The structural features of some proteins of the innate immune system involved in mediating responses to microbial pathogens are highly conserved throughout evolution. Examples include members of the Drosophila Toll (dToll) and the mammalian Toll-like receptor (TLR) protein families. Activation of Drosophila Toll is believed to occur via an endogenous peptide rather than through direct binding of microbial products to the Toll protein. In mammals there is a growing consensus that lipopolysaccharide (LPS) initiates its biological activities through a heteromeric receptor complex containing CD14, TLR4, and at least one other protein, MD-2. LPS binds directly to CD14 but whether LPS then binds to TLR4 and/or MD-2 is not known. We have used transient transfection to express human TLRs, MD-2, or CD14 alone or in different combinations in HEK 293 cells. Interactions between LPS and these proteins were studied using a chemically modified, radioiodinated LPS containing a covalently linked, UV light-activated crosslinking group ( 125 I-ASD-Re595 LPS). Here we show that LPS is cross-linked specifically to TLR4 and MD-2 only when co-expressed with CD14. These data support the contention that LPS is in close proximity to the three known proteins of its membrane receptor complex. Thus, LPS binds directly to each of the members of the tripartite LPS receptor complex. Bacterial endotoxin (lipopolysaccharide, LPS)1 okonp61 is a complex glycolipid composed of a hydrophilic polysaccharide moiety and a hydrophobic domain known as lipid A (1). LPS is an outer membrane constituent of all Gram-negative bacteria where it has indispensable barrier functions. LPS is also a potent activator of innate immune responses that result in the production of pro-and anti-inflammatory mediators from myeloid lineage and other cell types (2). LPS-induced cell activation depends on the presence of three proteins comprising a multiprotein cell surface receptor complex here termed the LPS receptor complex. One essential protein of the LPS receptor complex is CD14 (3), a 55-kDa glycoprotein present in soluble form (sCD14) in blood or as a membrane-bound form (mCD14) in myeloid lineage cells. This latter form is attached to the outer leaflet of the cell membrane via a glycosylphosphatidylinositol anchor. Multiple lines of biochemical and genetic evidence support the contention that CD14 principally acts to bind LPS and does not participate in signaling directly. Thus, others and we postulated that there must be at least one transmembrane protein that acts in concert with CD14 (2). This putative transmembrane protein is now identified as a member of the mammalian Toll-like receptor (TLR) family and is TLR4 (4, 5). Genetic and biochemical studies suggest that TLR4 plays an important role in LPS signaling under physiological conditions. Positional cloning and sequencing of the lps d locus localized the defect to the tlr4 gene (6). The importance of TLR4 in LPS signaling is further supported by the fact that TLR4-deficient mice are LPS hyporesponsive but resp...

Endotoxin (lipopolysaccharide; LPS) activates a wide variety of host defense mechanisms. In mammals LPS binding protein (LBP) and CD14 interact with LPS to mediate cellular activation. Using sucrose density gradients and a fluorescent endotoxin derivative we have investigated the mechanism of LPS binding to LBP and the soluble form of CD14 (sCD14). LPS binds to LBP to form two types of complex; at low ratios of LPS to LBP complexes with one molecule of LBP and 1-2 molecules of LPS predominate, while at high ratios of LPS to LBP a large aggregate of LBP and LPS predominates. Complexes of LPS with sCD14 do not form large aggregates, consisting of only 1-2 LPS bound to a single sCD14 even at high multiples of LPS to sCD14. LBP catalyzes LPS binding to sCD14. Catalysis by LBP apparently occurs because LBP provides a pathway for LPS to bind to sCD14 which avoids the necessity for LPS monomers in aqueous solution. The dissociation constants for LPS.LBP and LPS.sCD14 complexes were determined to be 3.5 x 10(-9) and 29 x 10(-9) M, respectively. These numbers suggest that when LBP and sCD14 are present at roughly equal concentrations as they are in normal human plasma and compete for limited LPS, the LPS will predominantly associate with LBP.

