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...oughout evolution as innate immunity, this type of activation of nonspecific defense mechanisms by infectious microorganisms uses germline-encoded receptors for the recognition of microbial pathogens =-=(25)-=-. A prominent example of such “pattern recognition receptors” is CD14, which is activated during the initial phase of bacterial infection in vertebrates (1, 2). CD14 binds LPS, which possesses a gener...

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...-1 and TNF as well as bacterial LPS are potent external stimuli regulating the activity of NF-�B transcription factors, which control the expression of numerous immune and inflammatory response genes =-=(26, 27)-=-. Members of the Toll receptor family share homology in their intracellular portions with the signaling domains of the IL-1R family, and Drosophila Toll and several mammalian TLRs have been shown to a...

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...nal transduction Bacterial LPS (also known as endotoxin), a constituent of the outer membrane of the cell wall of Gram-negative bacteria, is one of the main causative agents of septic shock in humans =-=(1, 2)-=-. Recognition of LPS is a key event in host antimicrobial defense reactions (1, 2). LPS is a complex glycolipid composed of a hydrophilic polysaccharide portion and a hydrophobic domain known as lipid...

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...greement with Toll signaling in Drosophila (3). In addition, overexpression of TLR4 (hToll) in 293 cells, which constitutively activates NF-�B, was recently found to involve IL-1 signaling components =-=(29, 30)-=-. In contrast to CD14, binding studies indicate that TLR2 does not bind LPS with high affinity (our unpublished observation). Concurrently, we demonstrate that TLR2 signaling requires CD14 to increase...

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...d in LPS signal transduction. Materials and Methods Cell Culture and Biological Reagents. A subclone of the human embryonic 293 cell line has been maintained at Tularik, Inc., since 1993 as described =-=(9)-=-. Mono-Mac-6 cells were licensed from Dr. H.W.L. Ziegler-Heitbrock (University of Munich, Munich, Germany). All other cell lines were obtained directly from the American Type Culture Collection (Rockv...

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...F-�B reporter gene activation (Fig. 3). This mutation precisely eliminates the most COOH-terminal block of amino acid homology within the cytoplasmic domains of members of the Toll and IL-1R families =-=(7)-=-. Serum Dependence of LPS Signaling through TLR2. To investigate the serum dependence of LPS signaling through TLR2, 293 cells transiently expressing TLR2 were stimulated with LPS over a wide concentr...

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...-1 and TNF as well as bacterial LPS are potent external stimuli regulating the activity of NF-�B transcription factors, which control the expression of numerous immune and inflammatory response genes =-=(26, 27)-=-. Members of the Toll receptor family share homology in their intracellular portions with the signaling domains of the IL-1R family, and Drosophila Toll and several mammalian TLRs have been shown to a...

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...ransducing component of the putative LPS receptor complex has remained elusive. The toll gene controls dorsoventral pattern formation during the early embryonic development of Drosophila melanogaster =-=(3)-=-. Interestingly, toll participates in antimicrobial immune responses upon infection in adult Drosophila (4). The Toll protein is a transmembrane receptor that displays resemblance in its intracellular...

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...r (TRAF)2, which suppresses NF-�B activation by TNF but not IL-1 in mammalian cells (22; Fig. 6). Conversely, a dominant-negative version of TRAF6, which inhibits NF-�B activation by IL-1 but not TNF =-=(22)-=-, also suppressed NF-�B activation by TLR2 (Fig. 6). In addition, a truncated version of MyD88, a constituent of the IL-1R complex (23, 24), inhibited both TLR2- and IL-1R–mediated NF-�B activation bu...

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...eporter gene induction was independent of serum conditions (Fig. 4). This is in agreement with observations that responsiveness to LPS at high concentrations bypasses the requirement for LBP and CD14 =-=(16)-=-. Role of CD14 in LPS Signaling by TLR2. The effect of CD14 on LPS signaling through TLR2 was further examined in transient transfection assays in 293 cells. When CD14 was coexpressed with TLR2, repor...

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...and IL-1–like receptors in signaling NF-�B activation. Elucidation of the NF-�B signaling cascades triggered by TNF and IL-1 has identified a number of distinct and shared components in both pathways =-=(28)-=-. Our findings indicate that LPS stimulation of TLR2 initiates an IL-1– rather than a TNF-like NF-�B activation pathway. This is in agreement with Toll signaling in Drosophila (3). In addition, overex...

