PURPOSE: These experiments were designed to study the effect of cytokines and nitric oxide (NO) on rat retinal pigment epithelial (RPE) cell tight junctions in vitro. METHODS: Cultures of confluent RPE cells from retinas of PVG rats (a strain susceptible to development of experimental uveitis) were prepared on filters and incubated with various stimulants. The function of the tight junction was evaluated by measuring the transepithelial electrical resistance (TER) of the cell monolayer and the passive permeation of [3H]inulin across confluent RPE cells. The morphology of the intercellular junctions was visualized by immunolocalization of the tight junction-associated protein zonula occludens-1 (ZO-1) and F-actin. RESULTS: Seventy-two hours after plating, the RPE cell monolayer showed a mean TER level of 67.6+/-18.8 omega/cm2. A decrease in TER was observed after treatment with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). The addition of tumor necrosis factor-alpha (TNF-alpha) accelerated the decrease of TER, whereas NG-monomethyl-L-arginine (L-NMMA) (an NO synthase [NOS] inhibitor) did not further modify the resistance decrease. In contrast, 3-morpholino-sydnonimine (SIN-1), a sydnonimine analog and NO donor, increased the TER. The variations of TER were correlated with the transepithelial fluxes of [3H]inulin and with tight junction morphologic changes of ZO-1 and F-actin immunostaining. CONCLUSIONS: Incubation with LPS associated with IFN-gamma and TNF-alpha induces alterations of RPE tight junctions, whereas NO is involved in the maintenance of their integrity. Cytokines and NO production could play a role in regulation of the blood-ocular barrier function and of the development of ocular inflammation.
PURPOSE: These experiments were designed to study the effect of cytokines and nitric oxide (NO) on rat retinal pigment epithelial (RPE) cell tight junctions in vitro. METHODS: Cultures of confluent RPE cells from retinas of PVG rats (a strain susceptible to development of experimental uveitis) were prepared on filters and incubated with various stimulants. The function of the tight junction was evaluated by measuring the transepithelial electrical resistance (TER) of the cell monolayer and the passive permeation of [3H]inulin across confluent RPE cells. The morphology of the intercellular junctions was visualized by immunolocalization of the tight junction-associated protein zonula occludens-1 (ZO-1) and F-actin. RESULTS: Seventy-two hours after plating, the RPE cell monolayer showed a mean TER level of 67.6+/-18.8 omega/cm2. A decrease in TER was observed after treatment with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). The addition of tumor necrosis factor-alpha (TNF-alpha) accelerated the decrease of TER, whereas NG-monomethyl-L-arginine (L-NMMA) (an NO synthase [NOS] inhibitor) did not further modify the resistance decrease. In contrast, 3-morpholino-sydnonimine (SIN-1), a sydnonimine analog and NO donor, increased the TER. The variations of TER were correlated with the transepithelial fluxes of [3H]inulin and with tight junction morphologic changes of ZO-1 and F-actin immunostaining. CONCLUSIONS: Incubation with LPS associated with IFN-gamma and TNF-alpha induces alterations of RPE tight junctions, whereas NO is involved in the maintenance of their integrity. Cytokines and NO production could play a role in regulation of the blood-ocular barrier function and of the development of ocular inflammation.