We have reported that human, murine, and lapine acute phase sera differ functionally from the respective normal sera in at least three ways (1, 2), insofar as interactions with bacterial LPS are concerned. First, the rate at which LPS binds to high density lipoprotein (HDL)' is much faster in normal sera than in acute phase sera. For example, in normal sera, the half time for binding of Salmonella minnesota Re595 LPS to HDL at 37°C is typically 2-3 min, whereas in acute phase sera, the same reaction can have a half time of up to 100 min . Second, when Re595 LPS is added to serum and spun to equilibrium in a CsCl density gradient, the LPS not bound to HDL is found at a density of 1 .33 g/cm3 in normal sera, whereas in acute phase sera the LPS forms a complex with a density of 1 .3 g/cm 3 . We refer to the form of LPS in acute phase serum as complex 1 .3 (C .1 .3). Finally, when sera containing LPS not yet bound to HDL are chilled rapidly to 4°C and dialysed against very low ionic strength buffer at 4°C an LPS-protein precipitate is formed from both normal and acute phase sera, but the LPS in the precipitate from normal serum redissolves readily in isotonic saline, while the LPS in the precipitate from acute phase serum does not. These phenomena have been seen after acute phase induction with either subcutaneous silver nitrate, intraperitoneal LPS, or casein (unpublished data). We have postulated the existence of a hitherto unrecognized acute phase reactant to explain these findings .In this report, a purification of the acute phase reactant from acute phase rabbit serum (APRS) responsible for the phenomena noted above is described, as are some of the properties of the reactant . As shown by experiments reported herein, the acute phase reactant does bind directly to LPS; therefore, we refer to it as LPS-binding protein, or LBP. Materials and MethodsMaterials . Biosynthetically tritiated LPS ([ 3H]LPS) and unlabeled LPS were isolated from Salmonella minnesota Re595 as described previously (2, 3) . Rabbit blood was collected

CD14 is a well-known pattern-recognition receptor in the innate immune system. Here, we show that CD14 enhances double-stranded RNA (dsRNA)-mediated Toll-like receptor 3 (TLR3) activation. Bone marrow-derived macrophages (BMDMs) from CD14-/- mice exhibited impaired responses to polyinosine-polycytidylic acid (pIpC) and reduced production of inflammatory cytokines. CD14-/- mice injected with pIpC also showed impaired cytokine production. When tested with [32P] labeled pIpC small fragments (pIpCsf) that maintain the inflammatory activity of crude pIpC, CD14 directly bound pIpCsf and mediated cellular uptake of pIpCsf. Our data show that TLR3 is intracellular and directly interacts with CD14. Internalized pIpCsf was localized in the lysosomes via the endosomes. In unstimulated cells, neither CD14 nor TLR3 was detected in the lysosomes. However, TLR3 was localized in the lysosomes as was CD14 once the cells took up pIpC. We also observed that internalized pIpCsf colocalized with CD14 and TLR3. Consequently, CD14 mediates pIpC uptake and enhances TLR3 signaling.

Many LPS binding proteins have been described, but the exact nature of the LPS receptors that signal cells remains unclear. MD-2 is a molecule that is found in association with Toll-like receptor 4, which has been shown to be a receptor for LPS. We have produced human MD-2 in baculovirus and tested it for LPS binding. MD-2 binds the lipid A region of LPS without the need for LPS binding protein. These data suggest that MD-2 may be binding LPS as part of the TLR4 receptor complex.

Endothelial cells are activated by microbial agonists through Toll-like receptors (TLRs) to express inflammatory mediators; this is of significance in acute as well as chronic inflammatory states such as septic shock and atherosclerosis, respectively. We investigated mechanisms of lipopolysaccharide (LPS)-induced cell activation in human coronary artery endothelial cells (HCAEC) using a combination of FACS, confocal microscopy, RT-PCR, and functional assays. We found that TLR4, in contrast to TLR2, is not only located intracellularly but also functions intracellularly. That being the case, internalization of LPS is required for activation. We further characterized the HCAEC LPS uptake system and found that HCAEC exhibit an effective LPS uptake only in the presence of LPS binding protein (LBP). In addition to its function as a catalyst for LPS-CD14 complex formation, LBP enables HCAEC activation at low LPS concentrations by facilitating the uptake, and therefore delivery, of LPS-CD14 complexes to intracellular TLR4-MD-2. LBP-dependent uptake involves a scavenger receptor pathway. Our findings may be of pathophysiological relevance in the initial response of the organism to infection. Results further suggest that LBP levels, which vary as LBP is an acute phase reactant, could be relevant to initiating inflammatory responses in the vasculature in response to chronic or recurring low LPS.

