Rat retinal pigment epithelial cells express an inducible form of nitric oxide synthase and produce nitric oxide in response to inflammatory cytokines and activated T cells.
In this report we show that rat retinal pigment epithelial (RPE) cells express an inducible form of nitric oxide synthase (iNOS) and secrete high levels of nitric oxide (NO.) when co-cultured with activated lymphocytes. We have previously shown that cultured rat RPE cells suppress syngeneic lymphocyte proliferation, an effect attributed to prostaglandin E2 (PGE2) secretion by the RPE cells. However supernatants from such co-cultures were also found to contain high levels of nitrite (NO2-), the stable end-product of NO. synthesis. RPE cell secretion of NO. was stimulated by the cytokines interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), an effect enhanced by endotoxin [lipopolysaccharide (LPS)], reduced by the competitive inhibitor of L-arginine metabolism, NG-monomethyl-L-arginine (L-NMMA) and inhibited by cycloheximide. These effects were dose dependent. Using reverse transcription (RT)/PCR a product of 1398 bp was amplified which showed sequence identity with iNOS cloned from rat vascular smooth muscle. Northern blot analysis of total RNA extracted from rat RPE before and after cytokine stimulation showed induction of a 4.5 kb (kilobase) transcript which hybridized with a 1398 bp (base pair) polymerase chain reaction (PCR)-generated cDNA probe derived from the sequence of rat RPE cell iNOS. These results indicate RPE cells express an inducible form of nitric oxide synthase (NOS) and that high levels of NO. may be produced locally in the eye by the RPE in the presence of activated lymphocytes. Given the cytostatic and cytotoxic properties of this molecule, NO. may play an important role as an inducible mediator of immunosuppressive mechanisms within the microenvironment of the eye at the site of lymphocyte activation.
Liversidge
J
J
Department of Ophthalmology, University of Aberdeen Medical School, UK.
Grabowski
P
P
Ralston
S
S
Benjamin
N
N
Forrester
J V
JV
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D017931:DNA Primers; D007371:Interferon-gamma / Q000494:pharmacology; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013601:T-Lymphocytes / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D017931:DNA Primers; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases