IFN{gamma} regulates retinal pigment epithelial fluid transport.

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.

CO2-induced ion and fluid transport in human retinal pigment epithelium.

In the intact eye, the transition from light to dark alters pH, [Ca2+], and [K] in the subretinal space (SRS) separating the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE). In addition to these changes, oxygen consumption in the retina increases with a concomitant release of CO2 and H2O into the SRS. The RPE maintains SRS pH and volume homeostasis by transporting these metabolic byproducts to the choroidal blood supply. In vitro, we mimicked the transition from light to dark by increasing apical bath CO2 from 5 to 13%; this maneuver decreased cell pH from 7.37 +/- 0.05 to 7.14 +/- 0.06 (n = 13). Our analysis of native and cultured fetal human RPE shows that the apical membrane is significantly more permeable (approximately 10-fold; n = 7) to CO2 than the basolateral membrane, perhaps due to its larger exposed surface area. The limited CO2 diffusion at the basolateral membrane promotes carbonic anhydrase-mediated HCO3 transport by a basolateral membrane Na/nHCO3 cotransporter. The activity of this transporter was increased by elevating apical bath CO2 and was reduced by dorzolamide. Increasing apical bath CO2 also increased intracellular Na from 15.7 +/- 3.3 to 24.0 +/- 5.3 mM (n = 6; P < 0.05) by increasing apical membrane Na uptake. The CO2-induced acidification also inhibited the basolateral membrane Cl/HCO3 exchanger and increased net steady-state fluid absorption from 2.8 +/- 1.6 to 6.7 +/- 2.3 microl x cm(-2) x hr(-1) (n = 5; P < 0.05). The present experiments show how the RPE can accommodate the increased retinal production of CO2 and H(2)O in the dark, thus preventing acidosis in the SRS. This homeostatic process would preserve the close anatomical relationship between photoreceptor outer segments and RPE in the dark and light, thus protecting the health of the photoreceptors.

Flagellin-stimulated Cl- secretion and innate immune responses in airway epithelia: role for p38.

Activation of an innate immune response in airway epithelia by the human pathogen Pseudomonas aeruginosa requires bacterial expression of flagellin. Addition of flagellin (10(-7) M) to airway epithelial cell monolayers (Calu-3, airway serous cell-like) increased Cl(-) secretion (I(Cl)) beginning after 3-10 min, reaching a plateau after 20-45 min at DeltaI(Cl) = 15-50 microA/cm(2). Similar, although 10-fold smaller, responses were observed in well-differentiated bronchial epithelial cultures. Flagellin stimulated I(Cl) in the presence of maximally stimulating doses of the purinergic agonist ATP, but had no effects following forskolin. IL-1beta (produced by both epithelia and neutrophils during infections) stimulated I(Cl) similar to flagellin. Flagellin-, IL-1beta-, ATP-, and forskolin-stimulated I(Cl) were inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) blockers GlyH101, CFTRinh172, and glibenclamide. Neither flagellin nor IL-1beta altered transepithelial fluxes of membrane-impermeant dextran (10 kDa) or lucifer yellow (mol wt = 457), but both activated p38, NF-kappaB, and IL-8 secretion. Blockers of p38 (SB-202190 and SB-203580) reduced flagellin- and IL-1beta-stimulated I(Cl) by 33-50% but had smaller effects on IL-8 and NF-kappaB. It is concluded that: 1) flagellin and IL-1beta activated p38, NF-kappaB, IL-8, and CFTR-dependent anion secretion without altering tight junction permeability; 2) p38 played a role in regulating I(Cl) and IL-8 but not NF-kappaB; and 3) p38 was more important in flagellin- than IL-1beta-stimulated responses. During P. aeruginosa infections, flagellin and IL-1beta are expected to increase CFTR-dependent ion and fluid flow into and bacterial clearance from the airways. In cystic fibrosis, the secretory response would be absent, but activation of p38, NF-kappaB, and IL-8 would persist.

Control of chemokine gradients by the retinal pigment epithelium.

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.

Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.

PURPOSE: Provide a reproducible method for culturing confluent monolayers of hfRPE cells that exhibit morphology, physiology, polarity, and protein expression patterns similar to native tissue. METHODS: Human fetal eyes were dissected on arrival, and RPE cell sheets were mechanically separated from the choroid and cultured in a specifically designed medium comprised entirely of commercially available components. Physiology experiments were performed with previously described techniques. Standard techniques were used for immunohistochemistry, electron microscopy, and cytokine measurement by ELISA. RESULTS: Confluent monolayers of RPE cell cultures exhibited epithelial morphology and heavy pigmentation, and electron microscopy showed extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. The mean transepithelial potential (TEP) was 2.6 +/- 0.8 mV, apical positive, and the mean transepithelial resistance (R(T)) was 501 +/- 138 Omega . cm(2) (mean +/- SD; n = 35). Addition of 100 microM adenosine triphosphate (ATP) to the apical bath increased net fluid absorption from 13.6 +/- 2.6 to 18.8 +/- 4.6 microL . cm(-2) per hour (mean +/- SD; n = 4). In other experiments, VEGF was mainly secreted into the basal bath (n = 10), whereas PEDF was mainly secreted into the apical bath (n = 10). CONCLUSIONS: A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied native human fetal and bovine RPE-choroid explants.

