv) measurements.
Figure 3.
Gene expression of CNTF, CT1, OsM, LIF and their receptors in hfRPE.
Relative amounts of messenger RNA were quantified using real time PCR. All data normalized to GAPDH = 106. Experiments were performed in triplicate using cells from four different donors.
Figure 4.
Constitutive expression of CNTF, CT1, OsM receptors in human RPE.
15 µg of enriched membrane proteins were electrophoresed and labeled with corresponding antibodies. Antibody specific bands (see arrows) for CNTFRα, LIFRβ, gp130 and OsMRβ are detected at approximately 41, 190, 130, and 200 kDa, respectively. Panel A (CNTFRα) - Lane 1: Magic Mark™ XP Western protein Standard; Lane 2: Brain (Human) membrane lysate-adult normal tissue; Lane 3: hfRPE membrane extract. Panel B (LIFRβ) - Lane 1: Magic Mark™ XP Western protein Standard; Lane 2: hfRPE membrane extract. Panel C (gp130) - Lane 1: HiMark™ Molecular Weight (HMW) Standard; Lane 2: hfRPE membrane extract; Lane 3: Magic Mark™ XP Western protein Standard; Lane 4: Hela whole cell lysate; Lane 5: NIH3T3 whole cell lysate. Panel D (OsMRβ) - Lane 1: HiMark™ Molecular Weight (HMW) Standard; Lane 2: hfRPE membrane extract; Lane 3: Magic Mark™ XP Western protein Standard.
Figure 5.
Immunofluorescence localization of CNTF, CT1, OsM receptors on hfRPE.
Central part of each panel is an enface view of cell culture monolayer, shown as a single optical section obtained from a Z-stack. Nuclei were stained with DAPI (blue) and ZO-1 tight junction marker stained green. Images of the cross section through the Z-plane are shown at the top of each panel. CNTFRα (A, red), LIFRβ (B, red), gp130 (C, red) and OsMRβ (D, red) were detected mainly on the apical membrane of confluent monolayer of hfRPE.
Figure 6.
CNTF, CT1, and OsM induced activation of STAT3 and ERK1/2 in human RPE.
A. Primary cultures of hfRPE and ARPE-19 were treated with CNTF, CT1, or OsM for a series of time points and the phosphorylation level of STAT3 and ERK1/2 were determined by Western blot analysis. B. CNTF, CT1, and OsM were added to the apical (Ap), basolateral (Ba), or both apical and basolateral (Ap/Ba) sides of confluent monolayers of hfRPE cells cultured on transwells for 30 minutes and phosphorylation levels of STAT3 were detected. C. Effects of JAK inhibitor I on CNTF induced phosphorylation of STAT3. Confluent monolayers of hfRPE cells were left untreated or treated with JAK inhibitor I (5 µM; both apical and basal sides) for 1 hour; 50 ng/ml of CNTF was added to the apical, basolateral, or both apical and basolateral side. Representative images from three separate experiments using cells from different donors. D. Effects of JAK inhibitor I on CNTF, CT1 and OsM induced phosphorylation of STAT3. Confluent monolayers of hfRPE cells were untreated or treated with JAK inhibitor I (5 µM; both sides) for 1 hour. 10 ng/ml of CNTF, CT1 or OsM was added to both apical and basolateral side and the phosphorylation levels of STAT3 were detected.
Figure 7.
Dose responses of CNTF, CT1, and OsM - induced hfRPE proliferation.
CNTF showed no significant effect on hfRPE proliferation, while CT1 significantly increased cell proliferation by 25% (50–100 ng/ml). In contrast, OsM produced a monotonic increase in its inhibitory effect on hfRPE proliferation between 1 and 100 ng/ml. There was a significant difference in inhibition between 1 and 10 ng/ml (P = 0.02) and between 10 and 100 ng/ml (P = 0.001). 5% FBS was used as positive control. (Summary data from two experiments using cells from different donors; * P<0.05, *** P<0.001 compared to 0% FBS negative control).
Figure 8.
Dose responses of CNTF, CT1 and OsM effects on hfRPE survivability.
CNTF showed significant protection on RPE cell survivability while OsM significantly decreased RPE cell survivability at concentrations starting from 0.1 ng/ml. (Summary data from experiments using cells from different donors; * P<0.05, ** P<0.01 *** P<0.001 compared to 0% FBS negative control).
Figure 9.
CNTF has no significant effect on hfRPE phagocytosis.
Confluent monolayers of hfRPE received FITC-labeled POS for 4 hour with or without CNTF (50 ng/ml) and phagocytosis rate were quantified using relative FITC fluorescence intensity normalized to Hoechst 33342 nuclei stain. MFG-E8 (0.6 µM) was used as the positive control. Bar graph A show relative binding+internalization and bar graph B shows internalization only. Bars represent mean ± SEM with triplicate technical repeats. * P<0.05, ** P<0.01, *** P<0.001 compared to negative control. NS: no statistical significance compared to each other. Practically identical results were obtained in another experiment.
Figure 10.
Effect of CNTF on polarized cytokine secretion from hfRPE.
A. Constitutive secretion of cytokine and neurotrophic factors from confluent monolayer of hfRPE. * P<0.05, ** P<0.01, *** P<0.001 comparison between apical and basal compartment; B–C. CNTF-induced secretion changes of NT3, IL8, VEGF, and TGFβ2. * P<0.05, ** P<0.01, compared to the apical compartment of untreated control; #<0.05, compared to the basal compartment of untreated control.
Figure 11.
CFTRinh-172 inhibits CNTF-induced fluid transport across RPE.
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; TEP (-) and RT (Δ) are plotted as function of time in the lower traces. For each set of experiments, left- hand panels (A, C, E), summary bar graphs are shown on the right-hand side (B, D, F, respectively) A. Addition of CNTF to the apical and basal bath increased JV across monolayer of hfRPE; subsequent addition of CFTRinh-172 decreased JV back to the baseline levels (n = 5). C. Pretreatment with JAK inhibitor I (5 µM) significantly inhibited the effect of CNTF on JV increase (n = 3). E. Subsequent addition of JAK inhibitor I decreased JV induced by CNTF (n = 3). * P<0.05, ** P<0.01 compared to each other.
Figure 12.
Schematic diagram of CNTF signaling in human RPE.
Human RPE constitutively secretes a range of growth factors, cytokines, and neurotrophic factors, including VEGF, IL8, TGFβ2, CNTF, and NT3 (blue arrows) to the apical bath. In addition, retinal damage can increase CNTF release toward the subretinal space. CNTF binding to its receptors at the RPE apical membrane activates JAK/STAT3 signaling and promotes RPE survivability. It also decreases VEGF, IL8, and TGFβ2 secretion into the apical bath and increases apical NT3 secretion along with fluid absorption (JV), mediated by the activation of basolateral CFTR chloride channels.