Institute of Dentistry, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, UK.
J Periodontal Res. 2016 Feb;51(1):16-25. doi: 10.1111/jre.12273. Epub 2015 Apr 13.
The multifunctional molecules adrenomedullin (AM) and nitric oxide (NO) are both involved in the host response to microbial challenge during periodontal disease. Whether they coexist in periodontal inflammation and if equally produced in the different forms of periodontal disease has not previously been investigated. The aims of this study were to describe the locations of AM and NO in healthy and inflamed gingival tissues and to determine and compare their levels in the gingival crevicular fluid and saliva of patients with gingivitis, chronic periodontitis and aggressive periodontitis.
AM and inducible nitric oxide synthase (iNOS) were immunolocalized in clinically healthy and inflamed gingival tissue sections. The cells expressing AM and iNOS were characterized using immunocytochemistry with different markers for macrophages [cluster differentiation (CD)68 and CD14)], dendritic cells (CD83), neutrophils [neutrophil gelatinase-associated lipocalin (nGAL)] and natural killer cells (CD56). In an initial study, the levels of AM and NO were also measured in samples of gingival crevicular fluid and saliva obtained from patients with a diagnosis of gingivitis (n = 9), chronic periodontitis (n = 9) and aggressive periodontitis (n = 9) using an ELISA and the nitrate/nitrite (NO metabolites) Griess assay, respectively.
Low levels of AM- and iNOS-expressing cells were detected in healthy gingival tissues in comparison with three-fold higher levels of these cells in inflamed tissues. These cells were localized mainly in the epithelial layer but were also present in deeper connective tissue. AM and iNOS were co-localized in particular cells within inflamed tissues, namely CD68(+) (52%) and CD14(+) (36%) macrophages, but also in nGAL(+) neutrophils (16%) and CD83(+) dendritic cells (14%). Interestingly, AM and NO levels in saliva were both found to be higher (p < 0.01) in patients with aggressive periodontitis than in patients with chronic periodontitis or gingivitis. In contrast, in gingival crevicular fluid, the levels of NO showed marked differences among patients with chronic periodontitis, aggressive periodontitis and gingivitis (p < 0.01), and the levels of AM were higher (p < 0.01) in both chronic and aggressive periodontitis compared with gingivitis alone.
The data presented demonstrate a functional linkage between AM and NO in periodontal disease, with salivary and gingival crevicular fluid levels possibly associated with different forms and severities of periodontal disease. Exacerbated production of both AM and NO in saliva suggests their potential use as salivary markers of aggressive periodontitis.
在牙周病中，多功能分子肾上腺髓质素（AM）和一氧化氮（NO）都参与宿主对微生物挑战的反应。它们是否同时存在于牙周炎炎症中，以及它们是否以同样的形式产生于不同形式的牙周病，这些问题以前尚未被研究过。本研究的目的是描述 AM 和 NO 在健康和炎症性牙龈组织中的位置，并确定和比较它们在牙龈炎、慢性牙周炎和侵袭性牙周炎患者的龈沟液和唾液中的水平。
在临床健康和炎症性牙龈组织切片中进行 AM 和诱导型一氧化氮合酶（iNOS）的免疫定位。使用针对巨噬细胞[簇分化（CD）68 和 CD14]、树突状细胞（CD83）、中性粒细胞[中性粒细胞明胶酶相关脂质运载蛋白（nGAL）]和自然杀伤细胞（CD56）的免疫细胞化学，对表达 AM 和 iNOS 的细胞进行特征描述。在初步研究中，还使用 ELISA 和硝酸盐/亚硝酸盐（NO 代谢物）Griess 测定法分别测量来自被诊断为牙龈炎（n = 9）、慢性牙周炎（n = 9）和侵袭性牙周炎（n = 9）患者的龈沟液和唾液中 AM 和 NO 的水平。
与炎症组织中高出三倍的水平相比，健康牙龈组织中 AM-和 iNOS 表达细胞的水平较低。这些细胞主要定位于上皮层，但也存在于更深的结缔组织中。AM 和 iNOS 共同定位于炎症组织中的特定细胞，即 CD68（+）（52%）和 CD14（+）（36%）巨噬细胞，但也存在 nGAL（+）中性粒细胞（16%）和 CD83（+）树突状细胞（14%）。有趣的是，侵袭性牙周炎患者的唾液中 AM 和 NO 水平均高于慢性牙周炎或牙龈炎患者（p < 0.01）。相比之下，在龈沟液中，慢性牙周炎、侵袭性牙周炎和牙龈炎患者之间的 NO 水平存在显著差异（p < 0.01），且慢性和侵袭性牙周炎患者的 AM 水平均高于单纯牙龈炎（p < 0.01）。
本研究结果表明，AM 和 NO 在牙周病中存在功能联系，唾液和龈沟液中的水平可能与牙周病的不同形式和严重程度有关。唾液中 AM 和 NO 的过度产生表明它们可能作为侵袭性牙周炎的唾液标志物。
