Muthukuru Manoj, Jotwani Ravi, Cutler Christopher W
Department of Periodontics, School of Dental Medicine, 110 Rockland Hall, Stony Brook University-SUNY, Stony Brook, NY 11794-8703, USA.
Infect Immun. 2005 Feb;73(2):687-94. doi: 10.1128/IAI.73.2.687-694.2005.
The oral mucosa is exposed to a high density and diversity of gram-positive and gram-negative bacteria, but very little is known about how immune homeostasis is maintained in this environment, particularly in the inflammatory disease chronic periodontitis (CP). The cells of the innate immune response recognize bacterial structures via the Toll-like receptors (TLR). This activates intracellular signaling and transcription of proteins essential for the induction of an adaptive immune response; however, if unregulated, it can lead to destructive inflammatory responses. Using single-immunoenzyme labeling, we show that the human oral mucosa (gingiva) is infiltrated by large numbers of TLR2(+) and TLR4(+) cells and that their numbers increase significantly in CP, relative to health (P < 0.05, Student's t test). We also show that the numbers of TLR2(+) but not TLR4(+) cells increase linearly with inflammation (r(2) = 0.33, P < 0.05). Double-immunofluorescence analysis confirms that TLR2 is coexpressed by monocytes (MC)/macrophages (mphi) in situ. Further analysis of gingival tissues by quantitative real-time PCR, however, indicates that despite a threefold increase in the expression of interleukin-1beta (IL-1beta) mRNA during CP, there is significant (30-fold) downregulation of TLR2 mRNA (P < 0.05, Student's t test). Also showing similar trends are the levels of TLR4 (ninefold reduction), TLR5 (twofold reduction), and MD-2 (sevenfold reduction) mRNA in CP patients compared to healthy persons, while the level of CD14 was unchanged. In vitro studies with human MC indicate that MC respond to an initial stimulus of lipopolysaccharide (LPS) from Porphyromonas gingivalis (PgLPS) or Escherichia coli (EcLPS) by upregulation of TLR2 and TLR4 mRNA and protein; moreover, IL-1beta mRNA is induced and tumor necrosis factor alpha (TNF-alpha), IL-10, IL-6, and IL-8 proteins are secreted. However, restimulation of MC with either PgLPS or EcLPS downregulates TLR2 and TLR4 mRNA and protein and IL-1beta mRNA and induces a ca. 10-fold reduction in TNF-alpha secretion, suggesting the induction of endotoxin tolerance by either LPS. Less susceptible to tolerance than TNF-alpha were IL-6, IL-10, and IL-8. These studies suggest that certain components of the innate oral mucosal immune response, most notably TLRs and inflammatory cytokines, may become tolerized during sustained exposure to bacterial structures such as LPS and that this may be one mechanism used in the oral mucosa to attempt to regulate local immune responses.
口腔黏膜暴露于高密度且多样的革兰氏阳性菌和革兰氏阴性菌中，但对于在这种环境下如何维持免疫稳态，尤其是在炎症性疾病慢性牙周炎（CP）中，我们知之甚少。固有免疫反应的细胞通过Toll样受体（TLR）识别细菌结构。这会激活细胞内信号传导以及诱导适应性免疫反应所必需的蛋白质的转录；然而，如果不受调控，它会导致破坏性的炎症反应。通过单免疫酶标记，我们发现人类口腔黏膜（牙龈）中有大量TLR2(+)和TLR4(+)细胞浸润，并且相对于健康状态，它们在CP中的数量显著增加（P < 0.05，学生t检验）。我们还发现TLR2(+)细胞的数量而非TLR4(+)细胞的数量随炎症呈线性增加（r(2) = 0.33，P < 0.05）。双重免疫荧光分析证实TLR2在原位由单核细胞（MC）/巨噬细胞（mphi）共表达。然而，通过定量实时PCR对牙龈组织进行的进一步分析表明，尽管在CP期间白细胞介素-1β（IL-1β）mRNA的表达增加了三倍，但TLR2 mRNA却有显著的（30倍）下调（P < 0.05，学生t检验）。与健康人相比，CP患者中TLR4（降低九倍）、TLR5（降低两倍）和MD-2（降低七倍）mRNA的水平也呈现类似趋势，而CD14的水平未变。对人类MC进行的体外研究表明，MC对牙龈卟啉单胞菌（PgLPS）或大肠杆菌（EcLPS）的脂多糖（LPS）初始刺激的反应是上调TLR2和TLR4 mRNA及蛋白质；此外，会诱导IL-1β mRNA并分泌肿瘤坏死因子α（TNF-α）、IL-10、IL-6和IL-8蛋白质。然而，用PgLPS或EcLPS对MC进行再次刺激会下调TLR2和TLR4 mRNA及蛋白质以及IL-1β mRNA，并使TNF-α分泌降低约10倍，这表明LPS可诱导内毒素耐受。IL-6、IL-10和IL-8比TNF-α对耐受的敏感性更低。这些研究表明，固有口腔黏膜免疫反应的某些成分，最显著的是TLR和炎性细胞因子，在持续暴露于诸如LPS等细菌结构期间可能会产生耐受，这可能是口腔黏膜试图调节局部免疫反应所采用的一种机制。
