Investigative ophthalmology & visual science · 1998
Suppression of nitric oxide generated by inflammatory macrophages by calcitonin gene-related peptide in aqueous humor.
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Taylor A W, Yee D G, Streilein J W
Аннотация
PURPOSE: Ocular immune privilege is mediated in part by the activity of constitutively produced immunosuppressive cytokines and neuropeptides. Aqueous humor was examined for content of calcitonin gene-related peptide (CGRP), and the potential of CGRP to mediate immunosuppressive activity within aqueous humor was determined.
METHODS: The concentration of CGRP in fresh, normal rabbit aqueous humor was assayed by competitive enzyme-linked immunosorbent assay. The ability of CGRP to suppress interferon (IFN)-gamma production by antigen-stimulated, primed lymph node cells was examined by assaying supernatants of stimulated CGRP-treated, primed T-cell cultures for IFN-gamma. The anti-inflammatory activity of aqueous humor and CGRP was assayed by treating IFN-gamma-lipopolysaccharide (LPS)-activated RAW 264.7 cells (macrophages) with aqueous humor, aqueous humor plus anti-CGRP antibody, or CGRP alone. Culture supernatants of the treated macrophages were examined for nitrite by Griess reagent. The production of inducible nitric oxide synthase (NOS2) protein was examined by immunoblotting cell lysates of treated activated macrophages.
RESULTS: The constitutive level of CGRP in fresh, normal rabbit aqueous humor was 5+/-1 x 10(-5) M. At its ocular concentration, CGRP did not inhibit IFN-gamma production by stimulated effector T cells, but it suppressed nitric oxide generation by activated macrophages. Neutralization of CGRP in normal rabbit aqueous humor prevented the aqueous humor from suppressing nitric oxide generation by macrophages. Neither CGRP nor aqueous humor suppressed NOS2 protein synthesis in activated inflammatory macrophages.
CONCLUSIONS: Calcitonin gene-related peptide is a constitutive neuropeptide in aqueous humor. Through CGRP, aqueous humor suppresses nitric oxide production by activated macrophages. This suppression appears to result from inhibiting NOS2 enzymatic activity, rather than from suppressing NOS2 synthesis. The results imply that the ocular microenvironment has diverse immunoregulatory mechanisms that suppress induction, activation, and mediation of immunogenic inflammation.
METHODS: The concentration of CGRP in fresh, normal rabbit aqueous humor was assayed by competitive enzyme-linked immunosorbent assay. The ability of CGRP to suppress interferon (IFN)-gamma production by antigen-stimulated, primed lymph node cells was examined by assaying supernatants of stimulated CGRP-treated, primed T-cell cultures for IFN-gamma. The anti-inflammatory activity of aqueous humor and CGRP was assayed by treating IFN-gamma-lipopolysaccharide (LPS)-activated RAW 264.7 cells (macrophages) with aqueous humor, aqueous humor plus anti-CGRP antibody, or CGRP alone. Culture supernatants of the treated macrophages were examined for nitrite by Griess reagent. The production of inducible nitric oxide synthase (NOS2) protein was examined by immunoblotting cell lysates of treated activated macrophages.
RESULTS: The constitutive level of CGRP in fresh, normal rabbit aqueous humor was 5+/-1 x 10(-5) M. At its ocular concentration, CGRP did not inhibit IFN-gamma production by stimulated effector T cells, but it suppressed nitric oxide generation by activated macrophages. Neutralization of CGRP in normal rabbit aqueous humor prevented the aqueous humor from suppressing nitric oxide generation by macrophages. Neither CGRP nor aqueous humor suppressed NOS2 protein synthesis in activated inflammatory macrophages.
CONCLUSIONS: Calcitonin gene-related peptide is a constitutive neuropeptide in aqueous humor. Through CGRP, aqueous humor suppresses nitric oxide production by activated macrophages. This suppression appears to result from inhibiting NOS2 enzymatic activity, rather than from suppressing NOS2 synthesis. The results imply that the ocular microenvironment has diverse immunoregulatory mechanisms that suppress induction, activation, and mediation of immunogenic inflammation.
Предметные рубрики MeSH
AnimalsAqueous HumorCalcitonin Gene-Related PeptideElectrophoresis, Polyacrylamide GelEnzyme-Linked Immunosorbent AssayImmune ToleranceImmunoblottingInterferon-gammaLipopolysaccharidesLymphocyte ActivationMacrophage ActivationMacrophagesMiceMycobacterium tuberculosisNitric OxideNitric Oxide SynthaseNitric Oxide Synthase Type IIRabbitsT-Lymphocytes