Retinal pigment epithelial function: a role for CFTR?

In the vertebrate eye, the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE) are separated by a small extracellular (subretinal) space whose volume and chemical composition varies in the light and dark. Light onset triggers relatively fast (ms) retinal responses and much slower voltage and resistance changes (s to min) at the apical and basolateral membranes of the RPE. Two of these slow RPE responses, the fast oscillation (FO) and the light peak, are measured clinically as part of the electrooculogram (EOG). Both EOG responses are mediated in part by apical and basolateral membranes proteins that form a pathway for the movement of salt and osmotically obliged fluid across the RPE, from retina to choroid. This transport pathway serves to control the volume and chemical composition of the subretinal and choroidal extracellular spaces. In human fetal RPE, we have identified one of these proteins, the cystic fibrosis transmembrane conductance regulator (CFTR) by RT-PCR, immunolocalization, and electrophysiological techniques. Evidence is presented to suggest that the FO component of the EOG is mediated directly or indirectly by CFTR.

P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia.

It has been reported that secretory mammary epithelial cells (MEC) release ATP, UTP, and UDP upon mechanical stimulation. Here we examined the physiological changes caused by ATP/UTP in nontransformed, clonal mouse mammary epithelia (31EG4 cells). In control conditions, transepithelial potential (apical side negative) and resistance were -4.4 +/- 1.3 mV (mean +/- SD, n = 12) and 517.7 +/- 39.4 Omega. cm(2), respectively. The apical membrane potential was -43.9 +/- 1.7 mV, and the ratio of apical to basolateral membrane resistance (R(A)/R(B)) was 3.5 +/- 0.2. Addition of ATP or UTP to the apical or basolateral membranes caused large voltage and resistance changes with an EC(50) of approximately 24 microM (apical) and approximately 30 microM (basal). Apical ATP/UTP (100 microM) depolarized apical membrane potential by 17.6 +/- 0.8 mV (n = 7) and decreased R(A)/R(B) by a factor of approximately 3. The addition of adenosine to either side (100 microM) had no effect on any of these parameters. The ATP/UTP responses were partially inhibited by DIDS and suramin and mediated by a transient increase in free intracellular Ca(2+) concentration (427 +/- 206 nM; 15-25 microM ATP, apical; n = 6). This Ca(2+) increase was blocked by cyclopiazonic acid, by BAPTA, or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbed fluid in the resting state, and ATP or UTP increased fluid secretion by 5.6 +/- 3 microl x cm(-2) x h(-1) (n = 10). Pharmacology experiments indicate that 31EG4 epithelia contain P2Y(2) purinoceptors on the apical and basolateral membranes, which upon activation stimulate apical Ca(2+)-dependent Cl channels and cause fluid secretion across the monolayer. This suggests that extracellular nucleotides could play a fundamental role in mammary gland paracrine signaling and the regulation of milk composition in vivo.

The P2Y(2) receptor agonist INS37217 stimulates RPE fluid transport in vitro and retinal reattachment in rat.

PURPOSE: To investigate the effects of INS37217, a synthetic P2Y(2) receptor agonist, on intracellular calcium signaling, electrophysiology, and fluid transport in vitro and on experimentally induced retinal detachment in rat eyes in vivo. METHODS: Freshly isolated monolayers of bovine and human fetal RPE were mounted in Ussing chambers for measurements of cytosolic calcium levels ([Ca(2+)](i)), membrane voltages and resistances, and transepithelial fluid transport. Retinal detachments were experimentally produced in Long-Evans rats by injecting modified phosphate-buffered saline into the subretinal space (SRS). Experimental or vehicle solutions were injected into the vitreous, and the size of blebs in the SRS was scored under masked conditions. RESULTS: Addition of INS37217 to Ringer's solution bathing the apical membrane transiently increased [Ca(2+)](i), altered membrane voltages and resistances and generally produced responses that were similar in magnitude to those of uridine triphosphate (UTP). In fluid transport experiments performed with the capacitance probe technique, INS37217 significantly increased fluid absorption across freshly isolated bovine and fetal human RPE monolayers. All in vitro results were blocked by apical 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), which has been shown to block P2Y(2) receptors in the RPE. Intravitreal administration of INS37217, but not UTP, in the rat model of retinal detachment enhanced the removal of SRS fluid and facilitated retinal reattachment when compared with vehicle control. CONCLUSIONS: These findings indicate that INS37217 stimulates the RPE fluid "pump" function in vitro by activating P2Y(2) receptors at the apical membrane. In vivo INS37217 enhances the rates of subretinal fluid reabsorption in experimentally induced retinal detachments in rats and may be therapeutically useful for treating a variety of retinal diseases that result in fluid accumulation in the subretinal space.