房水中降钙素基因相关肽对炎性巨噬细胞产生的一氧化氮的抑制作用。

Suppression of nitric oxide generated by inflammatory macrophages by calcitonin gene-related peptide in aqueous humor.

作者信息

Taylor A W, Yee D G, Streilein J W

机构信息

Schepens Eye Research Institute, Boston, Massachusetts 02114, USA.

出版信息

Invest Ophthalmol Vis Sci. 1998 Jul;39(8):1372-8.

PMID:9660485

Abstract

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.

摘要

目的

眼免疫赦免部分是由组成性产生的免疫抑制细胞因子和神经肽的活性介导的。检测房水中降钙素基因相关肽（CGRP）的含量，并确定CGRP在房水中介导免疫抑制活性的潜力。

方法

采用竞争性酶联免疫吸附测定法检测新鲜正常兔房水中CGRP的浓度。通过检测经CGRP处理的致敏T细胞培养物上清液中的γ干扰素（IFN-γ），来检测CGRP抑制抗原刺激的致敏淋巴结细胞产生IFN-γ的能力。通过用房水、房水加抗CGRP抗体或单独用CGRP处理IFN-γ-脂多糖（LPS）激活的RAW 264.7细胞（巨噬细胞），来检测房水和CGRP的抗炎活性。用格里斯试剂检测处理后巨噬细胞培养上清液中的亚硝酸盐。通过免疫印迹处理后的活化巨噬细胞的细胞裂解物，检测诱导型一氧化氮合酶（NOS2）蛋白的产生。