Trends in Microbiology
Innate recognition of lipopolysaccharide by Toll-like receptor 4–MD-2
Section snippets
Roles for Toll-like receptors (TLRs) in the decision to activate defense responses
A wide variety of bacterial components are capable of stimulating the innate immune system. These include lipopolysaccharide (LPS), peptidoglycan, lipoteichoic acid, lipoarabinomannan, lipopeptides and bacterial DNA. These microbial products activate keratinocytes, epithelial cells, macrophages, PMNLs and mast cells using innate immune receptors, leading to the production of inflammatory responses that activate adaptive immunity.
Innate immunity uses a variety of innate immune receptors
Bacterial infection causes sepsis
Bacterial infections can be associated with sepsis, a serious condition that results from a damaging host response to infection [9]. Inflammatory responses are advantageous for the eradication of bacteria, as long as they are under the control. However, once out of the control, deregulated inflammation leads to massive production of proinflammatory cytokines, such as tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6 by macrophages. Exaggerated production of cytokines leads to coagulation
LPS as a prototype of TLR ligands
LPS, a glycolipid component of the outer membrane of Gram-negative bacteria, exhibits the most potent immunostimulating activity of the TLR ligands [10]. Trace amounts of LPS are able to activate the innate immune system, leading to an array of proinflammatory mediators being produced, such as TNF, IL-1 and IL-6. Therefore, LPS is generally acknowledged to play a central role not only in eliciting inflammatory responses but also in causing septic shock during Gram-negative bacterial infection
LPS processing and signaling
LPS constitutes the outer layer of the outer membrane of Gram-negative bacteria and its composition changes according to the origin of the bacteria. LPS from Escherichia coli consists of three covalently linked regions: lipid A, a core oligosaccharide and an O side chain (Figure 1). The inner most layer, lipid A, which is responsible for LPS toxicity, consists of six fatty acyl chains linked in various ways to two glucosamine residues. The branched core oligosaccharide contains ten saccharide
LPS clearance by LBP and CD14
LPS processing facilitates not only the LPS response, but also LPS clearance (Figure 2). LBP can catalyze the movement of LPS from the outer membrane of Gram-negative bacteria directly to high-density lipoprotein (HDL) particles [23] in which LPS loses its biological activity. Soluble CD14 also facilitates LPS clearance by delivering LPS to HDL [24]. Neutralized LPS in HDL particles is excreted from the liver. LPS clearance consequently leads to the termination of LPS responses through
TLR4 delivers an LPS signal
TLR4, the first mammalian homologue of Drosophila Toll to be discovered [28], works downstream of CD14 and is responsible for delivering an LPS signal. Positional cloning analysis of the LPS-nonresponsive mouse strain C3H/HeJ revealed a point mutation that replaces proline712 with histidine in the signaling domain of the TLR4 protein [11]. Another LPS-nonresponsive mutant strain, C57BL10/ScCr, lacked the entire genomic region for the TLR4 gene. These results were confirmed by targeting of the
MD-2: a component of the LPS recognition complex
Although TLR4 is indispensable for LPS signaling, in vitro studies suggested that another molecule was also required. In vitro transfection of TLR4 cDNA did not confer LPS responsiveness to the human embryonic kidney (HEK)-derived 293 cell line or to a mouse IL-3-dependent pro-B cell line Ba/F3 [12]. LPS unresponsiveness of these TLR4-expressing cells was subsequently explained by the discovery of another component of the LPS recognition complex, MD-2. Molecular cloning of MD-2 was preceded by
The interaction between MD-2 and TLR4
Studies using mouse and human MD-2 mutants revealed amino acid residues that are crucial for interaction with TLR4 43, 44, 45. Binding of MD-2 to TLR4 was dependent on Cys95, Tyr102 and Cys105 in both mice and humans. It is possible that a region including these amino acids in the MD-2 protein constitutes the binding site for TLR4.
An important role for MD-2 in cell surface expression of TLR4 was revealed by in vitro studies using cells that lack mouse MD-2 [42]. In murine MD-2−/− fibroblast
Interaction between LPS and MD-2
A recent study using alanine-scanning mutagenesis revealed that MD-2 plays a role not only in cell surface expression of TLR4 but also in the LPS response. Mouse MD-2 mutants, in which a single alanine mutation was introduced at Tyr34, Tyr36, Gly59, Val82, Ile85, Phe126, Pro127, Gly129, Ile153, Ile154 and His155, showed normal ability to form the cell surface TLR4–MD-2 complex, but apparently had reduced ability to confer LPS responsiveness [44]. These amino acid residues might have a role in
LPS interaction with TLR4–MD-2 in responding cells
It is important to study where and how LPS interacts with TLR4–MD-2. Previous results with HEK 293 cells transiently expressing human CD14 and human TLR4–MD-2 demonstrated that LPS and TLR4–MD-2 were chemically cross-linked with each other, suggesting a direct interaction between the two [51]. In keeping with these human studies, Akashi et al. [52] demonstrated that the cell surface complex consists of LPS and mouse TLR4–MD-2. Membrane CD14 greatly enhances the formation of LPS–TLR4–MD-2
Roles for MD-2 in TLR4 signaling
LPS recognition begins with its interaction with TLR4–MD-2 and ends with an activation signal from TLR4. LPS signal is suggested to be triggered by the dimerization of the cytoplasmic domain (Figure 2), because chimeras that contained the self-interacting extracellular domains from integrins fused with the TLR4 cytoplasmic domain were constitutively active [54]. A lipid A antagonist, E5531, was shown to block LPS interaction with TLR4–MD-2 [52], probably through direct interaction with
Concluding remarks
Our immune system is able to sense the presence of LPS even at less than 1 ng per ml. Recent studies have revealed physical interactions between LPS and LPS recognition molecules, such as TLR4 and MD-2. It is, however, not fully understood how LPS interacts with TLR4–MD-2. Moreover, it is not clear how TLR4–MD-2 recognizes ligands other than LPS. Recently, progress has been made in understanding the complex recognition of LPS by TLR4, specifically that it requires additional molecules, such as
References (61)
The role of mannose-binding lectin in health and disease
Mol. Immunol.
(2003)Structure and function of the pentraxins
Curr. Opin. Immunol.
(1995)Pattern recognition receptors: doubling up for the innate immune response
Cell
(2002)Lipopolysaccharide binding protein-mediated complexation of lipopolysaccharide with soluble CD14
J. Biol. Chem.
(1995)Structure-function analysis of CD14 as a soluble receptor for lipopolysaccharide
J. Biol. Chem.
(2000)Identification of a lipopolysaccharide binding domain in CD14 between amino acids 57 and 64
J. Biol. Chem.
(1995)Regions of the mouse CD14 molecule required for toll-like receptor 2- and 4-mediated activation of NF-kappa B
J. Biol. Chem.
(2002)Resistance to endotoxin shock and reduced dissemination of gram-negative bacteria in CD14-deficient mice
Immunity
(1996)Lysines 128 and 132 enable lipopolysaccharide binding to MD-2, leading to toll-like receptor-4 aggregation and signal transduction
J. Biol. Chem.
(2003)Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4–MD-2–CD14 complex in a process that is distinct from the initiation of signal transduction
J. Biol. Chem.
(2002)