Expression of toll-like receptor 2, NOD2 and dectin-1 and stimulatory effects of their ligands and histamine in normal human keratinocytes
Expression of toll-like receptor 2, NOD2 and dectin-1 and stimulatory effects of their ligands and histamine in normal human keratinocytes
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DOI：
10.1111/j.1365-2133.2008.08897.x
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发表时间：
2009-02-01
影响因子：
10.3
通讯作者：
Tokura, Y.
中科院分区：
文献类型：
--
作者：
Kobayashi, M.;Yoshiki, R.;Tokura, Y.
表皮角质形成细胞参与皮肤先天免疫，并表达Toll样受体（TLR）和其他先天免疫蛋白。表皮持续暴露于致病性革兰氏阳性细菌或真菌。然而，很少有研究探讨天然免疫蛋白在角质形成细胞中的功能和表达。组胺因瘙痒和过敏而闻名，与先天免疫密切相关，但其对表皮先天免疫的影响尚不清楚。为了阐明配体病原体相关分子刺激角质形成细胞中先天免疫蛋白的表达，以及组胺在这一过程中的作用。我们研究了脂肽的作用（MALP-2，1-100 ng mL（-1）; TLR 2配体），肽聚糖（PGN，0.02-2 μ g mL（-1）; NOD 2的配体）和β-葡聚糖（1-100 μ g mL（-1）; dectin-1的配体）在组胺存在或不存在下对TLR 2的mRNA表达的影响，NOD 2和dectin-1以及人β-防御素2在培养的正常人表皮角质形成细胞中的定量实时聚合酶链反应。TLR 2的表达也在细胞表面和细胞内，通过流式细胞术和共聚焦显微镜测定。用酶联免疫吸附法测定角质形成细胞产生的白细胞介素（IL）-1 α和IL-8的量。在mRNA水平上，TLR 2被PGN增强，但不被其配体MALP-2或β-葡聚糖增强; NOD 2容易被所有三种配体诱导; dectin-1被其配体β-葡聚糖增强。这些增强的表达进一步增强组胺在1 μ g mL（-1）。而TLR 2的表面表达几乎检测不到流式细胞术，即使在刺激后，TLR 2的细胞内表达明显升高PGN和组胺进一步促进。共聚焦显微镜分析也揭示了TLR 2在细胞质中的表达增强。TLR 2、NOD 2和dectin-1的表达是功能性的，因为这些病原体相关分子诱导了IL-1 α、IL-8和防御素的产生，并且组胺再次极大地增强了这种产生。我们的研究表明，在角质形成细胞中，病原体相关分子以配体前馈或非相关的方式增强了功能性先天免疫受体的表达，组胺促进它们的表达和细胞因子和防御素的产生。
Epidermal keratinocytes are involved in the skin innate immunity and express toll-like receptors (TLRs) and other innate immune proteins. The epidermis is continuously exposed to pathogenic Gram-positive bacteria or fungi. However, few studies have examined the function and expression of innate immune proteins in keratinocytes. Histamine, which is well known for itch and allergy, is closely associated with innate immunity, but its influence on epidermal innate immunity is still unclear.To clarify the expression of innate immune proteins in keratinocytes stimulated by ligand pathogen-associated molecules, and the function of histamine in this process.We investigated the effects of lipopeptide (MALP-2, 1-100 ng mL(-1); ligand for TLR2), peptidoglycan (PGN, 0.02-2 mu g mL(-1); ligand for NOD2) and beta-glucan (1-100 mu g mL(-1); ligand for dectin-1) in the presence or absence of histamine on mRNA expression of TLR2, NOD2 and dectin-1 as well as human beta-defensin 2 by quantitative real-time polymerase chain reaction in cultured normal human epidermal keratinocytes. TLR2 expression was also examined at the cell surface and intracellularly, as determined by flow cytometry and confocal microscopy. The quantities of interleukin (IL)-1 alpha and IL-8 produced by keratinocytes were measured using enzyme-linked immunosorbent assay.At the mRNA level, TLR2 was enhanced by PGN but not by its ligand MALP-2 or by beta-glucan; NOD2 was easily induced by all three ligands; and dectin-1 was enhanced by its ligand beta-glucan. These enhanced expressions were further augmented by histamine at 1 mu g mL(-1). While the surface expression of TLR2 was barely detectable by flow cytometry even after stimulation, the intracellular expression of TLR2 was apparently elevated by PGN and further promoted by histamine. A confocal microscopic analysis also revealed the enhanced expression of TLR2 in the cytoplasm. The expression of TLR2, NOD2 and dectin-1 was functional, as these pathogen-associated molecules induced the production of IL-1 alpha, IL-8 and defensin, and again, histamine greatly enhanced this production.Our study demonstrated that the expression of functional innate immune receptors is augmented by the pathogen-associated molecules in a ligand-feed forward or nonrelated manner in keratinocytes, and histamine promotes their expression and the resultant production of cytokines and defensins.