Immune responses are initiated when molecules of microbial origin are sensed by the Toll-like receptors (TLRs). We now report the identification of essential molecular components for the trafficking of the lipopolysaccharide (LPS) receptor complex. LPS was endocytosed by a receptormediated mechanism dependent on dynamin and clathrin and colocalized with TLR4 on early/sorting endosomes. TLR4 was ubiquitinated and associated with the ubiquitinbinding endosomal sorting protein hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs. Inhibition of endocytosis and endosomal sorting increased LPS signaling. Finally, the LPS receptor complex was sorted to late endosomes/lysosomes for degradation and loading of associated antigens onto HLA class II molecules for presentation to CD4 þ T cells. Our results show that endosomal trafficking of the LPS receptor complex is essential for signal termination and LPS-associated antigen presentation, thus controlling both innate and adaptive immunity through TLR4.

Toll-like receptor 4 (TLR4) is indispensable for recognition of Gram-negative bacteria. We described a trafficking pathway for TLR4 from the endocytic recycling compartment (ERC) to E. coli phagosomes. We found a prominent colocalization between TLR4 and the small GTPase Rab11a in the ERC, and Rab11a was involved in the recruitment of TLR4 to phagosomes in a process requiring TLR4 signaling. Also, Toll-receptor-associated molecule (TRAM) and interferon regulatory factor-3 (IRF3) localized to E. coli phagosomes and internalization of E. coli was required for a robust interferon-β induction. Suppression of Rab11a reduced TLR4 in the ERC and on phagosomes leading to inhibition of the IRF3 signaling pathway induced by E. coli, whereas activation of the transcription factor NF-κB was unaffected. Moreover, Rab11a silencing reduced the amount of TRAM on phagosomes. Thus, Rab11a is an important regulator of TLR4 and TRAM transport to E. coli phagosomes thereby controlling IRF3 activation from this compartment.

Lipoteichoic acid (LTA) is a central inducer of inflammatory responses caused by Gram-positive bacteria, such as Staphylococcus aureus, via activation of TLR2. Localization of TLR2 in relation to its coreceptors may be important for function. This study explores the signaling, uptake, and trafficking pattern of LTA in relation to expression of TLR2 and its coreceptors CD36 and CD14 in human monocytes. We found TLR2 expressed in early endosomes, late endosomes/lysosomes, and in Rab-11-positive compartments but not in the Golgi apparatus or endoplasmic reticulum (ER). Rapid internalization of fluorescently labeled LTA was observed in human monocytes, colocalizing with markers for early and late endosomes, lysosomes, ER, and Golgi network. Blocking CD14 and CD36 with antibodies inhibited LTA binding and LTA-induced TNF release from monocytes, emphasizing an important role for both molecules as coreceptors for TLR2. Importantly, blocking CD36 did not affect TNF release induced by N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl-seryl-(lysyl)3-lysine or LPS. Expression of CD14 markedly enhanced LTA binding to the plasma membrane and also enhanced NF-kappaB activation. LTA internalization, but not NF-kappaB activation, was inhibited in Dynamin-I K44A dominant-negative transfectants, suggesting that LTA is internalized by receptor-mediated endocytosis but that internalization is not required for signaling. In fact, immobilizing LTA and thereby inhibiting internalization resulted in enhanced TNF release from monocytes. Our results suggest that LTA signaling preferentially occurs at the plasma membrane, is independent of internalization, and is facilitated by CD36 and CD14 as coreceptors for TLR2.

Toll-like receptor 4 (TLR4) signals the induction of transcription factor IRF3-dependent genes from the early endosome via the adaptor TRAM. Here we report a splice variant of TRAM, TAG ('TRAM adaptor with GOLD domain'), which has a Golgi dynamics domain coupled to TRAM's Toll-interleukin 1 receptor domain. After stimulation with lipopolysaccharide, TRAM and TAG localized to late endosomes positive for the GTPase Rab7a. TAG inhibited activation of IRF3 by lipopolysaccharide. Knockdown of TAG with small interfering RNA enhanced induction of the chemokine CCL5 (RANTES), but not of interleukin 8, by lipopolysaccharide in human peripheral blood mononuclear cells. TAG displaced the adaptor TRIF from TRAM. TAG is therefore an example of a specific inhibitor of the adaptor MyD88-independent pathway activated by TLR4. Targeting TAG could be useful in the effort to boost the immunostimulatory effect of TLR4 without causing unwanted inflammation.

Thioglucoside glucohydrolase 1 (TGG1) is one of two known functional myrosinase enzymes in Arabidopsis. The enzyme catalyzes the hydrolysis of glucosinolates into compounds that are toxic to various microbes and herbivores. Transgenic Arabidopsis plants carrying ␤-glucuronidase and green fluorescent protein reporter genes fused to 0.5 or 2.5 kb of the TGG1 promoter region were used to study spatial promoter activity. Promoter activity was found to be highly specific and restricted to guard cells and distinct cells of the phloem. No promoter activity was detected in the root or seed. All guard cells show promoter activity. Positive phloem cells are distributed in a discontinuous pattern and occur more frequent in young tissues. Immunocytochemical localization of myrosinase in transverse and longitudinal sections of embedded material show that the TGG1 promoter activity reflects the position of the myrosinase enzyme. In the flower stalk, the myrosinase-containing phloem cells are located between phloem sieve elements and glucosinolate-rich S cells. Our results suggest a cellular separation of myrosinase enzyme and glucosinolate substrate, and that myrosinase is contained in distinct cells. We discuss the potential advantages of locating defense and communication systems to only a few specific cell types.Arabidopsis ecotype Columbia has been shown to contain 23 different glucosinolates (Hogge et al., 1988). Myrosinase (EC 3.2.3.1), also known as ␤-thioglucoside glucohydrolase, catalyzes the hydrolysis of glucosinolates into Glc and an unstable intermediate that undergoes nonenzymatic rearrangement to form sulfate and isothiocyanates, thiocyanates, nitriles, epithioalkanes, or elementary sulfur dependent on the concentration of H ϩ , metal ions, epithiospecifier protein, and/or other cofactors (Bones and Rossiter, 1996; Foo et al., 2000). The complexity of the myrosinase-glucosinolate system suggests a diverse and multifunctional role in the cruciferous plants.Glucosinolates are a diverse group of sulfurcontaining glycosides that may serve as a sink for nitrogen and sulfur, and the hydrolysis products may have important roles in the defense of the plant against microorganisms and insects (Bones and Rossiter, 1996; Rask et al., 2000). Previous studies have shown that myrosinase is located in idioblasts named myrosin cells, which have been found in several species of Brassicacea, including oilseed rape (Brassica napus), white mustard (Sinapis alba), cauliflower (B. oleracea), and Chinese cabbage (B. campestris; Bones and Iversen, 1985; Thangstad et al., 1990 Thangstad et al., , 1991 Bones et al., 1991; Hö glund et al., 1991 Hö glund et al., , 1992 Geshi et al., 1998). Myrosin cells occur as scattered cells in radicles, stems, leaves, petioles, seeds, and seedlings (Bones and Rossiter, 1996; Rask et al., 2000). The myrosinase enzyme has been localized to myrosin cells by immunocytochemical methods (Bones et al., 1991; Thangstad et al., 1990 Thangstad et al., , 1991 Hö glund et al., 1991; Geshi et al., 1998), a...