TLR4 is the primary recognition molecule for inflammatory responses initiated by bacterial LPS (endotoxin). Internalization of endotoxin by various cell types is an important step for its removal and detoxification. Because of its role as an LPS-signaling receptor, TLR4 has been suggested to be involved in cellular LPS uptake as well. LPS uptake was investigated in primary monocytes and endothelial cells derived from TLR4 and CD14 knockout C57BL/6 mice using tritiated and fluorescein-labeled LPS. Intracellular LPS distribution was investigated by deconvolution confocal microscopy. We could not observe any difference in LPS uptake and intracellular LPS distribution in either monocytes or endothelial cells between TLR4−/− and wild-type cells. As expected, CD14−/− monocytes showed a highly impaired LPS uptake, confirming CD14-dependent uptake in monocytes. Upon longer incubation periods, the CD14-deficient monocytes mimicked the LPS uptake pattern of endothelial cells. Endothelial cell LPS uptake is slower than monocyte uptake, LBP rather than CD14 dependent, and sensitive to polyanionic polymers, which have been shown to block scavenger receptor-dependent uptake mechanisms. We conclude that TLR4 is not involved in cellular LPS uptake mechanisms. In membrane CD14-positive cells, LPS is predominantly taken up via CD14-mediated pathways, whereas in the CD14-negative endothelial cells, there is a role for scavenger receptor-dependent pathways.

Lipopolysaccharide (LPS)-binding protein (LBP) andbactericidal/permeability-increasing protein (BPI) are closely related LPS-binding proteins whose binding to LPS has markedly different functional consequences. To gain better insight into the possible basis of these functional differences, the physical properties of LBP-LPS and BPI-LPS complexes have been compared in this study by sedimentation, light scattering, and fluorescence analyses. These studies reveal dramatic differences in the physical properties of LPS complexed to LBP versus BPI. They suggest that of the two proteins, only LBP can disperse LPS aggegates. However, BPI can enhance both the sedimentation velocity and apparent size of LPS aggregates while inhibiting LPS-LBP binding even at very low (1:40 to 1:20) BPI:LPS molar ratios.The lipopolysaccharide (LPS)-binding protein 1 (LBP) and the bactericidal/permeability-increasing protein (BPI) are both LPS-interactive mammalian proteins with approximately 45% amino acid sequence identity (1, 2). LPS is considered to be the principal component of Gram-negative bacteria that alerts the host to invading bacteria and triggers defensive responses (3, 4). These responses are usually beneficial and effective but may also become excessive and lead to endotoxic shock (3-5). Both LBP and BPI modulate the bioactivity of LPS (2,3,5). LBP is a plasma protein that catalyzes the transfer of LPS from LPS aggregates to other LPS-binding proteins (3, 6 -9). Prominent among these is CD14, a surface molecule of myeloid cells that is also present in the circulation as a soluble protein. LBP and CD14 together represent the main pathway by which cells recognize low concentrations of LPS and are stimulated to respond to 10,11). In contrast to the LPS-stimulatory properties of LBP, binding of LPS by BPI results in inhibition of the bioactivities of LPS (2). BPI is produced by polymorphonuclear leukocytes and stored in its azurophilic granules (12, 13). It contributes substantially to both the intracellular and extracellular antibacterial activity of polymorphonuclear leukocyte-rich inflammatory exudates toward Gram-negative bacteria (14, 15). The high affinity of BPI for LPS accounts for the target-cell specificity of the antibiotic activity of BPI for Gram-negative bacteria (2, 16). In contrast to BPI, binding of LBP to bacterial envelope LPS does not produce detectable membrane alterations or other antibacterial effects.We have recently described the use of fluorescein-labeled LPS (FITC-LPS) as well as metabolically labeled ([ 3 H]LPS) and sucrose density gradients to characterize the formation and physical properties of complexes of LPS with LBP and the soluble form of CD14 (7). In this report we describe results obtained by studying the binding of FITC-LPS and [ 3 H]LPS to BPI using the techniques described earlier and in addition compare the effects of LBP and BPI on the light scattering properties of LPS. The results obtained reveal striking differences in the physical properties of LBP-LPS and BPI-LPS complexes. Thus, un...