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...l receptors share a characteristic repeating leucine-rich motif in their extracellular regions. Similar to IL-1Rs and Drosophila Toll, several TLRs have been shown to signal through the NF-�B pathway =-=(6, 8)-=-. In particular, functional analysis of TLR4 (also designated hToll) demonstrated that it is capable of inducing the expression of NF-�B–controlled genes for the inflammatory cytokines IL-1, IL-6, and...

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...nal transduction Bacterial LPS (also known as endotoxin), a constituent of the outer membrane of the cell wall of Gram-negative bacteria, is one of the main causative agents of septic shock in humans =-=(1, 2)-=-. Recognition of LPS is a key event in host antimicrobial defense reactions (1, 2). LPS is a complex glycolipid composed of a hydrophilic polysaccharide portion and a hydrophobic domain known as lipid...

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....5-cm dishes and transfected on the following day by the calcium phosphate precipitation method (11, 12) with the indicated amounts of expression vectors and 0.1 �g ELAM-1 luciferase reporter plasmid =-=(13)-=-. The cells were lysed for measurement of luciferase activity using reagents from Promega Corp. (Madison, WI). An RSV-�-galactosidase control plasmid (1 �g) was used for normalizing transfection effic...

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...ct composition may exist on different cell types. Recently, a biophysical model of LPS action has also been proposed which is dependent on LPS interactions with membrane lipids and endocytic movement =-=(32)-=-. It remains to be investigated if and how TLR2 might participate in such a signaling mechanism. Regardless of these considerations, the implication of TLR2 in LPS signaling supports an important role...

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...ably expressing Flag epitope–tagged TLRs or CD14 were generated by selection with G418 (GIBCO BRL) and subsequent FACS ® analysis as described (14). Nuclear extracts from 2 � 10 6 cells were prepared =-=(15)-=- and analyzed for NF-�B DNA-binding activity with a radiolabeled double-stranded oligonucleotide containing two NF-�B binding sites (15). Immunoprecipitation and Immunoblotting. For the detection of t...

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... TLR2 cDNA fragments. NF-�B Reporter Assay. Human embryonic kidney 293 cells (3 � 105 ) were plated in 3.5-cm dishes and transfected on the following day by the calcium phosphate precipitation method =-=(11, 12)-=- with the indicated amounts of expression vectors and 0.1 �g ELAM-1 luciferase reporter plasmid (13). The cells were lysed for measurement of luciferase activity using reagents from Promega Corp. (Mad...

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...GDNF), a member of the TGF-� ligand family. GDNF binds to the GPI-anchored GDNF receptor � and signals via the transmembrane receptor tyrosine kinase, RET, which itself does not appreciably bind GDNF =-=(31)-=-. Alternatively, the putative cellular LPS receptor may comprise additional components, or LPS receptors of distinct composition may exist on different cell types. Recently, a biophysical model of LPS...

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...oventral pattern formation during the early embryonic development of Drosophila melanogaster (3). Interestingly, toll participates in antimicrobial immune responses upon infection in adult Drosophila =-=(4)-=-. The Toll protein is a transmembrane receptor that displays resemblance in its intracellular portion to the signaling domains of members of the IL-1R family which mediate activation of NF-�B (3). Ind...

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... Electrophoretic Mobility Shift Assay. 293 cell clones stably expressing Flag epitope–tagged TLRs or CD14 were generated by selection with G418 (GIBCO BRL) and subsequent FACS ® analysis as described =-=(14)-=-. Nuclear extracts from 2 � 10 6 cells were prepared (15) and analyzed for NF-�B DNA-binding activity with a radiolabeled double-stranded oligonucleotide containing two NF-�B binding sites (15). Immun...

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... Electrophoretic Mobility Shift Assay. 293 cell clones stably expressing Flag epitope–tagged TLRs or CD14 were generated by selection with G418 (GIBCO BRL) and subsequent FACS ® analysis as described =-=(14)-=-. Nuclear extracts from 2 � 10 6 cells were prepared (15) and analyzed for NF-�B DNA-binding activity with a radiolabeled double-stranded oligonucleotide containing two NF-�B binding sites (15). Immun...