Effect of cytokines and nitric oxide on tight junctions in cultured rat retinal pigment epithelium.

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.

Tumor necrosis factor and nitric oxide production by resident retinal glial cells from rats presenting hereditary retinal degeneration.

The inherited retinal dystrophy observed in Royal College of Surgeons (RCS) rats is a widely used model for the study of the photoreceptor degeneration that occurs in retinitis pigmentosa and macular degeneration. The visual cell degeneration is accompanied by an abnormal accumulation of microglial cells in the retina of RCS rats presenting the dystrophy. In the present study, we show that combined stimulation of RCS dystrophic retinal Müller glial (RMG) cells with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) induced the release in culture supernatants of significantly higher amounts of tumor necrosis factor (TNF) and nitric oxide (NO) compared to nondystrophic congenic controls. In contrast, the levels of TNF and NO found in the supernatants from microglial cells were not significantly different in both strains. Interestingly, as shown by thymidine incorporation, microglial cells from RCS dystrophic rats have a prominent capacity of proliferation in culture medium compared to microglia isolated from RCS non dystrophic controls. Incubation of RMG cells and microglia with the stereoselective inhibitor of NOS, NG-monomethyl-L-arginine (L-NMMA), inhibited nitrite release in LPS + IFN-gamma-stimulated RMG cells and microglia. The addition of TGF-beta with LPS + IFN-gamma clearly inhibited TNF release in supernatants from both dystrophic and control rat RMG cells and microglia. While TGF-beta significantly inhibited nitrite synthesis in RMG cells, the effect on nitrite synthesis by microglia was very low. The retinal dystrophy observed in RCS dystrophic rats could result from an abnormal reactivity of RMG and microglial cells to release TNF and NO in response to stimulants. The immunomodulatory cytokine TGF-beta and inhibitors of NOS could be negative regulators in the cytokine network and nitrite synthesis thus interfering with the development of photoreceptor cell death.

Differential tumor necrosis factor and nitric oxide production in retinal Müller glial cells from C3H/HeN and C3H/HeJ mice.

Tumor necrosis factor (TNF) and nitric oxide (NO) have been shown to play a role in the pathogenesis of endotoxin-induced uveitis (EIU) in rats. Susceptibility to develop EIU in vivo is correlated with the extent of TNF production by retinal Müller glial cells (RMG). Moreover, RMG cells from the susceptible Lewis rat strain synthesize high amounts of nitrite under in vitro stimulation. Variations in susceptibility to endotoxin are observed among mice strains: C3H/HeN mice are known to be susceptible to develop EIU while C3H/HeJ are refractory. We show here that treatment of RMG cells from both strains with LPS + IFN-gamma does not induce TNF-synthesis in culture supernatants but produces high amounts of NO only in the supernatants from activated C3H/HeN RMG cells. The addition of TNF in the culture medium containing LPS/IFN-gamma further increases nitrite production in C3H/HeN RMG cells and allows the synthesis of low levels of nitrite in C3H/HeJ RMG cells. Addition of a specific NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), blocks NO release. We have previously shown that intraperitoneal injections of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) which inhibited nitrite and TNF release in the ocular media reduced EIU in rat. We conclude here that the in vivo susceptibility to develop EIU in mice is correlated with the extent of in vitro nitrite production by RMG cells confirming the implication of NO in the induction of ocular inflammation. The low level of retinal inflammation observed during EIU in C3H/HeN mice compared to rats could be related to the absence of TNF production by RMG cells.

Tumor necrosis factor and nitric oxide production by retinal Müller glial cells from rats exhibiting inherited retinal dystrophy.

The primary cause of the inherited retinal dystrophy observed in Royal College of Surgeons (RCS) rats is located in the retinal pigmented epithelium, which is unable to phagocytize photoreceptor outer segments. We have demonstrated here that retinal Müller glial (RMG) cells obtained from RCS dystrophic rats and stimulated in vitro with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) accumulated higher levels of tumor necrosis factor (TNF) and inducible nitric oxide synthase (NOS II) mRNA and released in culture supernatants significantly higher amounts of TNF and nitrite compared to cells derived from nondystrophic controls. The TNF and NOS II mRNA expression and TNF and nitrite synthesis induced in RMG cells from both strains by LPS + IFN-gamma was significantly prevented by including transforming growth factor-beta (TGF-beta) in the culture medium. Coincubation of the stimulants with an inhibitor of NOS II, NG-monomethyl-L-arginine (L-NMMA), while inhibiting nitrite synthesis, induced an increase of TNF production in supernatants from RMG cells without increasing TNF mRNA levels. The retinal dystrophy observed in RCS dystrophic rats could result from an abnormal susceptibility of RMG cells form RCS dystrophic rats to produce TNF and NO in response to stimulants. Administration of the immunomodulatory cytokine TGF-beta or inhibitors of NOS II would provide additional research avenues for photoreceptor rescue.