O'Brien W J, Heimann T, Tsao L S, Seet B T, McFadden G, Taylor J L
Medical College of Wisconsin, Department of Ophthalmology, The Eye Institute, 925 North 87th Street, Room 826, Milwaukee, WI 53226-4812, USA.
Invest Ophthalmol Vis Sci. 2001 Mar;42(3):713-9.
The purpose of these studies was to investigate the role of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and transforming growth factor-beta (TGF-beta) in the regulation of inducible nitric oxide synthase (NOS2) activity in rabbit corneal cells.
Rabbit corneal epithelial, stromal, and endothelial cells were grown in culture and treated with cytokines and growth factors, alone or in combination. NOS activity was measured at times up to 72 hours after treatment by assaying the culture medium for nitrite using the Griess reaction. Cell lysates were analyzed by Western blot analysis for NOS2 protein. RNA was isolated and amplified with NOS1-, NOS2-, and NOS3-specific primers by RT-PCR.
NOS2 expression was induced by combined cytokine treatment from nondetectable levels to abundant levels in low passage (<4) stromal cells and to low levels in corneal endothelial cells but not in corneal epithelial cells. In the absence of IFN-gamma, little or no nitrite accumulation was induced by TNF-alpha, IL-1beta, or lipopolysaccharide (LPS) treatment. The inductive effects of IFN-gamma were antagonized in a dose-dependent manner by the myxoma virus rabbit IFN-gamma receptor homolog, M-T7. rRaIFN-gamma, in combination with IL-1beta and TNF-alpha, induced the appearance of NOS2 mRNA within 24 hours but detectable nitrite did not accumulate in large amounts (>10 microM) until after 24 hours postinduction. NOS2 was identified as a 130 kDa protein on Western blot analysis using monoclonal antibody against murine NOS2. TGF-beta(1) and beta(2) inhibited the accumulation of cytokine-induced nitrite in a dose-dependent manner while not significantly reducing the steady state level of NOS2 mRNA. The activity of the induced NOS was inhibited by 1400W, a NOS2-selective inhibitor, but not 7-nitroindazole, a NOS1-selective inhibitor.
In cultured corneal stromal cells, NOS2 expression was upregulated by IFN-gamma in combination with IL-1beta and TNF-alpha but not by any of these cytokines alone, while TGF-beta downregulated the activity. Cultures of corneal epithelial cells could not be induced to express NOS2, yet cultures of endothelial cells produced low amounts of NO in response to cytokines. The NOS1 and NOS3 isoforms were not detected in any of these corneal cells.
这些研究的目的是调查干扰素-γ（IFN-γ）、肿瘤坏死因子-α（TNF-α）、白细胞介素1β（IL-1β）和转化生长因子-β（TGF-β）在兔角膜细胞中诱导型一氧化氮合酶（NOS2）活性调节中的作用。
兔角膜上皮细胞、基质细胞和内皮细胞在培养物中生长，并单独或联合用细胞因子和生长因子处理。在处理后长达72小时的时间点，通过使用格里斯反应检测培养基中的亚硝酸盐来测量NOS活性。通过蛋白质免疫印迹分析细胞裂解物中的NOS2蛋白。通过逆转录聚合酶链反应（RT-PCR）用NOS1、NOS2和NOS3特异性引物分离并扩增RNA。
在低传代（<4）基质细胞中，联合细胞因子处理可将NOS2表达从不可检测水平诱导至丰富水平，在角膜内皮细胞中诱导至低水平，但在角膜上皮细胞中未诱导。在没有IFN-γ的情况下，TNF-α、IL-1β或脂多糖（LPS）处理几乎不诱导或不诱导亚硝酸盐积累。黏液瘤病毒兔IFN-γ受体同源物M-T7以剂量依赖性方式拮抗IFN-γ的诱导作用。重组兔IFN-γ与IL-1β和TNF-α联合，在24小时内诱导NOS2 mRNA出现，但直到诱导后24小时才大量积累可检测到的亚硝酸盐（>10 microM）。使用抗鼠NOS2单克隆抗体通过蛋白质免疫印迹分析将NOS2鉴定为130 kDa蛋白。TGF-β(1)和β(2)以剂量依赖性方式抑制细胞因子诱导的亚硝酸盐积累，同时不显著降低NOS2 mRNA 的稳态水平。诱导的NOS活性被NOS2选择性抑制剂1400W抑制，但不被NOS1选择性抑制剂7-硝基吲唑抑制。
在培养的角膜基质细胞中，NOS2表达通过IFN-γ与IL-1β和TNF-α联合上调，而不是单独由这些细胞因子中的任何一种上调，而TGF-β下调活性。角膜上皮细胞培养物不能被诱导表达NOS2，然而内皮细胞培养物对细胞因子产生少量NO。在任何这些角膜细胞中均未检测到NOS1和NOS3同工型。
