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.