Toll-like Receptor-4 Mediates Lipopolysaccharide-induced Signal Transduction

Abstract

TLR4 is a member of the recently identified Toll-like receptor family of proteins and has been putatively identified as Lps, the gene necessary for potent responses to lipopolysaccharide in mammals. In order to determine whether TLR4 is involved in lipopolysaccharide-induced activation of the nuclear factor-B (NF-B) pathway, HEK 293 cells were transiently transfected with human TLR4 cDNA and an NF-B-dependent luciferase reporter plasmid followed by stimulation with lipopolysaccharide/CD14 complexes. The results demonstrate that lipopolysaccharide stimulates NF-Bmediated gene expression in cells transfected with the TLR4 gene in a dose-and time-dependent fashion. Furthermore, E5531, a lipopolysaccharide antagonist, blocked TLR4-mediated transgene activation in a dosedependent manner (IC 50 ϳ30 nM). These data demonstrate that TLR4 is involved in lipopolysaccharide signaling and serves as a cell-surface co-receptor for CD14, leading to lipopolysaccharide-mediated NF-B activation and subsequent cellular events. Lipopolysaccharide (LPS),1 a component of the outer membrane of Gram-negative bacteria, is a potent activator of a variety of mammalian cell types (1, 2). Activation by LPS constitutes the first step in a cascade of events believed to lead to the manifestation of Gram-negative sepsis, a condition that results in approximately 20,000 annual deaths in the United States (3). Activation of LPS-responsive cells, such as monocytes and macrophages, occurs rapidly after LPS interacts with circulating LPS-binding protein and CD14, a glycosylphosphatidylinositol-linked cell surface glycoprotein necessary for sensitive responses to LPS (1, 2). LPS has been shown to initiate multiple intracellular signaling events (4), including the activation of NF-B, which ultimately leads to the synthesis and release of a number of proinflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-␣ (1). However, since CD14 is not a transmembrane protein, it lacks the ability to transduce cytoplasmic signals (2), and before the recent discovery of Toll-like receptors (TLRs), the identity of a transmembrane protein that could relay LPS-induced signals across the cell-surface membrane remained elusive.Toll is a transmembrane receptor in Drosophila that is involved in dorsal-ventral patterning in embryos and in the induction of an anti-fungal response (5, 6). Activation of the Toll receptor by its ligand Spä tzle results in the interaction and stimulation of several signaling molecules that are homologous to proteins involved in NF-B activation by the IL-1 receptor in mammalian cells (7,8). The cloning of a family of human receptors structurally related to Drosophila Toll revealed five proteins that have extracellular domains that contain multiple leucine-rich repeats and cytoplasmic domains with sequence homology to the intracellular portion of the IL-1 receptor (9). Furthermore, constitutively active mutants of TLR2, TLR4, and TLR5 can induce the activation of NF-B (10, 11)...

PPARγ ameliorated LPS induced inflammation of HEK cell line expressing both human Toll-like receptor 4 (TLR4) and MD2

“…1L). (Peymani et al 2013).The effective concentration of LPS (1 lg/mL) was selected for further experiments as reported by Chow et al (1999). Cells were treated with LPS for 24 h, and then cells were harvested for expression analysis of two target genes, iNOS and TNFa by quantitative real time PCR analysis and Western blot analysis for NFjB.…”

“…Hence it is important for the activation of the host innate immune system (Poltorak et al 1998;Qureshi et al 1999;Hoshino et al 1999;Chow et al 1999;Shimazu et al 1999). Miyake's group reported an association between myeloid differentiation protein 2 (MD2) with TLR4 which was required for TLR4-mediated LPS-signaling (Wakabayashi et al 2006).…”

Vitamin D3 down‐regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen‐associated molecular patterns

“…As members of an evolutionary ancient first-line defence system, TLR detect the presence of conserved pathogen-associated molecular patterns (PAMP), which are produced by microorganisms. While lipopolysaccharide (LPS) signals mainly through TLR4 [4], TLR2 binds peptidoglycan, lipoteichoic acid (LTA), yeast-particle zymosan and the glycosylphosphatidylinositol anchor of Trypanosoma cruzi [5,6]. Moreover, binding of LPS is facilitated through the lipid-binding, glycosylphosphatidylinositolanchored membrane protein CD14 [7,8].…”

The PorB porin from commensal Neisseria lactamica induces Th1 and Th2 immune responses to ovalbumin in mice and is a potential immune adjuvant

“…Toll-Like receptors (TLRs) have recently been described as specific surface receptors expressed by APCs for recognition of pathogen associated molecular patterns (PAMPs) [37][38][39][40][41]. However, TLRs detect specific molecules which are not exclusive to pathogenic organisms.…”

“…However, TLRs detect specific molecules which are not exclusive to pathogenic organisms. For example, TLR4 recognizes LPS [37], which is present on all Gram-negative bacteria, including non-pathogenic bacteria, and TLR5 [42] recognizes flagellin [43], also expressed by most gut commensal [44]. Various TLR ligands have been shown to modulate the immune response [45][46][47] and act as vaccine adjuvants regardless of their pathogenic or non-pathogenic origin (i.e LPS, CpG DNA).…”