Toll-like receptor 4: the missing link of the cerebral innate immune response triggered by circulating gram-negative bacterial cell wall components.

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.