Laflamme N, Rivest S
Laboratory of Molecular Endocrinology, CHUL Research Center and Department of Anatomy and Physiology, Laval University, Québec, Canada G1V 4G2.
FASEB J. 2001 Jan;15(1):155-163. doi: 10.1096/fj.00-0339com.
The recent characterization of human homologues of Toll may be the missing link for the transduction events leading to NF-kappaB activity and proinflammatory gene transcription during innate immune response. Indeed, CD14 is not thought to participate directly in the cell signaling, but rather one or more of the mammalian Toll-like receptors (TLRs) acts in concert with the lipopolysaccharide (LPS) receptor to discriminate between microbial pathogens or their products and initiate transmembrane signaling. Mammalian cells may express as many as 10 distinct TLRs, although the importance of TLR4 in response to gram-negative bacteria and LPS is now supported by the fact that TLR4-mutated mice are LPS resistant. We investigated the expression of TLR4 across the rat brain under basal conditions and in response to systemic LPS and IL-1beta injection. We first cloned the rat TLR4 cDNA via RNA isolation and polymerase chain reaction (PCR) amplification with a proofreading polymerase. Total RNA was isolated from the rat liver tissue using Tri-Reagent and reverse transcribed into cDNA using Superscript II reverse transcriptase and an oligonucleotide primer with a degenerate 3' end of sequence 5'-T12(GAC)N-3'. Positive hybridization signal was found in the leptomeninges, choroid plexus (chp), subfornical organ, organum vasculosum of the lamina terminalis, median eminence, and area postrema. Scattered small cells also displayed a convincing hybridization signal within the brain parenchyma. Few well-defined nuclei exhibited positive TLR4 transcript: the supramamillary nucleus, cochlear nucleus, and the lateral reticular nucleus. The circumventricular organs, the leptomeninges, and chp also exhibited constitutive expression of the LPS receptor mCD14. In contrast to the strong up-regulation of the gene encoding mCD14 during endotoxemia, neither LPS nor IL-1beta caused a convincing increase in the TLR4 mRNA levels across the CNS. A down-regulation of the gene encoding TLR4 was found in the cerebral tissue of immune-challenged animals. The constitutive expression of both mCD14 and TLR4 may explain the innate immune response in the brain, which originates from the structures devoid of blood-brain barrier in presence of circulating LPS.
Toll蛋白人类同源物的最新特性，可能是天然免疫反应过程中导致NF-κB活性和促炎基因转录的转导事件中缺失的环节。实际上，人们认为CD14并不直接参与细胞信号传导，而是一种或多种哺乳动物Toll样受体（TLR）与脂多糖（LPS）受体协同作用，以区分微生物病原体或其产物，并启动跨膜信号传导。哺乳动物细胞可能表达多达10种不同的TLR，尽管TLR4在应对革兰氏阴性菌和LPS方面的重要性现在已得到如下事实的支持：TLR4突变的小鼠对LPS具有抗性。我们研究了基础条件下以及响应全身注射LPS和IL-1β后大鼠脑中TLR4的表达情况。我们首先通过RNA分离和使用校正聚合酶的聚合酶链反应（PCR）扩增克隆大鼠TLR4 cDNA。使用Tri-Reagent从大鼠肝脏组织中分离总RNA，并使用Superscript II逆转录酶和具有5'-T12(GAC)N-3'序列的简并3'末端的寡核苷酸引物将其逆转录为cDNA。在软脑膜、脉络丛（chp）、穹窿下器官、终板血管器、正中隆起和最后区发现了阳性杂交信号。散在的小细胞在脑实质内也显示出令人信服的杂交信号。少数明确的核显示TLR4转录本呈阳性：乳头体上核、耳蜗核和外侧网状核。室周器官、软脑膜和chp也显示出LPS受体mCD14的组成性表达。与内毒素血症期间编码mCD14的基因强烈上调相反，LPS和IL-1β均未导致整个中枢神经系统中TLR4 mRNA水平有明显升高。在免疫应激动物的脑组织中发现编码TLR4的基因下调。mCD14和TLR4的组成性表达可能解释了脑中的天然免疫反应，这种反应在循环LPS存在的情况下起源于缺乏血脑屏障的结构。
