Human Retinal Pigmented Epithelial Cells Produce Nitric Oxide in Response to Cytokines

doi:10.1006/bbrc.1994.1017

Biochemical and Biophysical Research Communications

Volume 198, Issue 1, 15 January 1994, Pages 120-126

https://doi.org/10.1006/bbrc.1994.1017 Get rights and content

Abstract

The present study demonstrates that human retinal pigmented epithelial cells produce nitric oxide (NO) upon co-treatment with interferon γ (IFNγ) and interleukin-Iβ (IL-Iβ). The biosynthesis of NO, which was measured by the accumulation of the stable end-product nitrite, requires an induction period of approximately 12 hours and continues for at least three days. The synthesis was abolished by the stereoselective inhibitors of NO synthase (NOS), N^G-monomethyl-L-arginine, Nω-nitro-L-arginine and Nω-nitro-L-arginine methyl ester and by cycloheximide. Transforming growth factor β suppressed cytokine-induced NOS. The results indicate that cytokines such as IFNγ and IL-1β are capable of inducing NOS, while TGFβ prevents this induction, in subcultured pigmented epithelial cells from the human retinal.

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L’objectif de ce travail est de situer le rôle des cytokines Th1 (IFN- γ et IL-12), Th2 (IL-10) et du monoxyde d’azote (NO) dans les mécanismes immuno-pathologiques des uvéites « Behçet » et des uvéites isolées « idiopathiques ». Les résultats de cette étude préliminaire auraient des retombées sur le suivi et le pronostic de l’évolution des deux pathologies.
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The aim of this study was to investigate the role of Th1 (IFN-γ and IL-12), Th2 (IL-10) cytokines and nitric oxide (NO) in the immunopathologic mechanisms of uveitis related to Behçet's disease and isolated idiopathic uveitis.
This study was conducted on 24 patients with Behçet's syndrome who had active uveitis, ten of whom showed isolated uveitis classified as idiopathic uveitis, and 13 healthy controls. The levels of IFN-γ, IL-12 and IL-10 in sera and supernatants of PBMC cultures stimulated by PHA were estimated using immunoenzymatic dosage (ELISA sandwich according to the methods recommended by Immunotech France). The production of NO was measured in vivo and in vitro for the same patients using the modified Griess method.
The induction of IFN-γ and IL-12 was higher in the two groups of patients than in the controls (P<0.001). Significant IL-10 levels were recorded in 56.5% of patients with Behçet's disease presenting uveitis versus 30% of patients with idiopathic uveitis. NO production was more pronounced in idiopathic uveitis than in Behçet's syndrome (P<0.02).
The predominance of the Th1 profile was associated with high production of NO in idiopathic uveitis. A Th1/Th2 profile with a moderated increase in NO production was observed in Behçet's disease. Our data have a clinical impact. The observation of combined Th1/Th2 cytokines and NO elevation in both in vivo an in vitro experiments could have a predictive value in characterizing uveitis associated with Behçet's disease.
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Citation Excerpt :
NOS has been identified by means of either NOS immunohistochemistry or nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry in the central nervous system, including the retina. In spite of a number of examinations, NOS had not been identified in retinal ganglion cells of adult animals (NADPH-diaphorase histochemistry [39,21,37,38]; nNOS immunohistochemistry [13,55,56,34,12,28,24]; iNOS [28,17,18,22]; eNOS [29]) or animals during postnatal development [34,42,33]. Recent studies have shown that nNOS is localized to retinal ganglion cells of adult animals [43,32,4].
The expression of isoforms of nitric oxide synthase (NOS), enzymes responsible for NO production, and the synthesis of nitric oxide (NO) in rat retinal ganglion cells (RGCs) during synaptogenesis for various phases of the pre- and postnatal developmental periods were investigated. The retinas from prenatal, lactating, young, and adult rats were fixed in paraformaldehyde. The cryosections or paraformaldehyde-fixed ganglion cells purified from rat pups were immunostained for constitutive isoforms of NOS (n and eNOS) and observed with a confocal laser scanning microscope. Synthesis of NO in the RGCs was achieved by in vitro stimulation with glutamate. The intracellular NO levels were measured in real time using diaminofluorescein-2 diacetate, a fluorescence indicator of NO. Immunohistochemical analysis revealed nNOS and eNOS expressed in retinal ganglion cells during the first 2 postnatal weeks. Cultured RGCs also expressed nNOS and eNOS in vitro. Intracellular NO levels in cultured RGCs showed spontaneous fluctuation during a 20-min observation. The presence of both a non-specific NOS inhibitor, l-NAME, and a specific nNOS inhibitor, 7-NI, significantly inhibited (P<0.001) the increase of intracellular NO 6 and 8 min after the introduction of l-arginine and glutamate to the medium. This study revealed that all constitutive NOS isoforms are expressed in RGCs and demonstrated that NO is produced by nNOS mainly through stimulation by glutamate in cultured RGCs.
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2001, Progress in Retinal and Eye Research
Vision is dependent on proper function of several intraocular structures. Immune responses to eliminate invading pathogens from the eye may threat vision by causing damage to these structures. Therefore, immunological defence of the eye should be carefully balanced between efficacy and maintenance of functional integrity. The eye is equipped with several regulatory mechanisms to prevent certain immune and inflammatory responses and is, therefore, regarded as an immune privileged site. The retinal pigment epithelium (RPE) contributes to the immune privileged status of the eye as part of the blood–eye barrier and by the secretion of immunosuppressive factors inside the eye. RPE cells, however, may also play an important role in the development of immune and inflammatory responses in the posterior part of the eye. During the last decade it has become clear that RPE cells are highly sensitive to a variety of inflammatory cytokines. Under inflammatory conditions, RPE cells produce a myriad of cytokines that may activate the resident ocular cells or attract and activate leukocytes. Cytokine stimulation of RPE cells causes profound effects, including nitric oxide secretion, cell surface expression of MHC class II and adhesion molecules and abrogation of barrier function. This article provides a comprehensive review of the literature concerning RPE cells and cytokines.
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Regular ArticleHuman Retinal Pigmented Epithelial Cells Produce Nitric Oxide in Response to Cytokines

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Regular Article
Human Retinal Pigmented Epithelial Cells Produce Nitric Oxide in Response to Cytokines