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...

Cytoplasmic innate immune receptors are important therapeutic targets for diseases associated with overproduction of proinflammatory cytokines. One cytoplasmic receptor complex, the Nlrp3 inflammasome, responds to an extensive array of molecules associated with cellular stress. Under normal conditions, Nlrp3 is autorepressed, but in the presence of its ligands, it oligomerizes, recruits apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc), and triggers caspase 1 activation and the maturation of proinflammatory cytokines such as IL-1β and IL-18. Because ischemic tissue injury provides a potential source for Nlrp3 ligands, our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in components of the Nlrp3 inflammasome (Nlrp3−/− and Asc−/− mice). To examine the role of the inflammasome in renal ischemia-reperfusion injury (IRI) we also tested its downstream targets caspase 1, IL-1β, and IL-18. Both Nlrp3 and Asc were highly expressed in renal tubular epithelium of humans and mice, and the absence of Nlrp3, but not Asc or the downstream inflammasome targets, dramatically protected from kidney IRI. We conclude that Nlrp3 contributes to renal IRI by a direct effect on renal tubular epithelium and that this effect is independent of inflammasome-induced proinflammatory cytokine production.

The lipopolysaccharide (LPS) receptor is a multi-protein complex that consists of at least three proteins, CD14, TLR4, and MD-2. Because each of these proteins is glycosylated, we have examined the functional role of N-linked carbohydrates of both MD-2 and TLR4. We demonstrate that MD-2 contains 2 N-glycosylated sites at positions Asn 26 and Asn 114 , whereas the amino-terminal ectodomain of human TLR4 contains 9 N-linked glycosylation sites. Site-directed mutagenesis studies showed that cell surface expression of MD-2 did not depend on the presence of either N-linked site, whereas in contrast, TLR4 mutants carrying substitutions in Asn 526 or Asn 575 failed to be transported to the cell surface. Using a UV-activated derivative of Re595 LPS (ASD-Re595 LPS) in cross-linking assays, we demonstrated a critical role of MD-2 and TLR4 carbohydrates in LPS cross-linking to the LPS receptor. The ability of the various glycosylation mutants to support cell activation was also evaluated in transiently transfected HeLa cells. The double mutant of MD-2 failed to support LPS-induced activation of an interleukin-8 (IL-8) promoter-driven luciferase reporter to induce IL-8 secretion or to activate amino-terminal c-Jun kinase (JNK). Similar results were observed with TLR4 mutants lacking three or more N-linked glycosylation sites. Surprisingly, the reduction in activation resulting from expression of the Asn mutants of MD-2 and TLR4 can be partially reversed by co-expression with CD14. This suggests that the functional integrity of the LPS receptor depends both on the surface expression of at least three proteins, CD14, MD-2, and TLR4, and that N-linked sites of both MD-2 and TLR4 are essential in maintaining the functional integrity of this receptor.A family of related genes encoding the 10 known Toll-like receptors (TLR) 1 is involved in innate immune responses in mammals (1-4). Engagement of TLRs by specific products of the pathogen, i.e. bacterial endotoxin or lipopolysaccharide (LPS) (5-7), bacterial DNA (8), outer membrane lipoproteins and lipopeptides (9 -11), and bacterial flagellin protein (12), results in activation of innate immune responses. Among these substances LPS is considered to be the prototypic activator (13,14). The functional membrane receptor for LPS is comprised of at least three proteins, CD14, TLR4, and MD-2. Binding of LPS to TLR4 and MD-2 is enhanced by CD14 and the plasma protein LPS-binding protein (LBP) (15).Although there have been a number of detailed structurefunction analyses for both , only limited information is available for MD-2 or TLR4. The mature human MD-2 and TLR4 sequences deduced from cDNA contain 160 and 839 amino acids residues, respectively. Both proteins have potential N-linked glycosylation sites. Sequence analysis suggests MD-2 has two potential N-linked glycosylation sites, whereas the ectodomain of TLR4 contains nine. Herein we describe the properties of MD-2 and TLR4 mutants lacking the potential N-linked glycosylation sites. We compared the mutated proteins with the parent...

The antiapoptotic properties of the inhibitor of apoptosis (IAP) family of proteins have been linked to caspase inhibition. We have previously described an alternative mechanism of XIAP inhibition of apoptosis that depends on the selective activation of JNK1. Here we report that two other members of the IAP family, NAIP and ML-IAP, both activate JNK1. Expression of catalytically inactive JNK1 blocks NAIP and ML-IAP protection against ICE-and TNF-␣-induced apoptosis, indicating that JNK1 activation is necessary for the antiapoptotic effect of these proteins. The MAP3 kinase, TAK1, appears to be an essential component of this antiapoptotic pathway since IAP-mediated activation of JNK1, as well as protection against TNF-␣-and ICE-induced apoptosis, is inhibited when catalytically inactive TAK1 is expressed. In addition, XIAP, NAIP, and JNK1 bind to TAK1. Importantly, expression of catalytically inactive TAK1 did not affect XIAP inhibition of caspase activity. These data suggest that XIAP's antiapoptotic activity is achieved by two separate mechanisms: one requiring TAK1-dependent JNK1 activation and the second involving caspase inhibition.

Current evidence indicates that the chronic inflammation observed in the intestines of patients with inflammatory bowel disease is due to an aberrant immune response to enteric flora. We have developed a lipid A-mimetic, CRX-526, which has antagonistic activity for TLR4 and can block the interaction of LPS with the immune system. CRX-526 can prevent the expression of proinflammatory genes stimulated by LPS in vitro. This antagonist activity of CRX-526 is directly related to its structure, particularly secondary fatty acyl chain length. In vivo, CRX-526 treatment blocks the ability of LPS to induce TNF-α release. Importantly, treatment with CRX-526 inhibits the development of moderate-to-severe disease in two mouse models of colonic inflammation: the dextran sodium sulfate model and multidrug resistance gene 1a-deficient mice. By blocking the interaction between enteric bacteria and the innate immune system, CRX-526 may be an effective therapeutic molecule for inflammatory bowel disease.